Decidua Formation Research Articles

The Role of Interleukins in Recurrent Implantation Failure: A Comprehensive Review of the Literature.

Recurrent implantation failure (RIF) is a multifactorial condition affecting 10–15% of in vitro fertilization (IVF) couples. Data suggest that functional dysregulation of the endometrial immune system constitutes one of the main pathophysiological mechanisms leading to RIF. The aim of this article is to provide a thorough presentation and evaluation of the role of interleukins (ILs) in the pathogenesis of RIF. A comprehensive literature screening was performed summarizing current evidence. During implantation, several classes of ILs are secreted by epithelial and stromal endometrial cells, including IL-6, IL-10, IL-12, IL-15, IL-18, and the leukemia inhibitory factor. These ILs create a perplexing network that orchestrates both proliferation and maturation of uterine natural killer cells, controls the function of regulatory T and B cells inhibiting the secretion of antifetal antibodies, and supports trophoblast invasion and decidua formation. The existing data indicate associations between ILs and RIF. The extensive analysis performed herein concludes that the dysregulation of the ILs network indeed jeopardizes implantation leading to RIF. This review further proposes a mapping of future research on how to move forward from mere associations to robust molecular data that will allow an accurate profiling of ILs in turn enabling evidence-based consultancy and decision making when addressing RIF patients.

A History of Neonatal Uterine Bleeding and Its Significance

Bleeding in newborns and young girls fascinated writers for more than a millennium. Initially, there was confusion between neonatal bleeding, early menstruation due to precocious puberty, and hemorrhage due to disease. During the 19th century descriptions appeared of what is referred to today as ‘neonatal menstruation’ or ‘neonatal uterine bleeding’. By the turn of the century, Halban linked bleeding to active substances present during pregnancy and hypothesized that, while the maternal uterus reacts with decidua formation, the “weaker” fetal uterus reacts only with menstrual-like changes. Despite this clear description, several alternative theories endured for decades. Bleeding was believed to be due to a ‘catarrhal’ or neoplastic state of the genital tract, pulmonary circulatory disorder, congenital heart malformations, closure of the umbilical cord or affections of the intestine. During the 1950s, progesterone response and resistance were proposed to explain the pathogenesis of bleeding and its low incidence. The fetal endometrium is resistant to the high circulating progesterone. A decidual response is infrequent and results in menstrual shedding upon progesterone withdrawal after birth. Further research linked fetal stress consequent to pregnancy complications and post-maturity to increased incidence and preterm birth to reduced incidence of neonatal uterine bleeding.

Possible Roles of Calreticulin in Uterine Decidualization and Receptivity in Rats and Humans.

Previous in vitro studies have suggested that calreticulin (CALR), which is responsible for the folding and quality control of glycoproteins, may be associated with decidualization. However, its precise role in regulating decidualization has not been explored in vivo. Here, we used pregnant rat models to examine endometrial CALR expression during the peri-implantation period. We also examined whether polypectomy, a procedure that could ameliorate infertility, alters the endometrial expression levels of CALR and several implantation factors in women diagnosed as infertile. In rats, uterine CALR was expressed at a high level at the implantation site, and a marked increase in CALR expression was observed in decidual cells of normal pregnancy. In addition, endometrial CALR expression was enhanced by either administration of estradiol-17β in the delayed implantation rat model or the artificial induction of decidualization in the pseudopregnant rat. In cultured stromal cells, siRNA-mediated silencing of CALR inhibited the decidual stimulus-induced expression of prolactin, decidual/trophoblast prolactin-related protein, and connexin 43. In humans, the endometrial expression levels of the mRNAs encoding CALR and the implantation-related factor insulin-like growth factor binding protein (IGFBP)-7 tended to increase after polypectomy. The strongest positive correlation between expression levels before polypectomy was observed for IGFBP-7 and CALR, and the strength of this correlation increased after the surgery. Thus, endometrial CALR may play a role in the formation of decidua, and the polypectomy of infertile patients may result in the co-operative expression of endometrial factors, including CALR, that could enhance endometrial receptivity.

