Journal of Reproductive Immunology

Editorial Board

2010-08-01

SCIENTIFIC PROGRAM

2010-06-21

Gamete immunology: achievements and perspectives

K. Koyama — 2010-08-01

Undoubtedly, the main task of our International Society for Immunology of Reproduction (ISIR) is to advance knowledge of reproductive immunology and medicine and to promote reproductive health, in collaboration with national and regional societies of reproductive immunology. Since the 1st ISIR Congress in Paris in 1980, impressive progress has been made in our understanding of the cellular and molecular basis of various reproductive phenomena and now reproductive immunology is one of the most rapidly advancing fields of biomedical science.

Stress during pregnancy: maternal endocrine–immune imbalances and fetal health

P. Arck — 2010-08-01

High stress perception is frequently mentioned when discussing causes of infertility and unexplained reproductive failures. However, less severe pregnancy outcomes are of particular importance to population health. An emerging area of research called ‘developmental origins of health and disease’ currently focuses on the identification of environmental insults during pregnancy. Such environmental insults have been proposed to induce permanent neuroendocrine and immunological functional changes to the offspring, causing an increased susceptibility to develop chronic disease later in life. Maternal stress perception during pregnancy has been confirmed as a potent environmental factor which can program the fetus leading to chronic diseases such as schizophrenia or depression later during post-natal life. Disorders of the immune system leading to allergies, inflammatory bowel disease, and atherosclerosis also merit consideration since epidemiological data evidence a steady rise in the incidence of such disorders over the past five decades. Our research pursued to date employing cohort studies and fundamental research reveals insights on how maternal stress perception during pregnancy generates a risk for the offspring to develop chronic diseases in later life, such as allergies, atherosclerosis and anxiety-like behaviour. The identification of relevant endocrine and immune mechanisms involved in stress-induced altered fetal/neonatal development will subsequently allow the development of primary prevention strategies aiming to abrogate the effect of maternal stress on the developing fetus.

Maternal antibodies and fetal brain development

L. Wang, J. Lee, P. Huerta, B. Volpe, B. Diamond — 2010-08-01

Brain-reactive potentially neurotoxic antibodies do not affect the adult brain because the brain is sequestered by the blood–brain barrier (BBB). The fetus is protected from all maternal antibodies until after the first trimester of gestation. At that time all fetal organs are exposed to maternal antibody, including the fetal brain. The mother may, therefore be unaffected by potentially neurotoxic antibody unless there is a break in barrier integrity while fetal brain development is impaired.

Preeclampsia and intra-uterine growth restriction: the role of the father

G.A. Dekker, C.T. Roberts — 2010-08-01

Preeclampsia is a disease of an individual couple with primarily maternal and fetal manifestations. Factors that are unique to a specific couple include the length and type of sexual relationship, the maternal acceptance of the invading cytotrophoblast and seminal fluid levels of transforming growth factor-β and other cytokines. The maternal and fetal genomes perform different roles during development. Heritable paternal, rather than maternal, imprinting of the genome is necessary for normal trophoblast development. Preeclampsia may relate to extreme genetic conflict, or a mother unable to cope with a ‘physiologic’ genetic conflict. The paternal contribution to preeclampsia is demonstrated by four factors. (1) The effect of the length of sexual relationship. The SCOPE consortium has recently published the results of a large prospective study in nulliparous women providing further evidence that short duration of sexual relationship prior to conception is an independent risk factor for both preeclampsia and ‘placental’ small for gestational age. (2) The concept of primipaternity versus primigravidity. In 2002, the primipaternity concept was challenged by Skjaerven et al. According to these authors, prolonged birth interval and not paternity change was the explanation for the increased preeclampsia risk in multiparous women with new partners. More critical analysis of their data and the results of various subsequent studies have however clearly demonstrated that the primipaternity concept still stands. (3) Existence of the so-called ‘dangerous’ father has been demonstrated in various large population studies. Men who have fathered one preeclamptic pregnancy are nearly twice as likely to father a preeclamptic pregnancy in a different woman. It is currently not certain how the father exerts this effect. (4) Paternal booking characteristics. Advanced paternal age (>45 years) has been demonstrated to almost double the risk of preeclampsia, while paternal obesity and central adiposity may be independent risk factors for infants who are small for gestational age by customized birth weight centiles.

The influence of fetal sex on the fetal–placental adaptation to stressors

V.L. Clifton — 2010-08-01

There are known sex specific differences in fetal and neonatal morbidity and mortality but the mechanisms are unknown. Similarly there are known sex differences in adult physiology relating to brain, immune, cardiovascular and liver function. These data indicate from in utero life to adult life, males and females respond differently to the same physiological and pathophysiological events. In the case of maternal asthma during pregnancy, we have identified that the fetal–placental unit institutes sex specific strategies to cope with the presence of the disease. From our prospective cohort studies, female fetal growth was reduced and placental function was altered in the presence of mild asthma and no inhaled glucocorticoid use. If asthmatic mothers were treated with inhaled glucocorticoids, female fetal growth was comparable to the non-asthmatic population. These data suggest the inflammatory effects of the disease affect female fetal growth. We have recently reported that inflammatory cytokine pathways are upregulated in the presence of maternal asthma and a female fetus which may contribute to reduced female fetal growth. Conversely we found the male fetus continues to grow normally in the presence of maternal asthma with no changes in placental function or inflammation. The mechanisms that confer a sexually dimorphic difference between the male and female fetus are currently being examined. In relation to placental inflammatory pathways, p38 MAPK activity was greater in females than males which may account for the greater cytokine expression of the placentae from females relative to males. However the regulation of cytokine production also varies by sex with females being hypersensitive to cortisol inhibition of cytokine production relative to males. These data suggest that the placental inflammatory response and the regulation of that response varies in a sex specific manner in the placenta and may contribute to the sexually dimorphic difference in fetal and neonatal outcome.

The uterine microenvironment in fertility and infertility

2010-08-01

Successful implantation requires synchronous development of the blastocyst and acquisition of endometrial receptivity. Complex embryo-maternal signaling via soluble mediators enables the early events of attachment and adhesion of the blastocyst to the endometrial epithelium. The fluid within the uterine cavity provides the microenvironment in which these events occur.

Embryo implantation: unravelling the functions of DC and NK cells

S.M. Blois, I. Tirado Gonzalez, G. Barrientos — 2010-08-01

Human pregnancy rates, even in the context of assisted reproduction protocols, remain remarkably low and often arise from dysregulations of the early events driving uterine receptivity and embryo implantation. The regulatory functions of decidual leukocytes during early pregnancy, particularly dendritic cells and NK cells, may be important not only for the establishment of maternal immunological tolerance but also in the regulation of stromal cell decidualization and the vascular responses associated with the implantation process. However, the specific contributions of dendritic cells and NK cells during implantation are still difficult to dissect mainly due to reciprocal regulatory interactions established between them within the decidual microenvironment. This talk will present current evidence on the regulatory pathways driving embryo implantation, suggesting that NK cells promote uterine vascular modifications that assist decidual growth but dendritic cells directly control stromal cell proliferation, decidual angiogenesis and the homing and maturation of NK cell precursors in the pregnant uterus. Thus, successful implantation appears to result from an interplay between cellular components of the decidua which is governed by the unique immunoregulatory and pro-angiogenic functions of dendritic cells.

Trophoblast cells as immune modulators in pregnancy

G. Mor — 2010-08-01

The placental immune response and its tropism for specific viruses and pathogens affects the outcome of the pregnant woman's susceptibility to and severity of certain infectious diseases. The generalization of pregnancy as a condition of general immune suppression or increased risk is misleading and prevents the determination of adequate guidelines for treating pregnant women during pandemics. There is a need to evaluate the interaction of each specific pathogen within the fetal/placental unit and its responses in order to design the adequate prophylaxis or therapy.

Functional role of uterine natural killer cells in early human pregnancy

G.E. Lash — 2010-08-01

Two key processes in the establishment of human pregnancy are invasion of extravillous trophoblast (EVT) into uterine decidua and inner myometrium and remodeling of uterine spiral arteries during the first and early second trimester. Failure or inadequate completion of these processes has been linked with several pregnancy complications, including second trimester miscarriage, pre-eclampsia and fetal growth restriction. Regulation remains incompletely understood, although it likely requires a balance between EVT and decidua derived factors. Leukocytes, namely uterine natural killer (uNK) cells, macrophages, T lymphocytes and dendritic cells, comprise around 30–40% of the cells within the decidual stroma in early pregnancy. The uNK cells are the most numerous of these leukocytes and recently have been shown to have potential roles in regulating EVT invasion and spiral artery remodeling.

The human decidual NK cell response to pathogens

2010-08-01

NK cells present in the peripheral blood respond rapidly to pathogens or pathogen-infected cells by various means including cytotoxicity and production of cytokines. Whether decidual NK (dNK) cells are able to play a similar role when the pregnant uterus is infected by viruses, bacteria or parasites is unknown. We have focussed on the cytotoxic effector function of dNK cells. Decidual NK cells are generally considered as poorly cytotoxic when compared to their peripheral blood counterparts. However, we have recently demonstrated that freshly isolated dNK cells from healthy early pregnant uterus do have a cytotoxic potential mostly mediated by the specific engagement of NKp46 activating receptor (). We further found that the co-engagement of CD94/NKG2A inhibiting receptor drastically inhibited the cytolytic function of dNK. This observation suggested that the in situ CD94/NKG2A receptor interaction with its HLA-E specific ligand is a dominant negative regulatory mechanism that prevents unwanted dNK cell cytotoxicity in non-infected pregnant uterus. How do dNK cells behave when they are activated by pathogens or pathogen-infected cells present at the maternal–fetal interface? We will briefly discuss the following questions:

HLA-C mediated immune regulation at the fetal–maternal interface

F.H.J. Claas, T. Tilburgs, M. Eijkmans, D.L. Roelen, S.A. Scherjon — 2010-08-01

During pregnancy maternal leukocytes at the fetal–maternal interface play a key role in the immune acceptance of the allogeneic fetus. So far most investigators have focussed on the role of decidual NK cells, which contain immune modulatory properties and facilitate trophoblast invasion into maternal tissue.

Cytoplasmic pattern recognition receptors in placental infection

V.M. Abrahams — 2010-08-01

There is a strong clinical association between infection and preterm labor. The placenta functions as an active barrier by the trophoblast recognizing and responding to microbes through pattern recognition receptors, in order to control pathogens at the maternal–fetal interface. Consequently, either inefficient clearance of an infectious agent, or an overactive placental response, may have a significant impact on pregnancy outcome. Recently, our lab has reported that the trophoblast express the cytoplasmic-based Nod-like receptors (NLRs). As a result of their restricted localization, NLRs function as intracellular receptors that respond to bacterial components which have gained access to the cytoplasmic compartment, either from the extracellular space, or from invasive intracellular bacteria. We have found that first trimester trophoblast cells express Nod1 and Nod2, and their signaling effector protein, RICK. We have also found that first trimester trophoblast cells express Nalp1 and Nalp3, and their signaling adapter protein, ASC, which together with caspase-1, form the inflammasome. Through these NLRs, the trophoblast mounts an inflammatory response. Following Nod1 and Nod2 activation by iE-DAP and MDP, respectively, first trimester trophoblast produce elevated levels of chemokines; while following Nalp3 activation IL-1β production is upregulated, suggesting activation of the inflammasome. To better understand the role of NLRs in the placental response to infection, we have exposed trophoblast cells to the intracellular bacterium, Chlamydia trachomatis. Upon Chlamydia infection, trophoblast cytokine/chemokine production is differentially modulated. Chlamydia infection leads to a strong suppression of constitutive MCP-1 and GROα secretion. This correlates with degradation of NFκ B p65, which could be mediated by a Chlamydia virulence factor. In parallel, Chlamydia infection of the trophoblast induces the upregulation of intracellular pro-IL-1β, and its processing into active, secreted IL-1β, again, indicative of inflammasome activation. Together our studies demonstrate that the trophoblast express functional NLRs which may be important for their responses to intracellular pathogens.

Pharmacological inhibition of inflammatory pathways for prevention of preterm birth

J.A. Keelan — 2010-08-01

The major cause of spontaneous preterm birth (sPTB) <32 weeks’ gestation is intrauterine inflammation (IUI). IUI typically arises in response to colonisation of the decidua and fetal membranes by pathogenic microorganisms. These trigger activation of the local innate immune system, resulting in release of inflammatory mediators, leukocytosis, apoptosis, membrane rupture, cervical ripening and onset of uterine contractions. Amniotic infection is confirmed in about half of all deliveries with IUI. However, recent PCR evidence suggests that in the majority of cases, microorganisms are present in the amniotic fluid, but are not always cultured by standard techniques. Conversely, some pregnancies have infected placental tissues without inflammation and sPTB. Hence, while intrauterine microbial infection can provoke sPTB, it is neither necessary nor even inevitable; it is the magnitude and nature of the inflammatory response that determines the progression to sPTB. Administration of antibiotics has been explored somewhat unsuccessfully to treat or prevent sPTB. While this may be partly due to pharmacokinetic/dynamic limitations, to be effective the antibiotic therapy would need to reduce both the infection and the inflammatory response. Furthermore, increased bacterial lysis in response to the antibiotic might actually enhance agonism of the innate immune system. In this talk I will discuss the proposition that in pregnancies at risk of inflammation-driven preterm labour, administration of anti-inflammatory drugs is a viable therapeutic option to prevent sPTB and improve fetal outcomes. Preventing fetal inflammation via administration of placenta-permeable drugs could also have significant perinatal benefits in addition to those related to extension of gestational age, as a fetal inflammatory response is associated with a range of significant morbidities. A number of potential drugs are available, effective against different aspects of the inflammatory process, which may be viable modalities in this context; these will be discussed, together with potential safety concerns associated with administration of anti-inflammatory agents in pregnancy.

Toll-like receptors and inflammatory signalling in spermatogenesis and testicular immune regulation

M.P. Hedger — 2010-08-01

While it is self-evident that infection and inflammation in the reproductive tract can inhibit male fertility, the observation that fertility is also compromised by systemic inflammation and disease is a basic conundrum of male reproductive biology. However, recent studies implicating microbial pattern-recognition receptors, such as the Toll-like receptors (TLRs), as well as inflammatory signalling pathways and cytokines in testicular function have cast new light on this mysterious link between inflammation and reproduction. Spermatogenesis is a complex, organized and highly regulated process involving intimate interactions between the developing germ cells and supporting Sertoli cells. Sertoli cells express several TLRs and respond to TLR ligands by producing a number of inflammatory cytokines and mediators, in particular interleukin-1α (IL-1α), IL6 and nitric oxide (NO), as well as immunoregulatory cytokines, such as activin A. Not only do these molecules regulate spermatogonial/spermatocyte development and many critical supportive functions of the Sertoli cells, but their production varies across the cycle of the seminiferous epithelium, with significant changes in expression coinciding with key events within the cycle. The possibility that this cyclical production may occur in response to endogenous TLR ligands expressed by the developing germ cells has important implications for understanding normal spermatogenic regulation. Inflammatory signalling pathways activated by TLR ligands appear to play fundamental roles in regulating Sertoli cell activity and responses to reproductive hormones, as well as regulation of immune events within the testis. Moreover, such relationships between inflammatory signalling and spermatogenesis provide a potential mechanism to account for the link between infection/inflammation and testicular dysfunction. At least some of the negative effects of inflammation on spermatogenesis may be attributed to elevated production of inflammatory mediators within the circulation and the testis, which exert disruptive effects on germ cell development and survival, and the ability of Sertoli cells to provide support.

Inflammatory pathways linking obesity and ovarian dysfunction

R.L. Robker — 2010-08-01

Obesity is characterized by chronic low-level inflammation. Abdominal fat is infiltrated by immune cells and releases adipose tissue-specific cytokines (adipokines) that affect systemic insulin sensitivity and lipid utilization. Obese women often experience reduced pregnancy rates, and even with assisted reproduction have fewer oocytes retrieved and lower success rates. We have sought to understand how alterations in the ovarian environment of obese females leads to impaired oocyte maturation and early embryo development. Female mice fed high fat diet exhibit weight gain, immune cell infiltration into adipose and elevated inflammatory markers. Intracellular fat also accumulates in ovarian cells; oocytes in particular are larger and contain dramatically more lipid than oocytes from mice fed control diet. Lipid accumulation is associated with induction of lipotoxicity responses, including endoplasmic reticulum (ER) stress, increased expression of TNFα, IL-6 and IL-1β, and apoptosis. Further, ovulation and fertilization rates are reduced. To determine whether similar events occur in ovaries of obese women, follicular fluid and granulosa/cumulus cells were obtained from women of varying body mass indices (BMIs) undergoing assisted reproduction treatment. Analysis of granulosa and cumulus cells revealed similar expression of genes involved in insulin signaling and lipid transport, but increased expression of ER stress marker ATF4 in granulosa cells from obese women compared to levels in non-obese women. Follicular fluid of obese women contained high levels of triglyceride, free fatty acids, cytokines, adipokines and C-reactive protein. When follicular fluid from obese women was used as maturation media for mouse oocytes, the oocytes accumulated lipid, ER stress was induced and oocyte maturation was blocked. Follicle fluid from moderate weight women or culture in lipid-rich media did not elicit these effects. Thus there are marked changes in the ovarian environment of obese women, with heightened inflammation likely contributing to defects in oocyte developmental competence.

Regulatory T cells and Th17 cells in immunity – function and plasticity

C. Dong — 2010-08-01

CD4+ T lymphocytes play important regulatory roles in the adaptive immunity. Upon activation, naïve CD4+ helper T (TH) cells differentiate into effector subsets with different cytokine expression profiles and immune regulatory function. Effector TH cells have been classified into TH1 and TH2 lineages: TH1 cells express IFNγ, and TH2 cells produce IL-4, IL-5 and IL-13. More recently, several other subsets of TH cells, including TH17 cells, regulatory T cells (Treg cells), TH9 cells and folliculate helper T cells (Tfh cells) have been identified and may play important roles in autoimmune diseases. In the meeting, I will discuss our recent work on the regulation of Treg and TH17 differentiation, the plasticity of T cell differentiation and the function of TH17 cells in autoimmune diseases.

Regulatory T cells in pregnancy

A. Betz — 2010-08-01

Regulatory T cells play a crucial role in maternal fetal tolerance. Immediately after conception, their number in the blood, spleen and uterus-draining lymph nodes substantially increases. Whilst this initial expansion is independent of exposure to paternal alloantigen, their accumulation in the gravid uterus appears to be constrained to alloantigen specific regulatory T cells. Clearly, the role of this shift in the composition and distribution of the T cell populations is the suppression of an extremely aggressive allogenic response directed against the fetus. However, in women suffering from certain autoimmune diseases, such as arthritis, this also correlates with a marked improvement in symptoms. Unfortunately, the remission only lasts until delivery when the disease invariably returns with a vengeance. The answers to where these regulatory T cells come from in the first place and whether there is something special about their function during pregnancy, making them more effective in the control of autoimmunity, are key to a better understanding of the underlying mechanisms.

The role of regulatory NK cells and regulatory T cells in the maintenance of pregnancy

S. Saito, T. Shima, A. Nakashima, Y. Lin — 2010-08-01

We have previously reported that NK cells expressing granulysin attack extravillous trophoblast (EVT) and induce apoptosis of EVT in miscarriage cases. On the other hand, we also reported that CD25+c-kit+Thy1++ NK cells in leukemic mice might be regulatory NK cells which inhibit MHC-class II expression on dendritic cells (DCs) and inhibit CTL induction. Interestingly, these NK cells are increased in the pregnant uterus, and most of the pregnant-uterine NK cells in NOD/SCID or NOD mice were regulatory NK cells.

Defects in IL-2-STAT5 signalling and reduced TGFβ and IL-2 cytokines account for regulatory T cell deficiency in women with recurrent spontaneous abortions

L. Fainboim, M. Arruvito, A. Billordo, A. Sotelo — 2010-08-01

We have previously shown a decreased frequency and function of regulatory T cells (Treg cells) in women suffering from recurrent spontaneous abortion (RSA). In the current study, we first investigated the expression of FOXP3 after T cell activation. We observed that expression of FOXP3 in activated PBMCs was already present above baseline before any cell division, indicating that it was induced in cells that were previously negative for this transcription factor. Because RSA women showed a more limited expansion of FOXP3-positive cells, we next assessed the role of IL-2 signalling through STAT5, which is known to be required for generation of inducible Tregs (iTregs). We demonstrated not only that TGFβ and IL-2 were diminished but also that the IL-2–STAT-5 signalling axis was down-regulated in RSA women. Finally, in addition to limited FOXP3+ cells expansion in vitro, iTregs from RSA women showed strikingly lower suppressor activity.

JAK/STAT signalling in placental trophoblast differentiation

U.R. Markert, D.M. Morales, S. Ospina — 2010-08-01

Trophoblast cells develop mannifold functions depending on progress of pregnancy, their localization and their environment. This environment is composed of a potpourri of maternal stroma and immune cells as well as of soluble factors which may be released locally or systemically and which include numerous hormones, cytokines and others. A variety of receptors on trophoblast cells receive extracellular signals and transform them into intracellular signals. Several cytokines of the interleukin-6 (IL-6) family are present in the placenta and trophoblast cells express gp130 which is the common receptor chain for these cytokines as well as receptor chains specific for several members of the IL-6 family. The receptor activates various intracellular signalling pathways, such as the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT), the Mitogen Activated Protein Kinase (MAPK) or the mammalian Target Of Rapamycin (mTOR) pathways, which are able to cross-talk between each other. Leukemia Inhibitory Factor (LIF), IL-6, IL-11 and oncostatin are major members of the IL-6 family which activate the mentioned pathways. A crucial function seems to play STAT3, which exists in at least two isoforms and has two phosphorylation sites (tyr 705 and ser 727). Homo- and heterodimers translocate into the nucleus and induce transcription of several genes and functions. Activation of STAT3 correlates with proliferation, motility and invasiveness of trophoblast cells similarly to tumor cells. Post-transcriptional gene silencing of STAT3 inhibits the functions of the above-mentioned cytokines. LIF and STAT3 also control a new class of regulatory molecules: microRNA. Expression of several microRNA which are involved in tumor invasion are regulated by LIF, which on the one hand underlines the role of LIF in governing trophoblast, but which on the other hand also demonstrates the involvement of miRNA in tuning placentation.

Regulation of mucosal immunity by a novel cytokine, interferon epsilon

P. Hertzog, K.Y. Fung, N. Mangan, H. Cumming, P. Hansbro, J. Horvath, D. Carr — 2010-08-01

We have recently discovered a new type I interferon gene, designated IFNɛ, located in the type I IFN locus in vertebrate genomes. Unlike other type I IFNs which have evolved multiple isoforms with diverse promoters that are activated by signaling from pattern recognition receptors, IFNɛ is not regulated by TLR or TLR ligands, nor interferon regulatory factors (IRFs) 3, 5 and 7. Furthermore, IFNɛ is constitutively and exclusively expressed in the female reproductive tract. Its expression is regulated during the estrus cycle and in pregnancy. We have generated a conditional IFNɛ gene targeted mouse to investigate the pathophysiological functions of this novel cytokine. We will present data on the role of this new IFN in sexually transmitted infections. The mechanism of IFNɛ-based protection involves regulation of early innate immunity and sculpting of adaptive immune responses. IFNɛ is thus a novel cytokine whose constitutive expression is well tolerated and not detrimental to reproductive tract physiology; but is crucial for optimal protection from viral and bacterial infection in that vital organ.

Cytokine regulation of mammary gland development

C.J. Watson, C.H. Oliver, W.T. Khaled, S. Pensa — 2010-08-01

Cytokine signalling is mediated by the Jak/Stat pathway. Members of the Stat family have important roles at different stages of mammary gland development in the mouse. In particular, Stat3 is critical for the initiation of post-lactational regression while Stat5 is essential for alveolar development during pregnancy and lactogenesis. We have discovered an unexpected role for Stat6 and the Th2 cytokines, IL-4 and IL-13, in lineage commitment during early pregnancy. Proliferation and alveolar morphogenesis are delayed in both Stat6 and IL-4/IL-13 doubly deficient mice at days 5 and 10 gestation. Furthermore, mammary epithelial cells in culture switch from production of the Th1 cytokines IL-12, interferon g and TNF a to secretion of the Th2 cytokines IL-4, IL-13 and IL-5 upon differentiation with lactogenic hormones. We are currently investigating the function of other transcriptional regulators that are components of the IL-13/Stat6 axis and have identified a novel zinc finger protein, which we have named ROMA, that is down-regulated by Stat6 and is expressed in progenitor cells. Genetic deletion studies in vivo indicate that ROMA may be a master regulator of lineage commitment. Thus, as epithelial cells commit to the luminal lineage, the cytokine milieu is dramatically changed adding an unexpected tier of complexity to the previously held paradigm that steroid and peptide hormones are the primary regulators of mammary gland development.

Progesterone and immune–endocrine cross-talk in fertility and infertility

J. Szekeres-Bartho — 2010-08-01

Progesterone is indispensable in creating a suitable endometrial environment for implantation, and also for the maintenance of pregnancy. Further to its endocrine effects, progesterone also acts as an “immunosteroid” and contributes to the establishment of a pregnancy protective immune milieu. A progesterone-induced gene PIBF encodes a protein which mediates several of the immunological effects of progesterone. The full length PIBF is associated to the centrosome and plays a role in cell cycle regulation, whereas secreted smaller forms exert cytokine-like effects. PIBF binds to a novel type of the IL-4 receptor and signals via the Jak/STAT system, to induce a number of genes, that not only affect the immune response, but might also play a role in cell cycle regulation and apoptosis as well as tumor and trophoblast invasiveness. PIBF negatively regulates trophoblast invasiveness, while it upregulates the invasive capacity of tumor cells. The signaling pathways involved in these differential effects as wells as their role in early and pregnancy and tumor growth will be discussed.

Peri-conceptual cytokine environment and consequences for human pregnancy

2010-08-01

Non-invasive assessment of oocyte competence and identification of endometrial markers of uterine receptivity would undoubtedly improve the efficiency of assisted reproductive technology. We documented 26 cytokines in individual follicular fluid (FF) using a bead-based multiplex sandwich immunoassay. Individual FF analysed were all issued from matured oocytes that were subsequently fertilized and transferred in both conventional ovarian hyperstimulation and monitored natural cycles. The granulocyte colony-stimulating factor (G-CSF) content of follicular fluid appeared in three distinct experiments (conducted by the European network EMBIC) as a non-invasive immune biomarker of oocyte competence able to predict potentiality of subsequent birth. Such tool may be a crucial tool to control multiple pregnancies in ART and define prognosis of success.

Treg cells and other leukocytes in the pathogenesis of endometriosis

I.S. Fraser, M. Berbic — 2010-08-01

Endometriosis is a very common and puzzling gynaecological condition which shows a great deal of variability from one woman to another. It may affect up to 15% of all women of reproductive age. There is a strong familial component, but aetiology and pathogenesis are still uncertain. Endometriosis is an ‘inflammatory’ condition with obvious infiltration of endometriotic lesions with numerous different leukocytes. There is increasing evidence to demonstrate marked changes in number and functions of these leukocytes in the eutopic endometrium and peritoneal fluid as well as in the lesions. Our hypothesis is that endometriosis is primarily an endometrial disease with underlying genetic disturbances which lead to a number of major molecular changes in function enhancing the likelihood that viable fragments of endometrial tissue will pass through the fallopian tubes and attach and grow on peritoneum.

Macrophages, TGFB superfamily cytokines, micro-RNAs and endometriosis

M.L. Hull — 2010-08-01

Endometriosis causes pain and subfertility in 6–10% of reproductive aged women. In a xenograft mouse model, human endometriosis-like lesions displayed necrosis and tissue breakdown at day 7 whereas by day 10 glandular and stromal remodelling had occurred. Host derived macrophages were present in the lesions and are likely to have contributed to this transition. In a similar model, the introduction of classically activated macrophages reduced lesion development, whereas alternatively activated macrophages, under the influence of TGFβ influence, enhanced ectopic endometrial lesion establishment.

Trophoblast deportation – just placental debris or fetal–maternal antigen sharing?

L.W. Chamley, Q. Chen, M. Abumaree, J.X. Ding, P.R. Stone — 2010-08-01

The surface layer of the human placenta is the multinucleated syncytiotrophoblast. As the syncytiotrophoblast ages or becomes damaged regions of it are extruded into the maternal blood in large multinucleated structures called syncytial knots. Syncytial knots are deported in the maternal blood to become trapped in the pulmonary capillaries as first described over 100 years ago (). There are at least 150,000 syncytial knots deported daily during normal pregnancy and greater amounts in preeclampsia. These syncytial knots expose foreign (fetal) antigens to the maternal immune system and are cleared from the maternal lungs on average in about 3 days (). Since they are trapped in the maternal lungs, there have been few studies of the biology of syncytial knots and their effects on maternal physiology. We recently developed a placental explant model that allows us to harvest and study syncytial knots (). Using this model we have shown that syncytial knots from normal placentae are mostly apoptotic. Macrophages that had phagocytosed apoptotic syncytial knots increased expression of indoleamine 2,3-dioxygenase, and IL-10 with decreased secretion of IL-1β (). Class II MHC expression was also increased but was significantly higher if the syncytial knots where necrotic. We have also shown that endothelial cells can phagocytose syncytial knots and that phagocytosis of apoptotic knots is silent but necrotic syncytial knots induce the activation of the phagocytosing endothelium significantly increasing secretion of IL-6, TNFα and TGFβ1. When PBMNCs were treated with conditioned medium from macrophages that had phagocytosed necrotic, but not apoptotic, syncytial knots, the PBMNCs proliferated. Our data suggest that deportation of apoptotic syncytial knots may be a mechanism to allow maternal immune tolerance to fetal antigens. Our data also suggest that phagocytosis of necrotic syncytial knots may lead to endothelial and/or immune activation with implications for pathological outcomes of pregnancies, such as preeclampsia and recurrent miscarriage.

