Monoamine oxidases activity maintains endometrial monoamine homeostasis and participates in embryo implantation and development.

BACKGROUND: Monoamine oxidases (MAOs) is an enzyme that catalyzes the deamination of monoamines. The current research on this enzyme is focused on its role in neuropsychiatric, neurodevelopmental, and neurodegenerative diseases. Indeed, MAOs with two isoforms, namely, A and B, are located on the outer mitochondrial membrane and are widely distributed in the central nervous system and peripheral tissues. Several reports have described periodic changes in the levels of this enzyme in the human endometrial tissue. RESULTS: The novel role of MAOs in endometrial receptivity establishment and embryonic development by maintaining monoamine homeostasis was investigated in this study. MAOs activity was observed to be enhanced during the first trimester in both humans and mice under normal conditions. However, under pathological conditions, MAOs activity was reduced and was linked to early pregnancy failure. During the secretory phase, the endometrial stromal cells differentiated into decidual cells with a stronger metabolism of monoamines by MAOs. Excessive monoamine levels cause monoamine imbalance in decidual cells, which results in the activation of the AKT signal, decreased FOXO1 expression, and decidual dysfunction. CONCLUSIONS: The findings suggest that endometrial receptivity depends on the maintenance of monoamine homeostasis via MAOs activity and that this enzyme participates in embryo implantation and development.

Feto-Maternal Interface Organ-on-Chip: A New Technology to Study Ascending Infection.

Modeling human pregnancy is challenging as two subjects, the mother and fetus, must be evaluated in tandem. To understand pregnancy, parturition, and adverse pregnancy outcomes, the two feto-maternal interfaces (FMi) that form during gestation (i.e., the placenta and fetal membrane) need to be investigated to understand their biological roles, and organ dysfunction can lead to adverse outcomes. Adverse pregnancy outcomes such as preterm rupture of the membranes, spontaneous preterm birth, preeclampsia, intra-uterine growth restriction, and gestational diabetes rates are on the rise worldwide, highlighting the need for future studies and a better understanding of molecular and cellular pathways that contribute to disease onset. Current in vivo animal models nor in vitro cell culture systems can answer these questions as they do not model the function or structure of human FMis. Utilizing microfabrication and soft-lithography techniques, microfluidic organ-on-chip (OOC) devices have been adapted by many fields to model the anatomy and biological function of complex organs and organ systems within small in vitro platforms.These techniques have been adapted to recreate the fetal membrane FMi (FMi-OOC) using immortalized cells and collagen derived from patient samples. The FMi-OOC is a four-cell culture chamber, concentric circle system, that contains both fetal (amniochorion) and maternal (decidua) cellular layers and has been validated to model physiological and pathological states of pregnancy (i.e., ascending infection, systemic oxidative stress, and maternal toxicant exposure). This platform is fully compatible with various analytical methods such as microscopy and biochemical analysis. This protocol will outline this device's fabrication, cell loading, and utility to model ascending infection-related adverse pregnancy outcomes.

Downregulation of miR-92a in Decidual Stromal Cells Suppresses Migration Ability of Trophoblasts by Promoting Macrophage Polarization.

Preeclampsia (PE) is a severe pregnancy complication that accounts for about 14% of maternal deaths. Its clinical manifestations commonly include hypertension and proteinuria. However, it is largely limited in understanding its pathogenetic mechanism. In this study, we used bioinformatics to compare differential gene expressions in decidual stromal cells from PE patients and healthy donors. The result indicated that higher levels of CCL5 and CXCL2 were expressed in decidual stromal cells of PE patients compared with healthy pregnancy. The bioinformatics analysis confirmed that decidual stromal cells derived from PE patients expressed significantly lower miR-92a compared with those derived from healthy donors. Transfection of miR-92a inhibitors upregulated IL-6, CXCL2, CXCL3, CCL5, and CXCL8 expressions in decidual stromal cells. Luciferase activity assay confirmed that miR-92a directly targeted the mRNA of IRF3 whose overexpression could promote the secretion of cytokines. The flow cytometric analysis demonstrated that M1 macrophage infiltration was higher in the placentas of PE patients than in those of healthy donors. We also observed that after transfection of miR-92a inhibitor, condition medium (CM) derived from decidual stromal cells significantly promoted M1 polarization of macrophages. In addition, the transwell migration assay and flow cytometric analysis together showed that decidual stromal cell-derived CM induced macrophages to suppress the trophoblast migration and proliferation. Taken together, our result indicates that downregulation of miR-92a in decidual stromal cells promotes the macrophage polarization and suppresses the trophoblast migration and proliferation.

Expression dynamics of Crry at the implantation sites in normal pregnancy and response against miscarriage induction.