Synergistic interactions of sperm impairing bacteria: Impact on pregnancy outcome in mouse model

Earlier in our laboratory, the role of various individual sperm impairing microorganisms on sperm parameters and female infertility has been elucidated at higher doses. As, multiple bacterial species tend to exert more pathogenic effect in comparison to single organism hence, present study was carried out to evaluate that if consortia of these sperm impairing organisms can lead to infertility in female mice at sub fertility dose. For this, impact of individual bacterial strains of Escherichia coli, Pseudomonas aeruginosa, Pseudomonas aeruginosa, Klebsiella pneumoniae and consortia of Escherichia coli and Pseudomonas aeruginosa, Escherichia coli and Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae was examined on the motility, viability of mouse spermatozoa and fertility outcome. The results showed that the individual bacterial strains of E. coli, S. marcescens and K. pneumoniae could led to immobilization of spermatozoa by agglutination and P. aeruginosa led to immobilization of spermatozoa without agglutination. Also, all of them led to 100% sperm death in 45 min of incubation. In case of consortia of bacterial strains, the results showed sperm agglutination in all the cases and they were able to induce 100% sperm death at 30 min of incubation time. Further, in vivo studies were carried out to evaluate the impact of individual bacterial strains and consortia of bacterial strains on the fertility outcome in female Balb/c mice. For this, female mice were administered intravaginally with 101 cfu/20µl of individual bacterial strains or consortia of strains for 10 consecutive days or PBS. The results showed that both individual bacterial strains and consortia of bacterial strains were able to efficiently colonize the mouse vagina. Further, control group receiving phosphate buffer saline and groups receiving individual bacterial strains showed all the pregnancy related changes viz. abdominal distension, string of pearls on palpation as well as delivery of pups on completion of gestation period and delivery of pups. The histological examination of reproductive organs viz. uterus and ovary, of the female mice receiving PBS or individual bacterial strains showed the formation of corpus luteum in the ovary and the formation of decidua’s in the uterus, indicative of pregnancy. However, mice receiving consortia of bacterial strains did not show any pregnancy related changes throughout the experiment. Thus, these results indicate that the presence of consortia of sperm impairing microorganisms in vaginal milieu is efficient in provoking infertility even at subfertility doses.

Selected Uterine Immune Events Associated With the Establishment of Pregnancy in the Dog.

In the dog, implantation takes place at approximately 17 days of embryonal life and, while exposed to relatively high circulating progesterone concentrations, embryos presence is required for the formation of decidua. Furthermore, a balance between pro- and anti-inflammatory responses in conceptus-maternal communication is crucial for the onset of pregnancy. Strikingly, the understanding of such immune mechanisms in canine reproduction is still elusive. Here, canine uterine samples from pre-implantation (day 10–12, E+) and corresponding non-pregnant controls (E–), implantation (day 17, Imp) and post-implantation (day 18–25, Post-Imp) stages of pregnancy were used to investigate the expression and localization of several immune-related factors. The most important findings indicate increased availability of CD4, MHCII, NCR1, IDO1, AIF1, CD25, CCR7, and IL6 in response to embryo presence (E+), while FoxP3 and CCL3 were more abundant in E– samples. Implantation was characterized by upregulated levels of FoxP3, IL12a, ENG, and CDH1, whereas CD4, CCR7, IL8, and -10 were less represented. Following implantation, decreased transcript levels of TNFR1, MHCII, NCR1, TLR4, CD206, FoxP3, and IL12a were observed concomitantly with the highest expression of IL6 and IL1β. MHCII, CD86, CD206, CD163, TNFα, IDO1, and AIF1 were immunolocalized in macrophages, CD4 and Nkp46 in lymphocytes, and some signals of IDO1, AIF1, and TNF-receptors could also be identified in endothelial cells and/or uterine glands. Cumulatively, new insights regarding uterine immunity in the peri-implantation period are provided, with apparent moderated pro-inflammatory signals prevailing during pre-implantation, while implantation and early trophoblast invasion appear to be associated with immunomodulatory and rather anti-inflammatory conditions.

Pregnancy success in mice requires appropriate cannabinoid receptor signaling for primary decidua formation.