How the immune system ‘Grows up’: evidence supporting the existence of a layered immune system

2010-08-01

The development of the mammalian immune system is typically thought to occur in a linear fashion, from immaturity to maturity, as a function of antigen exposure. Historical findings in avian species and mice suggest that this is an oversimplification and argue for the existence of a “layered” immune system. The “layered immune system” hypothesis argues that the developmentally ordered appearance of unique hematopoietic stem cells give rise to distinct lymphocyte lineages at different stages of development. Here we provide the first evidence in favor of this model in human beings. Our results suggest that fetal and adult T cells are distinct populations that arise from different HSC present during fetal development and adult life. Furthermore, our findings suggest that the fetal T cell pool may be biased towards generating immunological tolerance offering a mechanistic explanation for the tolerogenic properties of the developing fetus.

Translating AIRE-dependent tolerance to autoimmune ovarian disease

2010-08-01

Autoimmune ovarian disease (AOD) is a poorly understood disorder wherein immune-mediated destruction of oocytes leads to premature ovarian failure and infertility. Currently, diagnosis of AOD relies on its association with other autoimmune conditions since the known autoantibody tests lack sufficient sensitivity or specificity in identifying ovarian disease. Disease pathogenesis and the antigens targeted in AOD remain largely unknown, and directed therapies for intervention are lacking. We have generated a novel mouse model to study AOD using mice deficient for the Autoimmune REgulator (AIRE) gene, a gene first identified in the human Autoimmune Polyglandular Syndrome Type 1 (APS1). Like patients with APS1, Aire-deficient mice develop spontaneous disease of multiple organs, including the ovary, due to a breakdown in central tolerance. In the absence of Aire, thymic epithelial cells are deficient in presentation and tolerization of developing T cells to self-antigens, allowing autoreactive cells to escape into the periphery with potential for reactivation within target organs. Autoimmunity is manifested by the development of antigen-specific autoantibodies, organ infiltration, and transfer of disease by lymphocytes from affected Aire KO mice. Aire-deficient female mice develop autoimmune oophoritis, with 100% disease incidence in susceptible strains, and histologic disease is accompanied by frequent oligoclonal autoantibodies to ovarian tissue. This presents a valuable spontaneous disease model in which to identify ovarian antigens. Initial investigations have identified a putative 60kDa ovarian antigen that is targeted early in AOD. Further characterization of the cell-mediated and humoral mechanisms of disease and identification of the pathogenic antigen will allow the development of targeted immune therapies. Lessons from the study of Aire-mediated AOD will teach us how tolerance is broken in the ovary and will have potential for translation to patients in a significant cause of human ovarian failure and infertility.

Placental microvesicles and exosomes and systemic inflammation in pre-eclampsia

I.L. Sargent — 2010-08-01

During human pregnancy the placenta sheds a range of cellular debris into the maternal circulation including syncytiotrophoblast microvesicles (STBM) and exosomes. These have the potential to interact with maternal immune and endothelial cells and may have both proinflammatory and immunoregulatory effects. Increased shedding of STBM is associated with pre-eclampsia and may therefore be the cause of the systemic inflammatory response and endothelial dysfunction which characterize the maternal syndrome. We have investigated their function using vesicles prepared by perfusion of the maternal surface of normal placentas to mimic maternal blood flow (pSTBM). Characterisation of these pSTBM was carried out by western blotting and flow cytometry and a novel sizing method – Nanosight Tracking Analysis. The vesicles ranged in size from 20 to 600nm with a peak size around 200nm (which encompasses both microvesicles and exosomes) and express proinflammatory and anti-angiogenic molecules. We have investigated their functional effects on peripheral blood mononuclear cells from non-pregnant and normal pregnant women using cytokine arrays and demonstrated a range of proinflammatory cytokines and chemokines that are produced in response to pSTBM. Two of the cytokines (TNFα and IL-6) were significantly elevated in PBMC from normal pregnant women compared to the non-pregnant controls and another (IP-10/CXCL10) was significantly reduced. The pSTBM were also found to induce changes in cytokine secretion in microvascular endothelial cells and disrupt their growth as a monolayer. These findings support the hypothesis that excess shedding of syncytiotrophoblast vesicles in pre-eclampsia is a cause of the maternal syndrome.

Role of tissue factor in pregnancy complications

G. Girardi — 2010-08-01

Tissue factor (TF) is the major cellular initiator of the coagulation protease cascade but also plays important roles in inflammation. TF has important nonhemostatic functions in the development and/or maintenance of blood vessels during placental formation. However, aberrant expression on TF may lead to pregnancy complications and fetal death.

New perspectives in evaluation and treatment of recurrent pregnancy loss and multiple implantation failure

J. Kwak-Kim, S.K. Lee, K.M. Yang, A. Gilman-Sachs — 2010-08-01

In women with recurrent pregnancy losses (RPL) or multiple implantation failures (MIF), peripheral blood NK cells and Th1/Th2 cytokine producing cell ratios were significantly higher than those of normal fertile women. This implicates systemic immune regulation prior to and post-conception is directly related to reproductive outcome. Gonadal sex steroid and GnRH may be keenly related to systemic regulation of immune responses and indeed, peripheral blood T and NK cells are affected by ovarian cycle. Proportions (%) of CD3+ and CD3+CD4+ T cells were increased in the follicular phase compared to the luteal phase. The levels of CD3−CD56+ and CD3−CD56dim NK cells, and NK cytotoxicity were significantly increased in the luteal phase compared to the follicular phase. However, CD3+CD56+ NKT cells, Th1 cytokine producing T cell subsets, and ratios of Th1/Th2 cytokine producing T cells were not significantly changed during a menstrual cycle. Therefore, early follicular phase T and NK cell level may predict immune dysregulation at the time of implantation. In women with RPL or MIF, proportions (%) of activated T cells populations (CD4+/154+ of CD4+, CD8+/154+ of CD8+, CD3+/69+ of CD3+ and CD8+/69+ of CD8+cells) are significantly higher than in normal controls. CD8+/154+ and CD8+/69+ T cells are inversely correlated with IFN-γ/IL-10 producing CD3+/4+ Th cell ratios. These changes may be related with decreased Treg cell levels. Populations of Foxp3+ T regulatory cells are significantly decreased and levels of these cells are significantly correlated with TNF-alpha+ cells. These findings indicate that women with RPL and MIF have increased T cell activation in peripheral blood and T suppressor activation seems to be associated with decreased Th1 immunity. In conclusion, peripheral blood T cells have a significant role in idiopathic RPL or MIF, and regulation of T cell immunity may improve reproductive outcome in women with RPL or MIF.

Mouse models of preeclampsia—role of IL-10

S. Sharma — 2010-08-01

Pregnancy is a complex process and this complexity is further compounded by the fact that despite great advances made in clinical care, pregnancy conditions such as preeclampsia still remain enigmatic. Preeclampsia complicates 5–10% of all pregnancies worldwide and is a major cause of maternal, fetal and neonatal mortalities and morbidities. Delivery is still the only well accepted treatment. Why then our understanding of such serious pregnancy complications is still elusive? There are at least three reasons that possibly contribute to this dilemma. First, preeclampsia entails late pregnancy diagnosis and lack predictive biomarkers or assays. Second, this disease is associated with multi-factorial etiology. Lastly, no well defined animal models exist that can provide insights into the causative factors and pathways that program preeclampsia. Our recent research has focused on establishing in vitro predictive assays and mouse models for late pregnancy maladies. Based on the hemochorial placentation in mice and the important role of interleukin-10 (IL-10) in temporal maintenance of pregnancy, we have used mice with a null mutation in the Il10 gene (IL-10−/− mice) to study the effect of mimics of inflammation and infection on pregnancy. We have succeeded in establishing both in vitro and in vivo models that closely resemble the preeclampsia condition in humans. We will discuss our results in the context of underlying mechanisms and biomarkers that are likely to provide important insights into the pathogenesis of preeclampsia.

Innate immune protection in the human female reproductive tract against HIV and other sexually transmitted diseases

C.R. Wira, M. Ghosh, J.V. Fahey, S. Cu-Uvin — 2010-08-01

Previous studies from our laboratory have led to the hypothesis of a window of vulnerability in the female reproductive tract (FRT) that places women at risk for HIV infection (AIDS, 22 (2008) 1909–1917). We have also shown that secretions from isolated human FRT epithelial cells contain a spectrum of endogenous antimicrobials, some with anti-HIV-1 activity. Recognizing that sexual transmission per coital act is low, we hypothesized that cervical–vaginal lavages (CVL) of women contain endogenous antimicrobials that are protective against pathogens. In this study CVL were collected from 15 HIV(−) and 57 HIV(+) women by washing the cervical–vaginal area with saline. HIV(+) women were categorized by peripheral CD4 (cells/mm3) counts: Group 1 values set were >350, Group 2 were 200–350, and Group 3 were <100. None were receiveing anti-retroviral treatment. To measure anti-HIV activity, CVL were pre-incubated with R5/M-tropic, X4/T-tropic, and transmitted/founder HIV-1 viruses and added to indicator cell line TZM-bl to measure infection. Antimicrobials were measured by ELISA. In HIV(+) and HIV(−) CVL, we found a range of anti-HIV-1 activity against all viral strains. Overall anti-HIV activity and antimicrobial levels decreased with disease progression with no activity observed in samples with CD4<100. Anti-HIV activity in healthy HIV(+) CVL correlated with human β-defensin-2 (HDB2), MIP-3α and HIV IgG antibodies but not with Elafin or secretory leukocyte protease inhibitor (SLPI). Although HIV(−) CVL showed similar levels of anti-HIV activity, no significant correlations with the antimicrobials tested suggest that other immune factors are involved. Our findings indicate that both HIV(−) and healthy HIV(+) CVL contain antimicrobials capable of inhibiting HIV-1 infection and show that intrinsic activity is lost with disease progression. The presence of anti-HIV factors in CVL in early disease may in part explain why the frequency of infectious virus in CVL is low, and also the low rate of heterosexual transmission per coital act.(1) Supported by: AI071761 (CRW), AI40350 and AI066884 (SCU) from NIH and Lifespan/Tufts/Brown CFAR P30AI42853 and CDC CCU106795 (SCU).

Regulation of T cell responses against HSV

A. Ashkar — 2010-08-01

Genital herpes is one of the most common sexually transmitted diseases worldwide. Genital herpes infection, mainly caused by Herpes Simplex Virus type-2 (HSV-2), is associated with an increased risk of obtaining other sexually transmitted infections such as HIV. Currently there is no effective vaccine candidate for human use. While early innate immune responses, such as production of type I interferon, IL-15 and NK cell activation are essential to control initial HSV-2 replication, adaptive immune responses, particularly by T cells, are crucial to clear the infection. We have recently shown that a balance of both innate and adaptive immunities is required to control genital HSV-2 infection. Mice deficient in the innate immune system cytokine IL-15 (IL-15−/− mice), were able to generate antibody and T cell-mediated immune responses against HSV-2, but failed to provide protection against subsequent HSV-2 challenge, compared to wild-type B6 immunized mice. This was not due to the quality of the adaptive immunity generated in the absence of IL-15 since adoptive transfer of lymphocytes from immunized IL-15−/− to naive mice were able to provide protection against HSV-2 challenge similar to those seen with immunized cells from control mice. Mice overexpressing IL-15 (IL-15tg mice) have higher numbers of NK cells, greater NK-derived gamma interferon, and more CD8+ T cells. While IL-15 overexpression resulted in lower HSV-2 replication early on, surprisingly, IL-15tg mice immunized against HSV-2 were not protected against genital HSV-2 challenge compared to control immunized mice. Immunized IL-15tg mice had significantly fewer HSV-2-specific CD4+ T cells than B6 mice. We then confirmed that CD4+ T cells, but not CD8+ T cells, are essential for protection against genital HSV-2 challenge. Our data suggest that CD4+ T cell-mediated protection against genital HSV-2 requires IL-15 and NK cells.

Uropathogenic Escherichia coli (UPEC) and the inflammatory response of the testis

A. Meinhardt — 2010-08-01

Protection of the spermatogenic cells from the host immune response to pathogens is critical to male fertility. Central to these regulatory processes are the somatic cells of the testis including the testicular macrophages as the largest cohort of leukocytes in the testis. Failure of these protective mechanisms potentially caused by bacterial pathogenic factors bolstering their own survival may lead to inflammatory/infectious infertility which accounts for approximately 13–15% of all cases of male infertility. In this study, we aimed at investigating the host pathogen interaction using isolated testicular macrophages (TM) infected with UPEC as a relevant strain in urogenital tract infections in men. Whole genome expression analysis showed that the uropathogenic Escherichia coli (UPEC) strain CFT073, but not non-pathogenic E. coli (NPEC) activate calcium dependent noncanonical wnt5a and nuclear factor activated T cells (NFAT) signaling pathway in TM and peritoneal macrophages (PM, as control). UPEC, in contrast to NPEC contain hemolysin as a virulent factor, a pore forming complex inducing Ca2+ oscillations. Measurements of cytosolic-free calcium confirmed microarray results by a rapid increase of intracellular calcium upon treatment with UPEC, but not NPEC, in both TM and PM. Increased expression of wnt5a protein was observed after UPEC infection in TM and PM. In TM and PM, UPEC and hemolysin activate NFAT by dephosphorylation with subsequent translocation to the nucleus. Increased expression of downstream NFAT signaling pathway genes IL-3, IL-4, IL-10 and IL-13 was found by RT-PCR in TM and PM. Incubation with cyclosporin A (CsA), a pharmacological calcineurin inhibitor, blocked UPEC and hemolysin induced NFAT activation in TM and PM. Our data suggests that UPEC activate wnt5a and NFAT signal pathway in TM, which results in an anti-inflammatory response potentially allowing persistence of UPEC with subsequent damage to the testis. The results point to a role of hemolysin as responsible pathogen in UPEC.

Cell-associated HIV transmission: mechanisms and prevention

D. Anderson — 2010-08-01

HIV-infected leukocytes in genital secretions of HIV-infected men and women may be effective vectors of HIV sexual transmission because intracellular virus is protected from many antiviral innate and acquired defense mechanisms, and because intercellular HIV transmission via viral synapses is highly efficient. We are using confocal microscopy to study seminal leukocyte adhesion and apical to basal transmigration through a human vaginal epithelium organotypic model. Seminal T cells and macrophages adhere to the apical surface of the vaginal epithelium via LFA-1/ICAM-1 interactions, and migrate between epithelial cells through interactions between MAC-1 or LFA-1 on the leukocyte surface and complementary tight junction proteins JAM A and JAM B expressed on vaginal epithelial cells. The tissues express an array of chemokines including RANTES and MIP-1α which could attract leukocytes into the tissue. Leukocyte adhesion and infiltration are pronounced in tissues treated with TNFα which dramatically upregulates the expression of ICAM-1 and RANTES and makes the tissue more permeable. We are currently tracking the fate of cell-associated HIV in the vaginal epithelium and exploring approaches to block cell-associated HIV transmission including the use of monoclonal antibodies specific for HIV, CCR5 or cell adhesion molecules, and upregulated expression of type 1 interferons in the vaginal epithelium.

Immunological reconstitution of the female reproductive tract of humanized mice

J.V. Garcia-Matinez — 2010-08-01

The development of successful vaccines to prevent infection requires the use of effective in vivo models that recapitulate key aspects of the human immune system. Unfortunately, in many instances human specific pathogens do not infect mice or other tractable animal models where potential vaccine approaches can be evaluated. To address this specific issue significant effort has been made to develop and implement alternative models that faithfully recapitulate key aspects of the human mucosal immune response. In this regard, humanized mice represent a potentially viable alternative. Humanized mice are created by transplanting human hematopoietic stem cells into preconditioned immunodeficient mice. This results in systemic reconstitution of the entire human immune system. However, in order to have HLA-restricted immune responses, human thymic epithelum must be present. We addressed this issue by co-implanting a small piece of human liver and thymus under the kidney capsule. In these mice (designated bone marrow/liver/thymus or BLT mice) T cells are produced and educated in the context of human HLA on a bona fide human thymus. Production of human hematopoietic cells in situ in these mice results in the reconstitution of the entire female reproductive tract with human lymphoid cells. The presence of human lymphoid cells renders these animals susceptible to infection by human specific pathogens making them suitable to the evaluation of novel approaches to prevent infection.

Prevention of sexually transmitted viral infections: time for gender specific vaccine strategies?

C. Kaushic, V. Anipindi, F. Xiu, K. Roth — 2010-08-01

Sexually transmitted infections (STIs) are a major cause of morbidity and a tremendous burden on health care systems globally. Among strategies targeted to efficiently and inexpensively reduce the global burden of STIs, prophylactic vaccines present a logical first choice. However, despite decades of intensive efforts, the attempts to develop efficacious vaccines against STIs have failed repeatedly, with the exception of the HPV vaccine. However, some trends have emerged that provide learning opportunities for improved future strategies. One of the recurrent observations from clinical studies is the link between female sex hormones and altered susceptibility to STIs. More direct correlate between administration of female sex hormones and altered outcome of sexually transmitted viral infections comes from experimental models. Using a mouse model for genital HSV-2 infection we have shown that immunization under the influence of estradiol, especially via mucosal routes, results in better quality of protection against subsequent genital challenge. On the other hand, immunization under the influence of progesterone results in protection that is accompanied with chronic pathology. We are now examining the cellular mechanisms underlying these distinct outcomes. Our recent data shows that treatment with estradiol or progesterone results in alteration of the phenotype and function of antigen presenting cells in the genital tract prior to and following exposure to HSV-2. The changes in sex hormones also affects the T cell populations that respond to genital HSV-2 infection. Further, the innate antiviral responses of epithelial cell that the genital mucosa are potentiated by sex hormone treatments. Overall these studies indicate that in the female genital tract, sex hormones exert a powerful influence that can potentially be useful for modifying immune responses and even preventing sexually transmitted viral infections. If these approaches prove successful in experimental models, it may be time to consider customized, gender-specific vaccine strategies for human STIs.

Chlamydial vaccines: what do we need, what can we deliver

K.W. Beagley, D.P. Wilson, P. Timms — 2010-08-01

Chlamydia trachomatis infections are the most common bacterial sexually transmitted infection worldwide. Antibiotics have not halted the worldwide increase in incidence of infection, and currently there are no vaccines available. Vaccine studies, using the C. muridarum mouse infection model, have shown that it is almost impossible to elicit sterilizing immunity. Most studies result in only a shortening of the infection by 3–6 days, with or without a reduction in peak infection levels. Furthermore, most studies have not assessed protection against immunopathology. However studies in our laboratory have demonstrated protection against inflammatory damage in the absence of sterilizing immunity. Modeling the impact of vaccines based on animal data suggests that a vaccine that elicits even only partial immunity can greatly reduce the incidence of chlamydial infection. These studies showed that the most important requirements of a vaccine is to increase the infectious load required to establish an infection and/or to reduce the duration and peak load of infection. Both of these outcomes can be achieved in the mouse model. Other factors predicted to affect the effectiveness of a vaccine at a population level include whether or not both genders are vaccinated, the percent of the target population vaccinated and the longevity of protection. Recent studies in our laboratory also suggest that vaccines will not be effective in individuals who have an active infection or who have recovered from a natural infection, suggesting that human vaccines should be given prior to the onset of sexual activity. Overall, our animal and modeling studies suggest that aiming for a vaccine that elicits sterilizing immunity may not only be difficult to achieve, but also unnecessary in order to have a beneficial effect on the worldwide chlamydial epidemic. Because the consequences of acute C. trachomatis infection are relatively minor but the long-term consequences impose a significant health burden, it may be time to reevaluate the requirements for a human vaccine to target genital chlamydial infections.

Managing human population growth—utility of microbicides and spermicides

R.J. Aitken — 2010-08-01

There have been no radically new forms of contraception since the pill was introduced 1960. This technology was itself a direct result of research conducted in the 1950s based on endocrine advances that can be traced back to the 1920s. Whatever new forms of fertility regulation we introduce for the future they should exploit the massive advances that have been made in our understanding of the reproductive system over the last half century and be tailored to the needs of the 21st century. In this context, there is an urgent need to develop novel, safe, effective, dual-purpose contraceptive agents that combine the prevention of pregnancy with protection against sexually transmitted diseases (STDs). To achieve this aim we have researched a class of a topical contraceptive agent that selectively and instantaneously immobilizes millions of spermatozoa, while suppressing the infectivity of any pathogenic microbes that might also be present in the ejaculate. This approach is based on the ability of small molecular mass organic compounds to selectively and covalently adduct key proteins in spermatozoa and pathogenic organisms such as Chlamydia and disrupt their biological function. We have also successfully developed strategies for the preparation of latent formulations that would only become activated on contact with seminal plasma. The further development and refinement of these molecules should permit a radical rethink in the way that safe, effective topical protection is provided to regulate both fertility and the spread of STDs.

CD52 as a target for autoimmune antibodies to gametes

A. Hasegawa, S. Komori, K. Koyama — 2010-08-01

Many kinds of anti-sperm antibodies (ASA) have been detected in both men's and women's sera, but among them only a small number are related to infertility. In this study we examined the physiological function of sperm molecules that generate antibodies in females to clarify the mechanisms that mediate ASA-induced infertility. In general, male and female reproductive tissues are immunologically competent to protect from pathogens and they also have immune-suppressive functions so as not to impair sperm. However, if the balance is disrupted, undesired immune reactions to sperm may possibly occur. Previously, a monoclonal antibody (H6-3C4) was produced in our laboratory from an infertile patient's B-lymphocytes. It has been shown that H6-3C4 has strong sperm-impairing activity, detected by sperm immobilization test (SIT). The antibody recognizes the sperm-specific N-linked carbohydrate of CD52, which is a glycosylphosphatidylinositol (GPI-anchor) protein present in immune-related cells and male reproductive tissues. Although the core peptides are identical, the carbohydrate moieties are different between CD52 in immune cells and CD52 in male reproductive tissues (mrt-CD52). mrt-CD52 is synthesized and secreted by the epididymal epithelial cells for insertion into sperm membranes, but is not present in the testis. Recently, we found that CD52 was a suppressive factor in the immunological complement system. Anti-CD52 antibody would act to enhance complement activity and attack sperm by inhibiting CD52 function. Thus, the ASA associated with infertility would interfere with complement-regulatory molecules which protect sperm from complement attack. In addition, animal experiments revealed that mrt-CD52 generated antibodies in males (auto-antibody) and females (iso-antibody). Therefore, CD52 including sperm-specific antigen is a promising target for an immune-contraceptive vaccine in males and females.

Targeting endometrial IL11 and LIF: new generation contraceptives

E. Dimitriadis — 2010-08-01

Despite huge increases in access to contraceptives globally more than 700,000 maternal deaths, most in developing countries and related to unintended pregnancies, occurred between 1995 and 2000. Eighty million women have unintended or unwanted pregnancies annually. Steroidal contraceptives are unsuitable for many women, but pharmacological alternatives are not available. Leukemia inhibitory factor (LIF) and interleukin-11 (IL-11) are obligatory for implantation in mice and are dysregulated in endometrium of some women with infertility. As a novel contraceptive approach, new LIF and IL-11 antagonists were produced and their contraceptive efficacy tested in mice. Polyethylene glycol (PEG) was conjugated to LIF antagonist (LA) or IL-11 antagonist (IL-11A) to increase their serum half-life. Injection of PEGLA during early pregnancy blocked LIF action in the endometrium and resulted in total prevention of embryo implantation while having no embryo toxic effects (). Similarly, injection of PEGIL-11A blocked decidual cell formation resulting in pregnancy failure (). In women, vaginally administered drugs preferentially localise to the uterus suggesting that vaginal administration of PEGLA is an appropriate delivery method for contraceptive purposes. Further, vaginally administered PEGLA could be a ‘dual-role’ contraceptive, carried in a microbicide to also block sexually transmitted infections. PEGLA administered via vaginal gel was shown to prevent implantation. This is the first study to show the contraceptive efficacy of a PEGylated compound delivered vaginally. Contraceptive trials in non-human primates are currently underway to determine the effect of PEGLA on implantation. If effective, this will offer new opportunities as pharmacological, non-hormonal dual-role contraceptives for women.

Contraceptive vaccines for human and animal utility

2010-08-01

Contraceptive vaccines entail generating humoral and/or cell-mediated immune responses against hormones/proteins that are critical in reproductive processes and hence interference in their biological function(s) will result in a block of fertility. These can be designed to inhibit (i) production of the gametes, (ii) functions of gametes leading to block in fertilization, and (iii) gamete outcome (pregnancy). Mammalian egg is surrounded by a translucent matrix: zona pellucida (ZP), which in various species is composed of 3–5 glycoproteins. Immunization of female bonnet monkeys (Macaca radiata) with recombinant bonnet monkey ZP4 conjugated to diphtheria toxoid (DT) led to curtailment of fertility, which was associated with ovarian pathology. To minimize oophoritis, synthetic peptide based immunogens corresponding to ZP3 and ZP4 epitopes mapped by monoclonal antibodies have been used. DNA vaccine encoding chimeric peptide encompassing human ZP3 and ZP4 epitopes generated antibodies that reacted with native ZP of human oocytes and inhibited induction of acrosome reaction mediated by recombinant human ZP3 and ZP4. Immunization of mice with Virus Like Particles (VLPs) expressing fusion peptide comprising mouse ZP3 epitope and sperm antigen (YLP12) led to curtailment of fertility in the immunized female mice. However, ways to overcome the observed oophoritis associated with the zona proteins immunization are yet to be discovered, before it can be proposed for control of human population. Nonetheless, it is a very promising approach to control wildlife animal population. Immunization of female dogs with recombinant canine ZP3 conjugated to DT or recombinant canine ZP3 incorporating at N-terminus a promiscuous T non-B cell epitope of tetanus toxoid led to curtailment of fertility in the immunized animals. Porcine ZP3α and ZP3β have been expressed in E. coli whose potential to block fertility in wild horses will be evaluated in collaboration with Dr. Jay F. Kirkpatrick, Science and Conservation Center, USA.

Lymphatics in the human placental bed in endometrial decidualisation

2010-08-01

The mammalian placenta plays central roles in maternal tolerance of the semi-allogeneic fetus and fluid balance between maternal and fetal compartments. The lymphatics play a role in both immune function and fluid balance. The aim of this study was to describe the distribution of lymphatic vessels in human placental bed and decidua, with particular focus on the lymphatics that surround the remodelling spiral arterioles during decidualisation and trophoblast invasion. Placental bed, non-placental bed and decidual biopsies were obtained from women undergoing elective pregnancy termination (6–18 weeks gestational age) and from women undergoing elective caesarean section at term. Double immunohistochemical labeling was performed on serial sections to identify lymphatic vessels in conjunction with blood vessels, smooth muscle, epithelial and trophoblast cells, or proliferating cells. Using representative photomicrographs, descriptive findings of the organisation of the human placental bed lymphatics were made. Lymphatic vessels positive for podoplanin (D2-40) were abundant in hypersecretory endometrium (lacking stromal decidualisation) at all stages of gestation. By contrast, the decidua was nearly always devoid of lymphatics. In some samples, structures that appeared to be regressing lymphatics were observed at the boundary between hypersecretory and decidual tissues. Lymphatic vessels were notably absent from the vicinity of spiral arterioles that were surrounded by decidualised stromal cells. Lymphatic vessels in hypersecretory endometrium appeared larger and more elongated as gestation progressed. Proliferating lymphatic vascular endothelial cells were identified in both large vessels, and in streaks of D2-40 positive cells that could have been newly forming lymphatic vessels. In conclusion endometrial stromal cell decidualisation results in a loss of lymphatics, with this phenomenon being particularly apparent around the spiral arterioles; the functional consequences of this loss have yet to be elucidated.

Smoking influences local and systemic immune responses differently during pregnancy in mice

J.R. Prins, A. Lech, M.N. Hylkema, J.J.H. Erwich, M.M. Faas, B.N. Melgert — 2010-08-01

Smoking during pregnancy is associated with a decreased incidence of preeclampsia, lower birth weights, and an increased incidence of spontaneous abortions. Smoking has several effects on the immune system, including anti-inflammatory effects, which could explain the decreased incidence of preeclampsia in women who smoke. Smoking possibly also has direct effects on placental development that may explain the lower birth weights and increased abortion rates. This study aimed to analyse effects of smoking on local and systemic responses of the maternal immune system during pregnancy. Pregnant mice were exposed to smoke or air, 7h a day, from 7 days before mating until the day of sacrifice at gestational day 11.5. T-cell subsets were identified by flow cytometry in iliac (uterus-draining) and inguinal lymph nodes, and in the spleen. Placental tissue was fixed in paraformaldehyde for histological analysis. Slides were incubated in 0.5% diastase for 30min to remove endogenous glycogen and stained for NK cells using the Periodic Acid Schiff (PAS) staining. Smoke-exposed mice had significantly more resorptions and therefore significantly lower percentages of viable pups than non-smoking animals. This was accompanied by higher numbers of NK cells in decidua of smoke-exposed mice. Systemically, the ratio between CD25+Foxp3− effector and CD25+Foxp3+ regulatory T cells in these smoke-exposed mice was significantly lower due to a systemic decrease in effector T cells. However, locally in the uterine draining lymph nodes, this ratio was not different in smoke-exposed mice. We conclude that maternal smoke exposure during pregnancy affects local and systemic immune responses in different ways. Smoking increases decidual NK cell numbers, suggesting that these cells are involved in the increased resorption rates we found. Smoking also decreases systemic numbers of effector T cells which may explain the decreased incidence of preeclampsia in smoking women. Studying these immunological changes may help us explain altered pregnancy outcomes in smoking women.