In mammals, immune tolerance against fetal tissue has been established for normal pregnancy progression. It is known that Crry regulates complemental activity to prevent injury of the mouse embryo and extra-embryonic tissue. This study aimed to examine the expression appearance and normal localization sites of Crry in the mouse placenta. Also, the emergency responses of Crry were verified at the time of experimental miscarriage induction. Moreover, we investigated an existing similar protein of Crry in animal placentas other than mice. Crry expression level showed a peak at day 8.5 of pregnancy. Trophoblast giant cells and decidual cells indicated a positive reaction to anti-Crry antibody. After treatments of interferon-γ, Crry expression was increased significantly in the survived implantation sites as compared with the controls. However, there was no significant difference in the miscarriage-initiated sites. It disclosed that Crry distributes from the early to middle periods of the placentas and involves complement regulation at the extraembryonic tissue and decidua basalis. Crry also showed an ability to respond to risk against external initiation for urgent miscarriage. Finally, we found anti-mouse Crry antibody-bound proteins in the placenta of many animals. It suggests a potency of Crry to make an environment of immune tolerance in many types of mammal placentas.

Embryonic growth retardation and altered expression of IGF-II is reciprocal induced by phytocompounds during early gestation in mice.

Methanolic crude extract of Scoparia dulcis (CESD) was orally administered to female mice during the early gestation (day 4-day 8) at a dose of 500 mg/kg/day. It induces embryo resorption and morphological changes of fetal maternal tissue. Histomorphology was studied by routine hematoxylin eosin stain. In situ immunofluorescence localization of IGF-II using Texas red showed an ordered expression of the growth factor in the maternal decidual cells, trophoblast cells and the embryo. Western blot analysis showed a gradual increase of IGF-II from D4 to D8 of control females. In contrast, the CESD-treated females showed resorption of embryo on D8 with disorganized in situ expression and lowered IGF-II in fetal maternal tissue. The phytocompounds present in the CESD could modulate either the ER or IGF-II receptors causing reduced IGF-II expression in the target tissues which lead to the failure of embryonic growth during periimplantation.

Decorin production by the human decidua: role in decidual cell maturation.

Decidualization involves the proliferation and differentiation of fibroblast-like endometrial stromal cells into epithelioid-shaped and secretory 'decidual' cells in response to steroid hormones. Human decidual cells produce insulin-like growth factor-binding protein-1 and prolactin (PRL), two well-recognized markers of decidual cell maturation and a proteoglycan decorin (DCN). We reported that DCN restrains the human trophoblast renewal, migration, invasion and endovascular differentiation needed for uterine arterial remodeling during normal pregnancy. DCN overproduction by the decidua is associated with a hypo-invasive placenta and a serious pregnancy disorder, pre-eclampsia (PE). Furthermore, elevated maternal plasma DCN levels during the second trimester is a predictive biomarker of PE. While these paracrine roles of decidua-derived DCN on trophoblast physiology and pathology have been well-defined, it remains unknown whether DCN plays any autocrine role in decidual cell development. The objectives of this study were to examine: the kinetics of DCN production during decidualization of human endometrial stromal cells; gestational age-related changes in DCN production by the first trimester decidua; and a possible autocrine role of DCN on decidual cell maturation. We found that DCN production is enhanced during decidualization of both primary and immortalized human endometrial stromal cells in vitro and during early gestation in decidual samples tested ex vivo, and that it is important for endometrial stromal cell maturation into a decidual phenotype. Decorin-depleted human endometrial stromal cells exposed to decidualizing stimuli failed to mature fully, as evidenced by fibroblastoid morphology, reduced insulin-like growth factor-binding protein-1 and PRL expression, and reduction in cellular ploidy. We identified heart and neural crest derivatives-expressed protein 2, and progesterone receptor as potential downstream mediators of DCN effects.

Regulation of CX3CL1 Expression in Human First-Trimester Decidual Cells: Implications for Preeclampsia.

