Differentiation Of Endometrial Stromal Cells Research Articles

WNT4 is a key regulator of normal postnatal uterine development and progesterone signaling during embryo implantation and decidualization in the mouse

WNT4, a member of the Wnt family of ligands, is critical for the development of the female reproductive tract. Analysis of Wnt4 expression in the adult uterus during pregnancy indicates that it may play a role in the regulation of endometrial stromal cell proliferation, survival, and differentiation, which is required to support the developing embryo. To investigate the role of Wnt4 in adult uterine physiology, conditional ablation of Wnt4 using the PR(cre) mouse model was accomplished. Ablation of Wnt4 rendered female mice subfertile due to a defect in embryo implantation and subsequent defects in endometrial stromal cell survival, differentiation, and responsiveness to progesterone signaling. In addition to altered stromal cell function, the uteri of PR(cre/+)Wnt4(f/f) (Wnt4(d/d)) mice displayed altered epithelial differentiation characterized by a reduction in the number of uterine glands and the emergence of a p63-positive basal cell layer beneath the columnar luminal epithelial cells. The altered epithelial cell phenotype was further escalated by chronic estrogen treatment, which caused squamous cell metaplasia of the uterine epithelium in the Wnt4(d/d) mice. Thus, WNT4 is a critical regulator not only of proper postnatal uterine development, but also embryo implantation and decidualization.

NADPH oxidase-derived reactive oxygen species mediate decidualization of human endometrial stromal cells in response to cyclic AMP signaling.

Differentiation of human endometrial stromal cells into specialized decidual cells is critical for embryo implantation and survival of the conceptus. Initiation of this differentiation process is strictly dependent on elevated cAMP levels, but the signal intermediates that control the expression of decidual marker genes, such as prolactin (PRL) and IGFBP1, remain poorly characterized. Here we show that cAMP-dependent decidualization can be attenuated or enhanced upon treatment of primary cultures with a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor (diphenylen iodonium) or activator (apocynin), respectively. Time-course analysis demonstrated that cAMP enhances endogenous reactive oxygen species production, apparent after 12 h of stimulation, which coincides with a dramatic increase in decidual PRL and IGFBP1 expression. Knockdown of the Rho GTPase RAC1, which disables activation of the NADPH oxidase homologs NADPH oxidase (NOX)-1, NOX-2, and NOX-3, had no effect on PRL or IGFBP1 expression. In contrast, silencing of NOX-4, or its cofactor p22(PHOX), inhibited the expression of both decidual markers. Finally, we show that the NOX-4/p22(PHOX) complex regulates the DNA-binding activity of CCAAT/enhancer binding protein-β, a key regulator of human endometrial stromal cell differentiation. Thus, NOX-4 activation and reactive oxygen species signaling play an integral role in initiating the endometrial decidual response in preparation of pregnancy.

Characterization of endometrial mesenchymal stem-like cells obtained by endometrial biopsy during routine diagnostics

Endometrial cell clones derived from in vitro cultured purified stromal cells obtained by endometrial biopsy display characteristic stem cell features, including clonality; long-term culturing properties; multilineage differentiation potential; expression of CD146, CD105, CD90, CD73, MSI1, NOTCH1, and SOX2; and absence of CD34 and CD14 expression. We conclude that adult stem cells are present in endometrial biopsies performed in a routine clinical setting, offering new large-scale approaches for future research, diagnostics, and therapies involving adult stem cells.

The molecular basis of recurrent pregnancy loss: impaired natural embryo selection

Recurrent pregnancy loss (RPL) is a common and distressing disorder. Chromosomal errors in the embryo are the single most common cause, whereas uterine factors are invariably invoked to explain non-chromosomal miscarriages. These uterine factors are, however, poorly defined. The ability of a conceptus to implant in the endometrium is normally restricted to a few days in the menstrual cycle. A limited 'window of implantation' ensures coordinated embryonic and endometrial development, thereby minimizing the risk of late implantation of compromised embryos. In this paper, we review emerging evidence, indicating that RPL is associated with impaired differentiation of endometrial stromal cells into specialized decidual cells. From a functional perspective, this differentiation process, termed decidualization, is not only critical for placental development but also signals the end of the implantation window and bestows on the endometrium the ability to recognize, respond to and eliminate implanting compromised embryos. Thus, we propose that spontaneous decidualization of the human endometrium, which inevitably causes menstrual shedding in the absence of a viable conceptus, serves as functional 'window for natural embryo selection'. Conversely, impaired decidualization predisposes to late implantation, negates embryo quality control and causes early placental failure, regardless of the embryonic karyotype. This pathological pathway also explains the common observation that many RPL patients seem exceptionally fertile, often conceiving within one or two cycles. Thus, as the clinical correlate of inappropriate uterine receptivity, 'superfertility' should be considered as a genuine reproductive disorder that requires targeted intervention.