With implantation, mouse stromal cells begin to transform into epithelial-like cells surrounding the implantation chamber forming an avascular zone called the primary decidual zone (PDZ). In the mouse, the PDZ forms a transient, size-dependent permeable barrier to protect the embryo from maternal circulating harmful agents. The process of decidualization is critical for pregnancy maintenance in mice and humans. Mice deficient in cannabinoid receptors, CB1 and CB2, show compromised PDZ with dysregulated angiogenic factors, resulting in the retention of blood vessels and macrophages. This phenotype is replicated in Cnr1-/- but not in Cnr2-/-mice. In vitro decidualization models suggest that Cnr1 levels substantially increase in mouse and human decidualizing stromal cells, and that neutralization of CB1 signaling suppresses decidualization and misregulates angiogenic factors. Taken together, we propose that implantation quality depends on appropriate angiogenic events driven by the integration of CB2 in endothelial cells and CB1 in decidual cells.

Transforming growth factor beta signaling and decidual integrity in mice†.

Transforming growth factor beta (TGFβ) signaling regulates multifaceted reproductive processes. It has been shown that the type 1 receptor of TGFβ (TGFBR1) is indispensable for female reproductive tract development, implantation, placental development, and fertility. However, the role of TGFβ signaling in decidual development and function remains poorly defined. Our objective is to determine the impact of uterine-specific deletion of Tgfbr1 on decidual integrity, with a focus on the cellular and molecular properties of the decidua during development. Our results show that the developmental dynamics of the decidua is altered in TGFBR1 conditionally depleted uteri from embryonic day (E) 5.5 to E8.5, substantiated by downregulation of genes associated with inflammatory responses and uterine natural killer cell abundance, reduced presence of nondecidualized fibroblasts in the antimesometrial region, and altered decidual cell development. Notably, conditional ablation of TGFBR1 results in the formation of decidua containing more abundant alpha smooth muscle actin (ACTA2)-positive cells at the peripheral region of the antimesometrial side versus controls at E6.5-E8.5. This finding is corroborated by upregulation of a subset of smooth muscle marker genes in Tgfbr1 conditionally deleted decidua at E6.5 and E8.5. Moreover, increased cell proliferation and enhanced decidual ERK1/2 signaling were found in Tgfbr1 conditional knockout mice upon decidual regression. In summary, conditional ablation of TGFBR1 in the uterus profoundly impacts the cellular and molecular properties of the decidua. Our results suggest that TGFBR1 in uterine epithelial and stromal compartments is important for the integrity of the decidua, a transient but crucial structure that supports embryo development.

The effect of bone-marrow transplantation on decidua formation in pseudopregnant rats

A main cause of failed pregnancy is insolvency of the decidual reaction of endometrial cells. It is believed that bone-marrow cells (BMCs) are a partial source of decidual cells in endometrial tissue. In the present work, the effect is studied of transplantation of BMCs on the desidualization processes using the model of pseudopregnancy in rats. BMCs were flushed from rat femurs and tibias. On the fifth day of pseudopregnancy, a suspension of single BMCs was injected into one of the uterine horns. PBS injection into the contralateral horn without cells served as control. Rats were sacrificed on the 11th day of pseudopregnancy. The diameter in the meso-antimesometral direction in the experimental uterine horn increased by 1.5–2 times compared to the control horn. The weight of decidual tissue in the experimental horn was three times greater than the weight of the control horn. The presence of transplanted BMCs in decidual tissue was documented by preliminary double-staining of BMCs with membrane dye PKH26 Red and nuclear dye Hoechst 33342. Histological analysis of decidua sections after transplantation did not reveal any alterations in cell differentiation or tissue structure. We concluded that transplantation of BMCs stimulated decidualization in animals.

