Human chorionic gonadotropin contributes to maternal immunotolerance and endometrial apoptosis by regulating Fas-Fas ligand system

The first known hormonal signal of the conceptus during implantation is human chorionic gonadotropin (hCG). Interestingly, increased apoptosis in human endometrium coincides with the implantation window. Factors from the fetal or placental origin as well as maternal hormonal factors are likely to have a potential role in the regulation of apoptotic signaling molecules. We hypothesized that hCG may be a placental link for the development of local maternal immunotolerance. Fas-Fas ligand (FasL) system is one of the apoptotic signaling pathways, shown to be important in the development of local immune tolerance during and after implantation. We report that hCG treatment decreases cell proliferation and increases apoptosis in endometrial cells. Moreover, hCG stimulates FasL mRNA and protein expression without affecting Fas mRNA in these cells. Interestingly, in coculture experiments, hCG-treated endometrial cells induce an increase in T cell apoptosis. Our in vivo results reveal that cells of early pregnancy decidua express strong FasL immunoreactivity, and decidual areas containing interstitial cytotrophoblasts have numerous TUNEL-positive cells. Compared with decidual areas devoid of interstitial cytotrophoblasts, we observed in decidual areas containing interstitial cytotrophoblasts clearly less amount of TUNEL-positive cells. These results suggest that hCG may be a link in the development of peritrophoblastic immune tolerance and may facilitate the trophoblast invasion by regulating proapoptotic molecules such as FasL in endometrial cells.     

Direct effect of gonadotropins on decidualization of human endometrial stromal cells

Decidualization of stromal cells isolated from proliferative human endometrium was achieved by adding to the culture medium human gonadotropins (FSH, FSH + LH, hCG). In addition to changes in the morphology of the stromal cells to the decidual phenotype, decidualization was evident from the expression of prolactin (PRL), demonstrated immunocytochemically, by Western blotting analysis, and by measuring its output into the medium through solid phase enzyme immunoassay. Gonadotropins also induced cAMP formation in the endometrial stromal cells under the same experimental conditions. This finding suggests that the mechanism by which gonadotropins promote decidualization of human endometrial stromal cells in vitro involves the introduction of cAMP, a compound that we have found to elicit the expression of PRL in this system. PRL is likely to be a key intermediate in the process of decidualization since it is by itself capable of inducing differentiation of the endometrial stromal cells to the decidual phenotype. Awareness of direct actions of gonadotropins on the endometrial cells and, in particular, of the decidualizing effects of FSH (Metrodin), FSH + LH (Pergonal) and hCG may contribute to the understanding of physiologic as well as pathophysiologic conditions relevant to endometrial functions and fertility.     

Cytological distribution of chorionic gonadotropin subunit and placental lactogen messenger RNA in neoplasms derived from human placenta

Normal trophoblast of the human placenta elaborates at least two major protein hormones, chorionic gonadotropin (hCG), and placental lactogen (hPL). There are several gestational trophoblastic diseases of the placenta called hydatidiform mole, invasive mole, and choriocarcinoma. Molar and choriocarcinoma tissues characteristically synthesize large amounts of hCG and small quantities of hPL. To examine the role of trophoblast differentiation in the expression of the hCG and hPL genes, we studied the cytological distribution of their messenger RNA (mRNA) in tissue sections of human hydatidiform mole and choriocarcinoma by in situ hybridization. Histologically, these tissues are in different stages of cellular differentiation. In normal placenta, hCG alpha and - beta mRNA can be localized to some cytotrophoblasts and primarily to the syncytium, whereas hPL mRNA appears only in the syncytial layer. In hydatidiform mole, which still retains placental villous morphology, the hPL gene and hCG alpha and -beta genes are expressed but are poorly localized because of the admixture of cyto- and syncytiotrophoblasts. By contrast, choriocarcinoma, which is devoid of placental villous pattern but in which the cyto- and syncytiotrophoblast-like components are distinguishable, expresses hCG alpha and -beta in the syncytial- like areas but little, if any, hPL. These results suggest that a certain level of trophoblast differentiation, such as villous formation, is associated with hPL expression, while the hCG alpha gene and the hCG beta gene can be expressed in more disorganized tissues that contain cytotrophoblastic elements.     