The effect of DNA methylation inhibitor 5-Aza-2′-deoxycytidine on human endometrial stromal cells

Decidualization, the differentiation of endometrial stromal cells is a crucial step for successful implantation of an embryo, development of the placenta and completion of pregnancy to term. Epigenetic mechanisms are thought to be strongly involved in the regulation of processes controlling implantation, placentation, organ formation and foetal growth. Recent studies suggest that decreased DNA methylation facilitates a receptive endometrium. Hence, the aim of this project was to compare the transcriptional profile changes induced by the inhibitor of DNA methylation, 5-Aza-2'-deoxycytidine (AZA) to the transcriptional changes that happen during decidualization. When DNA methylation was inhibited in a human endometrial stromal cell (HESC) line with AZA, it resulted in the fibroblast-like stromal cells being transformed into decidual-like morphology after 9 days. Expression of both prolactin and insulin-like growth factor binding protein-1, the two established decidualization marker genes, were minimally up-regulated by AZA after 10 days of treatment. In a microarray of a three-way experiment between AZA-treated and oestradiol/progestin/cAMP-treated [medroxy-progesterone acetate (MPA)-mix] HESC and the untreated controls, we detected more than 1000 common genes that had a significant difference of expression compared with the controls. AZA-treated cells in the microarray significantly expressed 76 genes in common with the MPA-mix treated cells, and AZA treatment also differentially regulated 148 genes independently to that of MPA-mix treatment. The MPA-mix regulated at least 36 genes in the cell adhesion, extracellular matrix remodelling and RhoGTPase cytoskeletal reorganization pathway; AZA regulated 19 of these genes in common and 15 other RhoGTPase pathway genes. AZA induced some decidualization-like responses of endometrial stromal cells independently of progestins or cAMP, possibly via the cytoskeletal reorganization pathway of the RhoGTPase family.

A Role for the orphan nuclear receptor estrogen-related receptor alpha in endometrial stromal cell decidualization and expression of genes implicated in energy metabolism.

Context: Differentiation (decidualization) of endometrial stromal cells (ESC) is an essential prerequisite for successful implantation and establishment of pregnancy.Objective: The aim was to determine whether the orphan nuclear receptor estrogen-related receptor α (ERRα, NR3B1), and its target genes, medium chain specific acyl-CoA dehydrogenase (MCAD, ACADM), pyruvate dehydrogenase kinase 4 (PDK4), and phosphoenolpyruvate carboxykinase 2 (PEPCK, PCK2), play a role in the decidualization process.Setting: We conducted the study at a University Research Institute.Patients and Methods: Endometrial tissues were collected from women with regular menstrual cycles; tissues were used for recovery of primary ESC or RNA extraction or were fixed for immunohistochemistry. Primary ESC were decidualized in vitro; some cells were treated with XCT790 (ERRα inverse agonist).Results: Decidualization of ESC in vitro was associated with a significant increase in expression of transcripts encoding ERRα and its coactivator peroxisome proliferator-activated receptor γ coactivator-1 α. Expression of ERRα target genes was altered with increased expression of MCAD and PDK4 and reduced expression of PEPCK. Incubation of decidualized ESC with XCT790 reduced expression of ERRα and markers of decidualization such as IGFBP-1.Conclusion: Increased expression of ERRα may play a role in altering the bioenergetics of decidualized ESC in preparation for implantation of the embryo and successful establishment of early pregnancy.