C-X3-C motif ligand 1 (CX3CL1) mediates migration, survival, and adhesion of natural killer (NK) cells, monocytes, and T-cells to endothelial/epithelial cells. Aberrant numbers and/or activation of these decidual immune cells elicit preeclampsia development. Decidual macrophages and NK cells are critical for implantation, while macrophage-derived tumor necrosis factor-α (TNF-α), interleukin-1 ß (IL-1ß), and NK cell-derived interferon-γ (IFN-γ) are associated with preeclampsia development. Thus, serum and decidual levels of CX3CL1 from first-trimester pregnancy and preeclampsia-complicated term pregnancy were examined by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry, respectively. The effects of incubating primary human first-trimester decidual cells (FTDCs) with estradiol + medroxyprogesterone acetate + either IL-1ß or TNF-α and/or IFN-γ on CX3CL1 expression were also assessed by quantitative reverse transcription-polymerase chain reaction and ELISA. The inhibition of each signaling pathway with each kinase and nuclear factor κB (NFκB) inhibitors was evaluated by ELISA. Chemotaxis of CD56brightCD16- NK cells by various concentrations of CX3CL1 was evaluated. C-X3-C motif ligand 1 is expressed by both cytotrophoblasts and decidual cells in first-trimester decidua. C-X3-C motif ligand 1 expression is increased in term decidua but unchanged in first-trimester and term serum of patients with preeclampsia. Interferon-gamma and either IL-1ß or TNF-α synergistically upregulated CX3CL1 expression in FTDCs. Coincubation with IL-1ß or TNF-α or IFN-γ, mitogen-activated protein kinase kinase 1 and 2 (MEK1/2), c-JUN N-terminal kinase (JNK), and NFκB inhibitors suppressed CX3CL1 production. C-X3-C motif ligand 1 elicited concentration-dependent enhancement of CD56brightCD16- NK cell migration. In conclusion, the current study suggests that decidual cell-secreted CX3CL1 is involved in the later development of preeclampsia, whereas circulating CX3CL1 levels do not predict preeclampsia. Mitogen-activated protein kinase kinase 1 and 2, JNK, and NFκB signaling mediate IL-1ß-, TNF-α-, and IFN-γ-induced CX3CL1 production by FTDCs.

SFRP3 negatively regulates placental extravillous trophoblast cell migration mediated by the GCM1-WNT10B-FZD7 axis.

Migration of placental extravillous trophoblast (EVT) cells into uterine decidua facilitates the establishment of blood circulation between mother and fetus and is modulated by EVT-decidual cell interaction. Poor or excessive EVT migration is associated with pregnancy complications such as preeclampsia or placenta accreta. Glial cells missing 1 (GCM1) transcription factor is essential for placental development, and decreased GCM1 activity is detected in preeclampsia. To study whether GCM1 regulates trophoblast cell migration, here we showed that GCM1 promotes BeWo and JAR trophoblast cell migration through a novel target gene, WNT10B. Moreover, WNT10B signaling stimulated cytoskeletal remodeling via Rac1 and frizzled 7 (FZD7) was identified as the cognate receptor for WNT10B to up-regulate cell migration. We further showed that secreted frizzled-related protein 3 (SFRP3) is expressed in uterine decidual cells by immunohistochemistry and that SFRP3 expression in telomerase-transformed human endometrial stromal cells (T-HESCs) is elevated under decidualization stimuli and further enhanced by bone morphogenetic protein 2 via SMAD1. SFRP3 blocked the interaction between FZD7 and WNT10B to decrease BeWo cell migration, which corroborated the elevated BeWo cell migration when cocultured with decidualized and SFRP3-knockdown T-HESC monolayer. Our results suggest that GCM1 up-regulates EVT cell migration through WNT10B and FZD7, which is negatively modulated by decidual SFRP3.-Wang, L.-J., Lo, H.-F., Lin, C.-F., Ng, P.-S., Wu, Y.-H., Lee, Y.-S., Cheong, M.-L., Chen, H. SFRP3 negatively regulates placental extravillous trophoblast cell migration mediated by the GCM1-WNT10B-FZD7 axis.

Decorin over-expression by decidual cells in preeclampsia: a potential blood biomarker.

BACKGROUND: Decorin, a leucine-rich proteoglycan that is produced by decidual cells, limits invasion and endovascular differentiation of extravillous trophoblast cells during early placentation by binding to multiple tyrosine kinase receptors, in particular, vascular endothelial growth factor receptor-2. OBJECTIVE: Because many studies have reported an association between poor trophoblast invasion and endovascular differentiation with preeclampsia, the studies reported here tested (1) whether decorin over-expression in the chorionic villi and/or basal decidua is associated with preeclampsia and, if so, (2) whether this association results in a hypoinvasive placenta, and (3) whether elevated plasma decorin concentration in the second trimester is a predictive biomarker for preeclampsia. STUDY DESIGN: Decorin messenger RNA expression was measured with quantitative polymerase chain reaction at the tissue level and with in situ hybridization at the cellular level using (35)S-labeled antisense complimentary RNA probe in placentas from healthy control subjects and subjects with preeclampsia (14 each, 23-40 weeks of gestation). Tissue sections of the same placentas were also immunostained for decorin protein. A decorin over-expressing human endometrial stromal cell line was tested for invasion-regulatory effects on an invasive first-trimester extravillous trophoblast cell line HTR-8/SVneo plated in cocultures that were separated by a semipermeable membrane. Furthermore, we conducted retrospective measurements of plasma decorin levels during the second trimester (15-18 weeks of gestation) in a cohort of 28 body mass index-matched pairs of control subjects and subjects with preeclampsia before the onset of clinical disease. RESULTS: First, decorin messenger RNA expression at the cellular level measured with in situ hybridization exhibited profoundly higher expression levels in basal plate decidual cells within the placentas from preeclamptic subjects than those from control subjects at all gestational ages, whereas no difference between the 2 subject groups was noted in villus mesenchymal cells. Similarly decorin messenger RNA expression at the tissue level in chorionic villi (primarily resulting from fetally derived mesenchymal cells) did not differ significantly between control and preeclampsia placentas. These findings were validated with immunostaining for decorin protein. Second, knocking down decorin gene in a decorin over-expressing endometrial cell line (used as an in vitro surrogate of decorin over-expressing decidual cells) in cocultures with extravillous trophoblast cells abrogated its invasion-restraining actions on trophoblast cells, which indicated paracrine contribution of decorin over-expressing decidua to the poor trophoblast invasiveness in situ. Finally, retrospective measurement of plasma decorin levels during the second trimester in 28 body mass index-matched pairs of control subjects and subjects with preeclampsia revealed elevated plasma decorin levels in all subjects with preeclampsia in all body mass index groups. A receiver operating characteristic curve analysis revealed strong diagnostic performance of plasma decorin in the prediction of preeclampsia status. Although there was no significant gestational age-related change in decorin levels during the second trimester in control or subjects with preeclampsia, we found that plasma decorin had a significant inverse relationship with body mass index or bodyweight. CONCLUSION: We conclude that decorin over-expression by basal decidual cells is associated with hypoinvasive phenotype and poor endovascular differentiation of trophoblast cells in preeclampsia and that elevated plasma decorin concentration is a potential predictive biomarker for preeclampsia before the onset of clinical signs.