CELLULAR MECHANISM UNDERLYING EMBRYO-MATERNAL RELATIONSHIP IN INTRASPECIFIC AND INTERSPECIFIC PREGNANCY

Mouse and vole embryos were transferred into pseudopregnant CD-1 and scid female mice. Cellular changes involved in the formation of decidua in the pregnant mouse uterus up to day 8 of pregnancy were examined by histological, electron microscopic, and histochemical techniques. On day 6 of pregnancy, the vole embryos were laid in interstitium of antimesometrial side of the uterus, as well as in intraspecific pregnancy. Compared with intraspesific pregnant mouse, blood vessels were numerous in the decidua around the vole embryos in interspecific pregnancy. Both distribution and dilation of the blood vessels were increased on day 8. A part of cells in the inner cell mass had not nuclei, suggesting damaged vole embryos on day 8. At the implantation site, the uterine decidua was invaded by extravillous trophoblast (EVT) cells whose function is to destroy the walls of the uterine spiral. Moreover, this decidua was infiltrated by a population of natural killer (NK) cells and macrophage. These cells were particularly numerous in the decidua basalis at the implantation site where they come into close contact with invading EVT cells. These results suggest that interaction between NK, macrophage, and EVT provides the controlling relationship of embryo-maternal in intraspecific and interspecific pregnancy.

The progesterone receptor as a transcription factor regulates phospholipase D1 expression through independent activation of protein kinase A and Ras during 8-Br-cAMP-induced decidualization in human endometrial stromal cells

Decidualization is a biological and morphological process occurring in hES (human endometrial stromal) cells. Previously, we reported that PLD1 (phospholipase D1) plays an important role in cAMP-induced decidualization of hES cells. In the present study, we focused on how PLD1 expression is up-regulated during decidualization. Treatment with PKA (protein kinase A) inhibitors (Rp-cAMP or H89) or a Ras inhibitor (manumycin) partially inhibited PLD1 expression and decidua formation in response to cAMP treatment. Interestingly, dual inhibition of PKA and Ras completely inhibited PLD1 expression and cAMP-induced decidualization. These results suggest that PLD1 expression during decidualization is controlled additively by PKA and Ras. The use of inhibitors showed that extracellular-signal-regulated kinase, a downstream effector of Ras, was required for PLD activation and the morphological changes during decidualization, but not for the increase in PLD1 protein. Next, to investigate the regulator of the PLD1 gene at the transcriptional level, a promoter assay using deletion mutants of the PLD1 promoter was performed; the result indicated that PR (progesterone receptor) was a possible regulator of the PLD1 gene. In addition, chromatin immunoprecipitation assays on the PLD1 promoter identified PR as a transcription factor for PLD1 expression during 8-Br-cAMP-induced decidualization. Taken together, our findings demonstrate that PKA and Ras are novel regulators of PLD1 expression and also identify PR as a transcription factor for PLD1 expression during the decidualization of hES cells.

004. A NEW ERA IN CONTRACEPTIVE DEVELOPMENT: NON-HORMONAL OPTIONS THAT ALSO TARGET SEXUALLY TRANSMITTED INFECTIONS

Despite huge increases in access to contraceptives globally more than 700 000 maternal deaths related to unintended pregnancies occurred between 1995 and 2000 mostly in developing countries. Over 80 million women have unintended or unwanted pregnancies annually. Remarkably, there have been no new methods of contraceptives developed in the last 50 years. The extremely high incidence of sexually transmitted infections (STIs) indicates that it is desirable to develop contraceptives that also target STIs. Two interleukin (IL) 6-type cytokines, leukemia inhibitory factor (LIF) and IL11, are obligatory for implantation in mice and are dysregulated in endometrium of some women with infertility. Both LIF and IL11 are also thought to have roles in Chlamydia-induced inflammation which can lead to a multitude of pathologies. LIF and IL11 antagonists were produced and their contraceptive efficacy tested in mice. Polyethylene glycol (PEG) was conjugated to LIF antagonist (LA) or IL11 antagonist (IL11A) to increase their serum half-life. PEGLA injected during the peri-implantation period blocked LIF action in the endometrium and totally prevented embryo implantation while having no embryo toxic effects.1 Similarly, injection of PEGIL11A blocked decidua formation resulting in pregnancy failure.2 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 may be useful as a ‘dual-role’ contraceptive to also block STIs. PEGLA administered via vaginal gel was shown to prevent implantation having minimal effects on non-uterine LIF targets. This is the first study to show the contraceptive efficacy of a PEGylated compound delivered vaginally. It further indicates that PEGLA may be useful as a dual-role contraceptive. 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. (1) White CA, Zhang JG, Salamonsen LA, Baca M, Fairlie WD, Metcalf D, Nicola NA, Robb L, Dimitriadis E (2007) Proc Natl Acad Sci USA 104: 19 357–62.(2) Menkhorst E, Salamonsen LA, Robb L, Dimitriadis E (2009) Biol of Reprod 80: 920–927.