Mismatch between decidual DC-SIGN+ dendritic cells and Foxp3+ T regulatory cells in preeclampsia

P. Hsu, B. Santner-Nanan, M.J. Peek, J. Dahlstrom, R. Nanan — 2010-08-01

Normal pregnancy requires maternal immune tolerance towards the semi-allogeneic fetus. It is thought that preeclampsia might represent a form of maternal T cell-mediated intolerance towards the fetus. In line with this, we have reported a systemic increase of T regulatory cells (Tregs) in healthy pregnancies, which is not found in preeclampsia (J. Immunol. 183 (11) (2009) 7023–7030). Because the interaction between Tregs and dendritic cells (DCs) is important for the induction of immune tolerance we analysed the distribution and interaction between decidual dendritic cells (DCs) and decidual T regulatory cells (Tregs) in healthy pregnancy and in preeclampsia. Paraffin embedded sections of the decidua of 20 healthy pregnant and 20 preeclamptic women were obtained at term. Immunohistochemistry was performed on sections with double staining for DC-SIGN (marker of immature DCs) and Foxp3 (marker of Tregs) or CD83 (marker of mature DCs) and Foxp3. The number of each of cell type was counted and proximity between Tregs and DCs noted across 10 high power fields. There were significantly higher numbers of all cell types (DC-SIGN+, CD83+, Foxp3+) in preeclampsia (p-value=0.0002, 0.004, 0.006 respectively). 80% and 70% of the Foxp3+ Tregs were attached to DC-SIGN+ DCs in decidual tissues from women with normal pregnancy and preeclampsia respectively (p>0.05). Few Foxp3+ Tregs were attached to CD83+ DCs. There was a significant positive correlation between the number of Foxp3+ Tregs and DC-SIGN+ DCs in normal pregnancy (r2=0.68, p<0.001), but not in preeclampsia. We conclude that most Tregs are closely associated to DC-SIGN+ immature DCs (iDC) in both normal pregnancy and preeclampsia. However, the lack of correlation between Tregs and iDCs in preeclampsia implies that preeclampsia might be associated with ineffective tolerance induction at the decidual level.

Novel roles for macrophages in epithelial cell turnover in the cycling adult mammary gland

W.V. Ingman, A.C.L. Chua, L.J. Hodson, S.A. Robertson — 2010-08-01

The adult mammary gland responds to fluctuations in estradiol and progesterone over the course of the ovarian cycle. Following ovulation, rising levels of progesterone cause proliferation and differentiation of the mammary gland epithelium in preparation for potential pregnancy and lactation. If pregnancy does not proceed, serum progesterone drops, and apoptosis and remodelling of the newly developed epithelium occurs. The aim of this study was to investigate the physiological necessity of macrophages in the epithelial cell turnover associated with the ovarian cycle, using a mouse model of acute macrophage depletion (the Cd11b-Dtr transgenic mouse). As macrophages are known to regulate ovarian progesterone synthesis, the direct effect of macrophages on mammary gland development could not be investigated in ovary-intact mice. We developed a mouse model that mimics the epithelial cell turnover that occurs during the estrous cycle. Ovariectomised mice treated with exogenous estradiol and progesterone undergo proliferation and differentiation of epithelium into early alveolar structures, similar to that observed in the metestrus/diestrus phase of the cycle. Following alveolar bud formation, administration of the progesterone receptor antagonist mifepristone induces apoptosis and clearance of these new buds, similar to the diestrus/proestrus phase. Macrophage depletion during hormone-induced alveolar bud formation resulted in a 2-fold reduction in both ductal epithelial proliferation and the number of alveolar buds. In contrast, macrophage depletion during mifepristone-induced apoptosis and remodelling resulted in a 2-fold increase in the number of branch points, which are likely to be incompletely remodelled alveolar buds. These studies describe two novel roles for macrophages in the cellular turnover of epithelial cells in the cycling adult mammary gland. As progesterone rises during the metestrus/diestrus phase of the cycle, macrophages promote development of alveolar buds. When progesterone falls during the diestrus/proestrus phase, macrophages promote remodelling of the tissue back to its basic architecture.

Cross-talk between inflammation and stress reaction in toll-like receptor 4-mediated growth of endometriosis

K.N. Khan, M. Kitajima, K. Hiraki, A. Fujishita, M. Nakashima, H. Masuzaki — 2010-08-01

The pathogenesis of endometriosis is still unclear and is difficult to explain uniformly by a single factor. Hormonal factors and inflammation are commonly involved in the regulation of endometriosis. However, information on the combined role of inflammation and the stress reaction is lacking. We have investigated the mutual interaction between inflammation and the stress reaction in Toll-like receptor 4 (TLR4)-mediated growth of endometriosis. Biopsy specimens were collected from 30 women with endometriosis and 20 women without endometriosis. Endotoxin (LPS) and human heat shock protein-70 (Hsp70) levels in the menstrual fluid (MF) and peritoneal fluid (PF) were measured in these women. Gene and protein expression of TLR4 in macrophages and endometrial cells were examined. The single and combined effects of LPS and Hsp70 on the production of macromolecules by macrophages and Hsp70 expression by endometrial cells and on cell proliferation were investigated. Endotoxin and Hsp70 levels in MF and PF were significantly higher in women with endometriosis than in control women. A higher immunoexpression of Hsp70 was found in endometria of women with endometriosis than in control women. Gene and protein expression of TLR4 were detected in macrophages and endometrial cells. LPS was able to significantly stimulate TLR4-mediated production of a number of macromolecules by macrophages, induced expression of Hsp70 by endometrial cells and promote cell proliferation. This effect of LPS was significantly higher in women with endometriosis than in control women. Again, exogenous treatment with Hsp70 significantly induced TLR4-mediated cytokine production and cell proliferation. Pretreatment of cells with anti-TLR4 antibody significantly suppressed LPS+Hsp70-stimulated cytokine production and cell proliferation. A prominent inflammatory response and stress reaction was observed in women with endometriosis. We conclude that a cross-talk between inflammation and the stress reaction in pelvic environment may be involved in TLR4-mediated growth of endometriosis.

A role for autoimmune regulator (AIRE) in female fertility

S. Jasti, B.K. Petroff, M.G. Petroff — 2010-08-01

The discrimination between self and foreign antigens by developing T cells is a hallmark function of the immune system, and is established in the thymus during development of central tolerance. Along with ubiquitous antigens, many antigens otherwise restricted to peripheral tissues are expressed by medullary thymic epithelial cells under the control of the transcriptional regulator AIRE (AutoImmune REgulator). Functional deletion of AIRE in mice and humans causes a breakdown of self-tolerance and organ-specific autoimmune disease. Mice lacking AIRE have been reported to have defects in fertility, but whether this gene plays a role in immune tolerance to female reproductive tissues is not understood. We used AIRE-deficient mice on the Balb/c genetic background to evaluate a possible role of central tolerance in female reproduction. Mice lacking AIRE exhibited delayed puberty in comparison to wild-type controls. Only 37.5% of AIRE-deficient mice (n=8) gave litters, whereas 100% of wild-type females (n=6) delivered litters. Litter size in AIRE-deficient mice was also reduced in comparison to controls (4.6 pups vs. 6.8 pups, respectively). Following birth of the first litter, none of the AIRE-deficient mice delivered a second litter, whereas all control females delivered 2nd litters. To assess whether AIRE influenced ovarian follicular reserves, virgin females were sacrificed at 4, 8, 12, 16 and 20 weeks of age, and follicles were counted using histochemical techniques. While there was no clear relationship between follicular reserves and age, approximately 60% of mice exhibited ovarian inflammatory infiltrates; of these, 45% showed complete follicular loss (n=18). In conclusion, deletion of AIRE in mice severely impaired female fertility. This may be caused, at least in part, by a loss in ovarian follicular reserves and/or associated inflammation.Supported by KUMC Research Institute Lied Pilot Grant and NIH R21HD062879.

Rat testicular macrophages constitutively produce interleukin-10 and have an ‘alternatively activated’ response to inflammatory stimulation in vitro

W.R. Winnall, J.A. Muir, P. Hertzog, M.P. Hedger — 2010-08-01

The testis is a site of immune privilege, providing protection for the developing germ cells from innate and adaptive immune responses which can reduce fertility. Testicular macrophages (TMs) may play a role in the maintenance of testicular immune privilege, but the mechanisms by which they might do this are incompletely understood. Macrophages are classified into two general phenotypes based on their responses to stimulation; “classically activated” (M1) macrophages, which undergo strong inflammatory responses, and “alternatively activated” (M2) macrophages, which have anti-inflammatory and tissue repair properties. In order to characterise their phenotype, we have isolated rat TMs and cultured them for 2–16h, comparing them to bone marrow-derived macrophages (BMMs) by measuring inflammatory regulator production using real-time PCR and ELISA. M1 responses were stimulated with the bacterial product lipopolysaccharide (LPS) and interferon-γ (IFNγ). M2 responses were stimulated using the anti-inflammatory cytokine interleukin-4 (IL-4). TMs constitutively expressed mRNA for anti-inflammatory IL-10 and produced IL-10 protein, whereas BMMs produced no detectable IL-10. IL-10 mRNA was highest in TMs stimulated with LPS and IFNγ, at both 6 and 16h. TMs had a greatly diminished response to LPS/IFNγ at 2h, in terms of pro-inflammatory TNFα and IL-1β. TMs had little IL-10, TNFα or IL-1β response to IL-4 at 2h, but produced the suppressor of cytokine signaling, SOCS1, suggesting a novel role for this factor in the testis. Interestingly, after 16h of TM IL-4 treatment, there was a downregulation of IL-1β mRNA, but no significant change in IL-10 levels. TMs therefore exhibited an ‘alternatively activated’ phenotype, involving IL-10, consistent with a role in supporting testicular immune privilege. M2 macrophages contribute to fibrosis in other tissues, implicating a possible involvement of these macrophages in testicular fibrosis, which is a major cause of testicular damage in conditions such as vasectomy, cryptorchidism and infertility.

Microbial colonisation of follicular fluid: alterations in cytokine expression and adverse assisted reproductive technology outcomes

2010-08-01

Previous studies have measured cytokine expression within follicular fluid collected at the time of trans-vaginal oocyte retrieval and compared the profiles with the aetiology of infertility and/or successful or unsuccessful assisted reproductive technology (ART) outcomes. Seventy-one paired follicular fluid and vaginal swab specimens collected from ART patients were cultured to detect microorganisms and then were tested for the presence of cytokines by multiplex fluorescence bead assays. Specimen selection was based on two criteria: whether the follicular fluid specimen was colonised (with microorganisms prior to oocyte retrieval) or contaminated by lower genital tract microflora at the time of oocyte retrieval and; the aetiology of infertility. Patients included fertile women (infertile male partners) (n=18), women with a history of endometriosis (n=16) or polycystic ovary syndrome (n=14), or couples with a history of genital infection (n=9) or idiopathic infertility (n=14). Microorganisms and cytokines were detected within all follicular fluid and vaginal swab specimens tested. Cytokines were associated with successful fertilisation (IL-1α, IL-1β, IL-18 p=0.05–0.1; VEGF p=0.05–0.001) and embryo transfer outcomes (IL-1β, IL-6, IL-12p40, GCSF, IFNγ p=0.05–0.1) as well as the aetiology of infertility (IL-1β, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-18, GCSF, MCSF, LIF, TNFβ). Colonising microorganisms were associated with decreased fertilisation rates for fertile women (p=0.0002), and women with infertility due to endometriosis (p=0.0002) or polycystic ovary syndrome (p=0.002). Colonising microorganisms were also associated with decreased pregnancy rates in women from couples with idiopathic infertility (p=0.001).

Paternal antigen-specific regulatory T cells proliferate in the draining lymph nodes before the implantation and in the uterus after the implantation

T. Shima, M. Ito, Y. Sasaki, A. Nakashima, S. Saito — 2010-08-01

We have studied the role of paternal antigen-specific (PA-specific) regulatory T (Treg) cells for the maintenance of allogeneic pregnancy, using transgenic T cells expressing the T cell receptor Vβ6 that recognizes the Mls1a antigen expressed in DBA/2 mice. In BALB/c(♀)-DBA/2(♂) mating, Vβ6+CD4+Foxp3+Treg cells are recognized as PA-specific Treg cells. The percentage of Ki67+ cells in Vβ6+CD4+Foxp3+Treg cells increased in draining lymphnodes on day 3.5 pc (before-implantation) and day 5.5 pc (after-implantation) in allogeneic pregnant mice. The percentage of Ki67+ cells in uterus increased after day 5.5 pc in allogeneic pregnant mice, but did not change in syngeneic pregnant BALB/c. PA-specific Treg cells highly expressed CCR5, and PA-specific Ki67+ Treg cells highly expressed CCR4 on their surface.

GM-CSF regulates uterine macrophage and dendritic cell maturation and antigen presentation

L.M. Moldenhauer, S.N. Hudson-Keenihan, J.D. Hayball, S.A. Robertson — 2010-08-01

Dendritic cells (DCs) and macrophages are an abundant cell population in the uterine endometrium and are implicated in generating the maternal immune tolerance required to accommodate the conceptus. Granulocyte-macrophage colony-stimulating factor (GM-CSF, CSF2) is expressed by uterine epithelial cells under regulation by steroid hormones and male seminal fluid, and is a known regulator of DC and macrophage activation. We aimed to investigate the role of GM-CSF in uterine antigen presenting cell (APC) behavior using mice with a null mutation in the Csf2 gene. The number and phenotype of APCs in endometrial tissue and whole uterus were evaluated at estrus, on day 0.5 pc and day 3.5 pc by immunohistochemistry and flow cytometry. The functional competence of uterine APCs to activate maternal T cells was investigated by measuring proliferation of ovalbumin-reactive CSFE-labeled OT-I (CD8+) and OT-II (CD4+) recovered from female para-aortic lymph nodes on day 3.5 pc after mating with Act-mOVA males, which express ovalbumin ubiquitously in seminal fluid. Immunohistochemistry showed no effect of GM-CSF deficiency on the numbers of CD11c+ DCs and F4/80+ macrophages in the uterus at estrus or on day 0.5 pc and day 3.5 pc, but class A scavenger receptor+ cells were depleted, particularly after coitus. Flow cytometry revealed a reduction in MHC class II expression by uterine CD11c+ and F4/80+ cells from GM-CSF deficient mice. This was associated with an altered functional capacity of the APCs in the endometrium with their ability to activate OT-I and OT-II T cell proliferation reduced by 30% and 44% respectively (both p<0.05). These data show that GM-CSF regulates uterine APC expression of proteins required for effective antigen processing and presentation, and the absence of GM-CSF leads to a reduced ability for APCs to stimulate T cells. In conclusion, uterine epithelial cell GM-CSF expression during early pregnancy appears necessary to facilitate maternal immune changes, including T cell awareness of paternal antigens, conducive to the establishment of pregnancy.

Isolation and molecular studies of human HLA-G+ extravillous trophoblasts (EVT)

T. Tilburgs, B. Rybalov, L. Gardner, A. Moffett, J.L. Strominger — 2010-08-01

Invading fetal HLA-G+ EVT play a key role in the process of placental anchoring, opening of the uterine spiral arteries and prevention of a maternal immune attack on the foreign fetal tissue. However, HLA-G+ EVT are extremely difficult to study due to their low frequency and lack of methods to culture or expand them. In collaboration with Prof. Ashley Moffett we established methods to isolate HLA-G+ EVT from human 1st trimester tissue. Preparations yielded 0.5–5.0×106 cells with 8–20% HLA-G+ EVT and 50–70% HLA-G− EGFR1+ villous trophoblasts (VT). After culture on fibronectin the purity increased to 40–80% HLA-G+ cells, whereas purification using FACS sort provided 1.1–7.4×105 HLA-G+ EVT and 8.9–33×105 HLA-G− VT, both with >95% purity. After culture on fibronectin both FACS sorted HLA-G+ EVT and HLA-G− VT retained their phenotype and did not proliferate. FACS analysis showed that both trophoblasts subsets express growth factor receptors e.g. EGFR1, EGFR2, LIFR and/or IL-10R. However, thus far addition of (combinations of) EGF, HB-EGF, LIF or IL-10 did not induce proliferation or differentiation, demonstrating again the complexity of trophoblast biology. Furthermore, sufficient mRNA was obtained from 7 paired HLA-G+ EVT and HLA-G− VT preparations to perform RNA microarray analysis. Unique gene signatures based on a 4-fold expression difference were made and 329 and 341 genes uniquely expressed on HLA-G+ EVT and HLA-G− VT respectively were identified. Further analysis will focus on genes involved in HLA expression and the molecular pathways involved in cell migration, proliferation and invasion. Thereby valuable insights on how fetal trophoblasts differentiate and invade maternal tissue while preventing a maternal immune attack will be obtained.

Micro-RNA-profiles in response to LIF in trophoblastic cells

D.M. Morales, S. Ospina, U.R. Markert — 2010-08-01

Micro-RNAs are recently detected key regulators of the immune system and of carcinogenic processes, but their function in pregnancy is poorly investigated yet. Leukemia Inhibitory Factor (LIF) is a cytokine which plays a crucial role in trophoblastic cells and which triggers their invasion via Signal Transducer and Activator of Transcription 3 (STAT3). Therefore, we aimed to further analyze LIF induced signal transduction and LIF-regulated micro-RNAs in two trophoblastic cell lines. JEG-3 choriocarcinoma cells were stimulated with LIF for 1–24h, and total mRNA was isolated. Phosphorylation of signal transducers ERK1/2 (thr 702 and tyr 704) and STAT3 (ser 727 and tyr 705) was assessed by gel electrophoresis and Western blotting. STAT3 gene expression was analyzed as control of induction by RT-PCR. Expression of five different micro-RNAs, which are either known to be expressed in placental tissue or involved in tumor invasion, was quantified at six time points by qRT-PCR. Finally, by using a 7900HT Fast Real-Time PCR System combined with TaqMan micro-RNA assay, the expression profile of 760 micro-RNA was screened and compared in JEG-3 cells and HTR8/SVneo immortalized first trimester trophoblast cells stimulated or not with LIF for 4h. ERK1/2 and STAT3 were phosphorylated from 10 to 30min after LIF stimulation. Three out of five micro-RNA (miR-9, miR-141 and let-7g) were time-dependently down-regulated by LIF with a peak after 4h of treatment, which correlates with an increase of STAT3 expression. The micro-RNA profiling showed that expression of approximately 65% of all micro-RNA was at least 10-fold different in JEG-3 and HTR8/SVneo cells. LIF influenced around 10% of all micro-RNA, but with little coincidence between both cell lines. The results show that LIF modulates micro-RNA expression in trophoblastic cells, but different cell lines behave diversely.

Cyclic mechanical stretch augments neutrophil chemotactic chemokine and MMP production in cultured human uterine myometrial and decidual cells

Y. Zhao, K. Koga, Y. Osuga, Y. Taketani — 2010-08-01

To investigate the possible impact of uterine contraction in the immune environment within the myometrium and decidua during parturition. Myometrium smooth muscle cells (SMCs) were taken from cases of hysterectomy for benign disease. Decidual cells (DCs) were scraped from fetal membranes taken from elective Cesarean sections. Cyclic stretch which mimics uterine contraction was applied by Flexercell tension system for SMCs and DCs. IL-8 and GROα mRNA and protein expressions were measured by RT-PCR and ELISA. Neutrophil chemotactic activity in conditioned media was evaluated by migration assay. Pro-MMP1 was measured by ELISA. To evaluate the effect of progesterone, SMCs and DCs were pretreated with progesterone for 24h. The data showed the following: (1) In SMCs, cyclic stretch significantly increased IL-8 and GROα mRNA expression (7.11 and 3.92 times, P<0.05 for both) and their protein production (5.54 and 3.62 times, P<0.0001 and P<0.01, respectively). In DCs, cyclic stretch significantly increased IL-8 and GROα mRNA expression (3.61 and 2.22 times, P<0.05 for both) and their protein production (4.65 and 3.08 times, P<0.05 and P<0.01, respectively). (2) Supernatants from stretched cells had higher neutrophil chemotactic activity compared with control (2.53 and 6.88 times, in SMCs and DCs respectively) and this increase was canceled by anti-IL8 and GROα antibody. (3) Cyclic stretch significantly increased pro-MMP1 production both in SMCs (3.65 times) and DCs (5.26 times) (P<0.05 for both). (4) These effects of stretch were reduced by progesterone. These results suggest that during the labor, cyclic stretch induces chemokines in SMCs and DCs, which results in neutrophil infiltrations in the myometrium-decidual layer. Stretch also activates MMPs, which may cause cervical ripening and rupture of membrane. These indicate that uterine contraction per se contributes to the augmentation of parturition. The inhibitory effects of progesterone may explain the therapeutic property of progestin for preterm labor.

Viral infection sensitizes pregnancy to LPS leading to preterm labor

I. Cardenas, P. Aldo, R. Means, S. Lang, P. Stabach, G. Mor — 2010-08-01

Among pregnant women, an acquired viral infection with a concurrent bacterial infection is a detrimental factor associated to poor prognosis and death. Here, we used a murine model to evaluate the effect of a viral infection that does not lead to preterm labor on the response to low doses of LPS. Our objectives were: (1) to characterize the effect of a viral infection with a bacterial infection on pregnancy outcome, (2) to characterize the placental and fetal immune responses to the viral sensitization to LPS, and (3) to characterize the mechanisms of viral sensitization to LPS during pregnancy. C57B/6wt mice were injected intraperitoneally with murine gammaherpesvirus 68 (MHV68) at E8.5. Either PBS or LPS was injected i.p. at E15.5. Pregnancy outcome, fetal weight, viral load (plaques forming units/ml of tissue) at the placenta, were determined at E17.5. Placental morphology and immune cells infiltration were evaluated by immunohistochemistry. Cytokine profile and TLR expression were determined by Multiplex and qRT-PCR respectively. LPS treatment of MHV-68 infected animals was found to induce preterm delivery and fetal death in 100% of the mice in less than 24h compared to 29% in the LPS treated mice without a previous viral infection. Low doses of LPS (0.8μg/kg) are able to induce a significant increase on inflammatory cytokines only in the viral infected mice. Similarly, morphological evaluation of the placenta from MHV68 and LPS treated mice revealed necrosis and polymorphonuclear infiltration. The observed changes in LPS response correlated with viral induced upregulation of TLR2, TLR4 and TLR6. We describe for the first time that a silent viral infection in pregnant mice sensitizes to a bacterial infection leading to preterm delivery and IUGR. This effect is mediated by a specific regulation of TLRs in the trophoblast and increased expression of pro-inflammatory cytokines. Our data suggest that an ongoing viral infection may increase the risk for pregnancy complications due to subsequent bacterial infection.

Development of a vaginally applied, non-hormonal contraceptive: the contraceptive efficacy and impact on bone turnover of PEGLA, a long-acting LIF antagonist

2010-08-01

The WHO has called for the urgent development of pharmacological, non-hormonal contraceptives. Leukaemia inhibitory factor (LIF) is obligatory for embryo implantation in mice and associated with infertility in women. Injection of a long-acting LIF antagonist (PEGLA) blocks uterine LIF preventing implantation in mice, making PEGLA a promising non-hormonal contraceptive. LIF and LIFR null mice show decreased bone volume associated with increased osteoclast number and size, suggesting PEGLA may target bone. Vaginally administered PEGLA could be a ‘dual-role’ contraceptive: carried in a microbicide that blocks the vaginal transmission of sexually transmitted infections. We aimed to establish the contraceptive efficacy of vaginally administered PEGLA and identify non-uterine targets of PEGLA in mice. PEGLA was administered to mated female mice by intraperitoneal (IP) injection or vaginally (n=4/group) during the peri-implantation period to determine its effect on implantation and bone turnover. The tissue and blood accumulation of 125I-PEGLA or control was identified at various time-points following IP injection (≤120h) or vaginal administration (≤24h) (n=3/group). PEGLA administered via vaginal gel blocked implantation (0.0+0.0 vs 8.5+0.5) at a lower dose (500μg) than IP injection (1500μg). PEGLA administered by IP injection resulted in fewer (4.0+0.3% vs 7.7+1.5%; p<0.05) but larger (20.9+0.9μm vs 18.1+0.5μm; p<0.05) osteoclasts and increased trabecular bone volume (6.8+0.9% vs 3.1+1.1%; p<0.05) but vaginally administered PEGLA had no effect on bone (p>0.05). 125I-PEGLA accumulated more quickly (10min vs 30min) and was retained longer (96h vs 24h) in blood and tissue following IP injection compared to vaginal administration. This is the first study to show the contraceptive efficacy of a PEGylated compound following vaginal delivery. Local delivery of PEGLA decreased the required dose and eliminated the effect on bone, suggesting that local adminis tration would minimise the non-target effects of PEGLA. Contraceptive trials are now required in non-human primates to progress PEGLA towards human clinical trials.

Characterization of natural killer cells in implantation failure: effect of low-molecular weight heparin on endometrial natural killer cell counts

2010-08-01

Elevated levels of circulating NK cells have been linked to increased rates of implantation failure. However, the validity of peripheral blood NK cells has been discussed recently. In this sense, endometrial NK subsets are well characterized in recurrent abortion, but they are not enough studied in IVF failure. It has been demonstrated the efficacy of IVIG treatment in IVF failure when NK cell levels are elevated, but this treatment is expensive. On the other hand, heparin is able to modulate implantation, and invasion, having the potential to improve pregnancy rates and outcomes. However, the role of pre-conceptional heparin in modulating NK cell levels is still unknown. The goal of this work was to analyze the ability of NK cells to discriminate fertile and IVF failure patients in peripheral blood vs. endometrium, and also to determine the capacity of pre-conceptional low-molecular weight heparin to modulate NK cell counts. Thirty-six IVF failure patients and 16 fertile women were studied. Peripheral blood and endometrial tissue were obtained during implantation window w/o IVF-ET. Total counts and ratio of CD56+CD3−CD16+/− and CD56+CD9+CD16+/− subsets were analyzed by FACS. In the consecutive cycle infertile patients were treated with enoxaparin and NK subset analysis was repeated. Comparison of Medias, Spearman and Pearson correlation tests and ROC curve analysis were performed. The results show that IVF failure patients has elevated total count and ratio of CD56+CD9+CD16+ subset (p<0.05). These data did not correlate with peripheral blood values (p≥0.05). ROC curve analysis shown that total count of CD56+CD9+CD16+ is the parameter that best discriminated between fertile and infertile patients (p<0.01). Enoxaparin normalized counts of CD56+CD9+CD16+ (p<0.05). We conclude that endometrial CD56+CD16+ counts are a good parameter for immunological testing in IVF failure. Enoxaparin could be a potential immunomodulator in assisted conception of patients with elevated NK cell counts.

The physiological consequences of exposure to zona pellucida 3 antibody in the mouse ovary

M.L. Lloyd, J.M. Papadimitiou, S. O’Leary, S.A. Robertson, G.R. Shellam — 2010-08-01

We have established that a recombinant murine cytomegalovirus vaccine expressing murine ZP3 induces rapid and irreversible infertility in BALB/c mice. Mice receiving this vaccine demonstrate significantly reduced fertility by 14 days post inoculation (p.i.) and are infertile by 21 days p.i. The infertility was mediated by zona pellucida-specific antibodies that were detected in situ in infected mice, binding to the follicular zona pellucida by 14 days p.i. Blastocyst formation was significantly reduced by 14 days p.i. demonstrating the effect of zona pellucida-specific antibody on ovulated oocytes. The ovaries of infected mice demonstrated a loss of tertiary follicles by 21 days p.i. with a significant reduction in primary and secondary follicle numbers being demonstrated by 35 days p.i. Examination of ovarian sections by electron microscopy at 35 days p.i. revealed a structurally abnormal zona pellucida layer developing around remaining secondary follicles. In addition, abnormal gap junction formation was noted occurring directly between the oocytes and granulosa cells in follicles of infected mice. This corresponds with our previous finding of a surprising increase in the expression of connexin43, a gap junction protein, at early times post infection. Immunoglobulin-deficient μMT/μMT mice inoculated with the recombinant vaccine did not demonstrate similar pathology, indicating that antibody was the causative agent for the follicular abnormalities. Our results suggest that follicle loss in humans may occur via disrupted zona pellucida formation in developing follicles in women with zona pellucida-specific autoantibodies. Thus infertility may initially be due to fertilization failure, with a progressive follicle loss leading to premature ovarian failure in circumstances where high titers and/or chronic ongoing antibody occur.