Sphingolipid pathway regulates innate immune responses at the fetomaternal interface during pregnancy.

For a successful pregnancy, the mother's immune system has to tolerate the semiallogeneic fetus. A deleterious immune attack is avoided by orchestration of cellular, hormonal, and enzymatic factors. However, the precise mechanisms underlying fetomaternal tolerance are not yet completely understood. In this study, we demonstrate that sphingolipid metabolism constitutes a novel signaling pathway that is indispensable for fetomaternal tolerance by regulating innate immune responses at the fetomaternal interface. Perturbation of the sphingolipid pathway by disruption of the sphingosine kinase gene (Sphk) during pregnancy caused unusually high expression of neutrophil chemoattractants, CXCL1 and CXCL2, in the decidua, leading to a massive infiltration of neutrophils into the fetomaternal interface with enhanced oxidative damage, resulting in early fetal death. Sphk-deficient mice also exhibited neutrophilia in the peripheral blood, enhanced generation of granulocytes in the bone marrow, and a decrease in the number of decidual natural killer cells. The blockage of neutrophil influx protected Sphk-deficient mice against pregnancy loss. Notably, a similar result was obtained in human decidual cells, in which Sphk deficiency dramatically increased the secretion of CXCL1 and IL-8. In conclusion, our findings suggest that the sphingolipid metabolic pathway plays a critical role in fetomaternal tolerance by regulating innate immunity at the fetomaternal interface both in mice and humans, and it could provide novel insight into the development of therapeutic strategies to treat idiopathic pregnancy loss in humans.

Immunohistological study of the endometrial stromal fibroblasts in the opossum, Monodelphis domestica: evidence for homology with eutherian stromal fibroblasts.

Molecular phylogenetic studies suggest that the hemochorial placentation and decidualization are ancestral traits of eutherian mammals. While the origin of the placental tissue is well understood, the origin of the decidual cells is unclear. Here we address the origin of decidual cells by examining the expression patterns of six transcription factors (TFs) as well as four structural proteins in the endometrium of a marsupial, Monodelphis domestica, and compared them with the patterns known from eutherian species. We found a mesenchymal cell population in the subepithelial compartment of the opossum endometrium. These cells express a set of TFs, such as homeobox A11 (HOXA11), CCAAT/enhancer-binding protein beta (CEBPB), and progesterone receptor (PGR), that are important for eutherian endometrial stromal cells. On the other hand, we did not find the expression of a decidual cell marker desmin (DES) or of TFs that are important for decidual cell differentiation, such as forkhead box O1 (FOXO1), in those cells. Based on these results, we propose that opossum has cells homologous to eutherian endometrial fibroblasts but no decidual cells. In addition, we describe cellular changes associated with the progression of pregnancy: nuclear localization of CEBPB in luminal epithelial cells as early as 8 days postcoitum, expansion of endometrial glands, nuclear localization of FOXO1 in glandular epithelial cells, and expression of smooth muscle actin in luminal epithelial cells. These data show that the luminal and glandular epithelium react to the presence of the preplacentation conceptus and suggest a limited form of pregnancy recognition.

Antiphospholipid antibodies inhibit prostaglandin release by decidual cells of early pregnancy: possible involvement of extracellular secretory phospholipase A2.