Vascular Endothelial Growth Factor Receptor 2 (VEGFR-2) Functions to Promote Uterine Decidual Angiogenesis during Early Pregnancy in the Mouse

Implantation of an embryo induces rapid proliferation and differentiation of uterine stromal cells, forming a new structure, the decidua. One salient feature of decidua formation is a marked increase in maternal angiogenesis. Vascular endothelial growth factor (VEGF)-dependent pathways are active in the ovary, uterus, and embryo, and inactivation of VEGF function in any of these structures might prevent normal pregnancy development. We hypothesized that decidual angiogenesis is regulated by VEGF acting through specific VEGF receptors (VEGFRs). To test this hypothesis, we developed a murine pregnancy model in which systemic administration of a receptor-blocking antibody would act specifically on uterine angiogenesis and not on ovarian or embryonic angiogenesis. In our model, ovarian function was replaced with exogenous progesterone, and blocking antibodies were administered prior to embryonic expression of VEGFRs. After administration of a single dose of the anti-VEGFR-2 antibody during the peri-implantation period, no embryos were detected on embryonic d 10.5. The pregnancy was disrupted because of a significant reduction in decidual angiogenesis, which under physiological conditions peaks on embryonic d 5.5 and 6.5. Inactivation of VEGFR-3 reduced angiogenesis in the primary decidual zone, whereas administration of VEGFR-1 blocking antibodies had no effect. Pregnancy was not disrupted after administration of anti-VEGFR-3 or anti-VEGFR-1 antibodies. Thus, the VEGF/VEGFR-2 pathway plays a key role in the maintenance of early pregnancy through its regulation of peri-implantation angiogenesis in the uterine decidua. This newly formed decidual vasculature serves as the first exchange apparatus for the developing embryo until the placenta becomes functionally active.

Uterine DCs are crucial for decidua formation during embryo implantation in mice

Implantation is a key stage during pregnancy, as the fate of the embryo is often decided upon its first contact with the maternal endometrium. Around this time, DCs accumulate in the uterus; however, their role in pregnancy and, more specifically, implantation, remains unknown. We investigated the function of uterine DCs (uDCs) during implantation using a transgenic mouse model that allows conditional ablation of uDCs in a spatially and temporally regulated manner. Depletion of uDCs resulted in a severe impairment of the implantation process, leading to embryo resorption. Depletion of uDCs also caused embryo resorption in syngeneic and T cell-deficient pregnancies, which argues against a failure to establish immunological tolerance during implantation. Moreover, even in the absence of embryos, experimentally induced deciduae failed to adequately form. Implantation failure was associated with impaired decidual proliferation and differentiation. Dynamic contrast-enhanced MRI revealed perturbed angiogenesis characterized by reduced vascular expansion and attenuated maturation. We suggest therefore that uDCs directly fine-tune decidual angiogenesis by providing two critical factors, sFlt1 and TGF-beta1, that promote coordinated blood vessel maturation. Collectively, uDCs appear to govern uterine receptivity, independent of their predicted role in immunological tolerance, by regulating tissue remodeling and angiogenesis. Importantly, our results may aid in understanding the limited implantation success of embryos transferred following in vitro fertilization.

Interleukin 11 and Activin A Synergise to Enhance Medroxyprogesterone But Not cAMP-Induced Decidualization of Human Endometrial Stromal Cells.