Rabbit endometrium in organ culture: Morphological evidence for progestational differentiation in vitro

Summary This communication describes conditions for long-term organotypic culture of rabbit endometrium allowing progesterone-induced transformation, as typcial for early pregnancy, to continue in vitro. This system appears to compare favorably with in vitro models so far proposed for the study of hormonal control of uterine function or for the investigation of cell-biological aspects of embryo implantation. The specific aim in the presented system is to provide approximate normal epithelium-stroma interrelationships. Fragments of endometrium consisting of epithelium and stroma were obtained during early pseudopregnancy and cultured on a gyratory shaker. Morphology was investigated by light microscopy, transmission and scanning electron microscopy. During the first two days the epithelium grows over the exposed stroma regenerating a complete epithelial lining. No central necrosis is found in the stroma for up to 6 days, and the tissue keeps its organotypic architecture although certain morphological differences can be observed between regenerated versus original epithelium. In the regenerating portion a stage-specific cell differentiation and the reformation of a basal lamina are missing. Progesterone substitution preserves cell morphology and allows to maintain, in vitro, the stage-specific pattern of cell organelles. Most characteristic is the induction of extensive fusion of epithelial cells. These large symplasms are comparable in size and structure to those formed in pregnancy in the implantation chamber in vivo. Only the superficial parts of the original (not the regenerated) epithelium are capable of progesterone-induced large-scale fusion. This organotypical culture system appears to be of potential value for in vitro studies on hormone action and on endometrial receptivity for embryo implantation.Abbreviations d.p.c. dies post coitum - d p. hCG days after injection of hCG (dies post injectionem hCG) - FBS fetal bovine serum - hCG human chorionic gonadotropin Preliminary results were presented by Denker et al. (1984) and by Hohn et al. (1984)     

Glycodelin protein and mRNA is downregulated in human first trimester abortion and partially upregulated in mole pregnancy.

Glycodelin (Gd) is a major reproductive glycoprotein and a mediator for immunomodulatory effects directed to cellular, humoral, and innate immunity. Human pregnancy depends on a diversity of physiological processes including modulation of the maternal immunosystem. We evaluated the expression of Gd protein and mRNA in first trimester decidual tissue of normal pregnancies and spontaneous abortion and hydatidiform moles. Furthermore, in vitro experiments on endometrial cancer cells to analyze the effect of human chorionic gonadotropin (hCG) on Gd regulation were performed. In decidual tissue of abortion patients, Gd expression was significantly decreased compared with normal gestation, which was confirmed by in situ hybridization. In mole pregnancy, an upregulation of Gd in the first 8 weeks of pregnancy was present. Gd is a main product of decidual tissue in the first trimester of human pregnancy. Reduced Gd expression in abortive pregnancy could lead to an increased activation of the maternal immunosystem, thus causing rejection of the developing fetus. Moreover, Gd expression in endometrial cancer cells in vitro could be stimulated by addition of hCG. Therefore, we speculate that hCG could be one of the factors regulating Gd expression because hCG is downregulated in women with abortion and upregulated in mole pregnancy. In addition, we found a positive feedback loop in Gd and hCG expression in human pregnancy.     

Bone tissue formation from human embryonic stem cells in vivo

Although the use of embryonic stem cells in the assisted repair of musculoskeletal tissues holds promise, a direct comparison of this cell source with adult marrow-derived stem cells has not been undertaken. Here we have compared the osteogenic differentiation potential of human embryonic stem cells (hESC) with human adult-derived stem cells in vivo. hESC lines H7, H9, the HEF-1 mesenchymal-like, telomerized H1 derivative, the human embryonic kidney epithelial cell line HEK293 (negative control), and adult human mesenchymal stem cells (hMSC) were either used untreated or treated with osteogenic factors for 4 days prior to injection into diffusion chambers and implantation into nude mice. After 11 weeks in vivo chambers were removed, frozen, and analyzed for evidence of bone, cartilage, and adipose tissue formation. All hESCs, when pretreated with osteogenic (OS) factors gave rise exclusively to bone in the chambers. In contrast, untreated hESCs (H9) formed both bone and cartilage in vivo. Untreated hMSCs did not give rise to bone, cartilage, or adipose tissue in vivo, while pretreatment with OS factors engendered both bone and adipose tissue. These data demonstrate that hESCs exposed to OS factors in vitro undergo directed differentiation toward the osteogenic lineage in vivo in a similar fashion to that produced by hMSCs. These findings support the potential future use of hESC-derived cells in regenerative medicine applications.     