OBJECTIVE: To investigate the effect of antiphospholipid antibodies on eicosanoid production by human decidual cells and the in vitro interaction between antiphospholipid antibodies and secretory phospholipase A2. DESIGN: Cultures of human decidual cells from early pregnancy. SETTING: All decidual specimens were obtained from the Obstetrics and Gynecology Department of the Catholic University, Rome, Italy. PATIENT(S): Patients were undergoing operative laparoscopy for extrauterine pregnancy, with a period of amenorrhea ranging from 6 to 9 weeks. INTERVENTION(S): Decidual samples were collected at laparoscopy by routine uterine curettage. MAIN OUTCOME MEASURE(S): Decidual cells were incubated with antiphospholipid antibodies, and eicosanoids (prostaglandin [PG] E2, PGF2alpha, and thromboxane B2) were assayed by RIA after 24 hours of culture. In vitro interactions between antiphospholipid antibodies and secretory phospholipase A2 were investigated with use of a modified ELISA for phospholipase A2. RESULT(S): Antiphospholipid antibodies reduced eicosanoid release from decidual cells in a dose-dependent fashion. In vitro assays showed that antiphospholipid antibodies bound secretory phospholipase A2 and that a competition occurred between antiphospholipid antibodies and secretory phospholipase A2 for the common substrate cardiolipin. CONCLUSION(S): In light of the critical role played by eicosanoids in decidual function, we suggest that an interaction between antiphospholipid antibodies and secretory phospholipase A2 occurring in vivo might impair important cellular communications at the decidual level in the antiphospholipid antibody syndrome.

PIP: The presence of antiphospholipid antibodies is often associated with poor obstetric histories, including recurrent abortion, intrauterine growth retardation, and preeclampsia. While it has been suggested that these antibodies can affect the function of vascular endothelial cells at the decidual and placental levels, their cellular target and mode of action remain to be determined. Findings are presented from a study to investigate the effect of antiphospholipid antibodies upon eicosanoid production by human decidual cells and the in vitro interaction between antiphospholipid antibodies and secretory phospholipase A2. Specimens of decidualized endometrium were obtained by routine curettage from patients undergoing operative laparoscopy for extrauterine pregnancy in the Obstetrics and Gynecology Department of the Catholic University in Rome, Italy. Analysis determined that antiphospholipid antibodies reduce eicosanoid release from decidual cells in a dose-dependent manner, while in vitro assays showed that antiphospholipid antibodies bound secretory phospholipase A2 and that competition occurred between antiphospholipid antibodies and secretory phospholipase A2 for the common substrate cardiolipin. An interaction between antiphospholipid antibodies and secretory phospholipase A2 occurring in vivo may impair important cellular communications at the decidual level in the antiphospholipid antibody syndrome.

Morphological and functional features of endometrial decidualization following long-term intrauterine levonorgestrel delivery.

Irregular bleeding remains a common reason for the discontinuation of progestin-only contraception. The levonorgestrel releasing intrauterine system (LNG-IUS) has profound morphological effects upon the endometrium. Specific features are gland atrophy and extensive decidual transformation of the stroma. Morphological changes in the endometrium may be associated with perturbation of mechanisms regulating normal endometrial function. This study describes endometrial stromal and glandular features prior to and up to 12 months following insertion of the LNG-IUS. Comparison is made with first trimester decidua. In order to elucidate further mechanisms governing endometrial function with local intrauterine delivery of LNG, we here report histological features consistent with decidualization; a significant increase in granulocyte-macrophage colony stimulating factor (GM-CSF) immunoreactivity in decidualized stromal cells; glandular and stromal prolactin receptor expression and an infiltrate of CD56 + large granular lymphocytes and CD68 + macrophages. We are unaware of previous reports which have documented longitudinally both morphological and functional observations in endometrium exposed to local intrauterine levonorgestrel delivery. These studies demonstrate that long-term administration of intrauterine levonorgestrel results in features of altered morphology and function. No correlation was apparent between the end points in the study and the bleeding patterns described by the subjects. Further evaluation of these features in the context of menstrual bleeding experience may contribute to a better understanding of this troublesome side-effect which often leads to dissatisfaction and discontinuation of the intrauterine system.

PIP: The levonorgestrel-releasing intrauterine system (LNG-IUS) has profound morphologic effects on the endometrium, including gland atrophy and extensive decidual transformation of the stroma. The present study investigated these morphologic changes in tissue samples collected from 14 UK women up to 12 months after insertion of the LNG-IUS. Observed histologic features consistent with decidualization included a significant increase in granulocyte-macrophage colony stimulating factor immunoreactivity in decidualized stromal cells, glandular and stromal prolactin receptor expression, and an infiltrate of CD56+ large granular lymphocytes and CD68+ macrophages. The features of pseudo-decidualization closely resembled the morphology of early pregnancy decidua. These findings confirm that the stromal compartment of the endometrium undergoes changes consistent with decidualization for at least up to 12 months after insertion of an LNG-IUS. There was no correlation between the study endpoints and the menstrual patterns reported by study subjects. Further study of the decidualized nature of the stromal cells in the LNG-exposed endometrium should enhance understanding of the mechanisms responsible for breakthrough bleeding in users of progestogen-only contraceptives.