Decidualization of endometrial stromal cells is a critical requirement for embryo implantation and the establishment of pregnancy. The mechanisms of human endometrial stromal cell (HESC) decidualization are poorly understood although locally produced factors including interleukin 11 (IL-11) and Activin A enhance decidualization. We hypothesised that IL-11 and Activin A interact to progress decidualization. The aim of this study was to examine IL-11 and activin A interactions during HESC decidualization. Decidualization of HESC was assessed using an ex vivo model in which estrogen (E)+medroxyprogesterone (MPA) or cAMP was administered to HESC cells for up to 14 days (d). Medium was collected and replenished from d 114 for prolactin (PRL, a decidual marker), IL-11 and Activin A assay. HESC were treated with IL-11(100ng/ml) or Activin A (50ng/ml) for 24 hours and cellular RNA was isolated for real-time RT-PCR analysis and culture medium was collected for ELISA assays. HESC treated with cAMP or E+MPA secreted PRL from d 1 and 4 respectively. At d 1, HESC treated with cAMP secreted more IL-11 and Activin A compared to controls (IL-11 vs control, 100±20% vs 1000±85%, p<0.05; Activin A vs control, 100±10% vs 180±20%, p<0.05). At d 1 of cAMP treatment, there was a greater % increase in IL-11 compared to Activin A secretion compared to controls and suggested IL-11 may be produced earlier than Activin A during the decidualization process. By contrast, there was no increase in IL-11 or Activin A secretion during E+MPA-induced decidualization. Co-treatment of HESC with E+MPA ± IL-11 or Activin A increased PRL secretion compared to E+MPA alone (410±40% vs 100±5%, p<0.05; 170±10% vs 100±5%; respectively) at d 7-8 of culture. This was further enhanced when HESC were cultured with E+MPA+IL-11+Activin A compared to either cytokine alone and E+MPA. This suggested IL-11 and Activin synergised to further progress decidualization compared to each cytokine and E+MPA. By contrast, coculture of HESC with cAMP and either IL-11 or Activin A or IL-11+Activin A, did not significantly increase PRL secretion from HESC compared to controls at any time point. HESC treated with Activin A for 24 hours did not affect IL-11 mRNA expression but significantly stimulated IL-11 secretion compared to controls (100±25% vs 345±136%, p<0.05). HESC treated with IL-11 did not change Activin A mRNA expression or secretion compared to controls. These data suggest Activin A regulated IL-11 secretion from HESC post-transcriptionally. This study demonstrated that IL-11 and Activin A synergised to enhance MPA induced decidualization and that Activin A regulated IL-11 secretion but not mRNA expression by HESC. Interestingly, while cAMP was a potent inducer of IL-11 and Activin A secretion coinciding with decidualization, both cytokines did not synergise with cAMP to regulate decidualization. This knowledge is important in understanding the mechanisms by which two key locally produced cytokines, IL-11 and Activin A, promote decidualization of HESC and thus the formation of decidua, an essential component of a functional placenta.

Role of sympathetic nerves on early embryonic development and immune modulation of uterus in pregnant mice

To determine the role of sympathetic nerves in the early embryonic development and the immune modulation of maternal uterus during pregnancy, a model of chemical sympathectomy in mice was established by intraperitoneal injection of 6-hydroxydopamine (6-OHDA). The embryonic development and the distribution of maternal uterine immunocytes were investigated during early pregnancy (E1–E9) with methods of histology, immunohistochemistry and ELISA. Our data showed that in the 6-OHDA-treated group, the number of implanted embryos was only 64.4% of that in the control group at E7, and the development of uterine glands and vessels was poor in pregnant mice. In addition, in uterine tissues of 6-OHDA-treated mice, the number of CD8 + T cells increased ten-fold and the concentration of IL-2 increased 3.6-fold at E5. However, no obvious changes to the number of CD4 + T cells and IL-4 were observed. Thus, the CD4 +/CD8 + T cells ratio significantly decreased, while the IL-2/IL-4 ratio significantly increased. These findings indicated that the activation of sympathetic nerves might be favorable to fetal survival and development during early pregnancy through influencing on immune function and decidua formation of uterus.

