Patricia A. Mavrogianis

Decidual Stromal Cell Response to Paracrine Signals from the Trophoblast: Amplification of Immune and Angiogenic Modulators

Abstract During the invasive phase of implantation, trophoblasts and maternal decidual stromal cells secrete products that regulate trophoblast differentiation and migration into the maternal endometrium. Paracrine interactions between the extravillous trophoblast and the maternal decidua are important for successful embryonic implantation, including establishing the placental vasculature, anchoring the placenta to the uterine wall, and promoting the immunoacceptance of the fetal allograph. To our knowledge, global crosstalk between the trophoblast and the decidua has not been elucidated to date, and the present study used a functional genomics approach to investigate these paracrine interactions. Human endometrial stromal cells were decidualized with progesterone and further treated with conditioned media from human trophoblasts (TCM) or, as a control, with control conditioned media (CCM) from nondecidualized stromal cells for 0, 3, and 12 h. Total RNA was isolated and processed for analysis on whole-genome, high-density oligonucleotide arrays containing 54 600 genes. We found that 1374 genes were significantly upregulated and that 3443 genes were significantly downregulated after 12 h of coincubation of stromal cells with TCM, compared to CCM. Among the most upregulated genes were the chemokines CXCL1 (GRO1) and IL8,CXCR4, and other genes involved in the immune response (CCL8 [SCYA8], pentraxin 3 (PTX3), IL6, and interferon-regulated and -related genes) as well as TNFAIP6 (tumor necrosis factor alpha-induced protein 6) and metalloproteinases (MMP1, MMP10, and MMP14). Among the downregulated genes were growth factors, e.g., IGF1, FGF1, TGFB1, and angiopoietin-1, and genes involved in Wnt signaling (WNT4 and FZD). Real-time RT-PCR and ELISAs, as well as immunohistochemical analysis of human placental bed specimens, confirmed these data for representative genes of both up- and downregulated groups. The data demonstrate a significant induction of proinflammatory cytokines and chemokines, as well as angiogenic/static factors in decidualized endometrial stromal cells in response to trophoblast-secreted products. The data suggest that the trophoblast acts to alter the local immune environment of the decidua to facilitate the process of implantation and ensure an enriched cytokine/chemokine environment while limiting the mitotic activity of the stromal cells during the invasive phase of implantation.

The Altered Distribution of the Steroid Hormone Receptors and the Chaperone Immunophilin FKBP52 in a Baboon Model of Endometriosis Is Associated With Progesterone Resistance During the Window of Uterine Receptivity

This study examines the distribution of estrogen receptors (ESR), progesterone receptors (Pgr), and the chaperone immunophilin FKBP52 in the eutopic endometrium in a baboon model of endometriosis during the window of receptivity to determine if their aberrant distribution contributes to reduced fecundity. Endometriosis was induced by inoculation of menstrual endometrium into the peritoneal cavity. Eutopic endometrium was collected at 3, 6, 9, 12, and 15 months postinoculation. Western blot (WB) and immunohistochemical analyses were performed. Isolated endometrial stromal cells were cultured in the presence or absence of steroid hormones. In animals with endometriosis, ESR-1 (ER-α) decreased in endometrial stromal cells, while ESR-2 (ER-β) was reduced in both glandular epithelial (GE) and stromal cells. Immunoreactive total Pgr was markedly diminished in the GE, which was confirmed by WB analysis. Furthermore, treatment of isolated stromal cells from baboons with endometriosis with hormones did not increase levels of PRA or PRB as in control baboons. FKBP52 was also reduced in the eutopic endometrium of baboons with endometriosis. Endometriosis results in an aberrant distribution of ESR-1, ESR-2, Pgr, and FKBP52 in the eutopic endometrium. The authors propose that a dysregulation in the paracrine signaling between the endometrial stromal and GE cells reduces the responsiveness of Pgr, creating an endometrial environment that is unsuitable for implantation.

The Estrogen Early Response Gene FOS Is Altered in a Baboon Model of Endometriosis

Abstract Endometriosis, the presence of a functional endometrium outside of the uterine cavity, is associated with infertility. In our simulated model of pregnancy in baboons with experimental endometriosis, hCG infusion fails to induce expression of the immunoregulatory protein glycodelin. To test the hypothesis that the development of endometriosis is associated with an aberrant endometrial immunological environment, we examined the expression of a series of immunoregulatory genes in endometrium from baboons with and without endometriosis. Six months following intraperitoneal inoculation with menstrual endometrium, eutopic endometrium was surgically collected between Days 9 and 11 postovulation. Control endometrium was similarly collected from disease-free animals. Total RNA was extracted, and biotinylated cDNA probes were hybridized to the SuperArray GEArray Q series Th1/Th2/Th3 cDNA array, representing 96 genes. Gene expression levels were determined using ScanAlyze and GEArray Analyzer software. Seven genes were upregulated, including JUND, FOS, CCL11, NFKB1 and others, in the endometrium from baboons with endometriosis compared with the endometrium from disease-free animals; one gene, IL1R1, was downregulated. Quantitative RT-PCR confirmed upregulation of FOS and CCL11 in endometriotic eutopic endometrium. Immunohistochemical analysis revealed altered levels and distribution of FOS protein in the eutopic endometrium of baboons with induced endometriosis. These data suggest that in an induced model of endometriosis an aberrant eutopic immunological environment results in a decreased apoptotic potential and in rapid alterations in endometrial gene expression. We propose that the reduced fecundity associated with endometriosis has a multifold etiology in spontaneous and induced disease.