The use of recombinant vaccines to induce effective contraception in mice

M.L. Lloyd, L.M. Smith, A. Redwood, C. Hardy, G.R. Shellam — 2010-08-01

A recombinant murine cytomegalovirus (MCMV) vaccine expressing murine zona pellucida 3 (ZP3) induces rapid and irreversible infertility in BALB/c mice. Properties of MCMV, such as the large genome size (permitting foreign gene insertion) and the ability to induce persistent and latent infection (resulting in antigen expression over long periods of time) ensure that MCMV is an effective vaccine vector. MCMV has been extensively used in our laboratory to deliver a variety of reproductive antigens including murine ZP3, porcine zona pellucida C and the sperm antigen PH20 with contraceptive success varying from highly effective (ZP3) to completely ineffective (PH20 and porcine zona pellucida C) in reducing mouse fertility. The expression of these antigens by MCMV has not restricted in vitro growth; however, in vivo growth is severely compromised with salivary gland replication being virtually undetectable. Somewhat remarkably, the immunogenicity of the encoded antigen is not compromised and strong antibody responses are obtained from infected mice well after 100 days post inoculation, and are required for effective contraception. Interestingly, we have found that innate immunity to the specific MCMV strain being used as the vaccine vector influences contraceptive efficacy. Wild isolates of MCMV that differ in their engagement with the innate immune system have been used as vectors to overcome this problem. Future research will examine different insertion sites and the effect that this has on the kinetics of gene expression and the efficacy of the resultant immune response, with the aim of producing a vaccine that does not have impaired in vivo growth.

Immunocontraceptive vaccines and major histocompatibility complex variation in the brushtail possum

O.J. Holland, P.E. Cowan, D.M. Gleeson, J.A. Duckworth, L.W. Chamley — 2010-08-01

The possum is a major invasive pest in New Zealand. One option for its control is the use of immunocontraceptive vaccines. Initial trials of contraceptive vaccines have shown individual variation in responses. Given this variation in responses, the use of vaccines on wild populations could result in the evolution of a resistant population through selection for possums that remain fertile because of lack of response. Understanding the basis of this variation is therefore important. The major histocompatibility complex (MHC) is an important influence on the nature of immune responses. This research aimed to investigate the genetic variation in the MHC loci of the possum, and to identify possible variants associated with different zona pellucid-based immunocontraceptive vaccines. Five sets of polymerase chain reaction primers were developed to amplify possum MHC loci. These were used to determine sequences in 176 animals sampled from locations around New Zealand, and in 85 possums with known responses to vaccines. A high level of polymorphisms was found in both class I and class II MHC loci of the possum, 71 novel sequences were identified, and this brings the total number of MHC sequences known in the possum to 75. Putative MHC haplotypes were defined by linkage disequilibrium analysis. Haplotypes were compared between individuals with measured responses to immunocontraceptive vaccines. Two haplotypes were found to associate significantly with differences in vaccine response. Possums that carried haplotype 6 showed reduced responsiveness to one vaccine, while possums that carried haplotype 9 showed increased responsiveness to a separate vaccine. The identification of MHC haplotypes associated with different responses to immunocontraceptive vaccines confirms that variation in the MHC may contribute to non-responsiveness and the persistence of fertility in some individuals. A greater understanding of the MHC in these animals may allow vaccines to be optimised to minimise non-responsiveness.

Bait-delivered fertility control vaccines for brushtail possums in New Zealand

J. Duckworth, S. Scobie, P. Lubitz, W. Lubitz, P.E. Cowan — 2010-08-01

New Zealand has many wildlife management issues resulting from introduced mammals that threaten native biodiversity values and agricultural production. Our research on non-lethal management methods for possums is focused on developing zona pellucida (ZP) fertility control vaccines suitable for bait delivery to wild possums. Bacterial ghosts (BG), particulate vaccines derived from non-living empty cell envelopes of gram-negative bacteria, show potential for oral/aerosol vaccine delivery. We have demonstrated that BGs expressing possum ZP2-C administered by the ocular/nasal routes significantly reduced both the fertilisation rate of artificially inseminated possums and the number of offspring born in captive breeding trials. In collaboration with researchers in Austria, new constructs of BG vaccines, expressing 5–50 times more ZP2 protein, have now been developed by optimising the ZP nucleotide sequences for bacterial expression and using site-directed sequences to sequester the antigen into the periplasmic space. To improve vaccine efficacy, we are also evaluating formulations protecting bacterial ghosts from acid degradation in the stomach and from proteolytic enzymes in the intestine. Initial studies demonstrated that ZP2 BG vaccines delivered orally with gastric acid inhibitors reduced the conception rate of mated female possums. New BG formulations, including enteric coated granules, are being assessed in immunogenicity and natural breeding trials. Current research aims to improve the vaccine formulations to maximise the potency and longevity of the immune response and associated infertility, thus enabling vaccines suitable for field use to be developed.

Lipiodol alters the population of dendritic cells and regulatory T cells in the peritoneal cavity

G. Izumi, M. Takamura, K. Koga, Y. Osuga, Y. Taketani — 2010-08-01

Lipiodol is an oil-soluble contrast media using for hysterosalpingograpy (HSG). After the procedure, the medium is known to remain in the peritoneal cavity for several months. This study was performed to clarify the effect of lipiodol on the immunological environment in the peritoneal cavity. Forty-three women who underwent laparoscopy were recruited in this study. Of those, 12 had undergone HSG before the laparoscopy (HSG group) and 31 had not (control). Peritoneal fluid was corrected and mononuclear cells were isolated and analyzed by flow cytometry. Myeloid dendritic cell (DC) type 1 (MDC1), myeloid DC type 2 (MDC2) and plasmacytoid DC (PDC) were identified by BDCA1+CD19−, BDCA3+CD14− and BDCA2+, respectively. Mature DC was defined by positive staining of CD83. Frequency of regulatory T cell (Treg) was calculated by the ratio of CD4+CD25+Foxp3+ cells to CD4+cells. To evaluate the effect of lipiodol on the maturation of DC, we cultured monocyte-derived dendritic cells (Mo-DC, developed by treatment monocytes with GM-CSF and IL4) with or without 10% lipiodol for 2 days and frequency of CD83+ Mo-DCs were evaluated. The frequencies of mature MDC1 and mature MDC2 were significantly higher in HSG group than in control (10.7% vs 1.0%, p<0.001, 5.9% vs 3.5%, p<0.05, respectively), although total numbers of MDC1 and MDC2 were not significant different. Frequencies of PDC and Treg were significantly higher in HSG group than in control (0.52% vs 0.23%, p<0.05, 12.8% vs 7.0%, p<0.05, respectively). When cultured with lipiodol, the frequency of CD83+ DCs was increased. These results suggest that lipiodol remaining in the peritoneal cavity may lead the maturation of DC and further alters the immunological environment in the peritoneal cavity.

A novel approach into better understanding of the role of uterine leukocytes during the time of menstruation and in the presence of endometriosis

M. Berbic, A.J. Hey-Cunningham, C. Ng, A. Basten, R. Markham, I.S. Fraser — 2010-08-01

Uterine immune cell populations undergo substantial changes throughout the menstrual cycle and play crucial roles in the processes of endometrial tissue remodelling, breakdown and repair. Recent studies have suggested that antigen-specific responses may be playing important roles during the normal menstrual cycle and that their function may be altered in conditions like endometriosis. However, the function of uterine leukocytes has not been previously investigated from the perspective of the immune environment of uterine draining lymph nodes (LNs). We have demonstrated a range of subtle, but significant LN changes during the normal menstrual cycle and in endometriosis.

Uterine epithelial cell fucosylated structures are regulated by macrophages during early pregnancy in mouse

M.J. Jasper, A.S. Care, B. Sullivan, W.V. Ingman, J.D. Aplin, S.A. Robertson — 2010-08-01

Following seminal fluid exposure at coitus, macrophages accumulate within stromal tissue subjacent to the luminal epithelium in the mouse uterus. Intimate juxtaposition with the luminal epithelial cells at the endometrial surface affords macrophages a potential paracrine role in influencing epithelial cell function during early pregnancy. We aimed to investigate their role in regulating epithelial cell expression of fucosylated structures required for embryo attachment and implantation. Fucosyltransferase enzymes Fut1, Fut2 (EC 2.4.1.69), and Fut4 (EC 2.4.1.214) mRNAs were quantified by qRT-PCR in uterine epithelial cells after laser capture micro-dissection in situ, or after epithelial cell co-culture with macrophages or macrophage-secreted factors. When uterine macrophage recruitment was impaired by mating with seminal plasma-deficient males, epithelial cell Fut2 expression on day 3.5 post coitum (pc) was reduced compared to intact-mated controls. Epithelial cell Fut2 was upregulated in vitro by co-culture with macrophages or macrophage-conditioned medium (MCM). Macrophage-derived cytokines LIF, IL1B and IL12 replicated the effect of MCM on Fut2 expression, and MCM-stimulated expression was inhibited by anti-LIF and anti-IL1B neutralizing antibodies. The effects of acute macrophage depletion on fucosylated structures detected with lectins Ulex europaeus 1 (UEA-1) and Lotus tetragonolobus purpureas (LTP), or LewisX immuno-reactivity, were quantified in vivo in Cd11b-dtr transgenic mice. Acute depletion of macrophages caused a 30% reduction in luminal epithelial UEA-1 staining and a 67% reduction in LewisX staining in uterine tissues of mice hormonally treated to mimic early pregnancy. Together, these data demonstrate that uterine epithelial Fut2 mRNA expression and terminal fucosylation of embryo attachment ligands is regulated in preparation for implantation by factors including LIF and IL1B secreted from macrophages recruited during the inflammatory response to insemination. Our findings raise the possibility that altered endometrial macrophage populations might impact uterine receptivity in infertile women.

Seminal fluid regulates the uterine Foxp3+ T regulatory cell population through CCL19-mediated recruitment during the peri-implantation period in mice

2010-08-01

CD4+CD25+ Treg cells are a critical T cell subset that actively facilitates maternal immune tolerance of the semi-allogeneic conceptus in early pregnancy. The CD4+CD25+ cell population is expanded in the para-aortic lymph nodes (PALN) that drain the uterus in the pre-implantation period and we have shown that factors in the seminal fluid delivered to the female at coitus have a critical role in activating and expanding this cell population (). To investigate whether seminal fluid influences the number of Treg cells in the implantation site, we investigated the significance of seminal fluid exposure, and the relative contribution of the sperm and seminal fluid components, on uterine Treg cell populations at the time of implantation on day 3.5 post-coitum (pc). Using flow cytometry, immunohistochemistry and qRT-PCR for the signature Treg cell transcription factor Foxp3, an increase in the abundance of Foxp3+ cells and expression of Foxp3 mRNA in the uterus at the time of embryo implantation was identified. Seminal plasma, but not sperm, was necessary for uterine Treg cell accumulation since vasectomised but not seminal vesicle-deficient males induced a similar increase in uterine Treg cells as mating with intact males. In addition, we utilised qRT-PCR to explore the effect of seminal fluid on uterine expression of a range of chemokines on day 3.5 pc. Seminal plasma was shown to induce expression of mRNA encoding the chemokine Ccl19 (MIP3β), which targets CD62L+ Treg cells through the CCR7 receptor. Using immunohistochemistry and FACS, uterine epithelial cells were identified as the major cellular origin of this chemokine. Together, these results indicate that male seminal fluid is a key regulator of uterine Treg cell populations, and that seminal fluid operates through both expanding the availability of circulating Treg cells, and through stimulating their recruitment from the circulation into the uterine implantation site.

Modelling uterine macrophage-epithelial cell communication in vitro using peripheral blood monocytes

H. Nakamura, M.J. Jasper, S.A. Robertson, T. Kimura — 2010-08-01

In mice, macrophages are recruited into the endometrium by exposure to seminal plasma and these cells appear able to influence local cell communication and tissue remodeling events to promote uterine receptivity for embryo implantation. Our previous study showed that U937-macrophages regulate embryo adhesion molecule expression in human endometrial cells via LIF-mediated STAT3 signalling. In this study, we utilized an in vitro model comprising peripheral blood monocytes (PBMC) and Ishikawa cells to further explore regulation of macrophage-uterine epithelial cell communication. Initially, to investigate the effect of menstrual cycle stage on PBMC regulation of epithelial cell gene expression, Ishikawa cells were co-cultured with PBMCs recovered at the proliferative, peri-ovulatory (LH+1–2) and mid-secretory (LH+7–9) phases of the menstrual cycle, separated spatially using transwell inserts, and RT-PCR was used to quantify mucin and integrin mRNA expression. Co-culture with PBMCs recovered at ovulation did not elicit any change in gene expression, while PBMCs from proliferative phase increased MUC1 and MUC4 mRNA levels. Integrin β1 αV mRNA was increased by co-culture with PBMCs from the mid-secretory phase. Epithelial cells were then co-cultured with male PBMCs alone or in the presence of progesterone and/or 17β-estradiol, or PBMCs activated by seminal plasma. Co-culture with PBMCs activated by seminal plasma together with progesterone and 17β-estradiol significantly increased MUC1 mRNA levels and activated STAT3 in endometrial epithelial cells. These results suggest that the signaling characteristics of PBMCs are changed by exposure to ovarian steroid hormones in the menstrual cycle, as well as by male seminal plasma, and that these factors influence PBMC capacity to regulate uterine epithelial cell adhesion molecule expression. If similar changes are induced by seminal plasma and ovarian steroid hormones in resident uterine macrophages, these cells might have a role in facilitating development of an implantation-receptive endometrium via regulation of embryo adhesion molecules.

Indolamine 2,3-dioxygenase might contribute to the establishment of embryo tolerance in early bovine pregnancy

2010-08-01

A well-balanced embryo-maternal immune interaction in the endometrium during early pregnancy is of particular importance, since the conceptus represents a semi-allograft accepted by the mother. To analyse mechanisms of immune tolerance in early bovine pregnancy during peri-implantation, Simmental heifers inseminated with either cryo-preserved sperm or seminal plasma were slaughtered 12, 15 or 18 days post insemination. Uteri were flushed for the recovery of conceptuses, and ipsilateral intercaruncular endometrium was sampled. Gene expression analysis was performed using the Fluidigm® 96.96 Dynamic Array by simultaneously measuring the messenger RNA (mRNA) expression of 32 immune related genes in 94 preamplified endometrial and conceptus samples. In the endometrium, pro-and anti-inflammatory cytokines were determined by ELISA, and metabolites of the kynurenine pathway were analysed by tandem mass spectrometry. We detected neither increased mRNA expression of T- and NK-cell associated surface markers, nor elevated transcript levels of pro-inflammatory cytokines during the estrous cycle or due to pregnancy. TNF-α and INF-γ protein levels remained unchanged in pregnant animals as compared to controls. However, indoleamine 2,3-dioxygenase (IDO) mRNA, the initial enzyme of the tryptophan metabolizing kynurenine pathway, was significantly 18-fold (p<0.0001) more expressed in the endometrium of day 18 pregnant vs. non-pregnant animals. IDO may regulate detrimental immune responses towards the conceptus, since the activity of lymphocytes is dependent on tryptophan availability. In vivo, tryptophan levels declined over time (p=0.0008), while kynurenine levels ascended (p=0.005). IDO activity, estimated by the kynurenine/tryptophan ratio, revealed a significant interaction between pregnancy status and day of the cycle (p=0.03). Thus, elevated IDO expression in addition to increased enzyme activity might indicate a pivotal role for the kynurenine pathway during peri-implantation in order to protect the conceptus from the maternal immune system and to allow trophoblast attachment to the endometrium.

Programming embryo development with a pre-implantation inflammatory insult

P.Y. Chin, J.G. Thompson, S.A. Robertson — 2010-08-01

The cytokine milieu surrounding the pre-implantation embryo is essential in programming optimal embryo development. Perturbations to the maternal environment such as infection, inflammation and stress during the pre-implantation period can influence cytokine synthesis and may cause changes in embryo development that compromise pregnancy outcome. We aimed to investigate the effect of an inflammatory insult with LPS during the pre-implantation period on later fetal development, and to define the effect of IL10 on this response. LPS was administered i.p. to wildtype C57Bl/6 mice and mice with a null mutation in the Il10 gene (IL10−/− mice) on both day 3 and day 4 post coitum (pc). The effects of treatment on day 4 blastocysts and day 18 fetal development were analysed after treatments with various doses of LPS (0.5–62.5μg). Embryos flushed from LPS-treated mothers showed reduced viability and smaller total cell number, but were only significant when doses of >12.5μg LPS were administered. This was unlikely to be a direct effect of LPS since when embryos were cultured with LPS in vitro, no effect on development was seen even at very high doses (25μg/ml). At day 18 pc, pregnancy viability was reduced after low dose LPS treatment (0.5μg) in both IL10−/− mice (23%) and wildtype mice (61%) compared to control PBS-treated IL10−/− (44%) and wildtype (73%) females. Low dose LPS treatment also resulted in decreased fetal and placenta weights on day 18 pc in both genotypes. Our findings show that an inflammatory insult in the pre-implantation period programs the pre-implantation embryo for later adverse effects on fetal and placental development, and that IL10 protects embryos from the effects of LPS. This model will now be utilised to investigate the potential role of inflammatory cytokines such as TNFA and IFNG in mediating this response.

Regulatory interactions between DC and NK cells are not bidirectional during mouse implantation

G. Barrientos, I. Tirado González, P. Frank, P.C. Arck, B.F. Klapp, S.M. Blois — 2010-08-01

Uterine DC and NK cells appear to play a crucial role during early pregnancy regulating maternal immunity as well as decidual growth and neovascularization. However, complex regulatory interactions between DC and NK cells hinder the assessment of their specific functions within the decidual milieu. This study aimed to characterize the cross-talk between DC and NK cells in the decidua and investigate its relevance during the implantation process in mice. We used a transgenic mouse model which allows to deplete DC in vivo, and the asialo GM1 antibody strategy for ablation of NK cells to investigate the specific functions of these subsets during embryo implantation. Uterine tissue at gestation days 5.5 and 7.5 was analysed by RT-PCR and IHC for the expression of IL-11 and the cell cycle regulators cyclin D3, p21 and cyclin E at Gd 5.5 and 7.5 as hallmarks of stromal cell proliferation, differentiation and poliploidization. Decidual vascular development and remodeling was assessed based on the expression of PECAM-1, alpha smooth muscle actin and the VEGF/VEGFR system mediating angiogenesis. Even though uterine arterial remodeling was impaired, the individual depletion of NK cells did not affect stromal cell proliferation and differentiation. In contrast, the combined depletion of DC and NK cells led to severe impairments on decidual differentiation, reduced vascularization and angiogenic activity at the implantation sites similar to those described upon DC depletion. In conclusion, NK cells promote vascular modifications to support normal decidual development, but do not seem to directly affect stromal cell proliferation and differentiation. These functions are nevertheless insufficient to prevent implantation failure in the absence of DC, highlighting a hierarchical relationship in which NK cells are subordinate to the actions of DC driving embryo implantation.

Aberrant adhesion molecule and chemokine expression in chronic endometritis

K. Kitaya, T. Yasuo — 2010-08-01

Chronic endometritis (CE) is characterized by infiltration of plasmacytes within the endometrial stroma and potentially causes infertility, particularly embryo implantation failure. In parallel with stromal plasmacyte infiltration, the endometrial functional layer in CE is invaded by B cells, which are a rare leukocyte subset in the nonpathological endometrium. We investigated the molecular expression underlying this unusual increase of B cells in CE. Twenty-two out of 76 unexplained infertile women were diagnosed as CE by immunohistochemical detection of stromal plasmacytes in endometrial biopsy specimens using its specific marker syndecan-1. According to the endometrial dating criteria, seven of the chronic endometritis specimens showed out-of-phase morphology typically with delayed differentiation. Whilst the endometrium with CE contained numerous stromal B cell aggregates and glandular single B cells, other major leukocyte subsets, including T cells, NK cells, macrophages, and neutrophils were comparable in densities between CE and nonpathological endometrium. The microvascular endothelium showed immunoreactivity to adhesion molecule selectin E and chemokine CXCL13 along with immunoreactivity to CXCL1 in the glandular epithelium in CE, but not in the nonpathological endometrium. The expression pattern of other adhesion molecules and chemokines such as VCAM-1, MAdCAM-1, selectin P, CCL2, CCL19, CCL21, CXCL8, CXCL12 was similar between CE and nonpathological endometrium. Lipopolysaccharide induced surface selectin E expression and CXCL13 secretion in uterine microvascular endothelial cells, and CXCL1 secretion in endometrial epithelial cells in vitro. These findings indicate that the aberrant local microenvironment triggered possibly by Gram-negative bacterial infection plays a role in selective extravasation of circulating B cells in CE.

Follistatin-288 and follistatin-315 are regulated by progesterone in the mouse uterus

2010-08-01

Follistatin acts predominantly by binding and neutralising the activity of activin A, which plays key regulatory roles in development, reproduction and inflammation, including inflammation in the female reproductive tract. We are investigating the role of follistatin in the growth and development of the female reproductive tract. There are two isoforms of follistatin resulting from alternative gene splicing. Isoform FS288 binds spontaneously to heparan sulphate proteoglycans and is largely bound to cell surface proteoglycans. FS315 is the predominant circulating form and can only bind to heparan sulphate after binding activin A. In this study, our aim was to quantify the expression of both FS288 and FS315 mRNA in the mouse uterus during early pregnancy (days 1–4, pre-implantation), and in response to exogenous oestradiol-17β (single s.c. injection, 100ng/injection, dissection after 24h) and progesterone (three daily s.c. injections, 1mg/injection, dissection 24h after last injection) in ovariectomised mice. Gene expression was analysed using quantitative RT-PCR. Primers amplifying a product from exon 5 to 6a (unique to FS288) were used to detect FS288. Primers amplifying a product from exon 5 to 6b (unique to FS315) were used to detect FS315. In early pregnancy, expression of both FS288 and FS315 increased significantly (approximately 20-fold and 50-fold, respectively) on days 3–5, relative to days 1–2, corresponding with the increase in circulating progesterone levels that occur at this time. A significant increase in follistatin expression was also observed in ovariectomised mice in response to exogenous progesterone, but there was no increase in response to oestradiol-17β. In conclusion, our data suggest that both FS288 and FS315 mRNA are regulated by progesterone in the mouse uterus. As the response of each isoform was very similar, we further hypothesise that the mechanism regulating progesterone-induced uterine transcription of each follistatin variant is similar.

Implantation in pigs is associated with specific recruitment of endometrial T lymphocytes and activation of immune cells

R. Georgieva, T. Dimova — 2010-08-01

The involvement of the endometrial T cells in implantation and establishment of the epitheliochorial pig placenta, characterized by completely non-invasive trophoblast and lack of decidua development is not fully understood. Data about immunostimulation of maternal immune cells during implantation are lacking. The quantity and pattern of distribution of total T, Th, Tc and γ/δTCR cells as well as the expression of early (CD69) and late (SLA-DR) activation markers on maternal immune cells were evaluated in 10, 15, 20, 30 and 40 days pregnant endometrium, corresponding to the main implantation-related events; pre-receptive, attachment and early post-attachment phases of implantation, definitive placenta and the end of early pregnancy. Flow cytometric analysis and immunohistochemical methods were used. Total T, Tc and Th cells were in highest number at the time of trophectoderm attachment to the maternal epithelium. γ/δTCR cells present in relatively small and implantation uninfluenced proportions. In situ, T cells increased in amount in advance of implantation and formed T cell clusters (TCC) with implantation-stage dependent location. Early activation marker CD56 was expressed in situ by lymphocytes and granulocytes in pregnant endometrium only, and peaked in amount at pre-attachment phase. The quantity of the late activation marker SLA-DR on endometrial mononuclear cells was higher in pregnant endometrium compared to non-pregnant, and twice more in pre-attachment and attachment phase. In situ analysis showed that in pre-attachment stage of implantation more of the cells in TCC expressed late activation SLA-DR marker. The data showed that superficial implantation in pigs is associated with quantitative changes and recruitment of T cells from deep endometrial regions toward the subepithelial stroma as well as the activation of endometrial immune cells.

Embryo selection based on preimplantation factor (PIF) positivity and good morphology improve IVF success

2010-08-01

The selection of embryos for IVF is currently based on morphological criteria. The use of a quantitative method determining embryo's viability would optimize embryo selection. Preimplantation factor (PIF) is a novel embryo-derived peptide, detected during viable pregnancy in human and other mammals, absent/low in non-viable gestations.

Immunotropic diet suppresses natural killer cell number and activity—a case report

2010-08-01

Intralipid is a fat emulsion used for parental nutrition has been suggested as a therapeutic option to modulate abnormal natural killer (NK) cell activity in women with reproductive problems. As Intralipid provides a source of calories and essential fatty acids (linoleic acid, oleic acid, and palmitic acid) we designed an equivalent immunotropic diet and tested its effect in a woman with infertility problems and elevated numbers of peripheral blood NK cells and NK cell activity. The case study involved a 33 year-old woman who had undergone 5 intra-uterine inseminations and 4 IVF attempts, all unsuccessful and unexplained. Laboratory results revealed elevated numbers of peripheral blood NK cells in repeated measurements (CD3−CD15+CD56+ cells=26.7% and CD3−CD15+CD56+ cells=24.4%). The NK cell cytotoxic activity was also found to be elevated compared to controls (31.0% vs. 13.0% and 21.8% vs. 13.2%). All other hematological and biochemical tests were normal. An immunotropic and weight-maintenance diet was designed specifically for the subject, containing 30% fat with most of it coming from essential fatty acids. The diet included 70.3g of fat derived from daily intake and the other 8.7g from a liquid supplement administrated orally. After treatment with the diet for 15 days, the levels of peripheral blood NK cells and NK activity were reduced (CD3−CD15+CD56+ cells=18.5%, NK activity 18.4% vs. 13.9%). The patient continued the diet for another 24 days and a new measurement revealed a further reduction of NK cell number and activity (CD3−CD15+CD56+ cells=16.5%, NK activity 17.8% vs. 14.0%). NK cell reduction continued over time, and 8 weeks after discontinuation of the diet NK cells were 13.7% and NK activity was 15.3% vs. 13.5%. No other index was affected. The above case suggests that an immunotropic diet based on the substances composing Intralipid has a similar suppressive effect on NK cells and can be considered as an alternative therapeutic option to modulate NK activity in women with reproductive failure.

Kinetics of circulating preimplantation factor (PIF) levels during pregnancy

2010-08-01

Preimplantation factor (PIF) is a peptide secreted by viable human embryos, and it is detected in the maternal circulation in pregnancy. PIF appears to play an important role in maternal acceptance of the embryo by modulating the maternal immune system and promoting uterine receptivity. The aim of the present study was to investigate the kinetics of circulating PIF levels during normal pregnancy. Serially collected serum samples from 19 pregnant women (one sample per trimester (T)) were analyzed. All women completed a normal gestation without any complications during pregnancy or delivery. PIF was measured by Luminex (xMap technology) using Biotin-anti-PIFmAb (Bio-PIF-MAb*) and Ovalbumin-PIF conjugated microsphere beads. Sera from non-pregnant women as well as male sera tested under the same conditions used for negative controls. First trimester PIF levels were (mean±SE) 81.0±7.8ng/ml, (range 42.0–166.0), second trimester 98.0±11.8ng/ml (41.0–223.0) and third trimester 95.0±10.4ng/ml (41.0–213.0). A statistically significant increase was observed in second trimester compared with first trimester (p=0.01). Serial analysis of individual pregnancies revealed (a) in the majority of cases (68%) PIF increased in the second versus first trimesters; (b) in about half of cases (47%) PIF decreased during the thirds trimester, and (c) in 42% of cases the increase in the second trimester was followed by a decrease in the third trimester. The progressive increase in PIF in early and mid- pregnancy combined with the decreasing trend seen prior to delivery may reflect the embryo's contribution to modulation of the maternal immune response during pregnancy.

Microchimeric fetal cells migrate to the maternal injured tissues and trans-differentiate to the organ specific cells without maternal immunological elimination

R. Sunami, S. Hirata — 2010-08-01

Microchimeric fetal cells are present in the maternal body over a long period and thought to have the ability to colonize multiple tissues and organs. They are found in a wide range of maternal tissues affected with variety of diseases, thus, there is a possibility that they may contribute tissue repair and regeneration. To assess their possibility of use in regenerative medicine, we investigated whether fetal cells regenerate infracted maternal organs. We crossbred wild female mice with male transgenic mice, expressing enhanced green fluorescent protein (EGFP). On the day 60 after delivery, myocardial infarction was induced by ligation of the left main coronary artery. We also performed the ligation of carotid artery to induce the cerebral infarction in the other female mice. On the day 7,14 and 28 after each operation, maternal heart and brain were dissected. Detection and quantification of fetal cells in these organs were carried out by a fluorescent microscopic analysis and quantitative PCR amplification of the gfp transgene. Their characterization was assessed by the immunohistochemistry using myocardial cell or neural cell specific antibodies. Fetal DNA had been detected in the ischemic site of maternal heart and brain, since day 7 after operation. There was no significant difference among the amount of fetal DNA in these maternal organs dissected in the day 7, 14 and 28 after operation. Histological analysis showed that differentiated fetal cells were observed within the ischemic site of maternal heart and brain on the day 14 after the operation. Fetal cells in the ischemic maternal heart expressed troponin T and in the brain neuronal nuclear antigen (NeuN). Their morphological appearance was indistinguishable from their maternal counterparts. These results indicate that fetal cells migrate to the infarct portion of the maternal organ and trans-differentiate to the organ specific cells without maternal immunological elimination.