Induction of trophinin in human endometrial surface epithelia by CGbeta and IL-1beta

During embryo implantation, trophinin mediates cell adhesion by homophilic binding at the apical surfaces of trophectoderm and endometrium. Trophinin is expressed on the human endometrial epithelia in rare occasions. We developed hCG-coated agarose beads that mimic the physical and physiological features of an implantation-stage human blastocyst. When hCG-coated beads were applied to human endometrial epithelial cells in the presence of IL-1beta, endometrial cells acquired strong trophinin expression and the ability for apical cell adhesion with trophinin-expressing human trophoblastic cells. These results provide a mechanism for trophinin-mediated adhesion of human blastocyst to endometrium by a spatially and temporally restricted paracrine effect of hCG derived from the blastocyst.     

The role of steroid hormones in ART

Steroid hormones hold a major role in female fertility and their proper utilisation and monitoring in modern assisted reproduction protocols is important. Oocyte maturation and endometrial receptivity are the two major factors that appear to be related to a successful outcome in Assisted Reproductive Technology (ART). Many reports suggest that oocyte immaturity accounts for a considerable loss of efficiency in ART, mainly due to the poor quality of the obtained embryos and their inability to develop normally. Oestrogen appears to exert its effects on the cytoplasmic maturation of the oocyte, while progesterone has been shown to accelerate meiotic resumption. Moreover, ovarian stimulation appears to affect the normal luteal function and shifts in the window of implantation as a response to hormonal supplementation have also been observed. The ethical limitations in conducting in vivo studies of human implantation, have led to an indirect hormonal- and morphologic-oriented assessment of endometrial receptivity. The two main protocols of luteal support involve either progesterone supplementation or hCG administration, whereas the combined supplementation with oestradiol remains controversial. This brief review aims to summarize the current knowledge on steroidal actions during the above processes and to address their potential use in the improvement of current ART protocols.     

Direct involvement of HERV-W Env glycoprotein in human trophoblast cell fusion and differentiation

We recently demonstrated that the product of the HERV-W env gene, a retroviral envelope protein also dubbed syncytin, is a highly fusogenic membrane glycoprotein inducing the formation of syncytia on interaction with the type D mammalian retrovirus receptor. In addition, the detection of HERV-W Env protein (Env-W) expression in placental tissue sections led us to propose a role for this fusogenic glycoprotein in placenta formation. To evaluate this hypothesis, we analyzed the involvement of Env-W in the differentiation of primary cultures of human villous cytotrophoblasts that spontaneously differentiate by cell fusion into syncytiotrophoblasts in vitro. First, we observed that HERV-W env mRNA and glycoprotein expression are colinear with primary cytotrophoblast differentiation and with expression of human chorionic gonadotropin (hCG), a marker of syncytiotrophoblast formation. Second, we observed that in vitro stimulation of trophoblast cell fusion and differentiation by cyclic AMP is also associated with a concomitant increase in HERV-W env and hCG mRNA and protein expression. Finally, by using specific antisense oligonucleotides, we demonstrated that inhibition of Env-W protein expression leads to a decrease of trophoblast fusion and differentiation, with the secretion of hCG in culture medium of antisense oligonucleotide-treated cells being decreased by fivefold. Taken together, these results strongly support a direct role for Env-W in human trophoblast cell fusion and differentiation.     