Decidualization of Ovarian Endometriosis During Pregnancy Mimicking Malignancy

The purpose of this series is to present deciduosis (the formation of extrauterine decidua) as one of the differential diagnoses of a malignant tumor during pregnancy. Two cases are described in which pregnant patients had a pelvic tumor. The lesions, which were diagnosed in the early second trimester, consisted of complex masses with an extensive blood supply and had a sonographic appearance of a malignant tumor. The high suspicion for malignancy necessitated surgical intervention. During surgery, the lesions were observed to be of an ovarian origin with papillary excrescences covering their exterior. The lesions were excised and sent for histologic examination. The results showed a markedly decidualized endometriotic cyst in both cases. This phenomenon is a diagnostic challenge and should be considered in the differential diagnosis of a malignant mass during pregnancy.

266. Supressor of cytokine signalling 3 regulates IL-11 induced human endometrial stromal cell decidualization

Decidualization of endometrial stromal cells is critical for embryo implantation and establishment of pregnancy. Locally produced cytokines such as interleukin (IL)-11 enhance decidualization of human endometrial stromal cells (HESC). IL-11 signaling is negatively regulated by suppressor of cytokine signaling (SOCS) proteins. IL-11 stimulates SOCS3 in human pituitary cells. The aim of this study was to examine the role of SOCS3 on IL-11 induced HESC decidualization. Decidualization of HESC was assessed using an in vitro model in which estrogen (E)+progesterone (P) or cAMP was administered for 8 days to cells. Medium was collected for prolactin (PRL) assay (a decidual marker). Cellular protein was extracted for Western analysis and cellular RNA for real-time RT-PCR analysis. SOCS3 was overexpressed in HESC cells and the effect on decidualization assessed. HESC treated with E+P or cAMP secreted PRL from day 6. Treatment of HESC with E+P or cAMP increased the abundance of SOCS3 protein, coinciding with an increase in PRL secretion. cAMP maximally stimulated SOCS3 protein and mRNA during decidualization. Antiprogestin (onapristone) added to E+P or cAMP treated cells at day 6 reduced PRL secretion but had no influence on SOCS3 abundance suggesting that SOCS3 protein was not regulated via the P-receptor pathway. Addition of IL-11 to HESC increased SOCS3 abundance from 1 h. SOCS3 abundance returned to control levels following treatment of cells with IL-11 and IL-11 neutralising antibody. SOCS3 overexpression in HESC treated with cAMP reduced PRL secretion compared to mock- or non-transfected HESC. Furthermore, IL-11 mediated decidualization was diminished by SOCS3 overexpression. We have demonstrated for the first time that SOCS3 regulates IL-11 induced decidualization and that SOCS3 overexpression in HESC disrupts decidualization. This knowledge is important in understanding the mechanisms by which IL-11 promotes decidualization of HESC and thus the formation of decidua, an essential component of a functional placenta.

PGF2alpha induced differential expression of genes involved in turnover of extracellular matrix in rat decidual cells

In the rat, the decidual tissue is an important component for maternal recognition of pregnancy. Decidualization can be induced by either the implantation of the blastocyst or by artificial stimuli. The process of decidua formation or decidualization, is characterized by growth and differentiation of endometrial stromal cells. Prostaglandin F2alpha (PGF2α) has been shown to be involved in inhibition of implantation, alteration of embryo development, induction of luteal regression, and the mediation of pregnancy loss induced by microorganism infections. In order to establish a direct role for PGF2α in decidual function, we have evaluated its effects on the expression of an extensive array of genes using primary decidual cell culture. Upon treatment with PGF2α sixty genes were significantly down-regulated whereas only six genes were up-regulated (from a total of 1176 genes studied). Interestingly, the majority of the genes inhibited by PGF2α are either directly or indirectly involved in the turnover of the extracellular matrix (ECM). Genes such as gelatinase A (MMP2), cathepsin L, tissue inhibitor metalloproteinases 2 (TIMP2) and 3 (TIMP3), plasminogen activator inhibitor1 (PAI1), tissue type plasminogen activator (tPA), urokinase plasminogen activator (tPA), endothelin 1, calponin, carboxypeptidase D and calponin acidic were down regulated. The opposite effect was observed for prostromelysin 53 kDa (proMMP3), plasma proteinase I alpha and alpha 1 antiproteinase, all of which were significantly up-regulated by PGF2α. The results strongly suggest that the abortificient role of elevated levels of PGF2α after implantation is due, in large part, to inhibition of genes involved in the normal turnover of the extracellular matrix necessary for decidual formation.