Fetal derived cells in the maternal organs are eliminated by maternal immune system after mid gestation

R. Sunami, S. Hirata — 2010-08-01

Semiallogeneic fetus is protected from attack by its mother's immune system, however, it is not known yet whether the microchimeric fetal cells in various maternal organs are eliminated by maternal immune response. In this study, we evaluate the maternal immune system's interaction with microchimeric fetal cells. We crossbred male EGFP transgenic mice and wild type (C57BL/6 and BALB/c) or immuno-deficiency (NOD/SCID) virgin female mice and dissected maternal liver, kidney, pancreas, heart, lung, spleen and bone marrow on the day 7, 14, and 20 of pregnancy. Detection and quantification of fetal cells in these organs were carried out by a fluorescent microscopic analysis and quantitative PCR amplification of the gfp transgene. Mononuclear cells were extracted from maternal spleen, and cytotoxic activity on the fetal cells during pregnancy was evaluated in wile type mice. NOD/SCID and wild type mothers had equivalent levels of fetal cell containing on the day 7 of pregnancy. As the pregnancies progressed, significant differences in the incidence of microchimerism appeared; NOD/SCID mothers were progressively more likely to contain fetal cells than wild type mothers. More fetal cells were detected in C57BL/6 mother than BALB/c after the day 14 of pregnancy. There was no significant difference among the cytotoxicity of maternal cells on the fetus during pregnancy. The migration of microchimeric fetal cells to the mother is unaffected by the presence of a maternal immune system, however, there is a possibility that these fetal cells are eliminated by maternal immune response after the mid gestation.

Re-expression of NF-κB in T-cells from pregnant women promotes Th-1 cytokine production in response to stimulation

S.A. McCracken, K.A. Hadfield, A.W. Ashton, T.G. Nguyen, J.M. Morris — 2010-08-01

Normal pregnancy is associated with suppression of Th-1 immune responses, mediators of acute allograft rejection. We recently demonstrated that suppression of Th-1 cytokine production in T-cells from pregnant women is associated with reduced T-bet expression (a Th-1 transcription factor). Further, inhibition of NF-κB translocation in T-cells in non-pregnant women results in reduced T-bet expression and subsequent Th-1 cytokine production. In this study, we tested the hypothesis that T-bet regulates Th-1 cytokine production in T-cells from pregnant women, and that NF-κB transcriptional activity is central to the regulation of T-bet expression and thus Th-1 cytokine production. T-cells were isolated from pregnant women (n=5) by negative bead depletion. T-cells were transfected with 5μg of expression vectors for p65, a transcriptionally inactive p65 (E39I), T-bet or GFP as a control using electroporation. Cells were cultured in the presence of 20% autologous plasma at 37°C, 5% CO2 for 20h and then stimulated with PMA and ionomycin in the presence of Brefeldin A for a further 4h. p65 and T-bet expression was determined using Western blotting, and cytokine expression determined by PCR and flow cytometry. Post transfection with both p65 and E39I, NF-κB expression increased, T-bet expression was also increased in cells transfected with the T-bet vector. Post stimulation, T-bet expression increased in p65 and T-bet transfected cells, but did not change in cells transfected with E39I or in untransfected controls. Stimulation of T-cells transfected with p65 and T-bet resulted in significantly higher levels of IFNγ and IL-2 mRNA and protein, relative to untransfected and E39I transfected cells. These results demonstrate that NF-κB regulates T-bet expression and subsequent cytokine production, and that suppression of NF-κB in T-cells in pregnancy is seminal in maintaining the appropriate cytokine environment. Thus, pregnancy induced suppression of NF-κB in T-cells in pregnancy is paramount for pregnancy success.

Galectin-1 peripheral level increase during successful human pregnancy

2010-08-01

Galectin-1 (gal-1) is a soluble protein mainly expressed at sites of immune privilege such as the eyes, testis and placenta. Galectin-1 has been suggested to play an important role in conferring feto-maternal tolerance through multiple mechanisms like the induction of tolerogenic dendritic cells. It has been described that gal-1 is highly expressed in the feto-maternal interface and it is synthesized initially by trophectoderm immediately before the establishment of blastocyst, suggesting an important role in the attachment of the embryo to the uterine epithelium. This study aimed to investigate the role of gal-1 in human pregnancy evaluating the circulating levels of gal-1 during normal and pathological pregnancies. We collected serum from non-pregnant women, first and second trimester and term normal pregnancies and spontaneous abortion patients. Tissue gal-1 expression was investigated by IHC and serum gal-1 levels by ELISA. Serum from normal pregnancy (IR) showed a higher concentration of gal-1 compared with abortion (ABE) serum. Indeed, we could also show that non-pregnant women presented lower levels of gal-1 in comparison with first, second and term trimester, showing statistical differences in gal-1 levels. In addition, we observed higher intensity gal-1 expression in decidua, syncitiotrophoblast and extravillous trophoblast (EVT) from normal pregnancy compared with abortion samples. Our results suggest that gal-1 expression at the maternal interface is necessary for success and maintenance of human pregnancy. We conclude that decreased levels of gal-1 during spontaneous abortion could emerge as a predictive marker for pregnancy loss.

Receptor characterization of human pregnancy specific glycoprotein 1

G.S. Dveksler, F.A. Lisboa, J. Warren, M. Aparicio, E. Zudaire — 2010-08-01

Pregnancy specific b1 glycoproteins (PSGs), previously known as SP1, are the most abundant fetal proteins in the maternal bloodstream in late pregnancy. They are secreted by the syncytiotrophoblast and are detected early post-fertilization until term, when they reach a serum concentration of 200–400μg/ml. There are 11 human PSG genes (PSG 1-11) clustered on chromosome 19, which encode a family of proteins exhibiting greater than 80% identity at the amino acid level. PSGs belong to the carcinoembryonic antigen family and human PSGs are comprised of a leader peptide followed by one N-terminal immunoglobulin (Ig) variable region-like domain and two or three constant region Ig-like domains. PSGs are only expressed by species with hemochorial placentation, which include rodents and primates. We and others have proposed that PSGs have an immune-modulatory function as they induce the anti-inflammatory cytokine TGFβ1 in many cell types. In addition, we recently postulated that they are pro-angiogenic due to their ability to induce endothelial tube formation. The cellular receptor(s) for human PSGs remain unknown. Therefore we conducted studies to identify the receptor for PSG1, the highest expressed member of the family. We show that removal of cell surface glycosaminoglycans by enzymatic or chemical treatment of cells or competition with heparin completely inhibited binding of PSG1. In addition, PSG1 did not bind to cells lacking heparan or chondroitin sulfate on their surface and binding was restored upon transfection with all four syndecans. Importantly, the presence of glycosaminoglycans on the surface of endothelial cells was necessary for the ability of PSG1 to induce tube formation. Our results indicate that PSG1 uses syndecans as its receptors and this interaction mediates at least some of the PSG proposed functions.

Monocyte and T cell characterisation in pregnancies complicated by asthma

2010-08-01

We have shown that during pregnancy the asthma exacerbation rate is high, increasing the risk of an adverse neonatal outcome, including intrauterine growth restriction, preterm delivery and still birth. In a majority of cases exacerbations are un-resolvable with inhaled glucocorticoid treatment, suggesting pregnancy changes the asthma phenotype to a form that is non-responsive to asthma treatment. This may be related to maternal adaptations in immune pathways that occur with pregnancy. In normal pregnancy, maternal circulating leukocytes undergo modifications in cell concentration, phenotype and function over the course of pregnancy. However little is known about how this adaptation in pregnancy is influenced by the presence of maternal asthma. We aim to characterise leukocyte sub-populations and phenotypes in blood collected from pregnant non-asthmatic and asthmatic women. We hypothesised that maternal asthma worsens during pregnancy due to altered leukocyte phenotypes, including increased monocyte CD14dimCD16+ subset and disturbances in T cell subsets particularly Treg cells, Th1 and Th2 cells. Venous blood was collected from pregnant asthmatic subjects (n=10) and controls (n=10) at 12, 18 and 30 weeks gestation. Peripheral blood mononuclear cells (PMBCs) were isolated at all three time points. Multi-parameter flow cytometry analysis using appropriate antibody pairs was used to determine T cell and monocyte subsets. Our preliminary analysis demonstrated that there were no differences in T cell subtypes in control and asthma group as pregnancy progressed. However there were differences in monocyte subsets with differential expression of activation marker HLA-DR (P<0.05) in the asthmatic group. The expression of a ‘pro-inflammatory’ monocyte phenotype CD14dimCD16+ was also identified in some asthmatic subjects. We conclude that circulating monocytes are heterogeneous in phenotype and function. CD14dim CD16+ phenotype expands during infection and inflammatory response. The differential expression of leukocyte subsets in pregnancies complicated by asthma may be part of the mechanism contributing to worsening asthma during pregnancy.

No quantitative changes in the systemic immune response during pregnancy despite changes in cytokine production and composition of leucocytes

2010-08-01

During pregnancy the immune system of the mother is modulated in order to allow tolerance to the fetus and at the same time retain protective properties against infectious organisms.

Smoking alters monocyte subsets during pregnancy in mice

J.R. Prins, A. Lech, M.N. Hylkema, J.J.H. Erwich, M.M. Faas, B.N. Melgert — 2010-08-01

Alternatively activated macrophages (aaMF) have an important role in proper placental development. These macrophages develop from Gr1lo monocytes residing in peripheral blood. Smoking during pregnancy is associated with lower birth weights and an increased incidence of spontaneous abortions, which may be caused by altered placental development. Data on the effects of smoking on monocyte subsets during pregnancy and placental macrophages are lacking. This study aimed to analyse the effects of smoking on monocyte subsets during pregnancy in mice. Pregnant mice were exposed to smoke from 7 days before mating until the day of sacrifice, for 7h a day. Pregnant mice were sacrificed at gestational day 11.5 and spleens were harvested and thoroughly homogenised. Gr1hi and Gr1lo monocytes were identified by flow cytometry as F4/80+, CD11-b+, MHC-II+, and CD43+ with fluorescently labelled antibodies against these markers. Smoke-exposed mice had significantly smaller litter sizes, due to a higher number of resorptions. Furthermore, percentages and absolute numbers of Gr1lo monocytes were significantly decreased in spleens of smoking pregnant mice as compared to nonsmoking mice. No differences were found for Gr1hi monocytes between the groups. The decreased number of Gr1lo monocytes in the peripheral blood compartment after maternal smoke exposure may lead to decreased number of placental aaMF, which in its turn will hamper placental development. Further research on placentas of these mice will have to show whether the adverse pregnancy outcome and decreased number of Gr1lo monocytes as found in our smoking mice are also associated with decreased numbers of placental aaMF.

Progesterone modulates endocannabinoid and nitrergic systems of peripheral lymphocytes

A. Franchi, M.L. Wolfson, J. Aisemberg, C.A. Vercelli — 2010-08-01

Anandamide (AEA) is an endocannabinoid necessary for embryo implantation, but its exacerbated synthesis and/or decreased metabolism is related to early abortions in women. LPS induces AEA synthesis in murine macrophages and also inhibits the activity of the enzyme that degrades AEA (fatty acid amidohydrolase, FAAH). Nitric oxide (NO) is an essential molecule during pregnancy, but large quantities can cause septic embryonic resorption (ER). Previous studies from our laboratory indicate that LPS causes resorption in mice with an increase in the production of NO. Progesterone (P) serum levels were diminished in these animals and this hormone abrogated the augmentation of uterine and decidual synthesis of PGE, NO and TNFα induced in response to LPS. The aim of this work was to study if P modulates endocannabinoid and nitrergic system in murine peripheral lymphocytes. Peripheral lymphocytes from pregnant mice had higher activity of FAAH than those from non-pregnant animals (NP). In NP mice, our results show that treatment with LPS decreases both the activity and the expression of FAAH, and that P reverses the effects of the endotoxin. We observed that LPS increases the production of NO and the expression of inducible NO synthase in lymphocytes, a fact which is reversed by treatment with P. These results suggest that P, which is elevated in pregnancy, may have a protective effect on inflammatory processes mediated by NO and AEA in murine lymphocytes. All the protective effects of P could be abolished by the treatment with two P antagonist: RU-486 and lonaprizan. We then studied the expression of P receptor and we detected the expression of PRA and PRB and its modulation by LPS and P. We conclude that several inflammatory molecules can participate in the mechanism of LPS-induced resorption and their modulation by P could be a useful tool to prevent early pregnancy loss.

Quantitative and functional changes of peripheral natural killer cells in women with reproductive failure after artificial insemination with donor sperm

A.H. Liao, Y.C. Lu, B. Zeng, Y. Zhang, W.P. Xiang, L. Hu — 2010-08-01

The objective of this study was to determine if peripheral natural killer (NK) cells numbers and activity are altered in women with reproductive failures after artificial insemination with donor sperm (AID). Forty-five patients experiencing reproductive failure after AID cycles were recruited into this study. Women experienced at least 4 failures associated with negative pregnancy test (Group I, n=15), biochemical pregnancy loss (Group II, n=15), or they underwent embryo growth arrest or miscarriage in the first trimester (Group III, n=15). Twelve healthy non-pregnant women with successful delivery were selected as controls. NK cell subsets within lymphocytes and cytotoxicity in late follicular phase blood samples were evaluated by flow cytometry. A significantly increased percentage of NK cell cytotoxicity was detected in the peripheral blood of study groups compared with the controls (P<0.01). The percentage of both CD56+ and CD56+ CD16+ NK cells in the study groups was higher than that in the controls (P<0.05). Concerning the percentage of CD56+ CD16+ NK cells, there existed statistical differences in Group I compared with Group II and Group III (P<0.05). However, there was no statistically significant difference in the percentage of CD56+CD16− NK cells between the study and control groups (P>0.05). Our findings implicate that the quantitative and functional changes of human circulating NK cells might contribute to the etiology of AID failure.

Functional regulation of Th17 cells at maternal–fetal interface of early pregnancy in humans

D. Li, H. Wu — 2010-08-01

The embryo expresses antigens foreign to the mother, and thus has been viewed as an allograft. Now that Th17 cells are identified in human PBMC, we have investigated whether Th17 cells are in human peripheral blood and decidua during human first-trimester pregnancy, and how they affect trophoblast functions at maternal–fetal interface. We have found that the percentage of Th17 cells are significantly higher in PBMCs and decidua of first-trimester pregnancy than in healthy non-pregnant women, and they gradually decrease along with pregnancy continuation to the secondary and third trimester. Thereafter, it has been demonstrated that trophoblasts and decidual stromal cells, especially when co-cultured, can inhibit the differentiation of Th17 cells via soluble molecules. Decidual stromal cells other than trophoblasts recruit peripheral Th17 cells into the decidua via secretion of CCL20 and CCL2, which interact with their respective receptors on the Th17 cells. We have found that trophoblasts and decidual stromal cells express the IL-17 receptor. The supernatant from Th17 cells induces the proliferation and invasion of trophoblast mainly through secreting IL-17, with a similar effect to recombinant IL-17. It has also been found that the supernatant from Th17 cells inhibits apoptosis of trophoblasts via IL-17. These results suggest that Th17 cells are involved in human first-trimester placentation.

Immune mechanisms of Karakalpakian pregnant women with abnormalities of the fetal development uterine history

D. Musakhodjaeva, A. Kalandarova — 2010-08-01

In Uzbekistan there is one of the largest ecological tragedies of the last century – the Aral Sea. In parallel with deterioration of the ecological situation, deterioration of health of the population of the republic has been observed. In particular, the increase in frequency of occurrence of children with anomalies of intra-uterine development is observed in the Sub-Aral area. As immunological mechanisms at pregnancy have important value, we have carried out research to study the levels of cytokines in women with intra-uterine anomalies of the fetus. We examined 36 pregnant women at stages up to 22 weeks gestation with one or more intra-uterine anomalies of the fetus. Proinflammatory cytokines (IL-1, TNF, IL-6) and anti-inflammatory cytokines (IL-4, IL-10) were determined in serum. Our research has shown, that in women where fetal development with intra-uterine anomalies occurs, the number of CD3+ CD8+ T cells, and also the level of IL-4 and IL-10 during the given pregnancy is significantly reduced (P<0.05), and the level of IL-1 and TNFα is sharply raised in comparison with the data of women with normal pregnancy. These changes may be connected with the toxic and hypoxic influence of exogenous stimuli on neuro-endocrine regulation of the immune system, resulting in suppression of immunological reactivity and hence to oppression of the normal immune response in pregnancy.

Interferon epsilon regulates reproductive tract immunity to Chlamydia infection

K. Fung, H. Cumming, N. Mangan, J. Horvat, P. Hansbro, P. Hertzog — 2010-08-01

We recently identified a new cytokine in the Interferon (IFN) locus, which we designated IFNɛ because of its homology to other type I IFNs. In contrast to other type I IFNs, which are only expressed after induction, usually by pathogens, IFNɛ is expressed constitutively in reproductive organs. In order to determine the function of this novel gene, we generated mice with a null mutation in the IFNɛ gene. The uterus of IFNɛ−/− mice display lower levels of numerous Interferon Stimulated genes consistent with the constitutive IFNɛ signaling to maintain their basal levels of expression, presumable important for host defence. To determine the role of IFNɛ in genital tract infection, we examined the course and outcome of Chlamydia muridarum genital infection in IFNɛ−/− mice. Interestingly, IFNɛ−/− mice had an earlier onset of the disease and were unable to clear vaginal Chlamydial infections at the same rate as WT mice. Compared to the WT mice, IFNɛ−/− mice exhibited severe clinical signs of infections throughout the course of infection. Furthermore, IFNɛ−/− mice developed more chronic oviduct pathology in comparison to that in WT mice. Although IFNɛ−/− mice failed to completely clear Chlamydia muridarum from their genital tract, in out initial studies, there were differences in inflammatory cell infiltrates between IFNɛ−/− and WT mice. These data indicated that IFNɛ is important in maintaining constitutive innate immunity in the female reproductive tract, with potential considerations for human urogenital infections.

Persistent placental infection with lymphocytic choriomeningitis virus is modified by interleukin 10 deficiency in mice

K. Ray, V. Devi Negi, K. Oppenheimer, S. Khurana, E.A. Bonney — 2010-08-01

Infection of pregnant mice on day 10 of gestation with lymphocytic choriomeningitis virus (LCMV) results in an infection that is cleared from the systemic circulation in 7 days. However uterine and placental tissues remain infected. Two possible mechanisms may underlie this finding. One is specialized virus-cellular interaction. Another is immune modulation at this site. Interleukin 10 is an important modulator of immune responses, preterm birth and parasitic persistence in infections of non pregnant animals. IL-10 has also been suggested as an important mediator in models of chronic infection with LCMV. To test if IL-10 mediates decreased clearance at the maternal–fetal interface, normal C57BL/6 (B6) females and females of the same genetic background yet deficient in interleukin 10 (Il10−/−) were mated to same-strain males. On day 10 of gestation they received 2×105 plaque forming units (pfu) of LCMV. Seven days later their uteri were removed and analyzed by inspection for the presence of fetal and placental units, by plaque assay for viral burden and by QT-PCR for expression of gamma interferon, an important cytokine in the immune response to LCMV. Infected B6 females had a greater resorption rate as compared to Il10−/− females (0.3±.08, n=10 vs. 0.1±.03, n=11; p=0.01). However, normal-appearing placentas in infected B6 pregnancies had a significantly greater viral burden (1615±748×106pfu/g) as compared to those from IL10−/− pregnancies (23±11×106pfu/g, p=0.01). While the uteri of infected B6 and IL10−/− mothers expressed similar relative levels of gamma interferon (2±0.4 vs. 1±0.2, p=0.3), placentas of B6 pregnancies expressed significantly lower levels of this cytokine as compared to placentas found in IL10−/− pregnancies (1.2±0.3 vs. 4±1, p=0.04). These data support the potential role for IL-10 dependent, gamma interferon dependent or independent and tissue specific effects on viral persistence at the maternal–fetal interface. Further, they highlight the complex role of immunity and host–pathogen interactions in pregnancy success after infection.

Estradiol and selective estrogen receptor modulators (SERMs) alter antimicrobial production in the murine female reproductive tract in vivo

D.K. Hickey, C.R. Wira — 2010-08-01

Antimicrobial peptides possess broad-spectrum antibiotic activity against a number of pathogens including bacteria and viruses. Using ovariectomized mice as a hormonally unopposed in vivo model, we investigated the effect of estrogen receptor (ER) ligand interactions on the expression of α and β defensins, in addition to secretory leukocyte protease inhibitor (SLPI) in the female reproductive tract (FRT). Female BALB/c mice (6 weeks old) were treated subcutaneously with estradiol or SERMs (ICI 182780, Y134, PHTPP, Tamoxifen and Raloxifene), each with differing affinity to ERα or ERβ subtypes. Using real-time PCR, the relative expression of α defensin 2, β defensin 1, β defensin 2, β defensin 4 and SLPI mRNA expression was determined in uterine and vaginal tissues following 3 days of hormone/SERM treatment. B defensin but not α defensin mRNA expression was altered in estradiol treated mice. Estradiol had an inhibitory effect on β defensin 1 and 2 but a stimulatory effect of β defensin 4 in the vagina. In contrast, uterine mRNA expression of β defensin 1 was reduced while β defensin 2 was enhanced following estradiol treatment. In response to estradiol, SPLI expression was inhibited in the vagina and unaffected in the uterus. Following SERM treatment antimicrobial expression was altered in the upper and lower FRT in a peptide specific manner. Of note, SERM treatment significantly alters vaginal and uterine expression of antimicrobial peptides. In the vagina, Y134 (p<0.05), Tamoxifen (p<0.001) and Raloxifene (p<0.05) all significantly inhibited β defensin 2 mRNA expression. In contrast, PHTPP enhanced β defensin 2 (p<0.001). Interestingly, in the uterus, Tamoxifen and Raloxifene lowered uterine β defensin 2 mRNA expression, however this was not statistically significant. We conclude that ER stimulation with estradiol or SERMs alters antimicrobial mRNA expression in the FRT in vivo. Our studies suggest that SERMs may be used to enhance antimicrobial expression to protect FRT from infection with pathogenic organisms when antimicrobial peptides are reduced during the reproductive cycle.

Changes in innate immune parameters in the murine female reproductive tract throughout the estrous cycle

D.K. Hickey, C.R. Wira — 2010-08-01

Sex steroid hormone levels vary within the female reproductive tract (FRT) throughout the reproductive cycle. Estradiol has been shown to directly alter immunity in FRT. This study was undertaken to further our current knowledge of the baseline expression of estrogen receptor (ER) α and β, Toll-like receptors (TLRs), cytokines, chemokines and antimicrobial defensins in the upper and lower FRT throughout the reproductive cycle. The stage of estrous cycle of 6 week-old female BALB/c mice was determined by vaginal smears (proestrus, estrus and diestrus). Uterine and vaginal lavages were collected using sterile PBS (50μl). The concentrations (pg/ml) of secreted cytokines/chemokines (IL-1α , IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12(p40), IL-12(p70), IL-13, IL-17, TNFα, Eotaxin and G-CSF), chemokines (GM-CSF, MCP-1/CCL2, MIP-1α/CCL3, MIP-1β/CCL4 and Rantes/CCL5) were determined using luminex analysis. Whole vaginal and uterine tissue mRNA ERα, ERβ, TLRs and antimicrobial (α and β defensin) gene expression was determined by real-time PCR. Uterine secretion of cytokines and chemokines were significantly lower at diestrus when compared to proestrus. Furthermore, TLR gene expression also tended to be reduced at diestrus while ERα/β mRNA expression was enhanced. Vaginal production of cytokines was not altered during the estrous cycle except for IL-1β, which was enhanced at diestrus. Chemokine secretion, however, was significantly enhanced at diestrus and coincided with increased TLR expression. Unlike the uterus, vaginal ERα and β expression mRNA was significantly increased at estrus. Interestingly, antimicrobial α and β defensins were differentially regulated throughout the estrous cycle with mRNA expression both FRT site and peptide specific. This study shows that vaginal and uterine immunity is altered during the estrous cycle in BALB/c mice. In general, highly regulated changes in uterine tissue result in an inhibition of innate immunity at diestrus. Changes in vaginal innate immunity shows greater variation compared to the uterus, and responses tend to be enhanced during the diestrus phase of the reproductive cycle.

The role of interferon-epsilon in a viral infection of the reproductive tract

N.E. Mangan, K. Fung, S. Stifter, D.J. Carr, P.J. Hertzog — 2010-08-01

Sexually transmitted infections (STIs) represent a critical global health and socioeconomic problem with over 1 billion new cases per annum. Genital herpes, caused primarily by herpes simplex virus-2 (HSV-2) is one of the most prevalent STIs in the world and is associated with substantial morbidity, compounded by the lack of a current vaccine. This is partly due to our lack of a comprehensive understanding of how the immune system in the female reproductive tract functions. We have investigated the importance of interferon (IFN) signaling in HSV-2 infection in mice. Using IFN receptor (Ifnar) 1- and Ifnar 2-deficient mice we have addressed the role of differential Ifnar responses in the immune response to HSV-2. Significantly, we have discovered a new protein, designated IFNepsilon, that is exclusively expressed in the female reproductive tract. We have demonstrated that IFNepsilon acts via Ifnar 1 and Ifnar 2. However, IFNepsilon is unique, because unlike other related type I IFNs, it is constitutively expressed and regulated by hormones but not by pathogens. Importantly, using our IFNepsilon-gene targeted mice, we have demonstrated that this novel cytokine regulates innate immunity, protecting mice from STIs. IFNepsilon-deficient mice are more susceptible to intra-vaginal infection with HSV-2 compared to infected wildtype mice, as measured by clinical score of disease and increased viral titres in tissues. The associated immune response in IFNepsilon-deficient mice correlates with increased infection in these mice and mechanisms of IFNepsilon regulation of immune cells in infection will be discussed. We describe a new mechanistic role for IFNs in HSV-2 infection in mice.

Differential expression of intracellular innate immune cytosolic pathogen sensing receptors in the human female reproductive tract

M. Ghosh, Z. Shen, T.M. Schaefer, J.V. Fahey, C.R. Wira — 2010-08-01

NOD1, NOD2 (nucleotide binding oligomerization domain), RIG1 (retinoic acid inducible gene) and MDA5 (melanoma differentiation associated gene) comprise an intracellular pathogen sensing pattern recognition receptor (PRR) system. The NOD proteins recognize bacterial pathogens whereas the RIG1 and MDA5 recognize viral. Our study was aimed at determining the expression and induction patterns of all four PRR by epithelial cells from the female reproductive tract (FRT). We also determined the effects of estradiol and Neisseria gonorrhea, an intracellular FRT pathogen, on PRR expression. Epithelial cells (Ec) were isolated from Fallopian tube (FT), endometrium (EM), cervix (Cx) and ectocervix (Ecx) from hysterectomy patients by enzymatic digestion. Cells were treated with estradiol, Poly(I:C) (mimic of viral RNA), and heat-killed Neisseria gonorrhea. mRNA was isolated and real-time PCR was performed to measure NOD1, NOD2, RIG1, and MDA5. Cell culture supernatants were assayed for IL8 by ELISA. We found that epithelial cells throughout the FRT constitutively express NOD1, NOD2, RIG1, and MDA5. In a given patient, FT Ec expressed the highest levels of all four genes. Stimulation with Poly(I:C) upregulated all genes in the upper and lower tract at 6 and 24h which correlated with secretion of IL8. Unexpectedly, estradiol had no effect on gene expression. Treatment with Neisseria gonorrhea upregulated NOD2, and MDA5 by cervical Ec but not by FT, EM, and Ecx Ec. Upregulation correlated with IL8. Our data demonstrate that the Ec from FT, EM, Cx, and Ecx express PRRs NOD1, NOD2, RIG1, and MDA5 constitutively with FT expression the highest. All four receptors and IL8 were induced with Poly(I:C) showing that these receptors are functional. Further, receptors in the cervix were upregulated by Neisseria gonorrhea, a known cervical pathogen. To our knowledge, our finding of PRRs in the FRT indicates a new protective mechanism by epithelial cells.

Host immune and pathogen virulence factors that contribute to placental colonization by Salmonella typhimurium

T. Nguyen, A. Chattopadhyay, L. Krishnan — 2010-08-01

Pregnancy poses a high risk for infection with Salmonella enterica species causative agents of gastroenteritis and typhoid fever in humans. Normally resistant mice succumb rapidly to Salmonella Typhimurium (ST) infection during pregnancy due to rapid placental infection and an inflammatory storm evoked by the virulent bacterium. To determine the mechanisms of ST-induced placental pathology, we addressed the mode of entry of the bacterium and intracellular localization within placental cells. We have also elucidated the role of altered host immunity in placental infection.