Transient activation of c-myc protooncogene expression in Leydig cells by human chorionic gonadotropin

This study evaluated the effects of in vivo administration of human chorionic gonadotropin (hCG) on c-myc oncogene expression in Leydig cells. Sprague-Dawley rats (46-50 days old) were treated with hCG (10 units, i.p.), and purified Leydig cells were isolated 1-24 h later. HCG caused a transient elevation of c-myc mRNA in 4 h and returned to normal or lower than normal levels at 24 h. There was no change in c-fos or beta-actin mRNA levels. Our results suggest that the growth promoting effects of hCG on Leydig cells may be mediated by the transient expression of c-myc protooncogene.     

Hormonal regulation of differentiation of human fetal prostate and Leydig cells in vitro

Trowell type of organ culture was used for correlative study of the human fetal prostate and Leydig cell differentiation at the ultrastructural level. Androgens accelerated the differentiation of human urethral epithelial cells into secretory prostatic cells and the ultrastructure resembled that in vivo. Also Leydig cells retained in organ culture the same ultrastructural features as in vivo and human chorionic gonadotropin (hCG) accelerated their differentiation. It is concluded that this type of culture technic can be used in the study of early differentiation of human genital organ and androgenes and hCG take part of human prostatic and Leydig cell differentiation.     

Correlation of increased concentration of ovine endometrial cyclooxygenase 2 with the increase in PGE2 and PGD2 in the late luteal phase

The effect of intraoviductal embryos on endometrial receptivity was studied by intraendometrial and intrauterine embryo transfer. Five-week-old female ICR mice were mated after superovulation; a vaginal plug confirmed day 1 of pregnancy. On day 4 (90 h after hCG injection), blastocysts were collected and transferred to pseudopregnant female mice and to recipient mice in which the uterotubal junction had been ligated bilaterally on day 1 of pregnancy. Three embryos per uterine horn, a total of six embryos per recipient mouse at days 1-6, were transferred to the endometrium or uterine cavity and implantation and pregnancy rates were calculated. The implantation rate for intraendometrial embryo transfer to recipients of days 3, 5 and 6 was significantly higher for uterotubal junction-ligated mice (72.2, 20.8 and 9.7%, respectively) than for pseudopregnant mice (55.0, 8.3 and 0.0%, respectively). The implantation rate for intrauterine embryo transfer to recipients at days 2, 5 and 6 was significantly higher for uterotubal junction-ligated mice (11.1, 25.0 and 8.3%, respectively) than for pseudopregnant mice (0.0, 3.3 and 0.0%, respectively). Uterotubal junction-ligated mice achieved implantation and bore neonates by intrauterine embryo transfer on days 2 and 6, whereas no implantation was achieved in pseudopregnant mice. The difference in implantation rate could not be explained by a difference in progesterone concentration between the groups. The distribution of proliferating cells in the endometrium was also studied immunohistochemically by use of anti-proliferating cell nuclear antigen (PCNA) antibody in the recipient mice. PCNA-positive cells were more abundant in uterotubal junction-ligated mice and demonstrated a marked extension from the epithelium to the stroma over time, in contrast to those in pseudopregnant mice. These findings indicate that an intraoviductal embryo exerts a biological effect by sending a signal to the endometrial epithelium and stroma, thus facilitating endometrial receptivity to the embryo and improving the rate of implantation.     

hCG-dependent regulation of angiogenic factors in human granulosa lutein cells

As prerequisite for development and maintenance of many diseases angiogenesis is of particular interest in medicine. Pathologic angiogenesis takes place in chronic arthritis, collagen diseases, arteriosclerosis, retinopathy associated with diabetes, and particularly in cancers. However, angiogenesis as a physiological process regularly occurs in the ovary. After ovulation the corpus luteum is formed by rapid vascularization of initially avascular granulosa lutein cell tissue. This process is regulated by gonadotropic hormones. In order to gain further insights in the regulatory mechanisms of angiogenesis in the ovary, we investigated these mechanisms in cell culture of human granulosa lutein cells. In particular, we determined the expression and production of several angiogenic factors including tissue inhibitor of matrix metalloproteinases-1 (TIMP-1), Leptin, connective tissue growth factor (CTGF), meningioma-associated complimentary DNA (Mac25), basic fibroblast growth factor (bFGF), and Midkine. In addition, we showed that human chorionic gonadotropin (hCG) has distinct effects on their expression and production. hCG enhances the expression and production of TIMP-1, whereas it downregulates the expression of CTGF and Mac25. Furthermore it decreases the expression of Leptin. Our results provide evidence that hCG determines growth and development of the corpus luteum by mediating angiogenic pathways in human granulosa lutein cells. Hence we describe a further approach to understand the regulation of angiogenesis in the ovary.     