The Endometrial Response to Chorionic Gonadotropin Is Blunted in a Baboon Model of Endometriosis

Endometriosis-associated infertility has a multifactorial etiology. We tested the hypothesis that the endometrial response to the early embryonic signal, human chorionic gonadotropin (hCG), alters over time in a nonhuman primate model of endometriosis. Animals with experimental or spontaneous endometriosis were treated with hCG (30 IU/d), from d 6 after ovulation for 5 d, via an oviductal cannula. Microarray analysis of endometrial transcripts from baboons treated with hCG at 3 and 6 months of disease (n=6) identified 22 and 165 genes, respectively, whose levels differed more than 2-fold compared with disease-free (DF) animals treated with hCG (P<0.01). Quantitative RT-PCR confirmed abnormal responses of known hCG-regulated genes. APOA1, SFRP4, and PAPPA, which are normally down-regulated by hCG were up-regulated by hCG in animals with endometriosis. In contrast, the ability of hCG to induce SERPINA3 was lost. Immunohistochemistry demonstrated dysregulation of C3 and superoxide dismutase 2 proteins. We demonstrate that this abnormal response to hCG persists for up to 15 months after disease induction and that the nature of the abnormal response changes as the disease progresses. Immunohistochemistry showed that this aberrant gene expression was not a consequence of altered LH/choriogonadotropin receptor distribution in the endometrium of animals with endometriosis. We have shown that endometriosis induces complex changes in the response of eutopic endometrium to hCG, which may prevent the acquisition of the full endometrial molecular repertoire necessary for decidualization and tolerance of the fetal allograft. This may in part explain endometriosis-associated implantation failure.

Decidualization Regulates the Expression of the Endometrial Chorionic Gonadotropin Receptor in the Primate

Abstract Chorionic gonadotropin (CG) plays an important role in establishing a receptive endometrium by directly modulating the function of both endometrial stromal and epithelial cells in the baboon. The focus of this study was to characterize changes in CG receptor (LHCGR, also known as CG-R) expression during the menstrual cycle and early pregnancy, particularly during decidualization. LHCGR was localized by using a peptide-specific antibody generated against the extracellular domain. Immunostaining was absent in any of the cell types during the proliferative phase of the cycle. In contrast, during the secretory phase, both luminal and glandular epithelial cells stained positively. Stromal staining was confined to the cells around spiral arteries (SAs) and in the basalis layer. This stromal staining pattern persisted at the implantation site between Days 18 and 25 of pregnancy and after CG infusion. However, as pregnancy progressed (Days 40 to 60), staining for LHCGR was dramatically decreased in the stromal cells. These data were confirmed by nonisotopic in situ hybridization. To confirm whether the loss of LHCGR was associated with a decidual response, stromal fibroblasts were decidualized in vitro, and cell lysates obtained after 3, 6, and 12 days of culture were analyzed by Western blotting. LHCGR protein decreased with the onset of decidualization in vitro, confirming the in vivo results. Addition of CG to decidualized cells resulted in the reinduction of LHCGR in the absence of dbcAMP. We propose that CG acting via its R on stromal cells modulates SA in preparation for pregnancy and trophoblast invasion. As pregnancy progresses, further modification of SA by migrating endovascular trophoblasts and subsequent decidualization results in the downregulation of LHCGR. This inhibition of LHCGR expression also coincides with the decrease of measurable CG in peripheral circulation.

Species-specific effect of oviductal glycoproteins on hamster sperm binding to hamster oocytes

The secretory cells of the oviductal epithelium secrete a high‐ molecular‐weight glycoprotein (OGP). OGPs from different mammalian species show similar immunological characteristics, their cDNAs show high homologies, and they associate with the zona pellucida of oviductal oocytes in vivo. The purpose of this study was to determine the effect of OGP obtained from different species on the binding of hamster sperm to hamster oocytes. Hamster oocytes were inseminated (30 min) in the presence or absence of homologous or heterologous OGPs, and sperm bound/oocyte were counted after removing loosely attached sperm. Ovarian oocytes had an average of 2.9 ± 0.6 sperm bound/oocyte, whereas oviductal oocytes had 36.3 ± 2.7. Hamster OGP (0.1 mg/ml) significantly increased sperm binding to ovarian oocytes twofold and had no effect on sperm bound/oviductal oocytes. Human OGP (0.5 mg/ml) significantly decreased sperm binding to ovarian oocytes (0.9 ± 0.3 sperm bound/oocyte). This effect was dose dependent for oviductal oocytes and could be blocked by preincubating human OGP with a specific antibody to human OGP. The presence of baboon and cow OGP during the insemination of hamster oviductal oocytes also resulted in a significant decrease in sperm bound/oocyte, whereas the addition of hamster OGP to hamster oviductal oocytes had no effect. These results show that homologous OGP enhances sperm binding to the ZP, whereas heterologous OGP inhibits that effect. Thus, our results suggest that OGP plays a role in the species‐specific characteristics of sperm/ZP interaction, and that one must use a homologous system (OGP and gametes from the same species) to study the biological effect of OGP. Mol Reprod Dev 46:201–207, 1997.