Estradiol and selective estrogen receptor modulators (SERMs) regulate CCL20 and CXCL1 secretion in the murine female reproductive tract

D.K. Hickey, C.R. Wira — 2010-08-01

Chemokines play important roles in the immunology of the female reproductive tract (FRT) by recruiting immune cells and through their antimicrobial properties. This study demonstrates that estradiol regulates the secretion of CCL20 (MIP3α) and CXCL1 (mouse KC) in the FRT. Furthermore this work evaluated the role of selective estrogen receptor modulators (SERMs) in CCL20 and CXCL1 protein secretion. Female BABL/c 6 week old mice were used for in vitro and in vivo analysis. In vitro, single cell suspension of primary uterine epithelium, stromal and vaginal cells primary cells were used. In vivo, ovariectomized mice received three daily subcutaneous injections. Cultures and mice were treated with either vehicle alone (medium or saline), estradiol, ICI 182780 (ERα/ERβ antagonist), Y134 (ERα antagonist), Tamoxifen (ERα/ERβ antagonist/partial agonist) or PHTPP (ERβ antagonist). The concentrations of CCL20 and CXCL1 were determined in conditioned media (in vitro) and reproductive tract luminal secretions (in vivo) following estradiol/SERM treatment were determined via ELISA. Estradiol treatment inhibited CCL20 and enhanced CXCL1 secretion in vitro and in vivo. In vitro, SERMs ICI 182780 and Y134, in contrast to estradiol, stimulated CCL20 and inhibited CXCL1 secretion by purified uterine epithelial cells. In contrast, no chemokine secretion was observed by vaginal epithelial cells following SERM treatment. In contrast, in vivo, SERMs such as ICI 182780 and Tamoxifen, but not Y134, or PHTPP, increased the levels of CCL20 and CXCL1 respectively in vaginal secretions. Our study demonstrates that estrogen receptor–ligand interactions affect FRT chemokine secretions and that SERMs regulate FRT innate immunity in ways that are distinct from that seen with estradiol.

Single nucleotide polymorphisms in genes regulating inflammation and the risk of recurrent pregnancy loss in a Sinhalese population from Sri Lanka

2010-08-01

Recurrent pregnancy loss (RPL) affects 0.5–3% of couples and over half the cases have an unexplained etiology. Inflammatory processes are demonstrated at the maternal–fetal interface in many cases of unexplained RPL. The aim of this study was to investigate the association of functional single nucleotide polymorphisms (SNPs) in the interleukin family (IL-1A, IL-1B, IL-6, IL-4, IL-10, IL-1RN) and mannose-binding-lectin-2 (MBL2) genes in RPL. Sinhalese women with three or more recurrent pregnancy losses and no living children (n=212 cases) and women who have two or more living children and no history of pregnancy loss matched for age and ethnicity (n=202 controls) were recruited at the Human Genetics unit in Colombo. Peripheral blood was collected from the participants and DNA extracted. Genotypes of eleven SNPs were determined by the Sequenom MassARRAY system. Chi square test was used to test the genotypes at each polymorphic locus for Hardy–Weinberg equilibrium (HWE). Genotype results of cases were compared with controls using dominant and recessive genotype models by logistic regression analyses. All the genotype frequencies of cases and controls were in HWE. No significant difference was seen between the mean age of the RPL (31.9) and the control (32.3) group. Sixty percent of the women had experienced only first trimester pregnancy losses. MBL2 rs1800450 GG+GA genotypes were increased in RPL compared to controls (p=0.02, OR=4.8, 95%CI=1.0–22.5). The interleukin family SNPs were not associated with RPL. We conclude that the G allele of the MBL2 rs1800450 which is associated with increased MBL production confers an increased risk for recurrent pregnancy loss in Sinhalese women.

Investigating the mechanisms of action of anti-IgD (αIgD) as a potential immune regulator

V.M. Yenson, T.G.I. Nguyen, A.W. Ashton, J.M. Morris — 2010-08-01

Successful human pregnancy is associated with a shift away from Th1 immunity, as seen with the gestational remission of rheumatoid arthritis. Recurrent spontaneous miscarriage has been associated with dysregulation of this normal shift in immunity. Our lab has been investigating the potential effect of the anti-IgD antibody (αIgD) as an immune regulator. Little is known about the role of the αIgD in immune responses, other than a broad but profound effect on B-cell activation and polyclonal antibody production (). We have shown that therapeutic administration of αIgD alleviates both the incidence and severity of collagen-induced arthritis in the mouse model (), however its mechanism(s) of action is unknown. This study investigated the effect of αIgD on Th1 and Th2 cytokine production by human B- and T-cells in vitro. Isolated PBMCs, B-cell depleted PBMCs or isolated T-cells were treated with αIgD/isotype control (1 or 0.1mg/ml) overnight before artificial stimulation (PMA/Ionomycin). Cytokine production was analysed using flow cytometry. Cell–cell contact for the αIgD effect was investigated through media transfer and transwell experiments.

Peripheral blood Natural Killer and T cells in the menstrual cycle of women with recurrent unexplained miscarriage

I. Granne, J. Southcombe, I.L. Sargent, T. Child — 2010-08-01

Successful pregnancy is dependent on a T-helper 2 (Th2) immune bias, whilst T-helper 1 (Th1) responses are relatively inhibited. Natural Killer (NK) cells and cytotoxic T cells show a similar bias. The relevance of this bias in peripheral immune cells in implantation and miscarriage remains controversial. In this study 15 women with regular menstrual cycles and no history of miscarriage or subfertility and 10 women with a history of recurrent unexplained miscarriage were recruited. Blood samples were taken at baseline, mid follicular, peri-ovulation and mid-luteal time points. Flow cytometry was used to identify NKbright, NKdim, T-helper and cytotoxic lymphocytes. Cell surface markers were used to identify type 1 (IL-18 receptor) and type 2 (ST2-L) deviated cells. An absolute count of each cell population was determined. In the control group there was no significant variation in cell numbers over the cycle. When the type 1/type 2 ratio was calculated, there was a significant type 1 shift in the NKdim (p<0.01) and the cytotoxic T cell (p<0.05) populations in the mid-luteal phase compared to baseline. In the miscarriage group there was a significant increase in both the percentage and the absolute count of NKdim cells in the mid-luteal phase compared to baseline (p<0.05). However, the type 1 mid-luteal shift in NKdim cells identified in the control group was not seen in the miscarriage group. We conclude that the significant mid-luteal type 1 shift seen in the control group supports previous observations that implantation may favour a type 1 immune response, rather than the type 2 response favoured by subsequent pregnancy. This did not occur in the miscarriage group. It is possible that the inflammatory cytokine environment required for successful placentation is not present in the women with a history of recurrent miscarriage, and this is reflected in the peripheral blood.

Homozygocity for a 14-basepair insertion in the HLA-G gene is associated with recurrent miscarriage and low birthweight in children born before recurrent miscarriage

O.B. Christiansen, M. Dahl, H.S. Nielsen, A.M. Kolte, E.C. Larsen, T.V. Hviid — 2010-08-01

HLA-G expression on the trophoblast probably plays an important role in the immunological recognition of pregnancy and decreased HLA-G expression may impair normal trophoblast invasion. A 14-basepair (bp) long insertion in exon 8 of the HLA-G gene seems to decrease the stability of the HLA-G transcript, which may explain why plasma concentrations of soluble HLA-G seem to be lower in homozygous insertion carriers than in non-carriers. We investigated the HLA-G14bp insertion/deletion polymorphism in 316 Caucasian patients with three or more consecutive unexplained miscarriages and in 168 Caucasian women with children and no miscarriages. Investigations were undertaken by amplification of the HLA-G exon 8 using genomic DNA and primers GE14HLAG and RHG4. In all recurrent miscarriage (RM) patients, 60 (19.0%) were homozygous for the HLA-G14bp insertion compared with 19 (11.3%) of the controls (p<0.03). HLA-G14bp insertion homozygocity was found in 20.7% of patients with secondary (p<0.02) and 16.4% of patients with primary RM (not significant). Among the children born prior to a secondary RM diagnosis, the median birthweight decreased significantly (p<0.01) with increased number of HLA-G14bp insertions carried by the mother. The frequency of children with birthweights below the 50% percentiles for gestational age increased from 45.4% in mothers carrying 0–53.8% in mothers carrying 1% and 65.8% in those carrying 2 HLA-G14bp insertions (p=0.05). We conclude that homozygocity for the HLA-G14bp insertion seems to be associated with RM and especially secondary RM. In first pregnancies of patients with secondary RM, maternal carriage of this genotype seems to predispose to birth of children with significantly reduced birthweight compared with children born to patients with other HLA-G14bp genotypes. HLA-G14bp insertion homozygocity may affect maternal immune tolerance to the trophoblast thereby causing miscarriage or intrauterine growth restriction.

Etanercept immunotherapy in women with a history of recurrent miscarriage

M. Jerzak, M. Klochowicz, A. Górski, W. Baranowski — 2010-08-01

The aim of this study was to determine pregnancy outcome after etanercept immunotherapy in women with a history of at least three recurrent miscarriage (RM) or failed IVF. Etanercept is a tumor necrosis factor antagonist with anti-inflammatory effects. Its major mode of action is to suppress TNFα, a Th1 embryotoxic cytokine produced by activated natural killer (NK) cells. We studied pregnancy outcome in 30 women with increased NK cell number and/or activity before conception. Women received 4 doses (25mg) of etanercept twice weekly before conception. The consent for the study from the Bioethics Committee of the Military Institute of Medicine and from the patients was obtained. Natural killer cell activity was measured using flow cytometry. In addition, the peripheral blood NK cell surface antigens CD16 and CD56, and Treg cells were studied using flow cytometry, before treatment and 2 weeks after the last etanercept dose. We determined that there is a significantly lower natural killer cell activity after etanercept therapy in the study women (P<0.05). Moreover, there is a positive correlation between decreased natural killer cell activity after etanercept therapy and successful pregnancy in the study women (r>0.5, P<0.05). There were no significant differences in Treg level before and after etanercept therapy (P>0.05). We conclude that Etanercept therapy might be effective treatment for women with increased NK cell activity. Regulation of immune system activity may underlie possible effect of such therapy.

Proteome differences in unexplained recurrent pregnancy loss compared to normal placenta

B. Gharesi-Fard, J. Zolghadri, E. Kamali-Sarvestani — 2010-08-01

Recurrent pregnancy loss (RPL) is defined as at least two sequential abortion before the 20th week of gestation. Approximately 40% of RPL cases are caused by unknown etiological factors, and therefore are classified as unexplained RPL (URPL). Placenta is a pregnancy unique tissue and proper formation of the placenta is a key phenomenal for success a pregnancy or occurrence of URPL. Therefore, the aim of the present study was to compare the human placental proteome between URPL and normal gestational matched placentas. Total placental proteins were extracted and two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) technique was used to compare the proteome of five URPL and five gestational matched normal placentas. After staining, the gels were scanned and the protein spots were analysed using Image Master 2D Platinum Software. Non-parametric Mann–Whitney test was used for analysis of the mean intensity differences of spots between URPL and normal placentas. Differentially expressed protein spots were excised from coomassie brilliant blue stained gels and identified by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF/TOF) technique after in gel digestion.Though 19 spots showed statistically different expression (p<0.05), 12 out of them were successfully identified. Among 12 identified proteins only two were down-regulated (Calumenin, Enolase 1), while the remaining ten (Actin gamma 1 propeptide, Cathepsin D, HSPgp96, Tubulin beta, Tubulin alpha 1, Glutathione S-transferase, Vitamin D binding protein, Prohibitin, Actin beta, Apolipoprotein A-I) showed increased expression in URPL cases in comparison with normal placentas. In conclusion, the data of the present study indicated that the alteration in expression of proteins involves in endothelial dysfunction might play an important role in the pathogenesis of URPL.

Immunological cause of recurrent pregnancy loss complicated by genetic thrombophilic factors, and results of the treatment

Z. Ulcova-Gallova, I. Subrt — 2010-08-01

We investigated the frequency of pregnancies in 258 patients with repeated pregnancy loss (RPL) having positive antiphospholipid antibodies (aPLs) in serum and/or genetic thrombophilic factors (GTF). We had studied and then treated a total of 258 patients aged more than 33 years after three or more spontaneous pregnancy losses during 2004–2009. ELISA was used for detection of aPLs against phosphatidyl-serine, phosphatidyl-ethanolamine, phosphatidyl-inositol, DL-glycerol, phosphatidic acid, anti-annexin V, anti-cardiolipin, and anti-beta2-GPI. Thrombophilic factor genotypes were determined using a melting curve analysis of the PCR amplification product detected by the fluorescence resonance energy transfer (FRET). Low-molecular-weight heparin in combination with non-steroidal antiphlogistics, B vitamins, Acidum follicum and low doses of Prednisone was used as the treatment. In 96% of patients there was at least one risk factor found (either aPL positivity or thrombophilic factor). Both aPLs and GTFs were present in 43% of patients. Our results show significantly increased prevalence of IgG-aPLs against phosphatidyl-inositol (17–39% dependent on number of spontaneous miscarriages) and against phosphatidyl-serine (19–38%). Prevalence of the FV 1691G>A (Leiden mutation), MTHFR 677C>T and MTHFR 1298A>C variants was significantly higher than in common Czech population (p<0.001). No differences in frequency of FII 20210G>A mutation were observed. 183 women (71%) delivered healthy babies, 53 (20.5%) patients miscarried again, 22 (8.5%) patients did not become pregnant. In conclusion, analysis of autoantibodies against various kinds of phospholipids and genetic thrombophilic factors should be studied together in the diagnosis of RPL for appropriate treatment, which could be also directed against embryocytoxic cytokines on the future.This work was supported by a grant of Ministry of Education of the Czech republic MSM 002 162 0812.

Does Intralipid enhance VEGF secretion by peripheral blood mononuclear cells?

C. Coulam, R.S. Jeyendran, S. Ramu — 2010-08-01

Successful implantation requires the invading blastocyst to stimulate its own blood supply through angiogenesis. Vascular endothelial growth factor (VEGF) is the best characterized regulator of angiogenesis. Homozygosity of the VEGF −1154A gene has been shown to be a risk factor for reproductive failure including recurrent implantation failure and recurrent pregnancy loss. LDL enhances monocyte secretion of VEGF. We, therefore, evaluated the secretion of VEGF by peripheral blood mononuclear cells (PBMC) before and after treatment with Intralipid. PBMCs were isolated by a density gradient technique using Hystopaque-1077 from blood samples taken from 37 women experiencing reproductive failure. PBMCs (5×106) were incubated for 3h at 37°C in 5% CO2 with no Intralipid, Intralipid 10, 100 and 1000μg/ml. The specimens were centrifuged at 3500rpm for 15min, the supernatants were removed and analyzed for concentrations of VEGF by ELISA. The results are shown in the table.

Lymphoid cells of endometrium in early missed pregnancy with chronic inflammatory and immune disorders

L.V. Volkova — 2010-08-01

The problem of chronic inflammatory and immune disorders in the genesis of missed pregnancy is very important due to high incidence in structure of miscarriages. Variable changes of lymphoid cell population of endometrium are described in early pregnancies in the normal and pathological situation, but many aspects of practical diagnostics and interpretation of results are still unknown (). The aim of this investigation was to study lymphoid cells in scrapes of endometrium in early missed pregnancies with chronic inflammatory and immune disorders. Fourteen cases of early missed pregnancies of 4–11 weeks of gestation with chronic endometritis and 5 samples with immune disorders were examined in endometrial scrapes (H&E). The variants of lymphoid cell infiltration (diffuse, focal), size of infiltrates (aggregates, large lymphoid infiltrates, nodules), location in decidua, types of cell accumulations (perivascular, periglandular, stromal, in area of trophoblast invasion) were evaluated. Chronic endometritis was revealed in women with gynecological pathology and abortions in anamnesis (50% cases) and age more than 29 years (64.3%). Morphological changes in endometrium included large lymphoid infiltrates and nodules in decidua parietalis with perivascular, periglandular, stromal location and typical mesenchymal reactions. Structural manifestations of chronic inflammation were associated with the main types of pathological alterations: (1) endocrinopathy with predominance of combination of low decidualizaton and retardation of endometrial glands (50%); (2) decrease of trophoblast invasion (28.6%); (3) changes of chorionic villi (78.6%); and (4) rheological disturbances (7.1%). In cases with immunological disorders 3 women were younger than 24 years; histological manifestations included excessive lymphocytic infiltration (diffuse and focal, nodule-like), vasculitis and rheological changes with association to acute and chronic endometritis, endocrinopathy and pathology of chorionic villi. In some cases differential diagnosis between chronic endometritis and immunopathological disorders in endometrial scrapes was necessary.

Early missed pregnancy: pathohistological diagnostics of inflammatory and immune disorders

L.V. Volkova — 2010-08-01

The problem of etiological verification of early missed pregnancy of different genesis is very important for potential therapies. Causes of early abortions include genetic, endocrine, infectional, immunological and thrombophilic factors, antiphospholipid syndrome and anatomical defects. The total rate of local inflammatory and immune factors with interrelations to other causes of early miscarriages can be revealed by mean of special multi-step pathohistological examination of endometrial scrapes. The aim of this investigation was to evaluate histological verification of the aetiology of early missed pregnancy, incidence and morphological associations of endometritis and immune disturbances in the endometrial scrapes. Forty-two cases of early missed pregnancy of 3–12 weeks of gestation were examined. Pathohistogical changes in different zones of decidua parietalis, decidua basalis and chorionic villi in paraffin sections (H&E) were investigated according to special diagnostics scheme. Results: 92.9% cases of missed pregnancies were observed in 4–9 weeks of gestation in age from 21 before 42 years (71.4% cases of pregnancies – in 21–30 years). Anamnesis of 35.7% of women included medical abortions and gynecological diseases. The high total percentage of inflammatory and immune disorders in parietal endometrium was found: acute (28.2%) and chronic (35.9%) endometritis, immunologic disturbances (12.8%). Other variants of pathology in endometrium and early placenta were characterized by following incidences: endocrinopathy with decrease of decidualizaton (48.7%), endocrinopathy with retardation of endometrial glands (41%), rheological disorders (28.2%), villitis (12.8%), stopping of chorionic villi development before 3–4 weeks of pregnancy (38.5%), decrease of trophoblast invasion (35.8%). Interrelations and associations between inflammatory, immunopathological processes and different revealed variants of pathology in early missed pregnancies were analyzed.

Natural killer cell analysis for reproductive failure: madness, methods and more!

G.P. Sacks — 2010-08-01

The potential role of natural killer cell analysis in women with repeated reproductive failure is undefined, but difficult to ignore (). While it is clearly far too simplistic and unproven to claim that ‘high’ levels are the cause of implantation failure, it is also far too easy to simply reject the hypothesis for lack of sufficient evidence. Women with repeated reproductive failure often believe that they have an ‘immune problem’, and frequently see alternative practitioners to address those beliefs. It is in the interests of those women and their doctors that we must engage in the possibility of immune dysfunction. The assessment of NK cells requires a high degree of laboratory expertise. In the uterus, so far, most studies have used immunohistochemistry which is unable to assess subtypes. The counting of cells is hugely subjective, especially given the glandular nature of the tissue. And there has been inadequate accounting for the enormous increase in NK cell numbers in the late luteal phase. In peripheral blood, standard blood count flow cytometry cannot report on subtypes or levels of activation either. The arguments against NK analysis have largely been theoretical and based as much on scepticism as on fact. In the face of a demanding patient population, NK analysis needs to be addressed scientifically and logically. That means the development of reliable and robust assays of both uterine biopsy tissue and peripheral blood, the construction of potential hypotheses, the experimental application of testing and treatment protocols in pilot studies, and finally the design and completion of randomised trials. This presentation will discuss the significant progress that has been made, and while more progress is needed for general acceptance or rejection, this will not occur without it more (rather than fewer) studies.

Autoimmunity and perinatal outcomes in women with threatened miscarriages—a prospective case control study

G. Matthias — 2010-08-01

First trimester bleeding is a common complication which affects 15–25% of all pregnancies. Threatened miscarriage is diagnosed on the basis of documented fetal cardiac activity on ultrasound with a history of vaginal bleeding in the presence of a closed cervix. We aimed to investigate the hypothesis that autoimmunity in the presence of threatened miscarriage may indicate an underlying placental dysfunction. Furthermore it may manifest later in pregnancy with adverse obstetric and perinatal outcomes such as preeclampsia preterm labour, placental abruption and fetal intrauterine growth restriction. A prospective cohort study was performed on 428 women presenting with significant bleeding in the first trimester and 428 asymptomatic age matched controls. They were checked for auto immunity and followed up till delivery. Adverse perinatal and obstetric outcomes were recorded in subgroups of both cohorts. Knowledge about the outcome of ongoing pregnancies following threatened miscarriage is relevant to both women and their obstetricians in order to plan antenatal care and consider management and clinical interventions.

An essential role for macrophages in corpus luteum function and early pregnancy maintenance

A.S. Care, M.J. Jasper, W.V. Ingman, S.A. Robertson — 2010-08-01

The CD11b-DTR transgenic mouse enables conditional and transient systemic ablation of macrophages by administration of diphtheria toxin (DT). We have utilised this model to evaluate the importance of ovarian macrophages during early pregnancy. Macrophages are abundant within the ovary, and have been identified in the corpus luteum (CL) of most species studied, including in rodents and human. Through their secretory products, macrophages are thought to be involved in ovarian tissue remodelling, including luteinization, and in regulating steroidogenesis. Macrophages co-cultured with granulosa or luteal cells act to stimulate progesterone secretion in vitro. The effects of macrophage ablation during the pre-implantation phase of pregnancy were evaluated. Ablation of macrophages on day 1 pc or day 4 pc caused complete pregnancy loss in all DT-treated CD11b-DTR mice, while DT-treated wild-type mice or PBS-treated CD11b-DTR mice maintained viable pregnancies. Macrophage ablation on day 3 pc significantly reduced serum progesterone (P4) levels to 40% of control levels when measured 24h later. Administration of exogenous P4 on each of day 4–7 pc prevented fetal loss in DT-treated CD11b-DTR mice, and pregnancy progressed with viable pups at late gestation, while no pregnancies remained viable in DT-treated mice administered vehicle only. Immunohistochemical evaluation with anti-CD31 mAb showed that macrophage ablation in the CD11b-DTR mice resulted in disappearance of endothelial cells from within the CL. This suggests that macrophages are essential for support of endothelial cells within the CL, either by provision of trophic support and/or by macrophage transdifferentiation into endothelial cells. In conclusion, these data indicate a critical role for macrophages in corpus luteum development and steroidogenic function in early pregnancy. Given that various nutritional and immune stressors can profoundly affect macrophage numbers and behaviour, it seems reasonable to speculate that macrophage-regulated CL development is a vulnerable event and may contribute to some forms of unexplained infertility.

Molecular profiles of mouse ovarian folliculogenesis

Y. Hosoda, A. Hasegawa, K. Kumamoto, M. Ogino, Y. Ikeda, H. Tanaka, S. Komori — 2010-08-01

Significant progress has been made in the technology for assisted reproduction and medical treatments have been provided for many infertile couples. However, more advanced technologies are required for severe infertility such as premature ovarian failure and ovarian impairment due to adjuvant therapy for cancer. Ovarian tissue cryopreservation followed by in vitro growth of isolated follicles is a feasible proposition for such patients. The objective of this study is to clarify the close coordination of communication among follicle cells including oocytes, granulosa and theca cells required for follicle growth. To analyze differential gene expression in the ovary, DNA microarray was performed (Agilent DNA expression Array) using BDF1 mice with different ages of 7, 10, 13, 16 and 19 days, because juvenile mouse ovarian follicles grow concomitantly and develop according to chronology. Each mRNA was prepared from the mouse ovaries by miniprep kit (Quigen Corp.). Genes displaying significant expression were selected using a software program (IPA). ZP1, ZP2, ZP3, FIGLA, BMP-15 and GDF-9, which are oocyte-specific growth factors, showed strong intensities at day 7 and then gradually declined to day 19. KIT, KL, AMH and PDGF, which are known as granulosa-cell-secreting growth factors, showed relatively high expressions from day 7 to day 19. Some cytokines/chemokines, CXCL14, CCL21 and IL17 were also shown to have high-level expression at day 7, increasing until day 13. Together these findings suggest that some cytokines/chemokines as well as growth factors may play a functional role in early folliculogenesis. These data will facilitate identification of the regulatory factors involved in folliculogenesis and thereby enable establishment of in vitro culture systems for ovarian follicles.

Novel effects of COX-2-selective inhibitor NS-398 on IL-1β-induced COX-2 and IL-8 expression in human ovarian granulosa cells

Y. Wu, H. Ou — 2010-08-01

Upregulation of inflammatory cytokines, such as prostaglandins and interleukins (ILs), have been noted in the regulation of a variety of ovarian functions. Among the multiple cytokines that regulate ovarian activities, IL-1β is one of the early indicators of inflammation and it could induce the expression of several other pro-inflammatory factors, such as cyclooxygenase 2 (COX)-2 and IL-8, which in turn participate in the inflammatory cascades. In addition, non-steroidal anti-inflammatory drug (NSAID), such as NS-398, a selective inhibitor of COX-2 bioactivity, is a popular medicine to mitigate inflammatory response. Here we reported in a human ovarian granulosa cell line KGN, instead of attenuating the inflammatory response, NS-398 could enhance IL-1β-induced COX-2 and IL-8 mRNA and protein expression as well as the IL-8 secretion. Further enhancement of IL-1β-induced IL-8 promoter activation by NS-398 suggested that the acting stage appears to be the transcriptional stage. To pinpoint the involving signal pathways, by using inhibitors of MAPK (ERK, JNK, p38), NFκB as well as PKA and PKC, we primarily revealed that the MAPK and NFκB pathways may involve in the NS-398's augmenting effect on IL-1β-mediated IL-8 and COX-2 expression. Further biochemical analysis delineated that NS-398 could potentiate IL-1β-induced NFκB p65 subunit translocation from cytosol into nucleus but had no effect on IL-1β-induced MAPK phosphorylation. The alamarBlue and flow cytometry analyses demonstrated that cotreatment with IL-1β and NS-398 induced cell proliferation and cell cycle progression and the effect was neutralized by IL-8 antibody. Meanwhile, combination of NS-398 with IL-1β was noted to reduce the expression of p21 and p27, both of which are negative regulators of cell cycle. Altogether, our data identified a novel role of NS-398 that it could amplify IL-1β-induced COX-2 and IL-8 expression at least in part by promoting the IL-1β-activated NFκB signal pathway, and the resultant IL-8 elevation may therefore contribute to cell proliferation of granulosa cells.

Effective two-step culture method for development of early preantral mouse follicles

M. Ogino, A. Hasegawa, N. Mochida, H. Tanaka, Y. Hosoda, S. Komori — 2010-08-01

Application of ovarian tissue cryopreservation followed by in vitro culture of isolated follicles is a feasible method for fertility preservation in young female patients with cancer. For this purpose it is expected to develop culture systems that can be used for various stages of follicles. This study examines the effectiveness of a two-step culture system for producing fertilizable eggs from early preantral follicles. In experiment 1, medium-size (125–140μm: GroupA) and small-size (80–95μm: Group B) preantral follicles were collected by mechanical treatment from 16- and 7-day old mice, respectively. The isolated follicles were cultured in vitro for growth on a collagen-coated membrane (IVG-membrane) for 7 days. In the experiment 2, the small-size follicles were mounted in collagen gel for in vitro growth (IVG-gel) as a first step. After 9 days, oocyte–granulosa cell complexes (OGCs) were isolated from the IVG-gel and transferred to IVG-membrane as a second step and cultivated for induction of maturation (IVM), and 24h later they were subjected to in vitro fertilization (IVF). In experiment 1, 95.5% (128/134) of the follicles in Group A grew, and 59.7% (80/134) generated fertilizing oocytes by IVG-membrane culture, while no follicle in Group B grew at all. In experiment 2, 73.9% (91/123) of follicles in Group B grew in IVG-gel culture. When the growing OGCs were transferred to subsequent IVG-membrane culture, 43.9% (54/123) reached a mature stage after IVM. Some of the oocytes originated from Group B follicles fertilized successfully and developed into embryo stages. These results suggest that the combination culture of IVG-gel and IVG-membrane is a useful method for in vitro growth and development of small preantral follicles having 80–95μm diameters.

Comparative proteomic analysis of follicular fluids from patients with different infertility etiologies

M. Webber, S. Neubeck, I. Hoppe, U.R. Markert — 2010-08-01

The objective of this study was to investigate potential protein biomarkers for reproductive dysfunctions such as Polycystic Ovary Syndrome (PCO), endometriosis and low responders in follicular fluid by using explorative SELDI-TOF mass spectrometry and to clarify their possible correlations with infertility, ovarian response and IVF outcomes. Potential follicle specific proteins detected in earlier measurements were specially regarded. This is a prospective study in which four groups of patients were included: polycystic ovary syndrome patients, fertile egg donors, endometriosis patients and low responders on hormone therapy. Proteins in 122 follicular fluids from IVF cycles between May and August 2009 were analyzed by Surface Enhanced Laser Desorption Ionization Time of Flight Mass Spectrometry (SELDI-TOF-MS). P-values have been calculated by implemented software and correlations between the presence and levels of six previously described potentially follicular fluid specific proteins “P1” to “P6” with ovarian response and other clinical parameters were calculated with SPSS software. Comparative proteomic analyses showed significantly different protein pattern between the four groups (different intensity signals in 4 proteins from 2 to 20kDa and in 5 proteins from 20 to 200kDa). A significantly different expression of 9 proteins from 2 to 20kDa and 11 proteins over 20kDa was detected between donors and low responders and of 4 proteins between donors and endometriosis patients. Follicular fluid specific proteins P1 and P2 correlate significantly positively and P4 and P5 negatively with age. Additionally, there was an association between the expression of these proteins and FSH levels, estradiol at the day of hCG administration, number of oocytes retrieved and embryos obtained depending on the different patients groups. These results demonstrate that the oocyte environment during follicular maturation differs in women with different ovarian reserve and supports the idea to use protein biomarkers in follicular fluids to assess oocyte quality and IVF outcomes.