Natural Selection of Human Embryos: Impaired Decidualization of Endometrium Disables Embryo-Maternal Interactions and Causes Recurrent Pregnancy Loss

BackgroundRecurrent pregnancy loss (RPL), defined as 3 or more consecutive miscarriages, is widely attributed either to repeated chromosomal instability in the conceptus or to uterine factors that are poorly defined. We tested the hypothesis that abnormal cyclic differentiation of endometrial stromal cells (ESCs) into specialized decidual cells predisposes to RPL, based on the observation that this process may not only be indispensable for placenta formation in pregnancy but also for embryo recognition and selection at time of implantation.Methodology/Principal FindingsAnalysis of mid-secretory endometrial biopsies demonstrated that RPL is associated with decreased expression of the decidual marker prolactin (PRL) but increased levels of prokineticin-1 (PROK1), a cytokine that promotes implantation. These in vivo findings were entirely recapitulated when ESCs were purified from patients with and without a history of RPL and decidualized in culture. In addition to attenuated PRL production and prolonged and enhanced PROK1 expression, RPL was further associated with a complete dysregulation of both markers upon treatment of ESC cultures with human chorionic gonadotropin, a glycoprotein hormone abundantly expressed by the implanting embryo. We postulated that impaired embryo recognition and selection would clinically be associated with increased fecundity, defined by short time-to-pregnancy (TTP) intervals. Woman-based analysis of the mean and mode TTP in a cohort of 560 RPL patients showed that 40% can be considered “superfertile”, defined by a mean TTP of 3 months or less.ConclusionsImpaired cyclic decidualization of the endometrium facilitates implantation yet predisposes to subsequent pregnancy failure by disabling natural embryo selection and by disrupting the maternal responses to embryonic signals. These findings suggest a novel pathological pathway that unifies maternal and embryonic causes of RPL.

Natural Selection of Human Embryos: Decidualizing Endometrial Stromal Cells Serve as Sensors of Embryo Quality upon Implantation

BackgroundPregnancy is widely viewed as dependent upon an intimate dialogue, mediated by locally secreted factors between a developmentally competent embryo and a receptive endometrium. Reproductive success in humans is however limited, largely because of the high prevalence of chromosomally abnormal preimplantation embryos. Moreover, the transient period of endometrial receptivity in humans uniquely coincides with differentiation of endometrial stromal cells (ESCs) into highly specialized decidual cells, which in the absence of pregnancy invariably triggers menstruation. The role of cyclic decidualization of the endometrium in the implantation process and the nature of the decidual cytokines and growth factors that mediate the crosstalk with the embryo are unknown.Methodology/Principal FindingsWe employed a human co-culture model, consisting of decidualizing ESCs and single hatched blastocysts, to identify the soluble factors involved in implantation. Over the 3-day co-culture period, approximately 75% of embryos arrested whereas the remainder showed normal development. The levels of 14 implantation factors secreted by the stromal cells were determined by multiplex immunoassay. Surprisingly, the presence of a developing embryo had no significant effect on decidual secretions, apart from a modest reduction in IL-5 levels. In contrast, arresting embryos triggered a strong response, characterized by selective inhibition of IL-1β, -6, -10, -17, -18, eotaxin, and HB-EGF secretion. Co-cultures were repeated with undifferentiated ESCs but none of the secreted cytokines were affected by the presence of a developing or arresting embryo.ConclusionsHuman ESCs become biosensors of embryo quality upon differentiation into decidual cells. In view of the high incidence of gross chromosomal errors in human preimplantation embryos, cyclic decidualization followed by menstrual shedding may represent a mechanism of natural embryo selection that limits maternal investment in developmentally impaired pregnancies.

301. LEUKEMIA INHIBITORY FACTOR IS A CRITICAL REGULATOR OF DECIDUALIZATION OF ENDOMETRIAL STROMAL CELLS IN HUMANS AND MICE