Induction of Cytotoxic T-lymphocyte Antigen-2β, A Cysteine Protease Inhibitor in Decidua

During early pregnancy, the steroid hormone progesterone induces differentiation of uterine stroma to decidual cells, which regulate embryo-uterine interactions. The progesterone-induced signaling molecules that participate in the formation and function of decidua remain poorly understood. We recently utilized high-density oligonucleotide microarrays to identify several genes whose expression is markedly altered in pregnant uterus in response to RU486, a well characterized antagonist of the progesterone receptor (PR). Our study revealed that the gene encoding cytotoxic T-lymphocyte antigen-2beta (CTLA-2beta), a cysteine protease inhibitor, is expressed during PR-induced decidualization. The spatio-temporal expression of CTLA-2beta mRNA precisely overlapped with the decidual phase of pregnancy. Interestingly, administration of progesterone to estrogen-primed ovariectomized mice failed to induce CTLA-2beta expression. A concomitant artificial decidual stimulation was necessary to trigger this expression. Uteri of PR knockout mice failed to express this mRNA, even after a combined administration of steroid hormones and artificial stimulation. The uterine expression of CTLA-2beta was, therefore, dependent on PR as well as other unknown factor(s) associated with decidual response. To identify the molecular target(s) of CTLA-2beta,we analyzed its interaction with proteins present in soluble extracts prepared from day 7 pregnant uteri containing implanted embryos. A protein affinity strategy employing recombinant CTLA-2beta helped us to determine that cathepsin L, a cysteine protease, is one of its targets in the pregnant uterus. Consistent with this finding, expression of cathepsin L was detected in the giant trophoblast cells of the ectoplacental cone on day 7 of pregnancy. Collectively, our results support the hypothesis that expression of CTLA-2beta in the decidua may regulate implantation of the embryo by neutralizing the activities of one or more proteases generated by the proliferating trophoblast.

Cytomorphometric analysis and surface ultrastructure of developing decidua

A scanning electron microscopic (SEM) and morphometric analysis of the topographical changes occurring in the uterine luminal epithelial layer in association with decidual tissue (DT) formation in guinea pigs was undertaken in order to elucidate the surface ultrastructural characteristics which occur during the process of endometrial differentiation. Experimentally induced decidua formation was promoted by mechanical stimulation of the antimesometrial luminal surface during the period of maximal uterine sensitivity to stromal differentiation. DT-associated remodeling of the uterine epithelial layer was subsequently examined by light and SE microscopic analysis for apical epithelial and luminal contour alterations associated with decidua growth. Cytological changes in the luminal surface associated with DT induction included sparse microvillus growth from the apical epithelial surface, accompanied by the appearance of prominent apical membrane surface protrusions and endometrial gland openings as compared with non-DT-stimulated control samples. Decidua surface growth was characterized by a short, sparse epithelial microvillus pattern present over a highly contoured luminal uterine surface on which contoured gland openings were both numerous and prominent. These surface modifications contrasted with the flat, non-decidualized luminal surface contour which was covered by distinct, microvilli-laden, apical cell membranes, and defined by prominent intercellular membrane borders. The uterine surface at the time of maximal DT formation (i.e. growth) closely resembled that of a uterine luminal surface undergoing apoptosis and subsequent cellular reabsorption, characterized by disrupted cell surface membranes, sparse microvillus surfaces and prominent epithelial contours reflecting stromal tissue and vasculature involution. These data indicate that the alterations in the uterine luminal surface associated with DT formation are reminiscent of the endometrial changes associated with the initiation of early placentation, and may be used as a model for the analysis of the role of epithelial cell surface modifications associated with the induction and support of interstitial blastocyst implantation and early decidua formation.