Luteal cell-induced T cell responses depend on functional status of the CL and can be mediated by unconventionally secreted proteins

J.L. Pate, E. Brzezicka, K. Toyokawa — 2010-08-01

Lymphocytes residing within the corpus luteum are a dynamic population with changes elicited by the functional state of the CL. When αβ+ T cells (TC) were in contact with luteal cells, the proliferation of the physically separated γσ+ TC was significantly reduced. Thus, it was of interest to determine if αβ+ TC or their secretory products can alter luteal cell (LC) ability to stimulate γσ+ TC. The first objective was to determine if CD4+ can suppress LC-induced stimulation of γσ+ TC proliferation. Lymphocytes were cultured either in contact with LC or separated by culture inserts. In the absence of CD4+ cells, LC elicited a proliferative response in γσ+ TC, and the response were not dependent on cell contact. In contrast, after exposure to CD4+ TC, LC-induced proliferation of γσ+ TC required cell–cell contact. The second objective was to determine if CD4+ TC secreted products alter the responsiveness of γσ+ TC to luteal cells. αβ+ TC were separated from LC by a culture insert. After 48h, αβ+ TC were removed and were replaced by γσ+ TC either in contact or without contact with LC. The response of γσ+ TC to LC precultured with CD4+ TC that were separated by an insert was similar to what was observed in the first experiment when the CD4+ TC contacted LC. The third experiment tested whether TC mitogenic factor(s) secreted by LC were secreted by conventional or unconventional secretory pathways. LC were treated with brefeldin A to block conventional secretion, but upregulate unconventional secretion. LC+BFA conditioned media stimulated greater TC proliferation than LC conditioned media alone. It is concluded that αβ+ TC secrete a paracrine factor that modifies LC-stimulated proliferation of γσ+ TC, such that cell contact is required. Further, LC-secreted factors that stimulate TC proliferation may be secreted by unconventional secretory pathways.This project was supported by National Research Initiative Competitive Grant no. 2008-00551 from the USDA Cooperative State Research, Education, and Extension Service to JLP.

Interleukin-1 signalling in cord blood from preterm neonates

N.A. Hodyl, M.J. Stark, A. Osei-Kumah, V.L. Clifton — 2010-08-01

Increased concentrations of cord blood pro-inflammatory cytokines are associated with adverse conditions in preterm infants, including growth inhibition, chronic lung disease and cerebral damage. Their actions are, in part, self-regulated through the release of opposing cytokines and cytokine antagonists. While decreased levels of the endogenously released interleukin-1 receptor antagonist (IL-1ra) in preterm cord blood have previously been associated with adverse outcomes, these studies failed to assess simultaneously the balance between IL-1 receptor agonists and antagonists. The aim of this study was to characterise cord blood IL-1α, IL-1β and IL-1ra in cord blood collected from term (37–41 weeks, n=12), preterm (32–36 weeks, n=20) and very preterm (24–31 weeks, n=8) neonates. Plasma was separated from cord venous blood. Concentrations of IL-1α, IL-1β and IL-1ra, were determined by multiplex system. IL-1ra was detectable in 75% of term and 78% of preterm infants. IL-1α and IL-1β were detectable in 25% of term infants, and 14% of preterm infants. Cord blood concentrations of IL-1ra decreased across gestation, with significantly higher levels observed in very preterm compared to preterm, and preterm compared to term infants (p<0.05 in both instances). IL-1ra was significantly correlated with IL-1β in preterm (r=0.422, p=0.05) but not term cord blood. IL-1α and IL-1β concentrations were not significantly different in preterm compared to term, but were significantly correlated with each other (r=0.554, p=0.001). No sex specific differences in these cytokines were observed. Given that IL-1ra functions to regulate the agonist effects of IL-1α and -β in normal biologic processes, the significant correlation between IL-1ra and IL-1β in preterm cord blood suggests an attempt towards self-regulation in preterm parturition. The gestation specific decrease in cord blood IL-1ra levels suggests a greater role for this protein in protecting against inflammation in early development and in preterm compared to term infants.

Differential cytokine profiles in arterial and venous cord blood in preterm infants

M.J. Stark, N.A. Hodyl, A. Osei-Kumah, V. Clifton — 2010-08-01

Cord blood cytokines are markers of inflammation associated with preterm delivery and adverse neonatal outcome. However, few studies simultaneously examine cytokine profiles in venous and arterial cord blood despite different origins in the feto-placental circulation. We sought to describe the distribution of immune biomarkers in both venous and arterial cord blood in preterm neonates and investigate their potential as markers of fetal versus placental inflammation. Arterial and venous cord blood samples were collected from the chorionic plate of placentae from 28 infants delivered after spontaneous onset idiopathic preterm delivery (24–34 weeks gestation). Plasma was removed and cytokine profiles of 20 cytokines determined by multiplex array system. For both venous and arterial cord blood tumour necrosis factor-α (TNFα) and interleukin (IL)-8 concentrations were above detectable limits in all subjects while IL-12, IL-13, IL-1α, IL-1β, IL-7 and monocyte chemotactic protein (MCP)-3 were detectable in less than 15% of samples. Granulocyte colony stimulating factor (GCSF) was only above detectable limits in arterial cord blood and correlated with arterial IL-6 (r=0.56, p=0.013). Interferon (IFN) γ, IL-17, macrophage inflammatory protein (MIP)-1α, and TGFα were only detectable in venous cord blood. Median TNFα and IL-6 were 3-fold higher in venous versus arterial cord blood (p<0.05) while IL-8 levels were comparable. Cord blood cytokines have both fetal and placental origins which can be differentiated by arterial and venous sampling. This is an important consideration for interpretation of patho-physiologic processes in light of differing inter-relationships between pro-and anti-inflammatory cytokines as well as chemokines, dependent upon the site of sampling. The detection of GCSF only in arterial cord blood and its association with elevated pro-inflammatory cytokines suggests a capacity for the fetus to independently respond to inflammatory processes.

Specific microenvironment in the rupture zone of the fetal membranes at term delivery

2010-08-01

Human parturition represents an inflammatory response and two of its characteristics are the infiltration of leukocytes into the fetal membranes and the secretion of pro-inflammatory mediators at or around the time of parturition. The aim of this work was to study the histological differences between FM zones and relate them to the leukocyte density, leukocyte phenotype, leukocyte chemotactic activity and the secretion of pro-inflammatory mediators. Fetal membrane explants from the three zones (peri-placental (PZ), middle (MZ) and rupture (RZ)) were obtained from women following spontaneous vaginal delivery at term (n=5). Samples were obtained for histology, protein and mRNA abundance estimations. Histology monitored the collagen integrity and leukocyte phenotypes by the Red Sirius technique and immunohistochemistry, respectively. Leukocyte chemotactic activity was quantified in the protein extracts using Boyden chambers and flow cytometry, and 24 cytokines were quantified using a multiplex system. mRNA relative abundance of CXCL8, -10, CXCR1, -2, -3, -8 CAMs, IL-1β, TNF-α and MMP-9 was quantified. ANOVA and Kruskal–Wallis test were applied. The integrity of fetal membrane structure and its collagen distribution was PZ>MZ>RZ. In contrast, the leukocyte density and leukocyte chemotactic activity (p<0.05) increased PZ RZ=PZ (p=0.027); ICAM-1, -2 and P-SEL were MZ>RZ=PZ (p=0.039, 0.027 and 0.027), and TNFα and MMP-9 were MZ>RZ=PZ (p=0.027). IL-1β and CXCL8 were RZ>MZ>PZ (p=0.018 and p<0.001). We conclude that during labor a clear gradient exists in the fetal membranes, expanding from the placenta to the rupture site, where T cells, granulocytes and resident cells contribute to create a specific microenvironment that leads to rupture of these tissues.Funding: Alberta Innovates-Health Solutions Interdisciplinary Preterm Birth and Healthy Outcomes Team (PreHOT) (DMO).

Choriodecidua and amnion exhibit selective leukocyte chemotaxis during human labor

N. Gomez-Lopez, L. Vadillo-Perez, S. Nessim, F. Vadillo-Ortega, D.M. Olson — 2010-08-01

Leukocyte recruitment into the reproductive tissues is one of the processes that are involved in the onset of labor. Recruited leukocytes secrete mediators which contribute to the structural and biochemical changes that lead to the rupture of the fetal membranes at term. The purpose of this study was to assess the chemotactic activity of the amnion and the choriodecidua tissues at term and the phenotype of matched leukocyte subpopulations attracted by each membrane. Fetal membrane samples (amnion, choriodecidua and whole fetal membranes, n=5 in each case) were obtained from women at term without and with labor. Extracts of the membranes were prepared, and their chemotactic activity was determined using a modified Boyden chamber chemotaxis assay. Leukocytes were obtained from different term women not in labor or in labor and tested for chemotactic activity of membrane extracts from women not in labor or in labor. The number and phenotype of the leukocytes attracted were characterized by flow cytometry. Although all of the samples tested exhibited chemotactic activity, the number of attracted leukocytes by the choriodecidua and the whole fetal membrane extracts from laboring tissues was higher than those from non-laboring tissues (p=0.010, 0.008). In addition, the leukocyte chemotactic activities of laboring whole FM and choriodecidua were greater than those of the amnion (p=0.029, 0.016). Interestingly, during labor the major leukocyte chemotactic activity of whole fetal membranes for granulocytes, T cells, monocytes and NK cells depends on the choriodecidua, but in contrast, chemotactic activity for B cells depends on amnion exclusively. We conclude that the amnion and choriodecidua each exhibit chemotactic activity for selective leukocyte subpopulations and that both fetal membranes differentially regulate this process during labor. It could participate in the regulation of labor during human term gestation.Funding: Alberta Innovates-Health Solutions Interdisciplinary Preterm Birth and Healthy Outcomes Team (PreHOT).

The effects of apoptotic trophoblasts shed from placentae on monocyte differentiation: possible functions in modulating maternal immune response to the fetus

M.H. Abumaree, M.F. El-Muzaini, E.M. Mussaed, L.W. Chamley — 2010-08-01

During normal pregnancy, trophoblasts are shed from the placenta into the maternal blood () where these immunologically foreign cells are exposed to the maternal immune system. The effects of shed trophoblasts on maternal immune cells are poorly characterized, and therefore we conducted this study to examine the effects of shed trophoblasts on monocytes. An in vitro placental explant model () was used to harvest trophoblasts from 10 normal term placentae and their effect on monocyte differentiation determined by culturing 2×105 monocytes isolated from normal blood (using an untouched human monocytes kit) with or without 2×104 shed trophoblasts or PMA (10ng/ml) for 72h. Shed trophoblasts were then removed and adherent cells were characterized by microscopy, flow cytometry or ELISA. The levels of IL-10 and IL-1β were also quantified in the conditioned medium using Sandwich ELISA kits. Multinucleated syncytial knots and mononuclear cells were shed from the explants, and approximately 85% were apoptotic as determined by the activity of caspases 3 and 7. A cell-based ELISA showed that shed trophoblasts significantly increased the expression of indoleamine 2,3-dioxygenase (IDO) by monocytes (P<.001) exposed to shed trophoblasts. IDO expression was blocked by co-incubation with IL-4 (). In addition, shed trophoblasts increased the secretion of IL-10 (P<0.01) but decreased the secretion of the proinflammatory cytokine Il-1β (P<0.05) by monocyte cultures. Monocyte cultures treated with shed trophoblasts expressed higher levels of CD14 than untreated cultures (P<0.05). CD14 is a monocyte marker that is down-regulated during the differentiation of monocytes into macrophages (). These data suggest that apoptotic trophoblasts shed from the placenta may play an important role in modifying maternal immune responses to fetal antigens.

Characterisation of the surface proteome of the syncytiotrophoblast and syncytial knots from human placenta

O.J. Holland, P.R. Stone, L.W. Chamley — 2010-08-01

The human placenta is covered by the multinucleated syncytiotrophoblast (STB) which is bathed in maternal blood. Throughout pregnancy, multinucleated syncytial knots are shed into the maternal circulation. Maternal immune tolerance to fetal tissues may be influenced by exposure to factors in the STB or syncytial knots. Currently there is limited knowledge about the proteins present in syncytial knots, and whether these change in diseases of pregnancy such as preeclampsia. This study was undertaken to assess and compare the surface protein expression of the STB and syncytial knots. Syncytial knots were harvested from 15 first trimester placentae. Multiple methods were employed to extract membrane proteins, including; colloidal silica, triton X-114 and direct trypsinisation. Liquid chromatography/tandem mass spectrometry (LC–MS/MS) was used to identify proteins in the extracts. Over 800 proteins (including 109 unannotated proteins) were identified from placenta or syncytial knots. In silico analysis indicated proteins of known (83%) or unknown (17%) subcellular localisation. Of proteins with known subcellular localisation 40% were associated with the plasma membrane. The plasma membrane-associated proteins were sub-classified as structural, regulatory, trafficking, adhesion/migration, other function, or unknown function. Using the colloidal silica extraction method, a greater proportion of structural proteins were seen in syncytial knots (syncytial knots 66%, placenta 44%) and more regulatory proteins were seen in the placenta (syncytial knots 6%, placenta 10%). Also, a greater number of proteins with unknown function were identified in the placenta than syncytial knots (syncytial knots 2%, placenta 7%). Our data suggest there may be an increase in the relative proportion of plasma membrane-associated structural proteins in syncytial knots shed from the placenta. This change in protein expression profile could relate to the formation and extrusion of syncytial knots from the STB. Further characterisation of the proteome may identify proteins that are key to these processes.

The endogenous retroviral envelope protein syncytin-1 inhibits the release of TNFα and IFNγ in human blood cells

J.E. Schjenken, J.M. Tolosa, V.L. Clifton, R. Smith — 2010-08-01

A signature of retroviral envelope proteins is the presence of a fusogenic peptide and an immunosuppressive domain (ISD) within their sequences. The human placenta expresses endogenous retroviral (ERV) envelope proteins. One of them, syncytin-1 is highly expressed in the syncytiotrophoblast and is thought to be a key factor in regulating syncytialisation due to its fusogenic properties. Syncytin-1 also carries a sequence homologous to the consensus ISD. In light of this information, we hypothesised that syncytin-1 has, in addition to its fusogenic function, immunosuppressive properties and therefore may play a role in maternal immunotolerance. The recombinant ectodomain of syncytin-1 was purified as soluble cytoplasmic material using a combination of affinity chromatography and gel filtration. The immunosuppressive properties of syncytin-1 were tested using human whole blood/PBMC cultures challenged with LPS/PHA. Supernatants were assayed for TNFα and IFNγ release using commercial available ELISA kits. As a comparative positive control, the immunosuppressive placental mouse ERV envelope protein syncytin-B was produced in parallel and immuno-tested. Syncytin-1 recombinant ectodomain was purified as a multimeric protein with the characteristic SDS-PAGE protein profile of retroviral ectodomains. Syncytin-1 expression was confirmed using western blotting and LCMS sequencing. Syncytin-1 inhibited the production of TNFα in a dose dependent manner in whole blood cultures following maximal stimulation with LPS. A maximum of 50% TNFα inhibition was observed following 1μM syncytin-1 treatment. Syncytin-1 also inhibited the production of IFNγ by 30% in PHA stimulated PBMC at a concentration of 1μM. Syncytin-B inhibited the production of TNFα by 40% at a concentration of 1μM. Retroviral envelope proteins have previously been shown to inhibit Th1 responses and induce Th2 responses. Here we show for the first time a novel role for syncytin-1 in the inhibition of the Th1 cytokines TNFα and IFNγ. These data suggest that syncytin-1 may mediate immune tolerance toward feto-placental antigens.

Placental villous trophoblast supports vasculogenesis in vitro

W. Pfeiffer, J. Mostertz, F. Herr, G. Homuth, U. Völker, M.T. Zygmunt — 2010-08-01

Inadequate placental vascular development is associated with a number of pregnancy pathologies, such as intrauterine growth retardation, preeclampsia and early pregnancy losses. Therefore, an extensive analysis of the crosstalk between endothelial progenitor cells (e.g. CD133+ cells) and placental trophoblast is necessary for potential therapeutic interventions in the future. The effects of human trophoblasts on vasculogenesis were examined using peripheral blood mononuclear cells (PBMC) and CD133+ cells isolated from umbilical cord blood in a colony-forming-unit-endothelial-cell (CFU-EC) assay. Furthermore, a co-culture assay of CD133+ cells with placental villous trophoblast cells separated by a membrane of 0.4μm pore size was applied and compared to a co-culture of CD133+ cells with CD133+ cells. Total RNA isolation from CD133+ cells was performed with TriFast (PeqLab). Gene expression in the co-cultured CD133+ cells was analyzed using a Human Gene 1.0 ST Array (Affymetrix). Using CFU-EC assay, the number of colonies developing in trophoblast-conditioned media (TCM) was significantly increased compared to control conditions. Microarray analysis of CD133+ cells co-cultured with trophoblast cells showed an upregulation of leukocyte differentiation marker (CD1a, CD1c, CD11b, CD14, IL7R) as well as an induction of angio-/vasculogenesis associated cytokines and chemokines (e.g. IL-1beta, IL-8/CXCL8, CCL2, CCL3, CCL22, HB-EGF). We conclude that trophoblasts support vasculogenesis in a paracrine manner and induce angio-/vasculogenesis-related genes in CD133+ cells as well as leukocyte differentiation. To recapitulate, placental vasculogenesis is tightly connected to hematopoiesis and both have been shown to be enforced and controlled by trophoblast in vitro.

Increased EBI3 expression in placentas of preeclamptic patients

2010-08-01

Since fetal tissue is semiallogenic towards the mother, immunomodulatory properties are critical for the acceptance of the fetus. The cytokines IL-27 and IL-35 contain Epstein–Barr virus-induced gene 3 (EBI3). IL-27 has pro-inflammatory activities, but it also has several anti-inflammatory properties by the inhibition of Th1 responses and regulation of Th2 responses. IL-35 is expressed by regulatory T cells and contributes to the suppressive activity of these cells. This study investigates EBI3 and the EBI3 containing cytokines IL-27 and IL-35 in tissue of preeclamptic and control women. Plasma samples and placental biopsies of 10 control and 13 preeclamptic patients were collected. Plasma was analyzed for IL-27 and IL-35 using ELISA (IL-27 and IL-35 kit USCNLIFE). Placental biopsies were tested for EBI3 expression by RT qPCR. A significantly higher amount of EBI3 expression was present in preeclamptic placentas compared with control placentas (p=0.033). No difference is present in the amount of IL-27 and IL-35 in maternal plasma and umbilical cord plasma between preeclamptic patients and controls. In the placenta, significant differences are found in the EBI3 expression between preeclamptic and control patients. These differences are not found in the periphery by testing IL-27 and IL-35 which are EBI3 containing cytokines. These data suggest that EBI3 locally in the placenta plays a role in the etiology of preeclampsia.

Effects of anti β2-GPI antibodies on TLRmRNA expression and cytokine production in BeWo cells

2010-08-01

The antiβ2-GPI antibody was detected in recurrent fetal loss, intrauterine growth restriction and intrauterine death with strong pathogenic activity. The pathogenic activities of antiphospholipid antibodies consist of thrombosis and trophoblastic damage and so on. It has been reported that anti β2-GPI antibody increased NFκB via Toll like receptor (TLR)-4 on the endothelial cells. TLRs are expressed on the trophoblast. In order to investigate the effects of anti β2-GPI antibody for the placenta, the effects of anti β2-GPI antibody on the expression of TLR mRNA and cytokine production in choriocarcinoma cells were evaluated. The choriocarcinoma cells (BeWo) were cultured in 24-well tissue culture plate. The cells after incubation with purified β2-GPI were cultured with the IgGs taken from anti β2-GPI antibody positive and negative sera. The RNAs were purified from these cells 24h after. The expressions of TLR mRNA in BeWo cells were measured by using the real time PCR method (Applied Biosystems-7500). Cytokines such as IL2, IL4, IL10, TNFα, IFNγ and GM-CSF in cultured supernatant were measured by using the suspension array system Bio-Plex (BIO-RAD). The expression of TLR mRNA increased in BeWo cells cultured with the IgGs taken from anti β2-GPI antibody positive women. Especially, the expression of TLR-1, 2 and 4 by anti β2-GPI antibody positive IgG increased much more than that by negative IgG. Cytokine production such as IL6 and IL8 by anti β2-GPI antibody positive IgG increased much more than that by negative IgG. In conclusion, the anti β2-GPI antibody positive IgG increased the expression of TLR mRNA and cytokine production in BeWo cells. We suspect that the anti β2-GPI antibodies bind to trophoblast and increase the expression of TLR and cytokine production. The increased expression of TLR and cytokine production by anti β2-GPI antibody may play a role of increased inflammatory response in placental dysfunction.

STAT5 signaling in trophoblastic cells is induced by epidermal growth factor

S. Ospina, D.M. Morales, U.R. Markert — 2010-08-01

Epidermal growth factor (EGF) displays manifold functions in the placenta including tuning of invasion. Recent reports have demonstrated that activation of Signal Transducer and Activator of Transcription (STAT) proteins, including STAT3 and STAT5 actively participate in tumor development and progression. STAT5 is an intracellular protein downstream tyrosine-kinase receptors, which stimulates proliferation and cell cycle progression. Several cytokines involved in the regulation of trophoblast behaviour have been shown to mediate their effects though STAT3, but less is known about STAT5 activation in pregnancy. The aim of this study was to assess correlation of STAT5 and STAT3 phosphorylation. JAR, BeWo and JEG3 choriocarcinoma cells as well as HTR-8/SVneo immortalized trophoblast cells were stimulated with IL-2, IL-11, leukemia inhibitory factor (LIF), EGF or granulocyte macrophage-colony stimulating factor (GM-CSF) by applying diverse concentrations and times. The effect of the cytokines and growth factors on the expression, phosphorylation and localization of STATs was determined by Western blotting and immunocytochemistry. Only EGF induces STAT5 phosphorylation (Tyr 694) in all analyzed cell lines, while the other cytokines, except IL2, induce different intensity of STAT3 phosphorylation (Tyr 705) depending on cytokine and cell line. It can be concluded that cytokines that activate STAT3 and STAT5 in trophoblastic cells are different. The activating cytokines are highly expressed in the placenta. Therefore, it can be assumed that simultaneous activation occurs frequently and may further influence functions and behaviour of cells.

Effect of interferon-gamma on VEGF expression by trophoblasts in first trimester villous explant culture

H. Yoon, Y. Paek, G. Cho, Y. Yoon, M. Oh, H. Kim — 2010-08-01

The regulation of angiogenesis at the feto-maternal interface plays an important role in early pregnancy. It has been postulated that uNK cells play a role in spiral artery remodeling. In mice studies, activated uNK cell-derived interferon-gamma (IFNγ) has been shown to contribute to spiral artery modification. However, the role of IFNγ in human spiral artery remodeling remains unclear. Here we investigate the effects of IFNγ on angiogenesis in first trimester villous explant by analyzing VEGF protein and mRNA expression in trophoblasts. Villous explant culture was established from first trimester human placentas (6–8weeks of gestation, n=7) obtained from electively terminated pregnancies. Normal villous tissues were explanted on Matrigel and incubated under 3% O2 tension for 6 days. To evaluate the effect of IFNγ, 0.1, 1.0, and 10.0ng IFNγ was added per milliliter culture medium. The expression of VEGF in the villous explant cultures was evaluated by Western blot, RT-PCR, and immunohistochemistry. Levels of VEGF production significantly increased when trophoblasts were treated with 0.1ng/mL IFNγ, as compared with the control (P=0.048). The VEGF mRNA expression of the trophoblasts treated with 0.1ng/mL IFNγ was significantly increased, as compared with the control (P=0.012). However, VEGF production and VEGF mRNA expression decreased at higher concentrations of IFNγ. Immunostaining of VEGF increased in trophoblasts treated with IFNγ, as compared with the control, although the intensity of staining decreased in a dose dependent manner. These findings suggest that IFNγ plays a positive role in angiogenesis in early gestational trophoblasts at low concentrations. However, high concentrations of IFNγ are associated with a Th1 response, which is thought to be harmful in pregnancy.

Invasive activities of primary extravillous trophoblast via altered proteinases activity in low oxygen condition

K. Naruse, A. Onogi, T. Noguchi, T. Sado, T. Kitanaka, H. Oi, H. Kobayashi — 2010-08-01

Abnormal trophoblast invasion into the decidua, uterine myometrium and vascular system in early pregnancy is regarded as pathology of recurrent miscarriage and later hypertensive pregnancy or fetal growth restriction. Oxygen concentration may be a key factor for the invasiveness of trophoblast, as suggested by research on trophoblast cancer cell lines, similar to other cancer cells. However primary cell culture from human placental villi has been used less often in this research. We hypothesised that low oxygen conditions have effects on invasiveness and protease activity of extravillous trophoblasts from human early placental villi. Placental villi were taken at induced abortion of 7–15 weeks’ gestation with the patient's informed consent. Cytotrophoblasts were separated using a Percoll-based method and converted to invasive phenotype, a model of extravillous trophoblast, on Matrigel (Lash, G.E., Naruse, K., et al. Placenta, 2010). Invasion after 24h culture was quantified using an Invasion Chamber assay. Migration activity (wound healing assay), protease concentration (ELISA) and activity (gel zymography) in culture supernatants were also quantified. All experiments were performed in 20% and 5% oxygen, and in 3×1h severe anaerobic stimulation in 5% oxygen conditions. Cell viability and migration showed no difference between each oxygen condition. Invasion of the trophoblasts was increased in 20% oxygen conditions compared with 5% oxygen, and decreased in hypoxic stimulation. Concentrations of MMP-2, TIMP-2 and uPA in cell culture supernatants were not altered in hypoxic stimulation, but were increased in 20% oxygen compared with 5% oxygen. MMP-9 was not affected by anaerobic/aerobic conditions. We conclude that differently to cancer cell lines, invasive phenotype trophoblasts showed decreased protease activity and invasiveness in 5% oxygen compared with 20% oxygen, which may be similar to natural suppression of excess invasiveness of trophoblasts by moderate hypoxia in early pregnancy. On the other hand, increased HIF-1 was not seen to participate in invasiveness of primary trophoblasts, which suggests other signalling pathways exist for regulation of invasion.

Expression of ADAM-12 in the human placenta

I. Santillan, U.R. Markert, J.S. Fitzgerald — 2010-08-01

ADAM metallopeptidase domain 12 (ADAM 12; meltrin-α) is a catalytically active metalloprotease and disintegrin which plays a role in myoblast fusion and osteoclast formation. In the placenta, ADAM-12 has multiple functions, one of which seems to be regulation of trophoblast fusion. Therefore, we aimed to compare ADAM-12 expression in normal placentae and pathological placentae. We analyzed term (n=10) human placentae as well as placentae from patients with IUGR (n=10), choriocarcinoma (n=10) and mola (n=10). Tissue was fixed in formalin and processed for immunoperoxidase staining for detection of ADAM-12, cytokeratin-7, prolactin and CD56 (for control). ADAM-12 was detectable in all samples. ADAM-12 expression is more intensive in villous and extravillous trophoblast cells in normal placentae than in IUGR. In both groups ADAM-12 expression is stronger in wider areas of syncytiotrophoblast than in narrow areas. ADAM-12 expression pattern in mola appears heterogeneic (more and less intensively stained cells are neighbored), while expression in choriocarcinoma appears homogeneic. We conclude that ADAM-12 is widely expressed in normal and pathological placentae. Differences of expression intensity may be involved in trophoblast disorders.

Single nucleotide polymorphisms in genes modulating inflammation and the risk of preeclampsia in a Sinhalese population from Sri Lanka

2010-08-01

Preeclampsia (PE) is a common pregnancy complication contributing to maternal and neonatal morbidity and mortality. The maternal syndrome of preeclampsia is considered part of an increased systemic inflammatory response. We aimed to determine whether functional single nucleotide polymorphisms (SNPs) in pro-inflammatory (IL-1A, IL-1B, IL-6) and anti-inflammatory (IL-4, IL-10, IL-1RN) cytokine, mannose-binding-lectin-2 (MBL2) and cyclooxygenase-2 (Cox-2) genes are associated with preeclampsia. In this study 180 nulliparous Sinhalese women with preeclampsia and 180 normotensive women matched for age, ethnicity, parity and BMI were recruited from two tertiary care hospitals in Colombo. Women with a BMI ≥30kg/m2 were excluded. Preeclampsia was diagnosed using international guidelines. Early preeclampsia (n=73) was defined as onset of preeclampsia prior to 34 weeks of gestation. A small for gestational age baby was defined as having a birthweight below the 10th customised birthweight centile (n=102). Peripheral blood was collected from the participants and DNA extracted. Genotypes of 13 SNPs were determined by the Sequenom MassARRAY system. Genotype results of cases were compared with controls using dominant and recessive genotype models by logistic regression analyses. IL1A−889 TT+CT [OR (95% CI)=1.6 (1.1–2.5)], IL1A+4845 TT+GT [OR (95% CI)=1.8 (1.1–3.3)], MBL2+54 GG [OR (95% CI)=1.6 (1.0–2.6)], and COX-2-765 CC [OR (95% CI)=4.08 (1.0–17.5)] genotypes were increased in women with preeclampsia. IL6−634 CC+GC genotypes were increased in women who developed early onset preeclampsia [OR (95% CI)=1.9 (1.1–3.6)]. IL1B−511 AA+GA genotypes were increased in preeclamptic pregnancies resulting in the delivery of a small for gestational age baby [OR (95% CI)=1.7 (1.0–2.7)]. Our results demonstrate that single nucleotide polymorphisms in genes regulating inflammation are associated with preeclampsia in Sinhalese women. We conclude that genotypes leading to a pro-inflammatory state confer an increased risk for preeclampsia and may have a role in predicting preeclampsia.