Decidualization is the differentiation of endometrial stromal cells into decidual cells. It is a critical process in embryo implantation, placentation and the establishment of pregnancy. Inadequate decidualization can lead to infertility, abnormal placentation and recurrent miscarriage. Endometrial leukemia inhibitory factor (LIF) is indispensible in blastocyst implantation in mice and dysregulated in infertile women. LIF is produced by 1st trimester decidual cells but its role in decidualization is not known. This study aimed to examine the role of LIF in human and mouse decidualization. Primary human endometrial stomal cells (HESC) were isolated and decidualized (D) by treatment with estradiol (E) +medroxyprogesterone acetate (MPA) for 14 days. HESC were also treated with E+MPA+/–LIF (0.5, 5, 50, 100 and 200 ng/mL) for 14 days. Prolactin secretion was used to assess the extent of decidualization (n = 6). D and non-D HESC were also treated with LIF (0.5, 5, 50, 100 and 200 ng/mL +/– LIF inhibitor) for 15min and the phosphorylation (p) of signal transducer and activator of transcription (STAT)3/STAT3 abundance was detected by Western blot (n = 4). RNA was isolated for analysis of LIF and LIF receptor (R) mRNA expression during decidualization (n = 4). HESC treated with E+MPA+LIF (50, 100 and 200 ng/mL) secreted more prolactin compared to cells treated with E+MPA alone (P < 0.05). LIF increased pSTAT3/STAT3 abundance in D and non-D cells while LIF+LIF inhibitor abolished pSTAT3/STAT3. LIF mRNA was downregulated while LIF-R mRNA increased during decidualization. In vivo, mated mice (n = 5) were injected intraperitoneally with a unique long acting LIF inhibitor post-implantation at day 4.5 of pregnancy and resulted in reduced decidualization compared to control. This is the first study to demonstrate that LIF promoted decidualization of HESC possibly via pSTAT3. It further suggested that LIF regulated decidualization in mice demonstrating a newly identified critical role for LIF in the establishment of pregnancy.

Effect of relaxin on the decidual cell reaction in the Mongolian gerbil (Meriones unguiculatus).

Differentiation of endometrial stromal cells into decidual cells occurs during embryo implantation and pregnancy. Recently, it has been reported that relaxin affects the decidualization of cultured human endometrial cells in vitro; however, there has been no study on the decidualization of the Mongolian gerbil (Meriones unguiculatus). The authors demonstrated artificially induced decidualization, and the effect of relaxin on decidualization in gerbils. Ten-to-twelve-week-old female Mongolian gerbils were ovariectomized, treated with estradiol, progesterone, and relaxin, and the uterine horn was stimulated. On day 10, uterine horns were measured for weight, protein concentration, and the incorporation of 14C-methionine; tissue sections were examined. Interleukin-11 (IL-11) primers were used for RT-PCR to confirm decidualization. Decidualization can be induced artificially in gerbils. In general, the histological observations of gerbil decidual cells were very similar to those of rats. The uterine horn weight, protein content, and protein synthesis from 14C-methionine significantly increased in the relaxin-treated gerbils (P< 0.05). Mast cells in the relaxin-treated uterus had proliferated more than those of the non-relaxin-treated group, which was confirmed by IL-11 expression. We conclude that decidualization can be induced artificially, and relaxin increased weight of uterine horn, protein concentration, protein synthesis and IL-11 expression in gerbils.

Hsa-miR-222 Is Involved in Differentiation of Endometrial Stromal Cells in Vitro

Decidualization is a critical step during embryo implantation and characterized by the differentiation of endometrial stromal cells (ESCs) into decidual cells. Because miRNAs are important determinants of cellular fate specification, in this study, the miRNA expression in ESCs during in vitro decidualization was profiled by using a microarray. Significance analysis of microarrays revealed that 49 miRNA genes were differently (>2-fold) expressed between the noninduced ESCs and induced ESCs with a false discovery rate of 0. The expression variance of hsa-miR-222, 221, 143, 101, 30d, 30c, 181b, 27b, 29b, 507, and 23a was validated by using quantitative PCR (P < 0.05). Based on microRNA (miRNA) and mRNA expression variance and predicted target genes of miRNAs, a bioinformatic model of miRNAs controlling ESCs differentiation was formulated. Finally, we proved that down-regulation of has-miR-222 could decrease the number of cells in S phase during ESCs differentiation (P < 0.05). Antisense oligonucleotides of has-miR-222 could increase reporter gene expression by targeting the 3' untranslated regions of CDKN1C/p57kip2 mRNAs as well as increase CDKN1C/p57kip2 protein levels (P < 0.05). In conclusion, our results suggest that a subset of miRNAs play a key role in gene reprogramming during ESCs decidualization and that hsa-miR-222 participates in ESC differentiation by regulating ESCs terminally withdrawing from the cell cycle.

Wnt4 Acts Downstream of BMP2 and Functions via Canonical Beta-Catenin-Dependent Signaling Pathway to Regulate Human Endometrial Stromal Cell Differentiation.