Progesterone receptors: expression and regulation in the mammalian ovary.

The authors have briefly discussed the molecular structure, regulation, and function of progesterone receptors in the mammalian ovary. Particularly important is the contrast in the regulatory mechanisms of PR induction in the ovary (gonadotropins/membrane receptor mediated) and other well-known progesterone target tissues, such as the uterus and mammary gland (estrogen/nuclear receptor mediated). Future research will focus on how the PR gene responds to these hormonal regulatory signals in this cell-specific manner. Equally important in this discussion has been the mounting evidence indicating that PRs are an essential component of the ovulatory process. The observation that PR-/- knockout mice are incapable of undergoing ovulation, even in response to gonadotropin challenge, further supports the previous physiological evidence indicating that PRs in preovulatory follicles are induced before, and are necessary for, ovulation. Further studies are required to determine the identity of PR-regulated target genes during the periovulatory period. Although our knowledge of PR structure, regulation, and function has increased dramatically during the past decade, many exciting questions remain related to the regulation and function(s) of PRs in the ovary and other tissues.

PIP: Research into the structure, function, expression, and regulation of progesterone receptors (PRs) in the mammalian ovary has the potential to refine understanding of ovulatory processes. A consistent finding of animal studies is the periovulatory expression of the PR gene in the granulosa cells of preovulatory follicles. The presence of PRs in these follicles in the transition from the preovulatory to periovulatory periods may be crucial to successful ovulation given the finding that the blockage of progesterone action at the level of the ovary substantially decreases ovulation rates in hamsters and rats. Estrogen is the primary regulator of PR gene expression in uterine epithelial cells, but not in the ovary. On the other hand, estrogen action is important for preparing granulosa cells for gonadotropin induction of PR gene expression. This cell-specific pattern of gonadotropins/membrane receptor-mediated PR induction in the ovary and estrogen/nuclear receptor mediation in the uterus and mammary gland requires further study. Also needed are studies to determine the identity of PR-regulated target genes during the periovulatory period. The overall thrust of the existing literature, however, indicates that PRs are an essential component of the ovulatory process.

Endometrial endothelial cell proliferation in long-term users of subdermal levonorgestrel.

The aim of the present study was to quantify endothelial cell proliferation (a component of angiogenesis) using immunohistochemistry, in the endometrium of users of subdermal levonorgestrel (Norplant). It was postulated that the increased endometrial microvascular density seen in Norplant users, compared to normally cycling women, was associated with an increased rate of endothelial cell proliferation. The results, however, showed that the endometrial endothelial cell proliferative index of Norplant users (0.39 +/- 0.16%; mean +/- SEM) was significantly reduced compared to that seen in normally cycling women (8.99 +/- 1.64). At the same time, total numbers of endometrial endothelial cells per mm2 in Norplant users (317.40 +/- 13.88) were significantly higher than in normally cycling women (223.35 +/- 10.31). It is possible that in the endometrium with levonorgestrel use, there is either a reduced rate of regression of the blood vessels relative to the rest of the tissue, or there is a reduced rate of endothelial cell death or turnover. Peripheral oestrogen and progesterone concentrations, bleeding pattern over the previous 90 days, and the histological appearance of the endometrium did not appear to be associated with the endothelial cell proliferative index. The results suggest that subdermal levonorgestrel use affects the mechanisms that dictate the normal relationship between endometrial blood vessel growth and regression, and the surrounding non-vascular tissue.

PIP: Quantification of endothelial cell proliferation by use of immunohistochemical staining of routinely processed endometrium revealed minimal angiogenesis in Norplant implant acceptors. It was hypothesized that the increased microvascular density of the endometrium of Norplant users and their bleeding problems reflect a levonorgestrel-induced increase in endothelial cell proliferation. 34 endometrial biopsies from 32 women attending the Raden Saleh Clinic in Jakarta, Indonesia, were collected 3-12 months after Norplant insertion. The mean endothelial cell proliferation index for the 32 Norplant users was 0.39 +or- 0.16%; 24 of the 34 biopsies showed no evidence of cell proliferation. There was no significant variation in this index based on the histologic appearance of the biopsy, bleeding pattern, or peripheral blood estrogen and progesterone concentrations. This index is significantly lower than that recorded in another study (8.99 +or- 1.64) during a normal menstrual cycle. Also observed was a significantly increased number of endothelial cells per sq. mm in Norplant users (317.40 +or- 13.88) compared to normally cycling women (223.25 +or- 10.31). Although the study hypothesis was rejected, the factors behind the increased density of blood vessels and endothelial cells in the endometrium of Norplant users remain unclear. It is possible that levonorgestrel induces a reduced rate of regression of the blood vessels compared to the rest of the tissue, or that there is a reduced rate of endothelial cell death or turnover.