Bizarre expression of tissue inhibitor of metalloproteinase-3 in feto–maternal interface caused gestational hypertension in mice model

T. Kimura, Q. Zhang, H. Nakamura, K. Kumasawa, S. Koyama, C. Tabata, T. Tsutsui — 2010-08-01

Preeclampsia (PE) is believed to result from abnormal placentation in early pregnancy. Shallow endovascular cytotrophoblast invasion and endothelial cell dysfunction are two key features in the pathogenesis of PE. Matrix metalloproteinase-9 (MMP-9) is crucial for the invasive ability of trophoblast cells during placentation. Tissue inhibitor of metalloproteinase-3 (TIMP-3), which is known as a potent inhibitor of MMP-9, also plays a role in regulating trophoblast invasion to the uterine endometrium. However little is known about the role of matrix metalloproteases and their inhibitors on regulation of maternal and fetal circulation. In the present study, we applied in vivo gene transfer system with hemagglutinating virus of Japan envelope (HVJ-E) vector into feto–maternal interface of pregnant mice on day 6.5 post coitus (pc), during the period of placental development. Overexpression of TIMP-3 cDNA suppressed MMP-9 activity in the uterus. Maternal systolic blood pressure significantly rose from day 14.5 pc to parturition. The fetus and the placentae were significantly smaller, and serum soluble fms-like tyrosine kinase (Flt)-1 and soluble endoglin levels were significantly higher after TIMP-3 treatment than in control mice. These data suggest that the balance between matrix metalloprotease and its inhibitor may play a pivotal role in determining feto/maternal circulation.

Immunoregulatory gene polymorphisms in gestational hypertensive disorders

A. Highet, S. Thompson, D. Furness, V. Zhang, G. Dekker, C. Roberts — 2010-08-01

Pregnancy is a controlled state of inflammation. Deregulation of cytokine networks can lead to adverse pregnancy outcomes including preeclampsia (PE). We aimed to identify single nucleotide polymorphisms in immunoregulatory genes that signify an increased risk of the gestational hypertensive disorders PE and gestational hypertension (GH).

Immunoglobulin-D and the innate inflammatory response in preeclampsia

2010-08-01

Pregnancy is characterized by suppression of adaptive immune responses protecting the fetus from allograft rejection. Concomitant activation of the innate immune response during pregnancy protects the mother against infection. Preeclampsia is a serious complication of pregnancy characterized by systemic inflammation and endothelial dysfunction, indicating involvement of maternal innate immune cells. B cells bridge innate and adaptive immune responses by producing immunoglobulins such as IgD that bind innate immune cells modifying their function. Very little is known about IgD, though recently it was found to bind basophils, stimulating production of the pro-inflammatory cytokine TNFα (). The role of IgD in inflammatory responses in pregnancy and preeclampsia has not yet been investigated. In this study we aimed to (1) measure the amount of soluble IgD (sIgD) in plasma, (2) determine whether monocytes bind IgD in preeclampsia, and (3) determine the effect of IgD binding on monocyte function. PBMC and plasma were collected from healthy non-pregnant women of reproductive age (n=10), women with a clinically uncomplicated pregnancy (n=10) or preeclampsia (n=10). sIgD was measured in plasma using an in-house ELISA. Flow cytometry was used to measure sIgD bound to the surface of CD14+ monocytes and the effect of purified IgD on TNFα production in monocytes. Women with preeclampsia have a significantly greater amount of circulating sIgD compared with pregnant women (p<0.05). Monocytes from women with preeclampsia also have a greater density of IgD bound to their surface, compared with those from non-pregnant and healthy pregnant women (p<0.05). Incubating PBMC with purified IgD increases the density of this antibody on the cell surface of monocytes, indicating the as yet undefined monocyte “receptor” for IgD is not saturated. Purified IgD was found to augment TNFα production in monocytes, promoting a pro-inflammatory milieu. We conclude that IgD may be involved in the dysregulation of the innate inflammatory response in women with preeclampsia.

Soluble ST2 (sST2) and IL-33 in preeclampsia

I. Granne, J. Southcombe, J.V. Snider, T. Child, C.W. Redman, I.L. Sargent — 2010-08-01

ST2 is a member of the IL-1 receptor family and is expressed in both membrane bound (ST2L) and soluble forms (sST2). Soluble ST2 is thought to act as a decoy receptor for its recently identified ligand IL-33. sST2 has been shown to be increased in a number of inflammatory pathologies such as myocardial infarction, heart failure, allergic airways disease, asthma and sepsis. sST2 appears to be a promising biomarker in cardiovascular disease with raised levels predicting poor outcome. Preeclampsia is associated with an exaggerated maternal inflammatory response and we hypothesised that sST2 may be a potential biomarker for this disease. In this study, plasma samples were taken from 20 preeclamptic women (defined as ≥90mmHg diastolic blood pressure on 2 occasions and proteinurea ≥500mg in 24h). Maternal age, gestational age and parity matched normal pregnant controls were recruited. sST2 was measured by ELISA (using the Presage™ ST2 assay, Critical Diagnostics, NY, NY™). Western blotting was used to identify placental ST2 and IL-33. There was no difference in the mean age (31.55 vs 31.75), parity (0.35 vs 0.45) or gestation of the groups. Blood pressure was significantly higher in the preeclamptic group (p<0.001). In normal pregnancy, circulating sST2 increased towards term. There was a significant increase in sST2 in women with preeclampsia compared to controls (p≤0.001) with the largest differences seen in early onset preeclampsia (21–28 weeks). Levels were comparable to those seen after acute myocardial infarction. Both ST2 and IL-33 were identified in placental tissue by western blotting. This is the first study to show that plasma sST2 is increased in preeclamptic pregnancies and its source may be the placenta. Although its role in the disease process is unclear, sST2 is a potential novel biomarker for preeclampsia which may be independent of endothelial dysfunction.

Association of placentally derived biologically active microparticles with immune-dysregulation in adverse pregnancy outcomes (preeclampsia and recurrent pregnancy loss)

S. Baig, S. Ho, N. Kothandaraman, S. Vasoo, J. Lu, M. Wenk, M. Choolani — 2010-08-01

Preeclampsia is a significant clinical problem being a major cause of maternal mortality and morbidities, perinatal deaths, preterm birth, and intrauterine growth restriction. Current evidence supports a model of superficial placentation driven by immune maladaptation, with dysfunctional endothelium and increased placental debris in the maternal circulation. Unexplained recurrent pregnancy losses (RPL) may share a similar immunopathology. There is mounting evidence to suggest that placental debris consisting of syncytiotrophoblast membrane microparticles (STBMs) are potential contributors to altered systemic inflammatory responsiveness in normal and adverse pregnancies. However, to date, few studies have compared the inflammatory potential of placenta-derived purified STBMs generated from placentas of normal and adverse pregnancy outcomes. We hypothesize that STBMs in preeclampsia and RPL are more immunogenic compared to healthy pregnancies. We aim to investigate whether (a) STBMs in preeclampsia and RPL induce stronger activation of monocyte-derived dendritic cells (DCs) and (b) STBMs in adverse pregnancies possess unique protein and lipid composition contributing to their increased immunogenic potential. Placenta and maternal blood samples are collected from normal and complicated pregnancies. Microparticles are isolated from placental explant culture supernatants. Immunomodulation assays are conducted using STBMs as stimulant for dendritic cells. Cytokine ELISA and flow cytometry are conducted to assess functional and phenotypic maturation of DCs, respectively. Protein and lipid composition of STBMs are studied using mass-spectrometric approach (ESI LC–MS/MS). Together these preliminary data show immunogenicity of placental microparticles with regard to ability to induce functional and phenotypic maturation of dendritic cells. Proteomic and lipidomic analysis shows differentially expressed proteins and lipid species in preeclamptic STBMs. Analysis of biological replicates are underway. Knowledge gained from this study may allow for better elucidation of the role of placenta in immune-dysregulation of adverse pregnancies and development of novel diagnostic or interventional approaches.

Season, vitamin D-related genes and maternal circulating 25OH vitamin D3 associate with pregnancy complications in an Australian population

C.T. Roberts, S. Thompson, D. Furness, S. Lee, P. Anderson, H. Morris, G. Dekker — 2010-08-01

Vitamin D deficiency is associated with preeclampsia, as well as hypertension, type 2 diabetes, obesity, cancers, immune dysfunction, cardiovascular disease and bone disorders. We aimed to determine whether prevalence of pregnancy complications is seasonal, and if they are related to maternal serum 25 hydroxy vitamin D3 (25OHD3) or with single nucleotide polymorphisms in the vitamin D receptor (VDR) and CYP24A1 (Vitamin D metabolising enzyme) genes in the SCOPE cohort (latitude ∼35°S). Peripheral blood from 1169 nulliparous couples and cord blood from babies were collected. Genotyping was performed using the Sequenom MassARRAY system. Serum 25OHD3 was measured using the Roche Elecsys Immunoassay. Data for 900 Caucasian couples were analysed. Genotypes and vitamin D concentrations at 15 weeks gestation for preeclampsia (PE n=93), gestational hypertension (GHT n=92), preterm birth (PTB n=69), small for gestational age (SGA n=138), gestational diabetes mellitus (GDM n=38) were compared with normal controls (n=520) and analysed using Chi-square and ANOVA. The distribution of adverse outcomes was similar across seasons except for PTB, where 23% of babies were due to be born in September. Serum 25OHD3 at 15 weeks gestation was below 60nmol/L (level of vitamin D sufficiency) in 35% of women and below 80nmol/L in 75% of women, while just 4% of women had greater than 100nmol/L. Not surprisingly, serum 25OHD3 concentration was significantly different between months of sampling with lowest levels in late winter and early spring. Circulating 25OHD3 is higher in women who smoke and is inversely correlated with BMI. When adjusted for BMI, serum 25OHD3 was significantly different between outcome groups with the exception of PTB. Maternal VDR rs7975232 is associated with PE and GDM, while maternal CYP24A1 rs2248137 is associated with gestational hypertension. In Adelaide there is a seasonal prevalence of PTB that is not related to circulating 25OHD3 although the latter is associated with other pregnancy complications. 25OHD3 is hydroxylated in placenta so 1,25OH2 D3 measurements may be more pertinent. Polymorphisms in vitamin D related genes associate with adverse pregnancy outcomes.

Analysis of CD28 molecule in preeclamptic patients: preliminary report

S. Daher, K.P.T. Pendeloski, M.A. Dalboni, R. Mattar, N. Sass, M.R. Torloni — 2010-08-01

Preeclampsia (PE) is considered an inflammatory condition characterized by impaired T cell function. CD28, a co-stimulatory molecule, enhances T cell activation. The expression of CD28 is regulated by a complex cytokine network, including proinflammatory cytokines IL-12 and TNFA. CD195, a chemokine receptor, has been associated with Th1 type inflammatory response. CD28 gene polymorphism may influence the expression of this molecule and thus increase the risk for preeclampsia. The aim of this study was to assess CD28 gene polymorphism and evaluate CD195 and CD28 expression on CD4+ T cells in PE patients. This case–control study included 50 PE and 61 normotensive pregnant women (C). Polymorphisms genotyping were obtained by PCR-RFLP. We used mouse-anti-human mAbs to characterize lymphocytes (CD3-APC-Cy7) and Helper T cell (CD4-PerCP-Cy), co-stimulatory molecule (CD28-FITC) and chemokine receptor (CD195-PE). These expressions were detected by flow cytometry and measured by mean fluorescence intensity (MIF). The percentages of CD28null, CD195-, CD195+CD28−, CD195−CD28+, CD195+CD28+ were calculated within the CD3+CD4+ cell fraction. Data were analyzed by Mann–Whitney, Chi-square or Fisher tests. CD28 genotype frequencies were—CC: 4% PE vs. 3.5% C; CT: 28% PE vs. 24.5% C and TT: 68% PE vs. 72% C (p=0.89). Analysis of the CD3+CD4+ T cell fraction did not reveal significant differences in percentages of CD3+CD4+CD28null, CD3+CD4+CD195− and CD3+CD4+CD195+CD28− between PE and C (p>0.05). A significantly higher percentage of CD3+CD4+CD195+CD28+ T cells was identified in PE compared to C (p=0.04). Our data suggest that CD28 gene polymorphism is not associated with PE but these patients present a higher percentage of CD4+T cells expressing both CD195 and CD28 in peripheral blood. The study is ongoing to further investigate the role of this T cell population in PE.Financial support: FAPESP (#07/574446-0) and CAPES.

Gestational diabetes versus inflammatory mediators

2010-08-01

An abnormal inflammatory response is involved in the physiopathology of gestational diabetes (GD). Specific mediators such as adiponectin, TNFA, IL-6 and IL-10 seem to play a central role in this disorder. Polymorphisms in these genes may influence the expression of these molecules. The aim of our study was to investigate a possible association between adiponectin +45 T/G (rs2241766), adiponectin −11377 G/C (rs266729), IL-6 −174 G/C (rs1800795), IL-10 −1082 G/A (rs1800896) and TNFA −308 G/A (rs1800629) gene polymorphisms and assess adiponectin, TNF A, IL-6 and IL-10 levels in women with GD. This case–control study included 45 patients with GD and 81 healthy pregnant women without any obstetric or systemic disorders (C, control). Genomic DNA was extracted from peripheral blood, and genotyping was performed by PCR, followed by digestion with restriction enzyme. Adiponectin serum levels and culture supernatant TNFA, IL-6 and IL-10 concentrations were determined by ELISA. Data were analyzed by Mann–Whitney, Chi-square or Fisher tests. There were no associations between GD and the analyzed gene polymorphisms. There was a significant association between adiponectin −11377 gene polymorphism and adiponectin levels in GD women. Adiponectin serum levels were higher in C than in women with GD (8280ng/mL vs 5490ng/mL, p≤0.0001). TNFA supernatant levels were lower in C than in GD patients (27.6pg/mL vs 47.4pg/mL, p=0.0468). IL-6 and IL-10 levels did not differ between the two groups (1254pg/mL vs 1141pg/mL, p=0.7146 and 67.02pg/mL vs 76.31pg/mL, p=0.6399, respectively). Together these results show there were no associations between GD and the analyzed gene polymorphisms but a significant association between adiponectin −11377 gene polymorphism and adiponectin levels in this group. Women with GD have decreased levels of adiponectin and increased TNFA concentrations. GD women have normal IL-6 and IL-10 levels.Financial support: FAPESP (08/56718-9, 08/55888-8, 08/58548-3) and CNPq.

Adiponectin in women with gestational diabetes: relationship with obesity

2010-08-01

Obesity is associated with gestational diabetes (GD). Adiponectin is involved in glycemic regulation and in the physiopathology of obesity. Adiponectin gene polymorphisms may influence the expression of this molecule. The aims of this study were to assess adiponectin +45 (rs2241766) and −11377 (rs266729) gene polymorphisms, adiponectin and adiponectin receptor 1 (ADIPOR1) levels in GD according to the patients’ prepregnancy body mass index (BMI). We undertook a case–control study involving 45 GD patients and 81 healthy pregnant controls (C) grouped according to BMI (normal<25 and obese ≥25). Polymorphism genotyping was obtained by PCR-RFLP. Adiponectin serum levels were determined by ELISA and ADIPOR1 mRNA levels by RT-PCR. Data were analyzed by Mann–Whitney, Chi-square or Fisher tests. There were no associations between GD and analyzed gene polymorphisms. There was a significant association between adiponectin −11377 gene polymorphism and adiponectin levels in GD (p<0.05). When both groups were analyzed, adiponectin serum levels were higher in C than in GD women (8280ng/mL vs 5490ng/mL, p≤0.0001). Adiponectin serum levels were higher in normal weight versus obese women, both in the C (9030ng/mL vs 7308ng/mL, p=0.01) and in GD (5988ng/mL vs 4638ng/mL, p=0.03) groups, as well as in normal weight C versus normal weight GD (9030ng/mL vs 5988ng/mL, p=0.02) and in obese C compared to obese GD women (7308ng/mL vs 4638ng/mL, p=0.008). Regardless of BMI, ADIPOR1 mRNA levels did not differ between C and GD (p=0.25). Together these data show that there was no association between GD and the gene polymorphisms analyzed. However, there was a significant association between adiponectin −11377 gene polymorphism and adiponectin levels in women with GD. These women have decreased adiponectin levels and obesity was associated with adiponectin serum levels. ADIPOR1 mRNA levels did not differ significantly in women with GD.Financial support: FAPESP (08/56718-9, 08/55888-8, 08/58548-3) and CNPq.

Placental catechol-O-methyltransferase expression in the patients with preeclampsia

H. Seol, G. Cho, S. Hong, M. Oh, H. Kim — 2010-08-01

Placental hypoxia is speculated to contribute to the development of preeclampsia. 2-Methoxyoestradiol (2-ME) which is generated by catechol-O-methyltrasnferase (COMT) in the placenta was found to restore hypoxia-induced disruption of the angiogenic balance in an in vivo study. Circulating 2-ME is known to be decreased in patients with preeclampsia compared to normal pregnant women. However, placental COMT expression is not well understood in preeclampsia. The aim of this study was to investigate placental COMT expressions in patients with preeclampsia and normal pregnant women. Nine normal pregnant women and 11 patients with preeclampsia were included in this study and all participants were in the third trimester of pregnancy. Placental tissues were obtained immediately after delivery. COMT expression in the placenta was determined by Western blot analysis. In Western blotting, densitometric analysis revealed that protein expression of COMT is significantly decreased in the placenta of patients with preeclampsia compared to normal pregnant women (P=0.03). This result suggests that decreased placental COMT expression in preeclampsia might contribute to the pathophysiology of preeclampsia by decreasing production of 2-ME, which in turn may result in further placental hypoxia.

Maternal and fetal consequences of preeclampsia

Z.C. Nwosu — 2010-08-01

About 6–8% of all pregnancies especially in primigravidae is complicated by preeclampsia, a classical triad of elevated blood pressure, proteinuria and oedema. Maternal and neonatal morbidity and mortality are of utmost concern and the etiology of preeclampsia is uncertain. At-risk pregnant women can be suspected if they are primigravidae, multiparous, obese, diabetic, hypertensive, or if they have familial history of hypertension, diabetes, or renal vascular disease. Progression to severe preeclampsia is usually followed by devastating consequences. A patient's chance of survival is maximized if they are delivered of the baby, but survival of the baby is a function of the extent of maturity for the gestational age. Early identification of high-risk pregnant women and subsequent monitoring are pivotal steps in prevention, while subsidization of antenatal services especially in developing countries will significantly reduce the global burden of this condition in years to come.

Sperm and seminal plasma differentially regulate cytokine and chemokine protein expression by human cervical epithelial cells

D.J. Sharkey, S.A. Robertson — 2010-08-01

In the human cervix, introduction of seminal fluid at coitus induces cytokine expression and a local inflammation-like response characterised by induction of mRNAs encoding several pro-inflammatory cytokines including GM-CSF, IL-1α and IL-6, as well as chemokines IL-8, MIP-3α and MCP-1. This response is at least partly elicited by seminal plasma, where the major active signalling agents are TGFβ and 19-hydroxy PGE1. The current study aimed to determine whether sperm also contribute to seminal fluid signalling, and to examine whether seminal plasma and sperm induce different responses. To investigate this we utilised immortalised Ect1 ectocervical epithelial cells, which mimic the response of primary ectocervical epithelial cells. Ect1 cells were incubated with either 10% whole semen, 10% seminal plasma or washed sperm (20×106/ml) and their cytokine and chemokine production was assessed in supernatants 24h later using Luminex microbead technology. Distinct differences in the profile of cytokines and chemokines were elicited by the different seminal fluid components. Washed sperm stimulated greater increases in GM-CSF, IL-6, IL-8 and IL-1α production by Ect1 cells than seminal plasma. In contrast, seminal plasma stimulated marked increases in MCP-1 and MIP-3α, while sperm failed to induce any change. Washed sperm was ∼10 times more potent at stimulating IL-1α expression compared to seminal plasma. These data demonstrate that the sperm and seminal plasma components of semen act to stimulate distinct cytokine and chemokine profiles in Ect1 cells. The molecular basis of sperm signalling to epithelial cells, and the physiological significance of the distinct actions of sperm and seminal plasma, remain unknown. The composition of seminal fluid may therefore influence the character of the inflammatory response in the female partner, regulating immune responses to male seminal antigens as well as sexually transmitted pathogens.This work was supported by Project Grant 565368 from the NHMRC of Australia.

TGFβ superfamily members BMP-15, GDF-9 and Activin A act as seminal fluid signalling agents in human cervical epithelial cells

D.J. Sharkey, S.A. Robertson — 2010-08-01

Transmission of seminal fluid at coitus initiates a local inflammatory-like response, associated with increased cytokine expression and leukocyte recruitment in the human cervix. mRNAs encoding several pro-inflammatory cytokines including GM-CSF, IL-1α and IL-6, as well as the chemokines IL-8, MIP-3α and MCP-1 are induced. The full identity of the signalling agents in seminal fluid remains obscure. To date we have shown that all three mammalian isoforms of TGFβ (TGFβ1, TGFβ2 and TGFβ3) and 19-hydroxy PGE1 are key regulators of this response. The current study aimed to establish whether additional members of the TGFβ superfamily including BMP-15, GDF-9 and Activin A are also active signalling molecules. Immortalised Ect1 ectocervical epithelial cells were utilised to investigate this, as they closely mimic the response of primary ectocervical epithelial cells. Ect1 cells were incubated with rBMP-15, rGDF-9 or rActivin A (0.5–200ng/ml) and production of pro-inflammatory cytokines and chemokines was assessed 24h later using Luminex microbead technology. BMP-15 and Activin A acted to stimulate modest (80%) increases in GM-CSF and MIP-3α production respectively, while GDF-9 and Activin A both stimulated IL-6 production (2.4-fold and 2-fold increases respectively), in a dose-dependent manner. All three factors inhibited IL-8 and did not alter G-CSF, GRO, IL-1α, IL-17, IP-10, MCP-1, MDC, MIP-1α, RANTES, TNFα and VEGF production by Ect1 cells. These data demonstrate that BMP-15, GDF-9 and Activin A are additional seminal fluid signalling agents capable of targeting female reproductive tract epithelial cells and inducing different response profiles compared with TGFβ and 19-hydroxy PGE. The relative bioavailability of these factors in seminal fluid would therefore influence the profile of inflammatory response in the female partner, regulating immune responses to male seminal fluid antigens as well as sexually transmitted pathogens.This work was supported by Project Grant 565368 from the NHMRC of Australia.

In vitro model of semen inflammation; link to sperm membrane status, mitochondria potential and innate immunity

M. Fraczek, M. Piasecka, A. Szumala-Kakol, P. Jedrzejczak, M. Kurpisz — 2010-08-01

We have instrumented an in vitro reconstruction system of semen inflammation composed of selected bacterial strains (Escherichia coli, Staphylococcus haemolyticus, Streptococuss oralis, Bacterioides ureolyticus, Ureaplasma ureakyticum), leukocytes and cytokines (IL-1α, IL-6, IL-8, TNFα, IL-12 and IL-18) alone or in combinations. Peroxidative lipid production (oxidative stress) on spermatozoal membranes was evaluated through determination of malonylodialdehyde (MDA) levels with high-performance lipid chromatography (HPLC). Sperm DNA fragmentation was evaluated using Comet assay and flow cytometry with TUNEL labeling. Sperm plasma membrane stability was measured using LIVE/DEAD Sperm Viability Kit (SYBR14 and PI) and merocyanine 540 (M540) test. Mitochondrial activity was evaluated by JC-1 and NADH-dependent NBT assays (DT-diaphorase). Among the tested pro-inflammatory factors, leukocytes alone or selected bacteria directly affected peroxidation of sperm membranes in a statistically significant fashion (MDA – 0.48±0.24 and 0.45±0.22μM/l). None of the cytokines used alone or in combination exerted a direct pro-oxidative effect (compared to controls), however their incubation together with leukocytes or selected bacteria provided an additive effect to either of these components alone. Some combinations of cytokine (IL-6+IL-8 or IL-12+IL-18) seemed to provoke greater effects than others. Among bacteria studied, E. coli and B. ureolyticus had the strongest influence on MDA levels (p<0.001). Those bacteria strains as well as the above cytokine combinations affected DNA fragmentation in a statistically significant fashion (p<0.05). All the bacteria strains applied affected sperm plasma membrane architecture as measured by the M540 test (p<0.01). Moreover, the presence of E. coli and B. ureolyticus was associated with a significant decrease in both the number of cells with high mitochondrial transmembrane potential (JC-1) and the percentage of cells with normal oxidoreductive mitochondrial potential (DT-diaphorase) (p<0.05). Altogether, induced oxidative stress through the direct influence of leukocytes or bacteria and indirect cytokine action influenced sperm mitochondrial potential and the cell membrane system, making sperm dysfunctional with consequences for male infertility.

Follistatin and activin A in the male reproductive tract of adult mice under normal conditions and during acute inflammation

H. Wu, Y. Chen, W.R. Winnall, D.J. Phillips, M.P. Hedger — 2010-08-01

Inflammation plays an important role in the pathology of the male reproductive tract. Local and systemic inflammation suppresses androgen production and spermatogenesis, and can lead to testicular fibrosis. Elevated inflammatory cytokines in seminal plasma are associated with sperm damage and infertility. Activin A, a member of the transforming growth factor-β superfamily, is a critical component of the inflammatory response. Activin A levels increase in the circulation and various tissues during inflammation, while administration of follistatin, the activin-binding protein, ameliorates the severity of inflammation by blocking activin activity. The relative distribution of these two proteins and their response to inflammation within the male reproductive tract has not been examined in detail. Consequently, male reproductive tract tissues (testis, epididymis, vas deferens, prostate and seminal vesicles) and several control tissues were collected from C57BL6/J male mice injected with saline or lipopolysaccharide (LPS; 100μg) 1h previously. At this time, circulating concentrations of activin A are at their peak. Tissues were processed for mRNA measurements using quantitative RT-PCR and lysates were assayed by activin A ELISA and follistatin RIA. Activin A and follistatin mRNA and protein were highest in the vas deferens. Expression of activin A and follistatin mRNA was also significant in the testis and epididymis, but barely detectable in the accessory organs (seminal vesicles and prostate). However, activin A and follistatin protein levels in the accessory organs were comparable to those found in most other tissues. LPS treatment had no effect on activin A or follistatin mRNA or protein within 1h in any tissues of the male reproductive tract, consistent with the fact that these proteins are not regulated at the genomic level during acute inflammation. These data indicate a potential role for activin/follistatin in regulating inflammation and immunity in the male reproductive tract, particularly in the vas deferens.

Production of sperm immobilizing antibodies is not related with systemic autoimmune diseases in women

2010-08-01

It has been shown that sperm immobilizing antibodies are closely associated with infertility, by inhibiting sperm passage through female genital tract and fertilization. Recently, we experienced the first case of an infertile woman complicated by systemic autoimmune disease, Sjogren syndrome. In the present study, the incidence of sperm immobilizing antibodies in the sera of women with systemic autoimmune diseases was investigated to determine whether production of sperm immobilizing antibodies is associated with systemic autoimmune disease. Sperm immobilizing antibodies in the sera of 110 women with systemic autoimmune diseases, collected at the outpatients’ clinic of the Division of Rheumatology and Clinical Immunology in our university, were examined using Isojima's semi-quantitative sperm immobilization test (SIT). The autoimmune diseases were as follows; 36 patients with SLE, 10 with vasculitis, 50 with arthritis, 3 with PSS, 5 with dermatomyositis, 5 with MCTD, and one with Sjogren syndrome. Among 110 women with systemic autoimmune diseases, there was no one who was positive for SIT. These findings suggest that production of sperm immobilizing antibodies seems not to be associated with systemic autoimmune diseases in women.

Husband's sperm auto-agglutination test (HSAaT) as a specific method to measure antisperm antibody

I. Mansur, V.N. Indriani, I.A. Rachman — 2010-08-01

Sperm is a foreign antigen and every women exposed to sperm will potentially stimulate her immune response against sperm. Sexual contact between husband and wife causes sperm antigen exposure in the wife. One of the immune responses is antisperm antibody (ASA). For a wife, the antisperm antibody she forms will specifically target the husband's sperm and it may not react with sperm of other males. Each female has a different level of tolerance against sperm antigen. A wife may give higher response against her husband's sperm. This may result infertility. In this research, we have been developing a specific method to measure the antisperm antibody of the husband, which we call “Husband's Sperm Auto-agglutination Test” (HSAaT). We only measure the antisperm antibody reactive with the prepared husband's sperm, not for sperm from a non-husband. Evaluation of the titer of antisperm antibody after Paternal Leukocyte Immunization (PLI) treatment always uses husband's sperm. In this method, the sperm must be washed with buffer medium, then we observe the auto-agglutination reaction under the microscope. We found that the titer of antisperm antibody of the 18 samples of blood serum from virgin women (women unexposed to sperm) results in a 1:8 titer at most (control) while the antisperm antibody titer of infertile women, including women in the unexplained infertility category, is at least 1:4096 (before treatment). The titer in infertile women was lower after the PLI procedure. One woman was observed to become pregnant at an ASA titer of 1:1024 (sample 14) and other women also became pregnant with a titre of 1:512 or less after treatment with PLI (and in the absence of other treatments).

Authors Index

2010-06-21