Differentiation of endometrial stromal cells into decidual cells is a prerequisite for successful embryo implantation. Our previous studies have shown that bone morphogenetic protein 2 (BMP2), a morphogen belonging to the TGFβ superfamily, is markedly induced in primary cultures of human endometrial stromal cells as they undergo differentiation in response to a hormonal cocktail containing progesterone, estrogen, and a cyclic AMP analog. Using loss-of-function and gain-of-function approaches we further showed that BMP2 plays a key role during human endometrial stromal cell differentiation. The present study was undertaken to identify the downstream targets of BMP2 in human endometrium during decidualization. Gene expression profiling experiments identified Wnt4 as a downstream target of BMP2 regulation during differentiation of human endometrial stromal cells. To determine the function of Wnt4 during this differentiation process, we employed siRNA targeted to its mRNA. Our study showed that siRNA-mediated down-regulation of Wnt4 expression in the stromal cells efficiently blocked their differentiation, as indicated by the loss of expression of well-known biomarkers of decidualization such as prolactin and insulin-like growth factor binding protein 1. Conversely, adenovirus-mediated overexpression of Wnt4 in human endometrial stromal cells markedly advanced the differentiation program. Interestingly, the stimulatory effect of Wnt4 was accompanied by the accumulation of an active form of β-catenin in the nuclei of decidualizing stromal cells, indicating the involvement of the canonical Wnt signaling pathway in the differentiation process. Furthermore, overexpression of Dickkopf-1 (Dkk1), a known inhibitor of the β-catenin-dependent canonical Wnt signaling pathway, led to a marked suppression of BMP2- and Wnt4-mediated decidualization. We also performed gene expression profiling to identify downstream targets of Wnt4 in human endometrial stromal cells. Our studies identified Foxo1, a member of the Forkhead/Winged helix family of transcription factors, as a downstream target of Wnt4 in decidualized human endometrial stromal cells. Collectively, these studies uncovered a unique pathway involving BMP2, Wnt4, and Foxo1 that operates in the human endometrial stromal cells and is critical for their transformation into decidual cells. (Supported by NIH grants to RNT, FJD, MKB, and ICB). (poster)

The Androgen and Progesterone Receptors Regulate Distinct Gene Networks and Cellular Functions in Decidualizing Endometrium

Progesterone is indispensable for differentiation of human endometrial stromal cells (HESCs) into decidual cells, a process that critically controls embryo implantation. We now show an important role for androgen receptor (AR) signaling in this differentiation process. Decreased posttranslational modification of the AR by small ubiquitin-like modifier (SUMO)-1 in decidualizing cells accounted for increased responsiveness to androgen. By combining small interfering RNA technology with genome-wide expression profiling, we found that AR and progesterone receptor (PR) regulate the expression of distinct decidual gene networks. Ingenuity pathway analysis implicated a preponderance of AR-induced genes in cytoskeletal organization and cell motility, whereas analysis of AR-repressed genes suggested involvement in cell cycle regulation. Functionally, AR depletion prevented differentiation-dependent stress fiber formation and promoted motility and proliferation of decidualizing cells. In comparison, PR depletion perturbed the expression of many more genes, underscoring the importance of this nuclear receptor in diverse cellular functions. However, several PR-dependent genes encode for signaling intermediates, and knockdown of PR, but not AR, compromised activation of WNT/beta-catenin, TGFbeta/SMAD, and signal transducer and activator of transcription (STAT) pathways in decidualizing cells. Thus, the nonredundant function of the AR in decidualizing HESCs, centered on cytoskeletal organization and cell cycle regulation, implies an important role for androgens in modulating fetal-maternal interactions. Moreover, we show that PR regulates HESC differentiation, at least in part, by reprogramming growth factor and cytokine signal transduction.

A distinct cohort of the TGFβ superfamily members expressed in human endometrium regulate decidualization