Synthesis of 11-substituted androstenediones and testosterones as human decidual cell growth inhibitors.

11 alpha-Hydroxytestosterone (1a), 11 beta-hydroxytestosterone (1b), 11 alpha-methoxytestosterone (1c), 11 beta-methoxytestosterone (1d), 11-ketotestosterone (1e), and delta 9(11)-testosterone (1f) were synthesized from hydrocortisone (4b) or 11-epi-hydrocortisone (4a). The six target compounds, together with 11 alpha-methoxyandrostenedione (2c), 11 beta-methoxyandrostenedione, (2d) and their lead compound, testosterone (1), were found to effectively inhibit the growth and differentiation of human decidual cells in culture. There is no observable binding of these compounds to estrogen receptor of rabbit uterus. The introduction of a polar group (e.g., hydroxyl and carbonyl) to C-11 of androstenes decreases both the relative binding affinities to progesterone receptor and the inhibitory effects on human decidual cell growth, while the methylation of 11-hydroxyl group minimizes these effects. The similar effects of a polar group at C-11 of testosterone (1) on the inhibitory effects on human decidual cell growth and the relative binding affinities to progesterone receptor of rabbit uterus may suggest that one of the mechanisms of human decidual cell growth inhibition by these compounds is the anti-progestational activity of these androgens.

Quantitative comparison of prolactin production by decidua compacta and spongiosa from the first trimester elective termination of pregnancy.

Prolactin (PRL) producing capacity was studied in explants of decidua compacta and decidua spongiosa obtained from 41 patients undergoing termination of pregnancy at gestation 6 to 12 weeks. In vitro PRL producing capacity, expressed as mIU/g protein, of the decidua compacta was significantly higher (P < 0.05) than those of decidua spongiosa. Production of PRL increased with gestation from 6 to 12 weeks with a more rapid rate at the later gestation. The pattern of increase fitted significantly (P < 0.0001) to the exponential model for both decidua compacta and decidua spongiosa. The exponential regression equations for decidua compacta and decidua spongiosa were (ln y = 4.25 + 0.19x) and (ln y = 2.80 + 0.31x) respectively. Hence, although both decidua compacta and decidua spongiosa had a similar pattern of increase in PRL production, the rate of increment was significantly greater in decidua spongiosa than in decidua compacta. These findings suggest that separating decidua compacta and decidua spongiosa of the first trimester would reduce the heterogeneity of decidual tissue and offer a new approach to the studies of the synthesis, release and regulation of PRL production by human decidua.

Estrogen and progesterone receptors in human decidua after RU486 treatment.

OBJECTIVE: To examine RU486 action on decidua at the level of cellular estrogen receptor (ER) and P receptor (PR). DESIGN: Controlled basic study for contragestion mechanism of mifepristone. SETTING: Normal human volunteers in an academic research environment. PATIENTS: Sixty women with 6 to 7 weeks of gestation who voluntarily requested termination of pregnancy were recruited and randomly divided into three groups. INTERVENTION: A single dose of 200 mg RU486 was orally administered to the two treatment groups 12 and 24 hours, respectively, before surgical interruption of pregnancies. Placebo was used for control group. Decidual tissues were collected right after operation. MAIN OUTCOME MEASURE: Immunocytochemical reactions of PR and ER in decidua after RU486 treatment were compared with the control subjects. The differences of the reaction in decidual area with or without trophoblast invasion were noted. RESULTS: RU486 treatment increased PR and ER staining in vessel and stroma of decidua without trophoblast invasion (decidua parietalis) but not in decidua with trophoblast invasion (decidua capsularis or basalis). Chi-squared analysis indicated a significant increase in the number of ER-positive samples after RU486 treatment. CONCLUSION: The decidua parietalis was the primary target site of RU486. The lack of RU486 effect on decidua capsularis implied that trophoblast invasion prevented against antiprogestin impact.

PIP: In China, physicians randomly placed 60 pregnant women of 6-7 weeks gestational age into a group receiving one oral dose of RU-486 12 or 24 hours before undergoing vacuum aspiration or into a group receiving a placebo. Aspirate samples were submitted to a laboratory to determine the effect of Ru-486 on progesterone (P) and estrogen (E) receptors in human decidua. RU-486 multiplied P and E receptor staining in vessel and stoma cells of decidua (decidua parietalis) without fetal trophoblast invasion (89% for 12-hour group and 84% in 24-hour group vs. 26%; p M .005). This did not occur, however, in decidua with fetal trophoblast invasion (decidual capsularis or basalis). These findings confirmed the hypothesis that the P antagonist, RU-486, increases P and E receptor levels in decidual tissue. Further, the findings indicated that the contragestive mechanisms of RU-486 is directed at the maternal endometrium and not at the developing embryo or those interfacing cells which anchor it to the endometrium. In fact, the cytological barrier inhibits antiprogestin effects and may even play a role in protecting against spontaneous abortion.