Endometriosis Is Associated with Progesterone Resistance in the Baboon (Papio anubis) Oviduct: Evidence Based on the Localization of Oviductal Glycoprotein 1 (OVGP1)

Abstract Endometriosis has been associated with a reduced response to progesterone in both the eutopic and ectopic endometrium. In this study we evaluated OVGP1 and steroid receptor expression in oviducts of baboons with endometriosis during the midsecretory phase and determined whether progesterone resistance associated with endometriosis also occurs in the oviduct. Oviducts obtained during the window of uterine receptivity (Day 10 postovulation [PO]) from animals with induced and spontaneous disease were compared to control animals during the proliferative stage and in the implantation window as well as animals treated with the progesterone receptor (PGR) antagonist ZK 137.299 (ZK). OVGP1 was significantly higher in animals with endometriosis compared with Day 10 PO controls and was similar to that seen in the late proliferative phase and in ZK-treated animals. Baboons with spontaneous endometriosis also showed a similar persistence of OVGP1, which was correlated with the maintenance of estrogen receptor 1 (ESR1) in the epithelial cells of animals with endometriosis. However, epithelial cell height and the percentage of ciliation were not affected by endometriosis. These data imply that the normal antagonism of progesterone on ESR and OVGP1, which results in their downregulation during the window of implantation, is absent in animals with endometriosis. This was confirmed further when the action of PGR was antagonized in animals without disease, which also resulted in the persistence of ESR1 and OVGP1. These studies suggest that an aberrant oviductal environment may be an additive factor that contributes to endometriosis-associated infertility.

Immunocytochemical localization of transforming growth factor α, epidermal growth factor and epidermal growth factor receptor in the cat endometrium and placenta

This study was undertaken to determine the immunocytochemical localization of transforming growth factor α, epidermal growth factor and epidermal growth factor receptor in the endometrium of ovariectomized cats treated with oestradiol-17β and/or progesterone and in the endometrium and placenta of pregnant cats. Specific immunostaining was observed for all three antibodies. Moderate immunostaining for transforming growth factor α was observed in the epithelium of ovariectomized and oestrogen-treated cats. Dark epithelial staining was observed throughout pregnancy. The epithelial cells in progesterone-treated and peri-implantation animals contained dense deposits of reaction product, which were not reduced in intensity when immunoabsorbed antiserum was used. For epidermal growth factor, light--moderate epithelial staining was observed in ovariectomized and steroid-treated animals, and this increased in pregnant cats. Stromal staining for both the transforming and the epidermal growth factors was limited in steroid-treated animals and increased as pregnancy continued. Dark staining for epidermal growth factor receptor was observed in the epithelium and stroma in all the animals studied. The tips of surface epithelial convolutions in the non-implantation sites were always more darkly stained than in other regions of the surface epithelium. Staining in the placental trophoblast was limited to the syncytiotrophoblast for the two growth factors and the cytotrophoblast for the receptor during most of pregnancy and was absent late in pregnancy. The placental maternal giant cells contained specific immunoreactivity for all the immunogens from the middle of pregnancy to term. This study demonstrates that the two growth factors and the epidermal growth factor receptor are present in the endometrium and placenta of cats and suggests that these growth factors may play an autocrine/paracrine role during reproduction

Characterization of Antibodies Generated Against a Conserved Portion of Oviductal Glycoprotein (OGP) and Endogenous Hamster OGP and Their Ability to Decrease Sperm Binding to the Zona Pellucida In Vitro

PROBLEM: The effect of antibodies generated against hamster oviductal glycoprotein (OGP) on sperm binding to the zona pellucida (ZP) was evaluated.

Interactions Between the Embryo and Uterine Endometrium During Implantation and Early Pregnancy in the Baboon (Papio anubis)

The establishment of pregnancy in all mammalian species requires a synchronous interaction between the implanting embryo and the maternal endometrium. The mammalian uterus is receptive to the implanting blastocyst for a specific period of time and this receptive window appears to be regulated primarily by ovarian steroids. Embryo implantation is the natural culmination of this period, and successful nidation requires the precise preparation of both the blastocyst and endometrium. A remarkable synchrony is achieved by continuous maternal/conceptus interaction even prior to trophoblast invasion. The internal lining of the uterus is a specialized interface where a complex combination of anatomic, biochemical, endocrinologic, and immunologic events occur to ensure successful embryonic development. It is apparent, therefore, that the biological requirements of the early mammalian conceptus must be met by uterine and oviductal secretions since they constitute the primary environmental contact between the developing embryo and its mother prior to implantation.