BACKGROUNDSuccessful blastocyst implantation requires the differentiation of human endometrial stromal cells (HESC), a process known as decidualization. Activin A, a transforming growth factor β (TGFβ) superfamily member, enhances HESC decidualization and localizes to decidual cells in human endometrium. Other TGFβ superfamily members, including BMP2, BMP4, BMP7, GDF5, GDF8, GDF11, TGFβs and Nodal, may also play a role during decidualization. This study aimed to identify these TGFβ family members in human endometrium, and to determine whether they are involved in human decidualization.METHODSProtein localization of TGFβ family members was examined in secretory phase human endometrium and first trimester decidua by immunohistochemistry. mRNA expression was examined in HESC. Activin inhibitors (Activin-M108A/SB431542) with differing specificities for the other TGFβ members under consideration were applied during HESC decidualization in vitro. The secretion levels of potential TGFβ superfamily members were measured during decidualization, and recombinant proteins added to examine their effect.RESULTSThis study has identified BMP2, BMP4, BMP7, GDF5, GDF8 and GDF11 but not Nodal in secretory phase human endometrium, but only BMP2, GDF5 and TGFβ1 protein were detected in decidual cells. All ligands except Nodal were expressed by cultured HESC. Both inhibitors significantly reduced decidualization validating the role of activin, but potentially also other TGFβ members, during decidualization. BMP2 and TGFβ1 secretion increased during HESC decidualisation and exogenous administration of these proteins significantly enhanced decidualization in vitro.CONCLUSIONSLike activin, BMP2 and TGFβ1 are likely to be involved in HESC decidualization. This is the first study to identify and localize BMP4, BMP7, GDF5, GDF8 and GDF11 in secretory phase human endometrium. Understanding the factors critical for the implantation process is needed for improving fertility and pregnancy outcomes.

Cyclooxygenase-2 Derived PGE2 and PGI2 Play an Important Role Via EP2 and PPARδ Receptors in Early Steps of Oil Induced Decidualization in Mice

Differentiation of endometrial stromal cells into decidual cells (decidualization) is prerequisite for blastocyst implantation. Different prostanoids are shown to be involved in the cascade of events found in implantation and decidualization. Previous reports described that cyclooxygenase-2 (COX2) derived prostacyclin (PGI2) plays an important role via peroxisome proliferator activated receptor (PPARδ) nuclear receptor in implantation and decidualization. Herein, we investigated the role of COX2 derived PGE2 and PGI2 and examined the protein expression and regulation of COX1, COX2, membrane-bound prostaglandin E synthase (mPGES-1), prostaglandin I synthase (PGIS), PGE2 receptor (EP2) and PPARδ in hormone primed oil infused uterine horn as well as in non-infused uterine horn (control horn). Our results show that selective COX2 inhibitor (Nimesulide) inhibits decidualization while COX1 inhibitor (SC560) does not affect decidualization. COX2, mPGES-1, PGIS, EP2 and PPARδ immunostaining are strongly observed at 24 h and 48 h in oil-induced horn and than significantly reduced at 72 h and 120 h and absent in non-infused horn. However COX1 immunostaining is observed in infused as well as in non-infused horn. Our immunohistochemical studies corroborated well with follow up western blotting of the same proteins. PGE2 and PGI2 products were also elevated at 24 h and 48 h after oil induction in infused horn in comparison to control horn. Our data suggest that COX2 derived both PGE2 and PGI2 mediate its function via EP2 and PPARδ receptors in early steps of decidualization in mice.

Roles of Implantation-related Factors, Stathmin and Insuline-like Growth Factor-binding Protein 7 in Reproductive Endocrinology

Successful implantation and placentation require that trophoblasts adhere to the uterine epithelium and penetrate the decidualized endometrium. However, the biochemical mechanisms of the establishment of pregnancy including these phenomena have not yet to be definitively elucidated. We have found that stathmin, a cytosolic phosphoprotein that regulates microtubule dynamics, and insulin-like growth factor-binding protein (IGFBP)-related protein 1 (IGFBP-rP1, now called IGF-binding protein 7) were highly expressed in the endometrium around the time of implantation and decidualization. In this article, we review our recent findings of the research regarding the functions of these implantation-associated proteins in endocrine physiology and pharmacology. Analysis of the expression of both factors in rodent and human uterus has revealed that both factors are crucial for the process of endometrial stromal cell differentiation.

Promotion of Angiogenesis by Human Endometrial Lymphocytes

The human endometrium is a unique tissue that undergoes dramatic monthly remodeling during the menstrual cycle in preparation for an implanting conceptus. This remodeling involves sequential proliferation and differentiation of endometrial stromal and epithelial cells, coupled with extensive angiogenesis and infiltration of a specific specialized immune cell subset. Increasing evidence points to an essential role for these maternal leukocytes in stimulating the endometrial angiogenesis, and we propose that they also play a key role in the decidual vascular transformation. Aberrant endometrial angiogenesis, decidualisation and vascular transformation is thought to underlie many pathologies of pregnancy, from infertility to the development of preeclampsia and Intra Uterine Growth Restriction. In this chapter we review the cellular processes associated with each stage of endometrial and decidual transformation, detailing the role of the immune cell populations and the angiogenic and chemotactic factors secreted by them.