Effects of hormone therapy on the endometrium.

Hormone therapy induces a variety of histologic changes in the endometrium. Histologic patterns encountered in the most commonly used hormonal regimens are described. Oral contraceptives are associated with inactive, atrophic, or pseudosecretory glands and edematous stroma, decidual reaction without spiral arterioles, and stromal granulocytes. A high-potency progesterone may induce marked stromal and vascular hyperplasia and stromal myomatous nodules. Ovulation induction therapy accelerates the maturation of the stroma and is often associated with a discrepancy between the glands showing early secretory changes and an edematous, decidualized stroma. Hormone replacement therapy may stimulate endometrial proliferation if estrogens are used alone and produce endometrial hyperplasia and neoplasia. When estrogen and progesterone regimens are used, a wide range of histologic pattern may be found in various combinations: proliferative and secretory endometrium, glandular and adenomatous hyperplasia, stromal hyperplasia and decidual transformation, glandular metaplasia, atrophic endometrium, and any of the above with endometrial atrophy. Progesterone therapy for endometrial hyperplasia and neoplasia is followed by secretory changes of the endometrium, mostly subnuclear vacuoles, decidual reaction, and sometime squamoid "morules." Secretory changes seen after progesterone therapy in the endometrium do not rule out residual carcinoma. For hormone therapy for breast carcinoma, tamoxifen acts as an antiestrogen on the breast but often acts as an estrogen agonist on the endometrium; tamoxifen therapy may be associated with endometrial hyperplasia, polyps, adenomyosis, adenomatous hyperplasia, and adenocarcinoma.

PIP: Steroid sex hormones cause immediate changes in the endometrium. The histologic effect depends on the hormone, the potency, dosage, and the host receptor status. Oral contraceptives (OCs) containing a low-dose, low-potency progesterone and low-dose estrogen stop proliferation of the glands during the 1st few cycles and the glands are straight and unevenly distributed, with considerable stroma between them. The minimal secretory features of the glands disappear with longterm use of these OCs, resulting in complete atrophy of the glands and of the stroma. These OCs also cause decidual reaction without spiral arterioles and stromal granulocytes. OCs with high potency progesterone cause distinct hyperplasia of the endometrial stroma and vessels, atrophy of the glands, and stromal myomatous nodules. Ovulation induction therapy consists of various combinations of some drugs (clomid, human menopausal gonadotropins, gonadotropin-releasing hormone, and human chorionic gonadotropin). This therapy accelerates secretory changes in the stroma, resulting in enhancement of the endometrial maturation process. It makes it difficult to differentiate between glands displaying early secretory changes and an edematous, decidualized stroma. Hormone replacement therapy using estrogens and progesterone cause different histologic patterns, including stromal hyperplasia and decidual transformation, glandular and adenomatous hyperplasia, glandular metaplasia, proliferative and secretory endometrium, atrophic endometrium, and any of these with endometrial atrophy. Progesterone therapy for endometrial hyperplasia and neoplasia produces secretory changes of the endometrium, such as subnuclear vacuole, decidual reaction, and squamoid "morules." These changes can result in residual carcinoma. Tamoxifen therapy for breast cancer is linked with endometrial hyperplasia, polyps, adenomyosis, adenomatous hyperplasia, and adenocarcinoma.

Early changes of affinity and binding sites of progesterone and oestrogen receptors in decidua exposed to RU 486.

Sixty patients with 6-7 weeks of amenorrhoea were randomly allocated to three groups. The women in the first group (control) took a placebo 24 h before undergoing a vacuum aspiration. The patients in the second and third groups were given 200 mg of RU 486 orally, 12 or 24 h before surgical interruption of their pregnancy. Decidua were collected and frozen in liquid nitrogen. By Scatchard plot analysis, the number of cytosolic binding sites (1798 +/- 803 fmol/mg DNA) of progesterone in decidua in the control group was significantly reduced (P less than 0.01) to 696 +/- 408 or 626 +/- 179 fmol/mg DNA by RU 486 treatment for 12 or 24 h respectively. The dissociation constants of both cytosolic and nuclear progesterone receptors in RU 486-exposed decidua were increased (P less than 0.01). The number of nuclear binding sites of oestrogen receptor was significantly higher (P less than 0.05) in decidua with RU 486 treatment for 12 h (178 +/- 77 fmol/mg DNA) compared to the control (89 +/- 32 fmol/mg DNA). The results suggest that RU 486 might regulate progesterone and oestrogen receptors in the decidua of early human pregnancy, either directly or indirectly.