Transmembrane and truncated (SEC) isoforms of MUC1 in the human endometrium and Fallopian tube

The cell surface mucin MUC1 is expressed by endometrial epithelial cells with increased abundance in the secretory phase of the menstrual cycle, when it is found both at the apical cell surface and in secretions. This suggests the presence of a maternal cell surface glycoprotein barrier to embryo implantation, arising from the anti-adhesive property of MUC1. In previous work, we demonstrated alternatively spliced MUC1 variant forms in tumour cells. The variant MUC1/SEC lacks the transmembrane and cytoplasmic sequences found in the full-length variant. We now show that MUC1/SEC mRNA is present in endometrial carcinoma cell lines, endometrial tissue and primary cultured endometrial epithelial cells. The protein can be detected using isoform-specific antibodies in uterine flushings, suggesting release from endometrium in vivo. However, on the basis of immunolocalisation studies, MUC1/SEC also remains associated with the apical epithelial surface both in tissue and in cultured cells. Transmembrane MUC1 and MUC1/SEC are both strikingly localised to the apical surface of tubal epithelium. Thus MUC1 may contribute to the anti-adhesive character of the tubal surface, inhibiting ectopic implantation. The mechanism by which this barrier is overcome in endometrium at implantation is the subject of ongoing investigation.     

Inhibitory effect of human chorionic gonadotropin (hCG) on HIV-1 transmission from lymphocytes to trophoblasts.

It has been demonstrated that human chorionic gonadotropin (hCG) inhibits HIV production in vitro, suggesting that this soluble placental glycoprotein can control viral replication and spread in vivo. hCG--the major product of fetal trophoblasts--was tested on an in vitro model consisting of choriocarcinoma-derived ENAMI trophoblasts exposed to HIV-infected MOLT-4 lymphocytes. The results show a U-shaped antiviral dose-effect and suggest that hCG may contribute to protection against intrauterine transmission of HIV-1.     

Leucocyte populations and cytokine regulation in human uteroplacental tissues

Human endometrial tissue and the decidualized endometrium in pregnancy contain relatively large numbers of leucocytes, the proportions of which vary during both the menstrual cycle and early pregnancy. CD3+ T-cells, CD14+ macrophages and a population of phenotypically unusual CD3-CD16-CD56++ large granular lymphocytes (LGL) are present, whereas B-cells are virtually absent. Relative T-cell numbers decrease in first-trimester decidua; T-cells are therefore unlikely to have an important role in the immunological maintenance of pregnancy but could be more important in implantation, when their relative numbers are greater. Extensive numbers of class II MHC-positive tissue macrophages in both the endometrium and placenta will provide an immediate antigen non-specific host defence to infection at this important site. Nevertheless, most attention has focused on a role for the predominant LGL endometrial cell population in the implantation and maintenance of pregnancy because, at the time of implantation, these LGLs comprise 70-80% of all leucocytes in the endometrium. It is now well recognized that there is substantial and complex cytokine activity within human uteroplacental tissues; both leucocytic and non-leucocytic cells have been shown to be capable of producing a significant array of cytokines. However, to avoid excessive pathological sequelae, such cytokine activity must be locally regulated. This has been highlighted by recent reports indicating that abnormal Th1-type cytokine responses could be a reason for immunological reproductive failure in women. Key cytokines controlling differentiation into a Thl (interleukin 12) or Th2 (interleukin 4) type pattern both exist in unusual molecular forms at the human maternal-fetal tissue interface and hence might be fundamental regulatory elements controlling cytokine action locally by an antagonistic action.