414. The contraceptive potential of a long-acting IL-11 inhibitor

Interleukin 11 (IL-11) signalling is essential for the establishment of pregnancy in mice, through its action on the differentiation of uterine endometrial stromal cells (decidualisation), a critical process during embryo implantation. IL-11Rα deficient mice are infertile due to defective decidualisation1. IL-11 expression peaks between days (D) 4.5–9.5 of pregnancy (D0: day of plug) in mouse decidua. We examined the effect of administering (intraperitoneal [IP] injection or vaginal gel) a PEGylated IL-11 antagonist (PEGIL-11A) on decidualisation and pregnancy outcome in mice. The sera half-life of PEGIL-11A (IC50 2.8nM) following IP injection was 24h, compared with &lt;1 h for the non-PEGylated antagonist (IC50 0.26nM). Following IP injection, PEGIL-11A localised to decidual cells and blocked the IL-11 decidual target protein, cyclin D3. IP injection of 600µg/application PEGIL-11A (or PEG control) at 1000 h and 1600 h on D3 and 1000 h on D4 (n = 4/group), resulted in smaller implantation sites than controls on D6 due to retarded mesometrial decidual formation. On D10, severe decidual destruction was visible: implantation sites contained regions of haemorrhage and the uterine luminal epithelium had reformed, suggesting a return to oestrous cycling. Following vaginal application in aqueous placebo gel, PEGIL-11A localised to decidual cells. Vaginal application of 200µg/application PEGIL-11A (or control) twice daily from D2 to D5 (n = 4/group), resulted in smaller implantation sites than controls on D6 due to partial inhibition of mesometrial decidual formation. This study demonstrates that PEGIL-11A blocked IL-11 action in the uterus, resulting in total pregnancy loss, equivalent to the IL-11Rα deficient mouse. In women, IL-11 and its receptor are produced by the uterine luminal and glandular epithelium during the period of uterine receptivity2, suggesting that IL-11 may act during initial blastocyst attachment to the luminal epithelium as well as stromal decidualisation. This study provides proof-of-principle for the development of a novel, non-hormonal contraceptive for women. (1) Robb L et al. Nature Medicine 1998; 4: 303–308. (2) Dimitriadis E et al. Molecular Human Reproduction 2000; 6: 907–914.

Differential effects of human chorionic gonadotropin and decidualization on insulin-like growth factors-I and -II in human endometrial stromal cells

To investigate insulin-like growth factors (IGF)-I and -II in human endometrial stromal cells during decidualization in vitro and under the influence of hCG. In vitro experiment. Research laboratory at a medical university center. Premenopausal women undergoing hysterectomy for benign reasons. Endometrial stromal cells from hysterectomy specimens were isolated and incubated with 17beta-E(2), P, and recombinant hCG. Insulin-like growth factor-I and -II messenger RNA and protein were measured by semiquantitative real-time reverse transcriptase polymerase chain reaction and ELISA. During decidualization in vitro there was a significant increase of IGF-I level between day 0 and 3, followed by a decrease from day 6 to 9. Insulin-like growth factor-II level, however, was significantly reduced during all days of decidualization compared with day 0. In decidualized endometrial stromal cells, hCG decreased IGF-I in a dose-dependent manner without influencing IGF-II. Endometrial IGF-I and -II are differentially regulated during decidualization in vitro, suggesting different roles during the differentiation of endometrial stromal cells. Human chorionic gonadotropin as one of the first hormonal signals of the embryo selectively reduces IGF-I and may therefore contribute to the modulation of endometrial receptivity and differentiation during the process of early implantation.

Cyclic regulation of transcription factor C/EBPβ in human endometrium

The transcription factor CCAAT/enhancer-binding protein β (C/EBPβ) is a critical mediator of murine endometrial epithelial and stromal cell proliferation and differentiation. Consequently, female mice lacking the C/EBPβ gene are infertile. Previous studies have demonstrated the presence of C/EBPβ mRNA in human endometrium and upregulation of C/EBPβ during in vitro stromal decidualization. In order to further define the role of C/EBPβ in human endometrium, we characterized temporal changes in C/EBPβ mRNA abundance over the normal human menstrual cycle.