The feasibility of a less invasive method to assess endometrial maturation--comparison of simultaneously obtained uterine secretion and tissue biopsy.

OBJECTIVE: To compare the assessment of endometrial maturation parameters in endometrial secretion samples obtained by a novel minimally invasive technique with those assessed in tissue biopsies. DESIGN: Prospective study. SETTING: University Hospital. POPULATION: Healthy female volunteers attending a gynaecological outpatient clinic. METHODS: Endometrial secretion fluid and tissue sampling 5 days after a spontaneous ovulation assessed with ultrasound. MAIN OUTCOME MEASURES: Progesterone (P) receptor, Ki-67 expression and the Noyes criteria were used to date endometrial biopsies. In the endometrial fluid samples, glycodelin A (GdA), leukaemia inhibitory factor (LIF) and P levels were analysed, and protein content and electrophoresis patterns were determined. RESULTS: All data were correlated to estradiol (E2) and P serum concentrations. The dating according to histology and immunohistochemical staining patterns correlated significantly with GdA levels (r=0.376, P=0.048) in endometrial fluid samples as well with serum levels of E2 (r=0.568, P=0.001) and P (r=0.408, P=0.023). No correlation was observed between tissue dating and LIF levels and protein content in endometrial fluid samples. CONCLUSIONS: The measurement of GdA in endometrial secretion samples may provide a less invasive method for assessing endometrial maturation in potential conception cycles without disrupting implantation.

Impact of ovarian stimulation on mid-luteal endometrial tissue and secretion markers of receptivity.

The objective of this study was to investigate the effect of ovarian stimulation for IVF on endometrial secretion and tissue markers of receptivity in the mid-luteal phase. In 10 oocyte donors, endometrial secretions and biopsies were sampled 5 days after spontaneous ovulation and oocyte retrieval in consecutive cycles. Four subjects received progesterone in the luteal phase of the stimulated cycles. Mid-luteal endometrial maturation in the stimulated cycle was compared with the spontaneous cycle, by histological dating, Ki-67, oestrogen receptor (ER) and progesterone receptor (PR) expression, secretion levels of leukaemia inhibitory factor (LIF), glycodelin A (GdA) and progesterone, and protein profile. No significant differences in histological markers, expression of Ki-67, PR, ER, secretion protein profiles or concentrations of LIF, GdA, or progesterone were observed when comparing natural with stimulated cycles. Progesterone supplementation of stimulated cycles was associated with significantly lower Ki-67 (P = 0.03) and ER (P = 0.04) expression compared with the non-supplemented stimulated cycle. In this pilot study, ovarian stimulation was not demonstrated to alter the studied markers of endometrial maturation in the mid-luteal phase.

Indoleamine-dioxygenase is expressed in human decidua at the time maternal tolerance is established.

The semi-allogeneic fetus has to be tolerated by the maternal immune system. In mice, it has been shown that inhibiting indoleamine-dioxygenase (IDO) leads to fetal rejection, suggesting a central significance for IDO in establishing maternal tolerance. Consequently, we have analyzed IDO expression in human endometrium and decidua to determine whether it may be of significance in human reproduction. Endometrial (n=60) and decidual (n=68; first and second trimester) tissue samples and isolated cells were analyzed for IDO mRNA and protein expression by real-time PCR, Western blot and immunohistochemistry. IDO expression in the decidua of proven fertile women (n=34) was compared to women presenting with their first pregnancy (n=22) and women with a history of miscarriages (n=12). Expression of IDO was localized in glandular epithelial cells and scattered stromal leukocytes. Expression started at the mid-luteal phase in the menstrual cycle and was high until the second trimester of pregnancy. However, glandular expression of IDO decreased during the second trimester, whereas expression in villous trophoblast started at this time. There were no significant differences in decidual IDO expression between proven fertile women and women presenting with their first pregnancy or women with a history of miscarriages. From the expression pattern we conclude that IDO may play a central role in human pregnancies for the establishment of maternal tolerance of fetal antigens. Thereby, IDO expression may be needed in each pregnancy independently from prior pregnancies, and a history of miscarriage may not reflect a general deficiency in IDO expression.

Estradiol and medroxyprogesterone acetate regulated genes in T47D breast cancer cells.

Many mammary tumors express estrogen receptors (ER) and progesterone receptors (PR), and there is increasing evidence that progestins influence gene expression of breast tumor cells. To analyse the impact of progestins on breast cancer cells, we compared (a) the expression of two cytokines, involved in tumor progression, and searched (b) for differentially regulated genes by a microarray, containing 2400 genes, on T47D breast cancer cells cultured for 6 days with 17beta-estradiol (E2) or E2+medroxyprogesterone acetate (E2+MPA). Lower amounts of PDGF and TNFalpha were found in culture supernatants of E2+MPA treated T47D cells. MPA addition induced a 2.8-3.5-fold increase of the mRNA expression of (a) tristetraprolin, which is involved in the posttranscriptional regulation of cytokine biosynthesis, and (b) zinc-alpha2-glycoprotein and Na, K-ATPase alpha1-subunit, which both resemble differentiation markers of breast epithelium. In contrast, the mRNA expression of lipocalin 2, which promotes matrixmetalloproteinase-9 activity, was decreased five-fold in E2+MPA treated cells. Our data show that the expression of genes from various functional gene families is regulated differentially by E2 and E2+MPA treatment in T47D cells. This suggests that exogenous progestins applied for therapy and endogenous changes of the progesterone levels during the menstrual cycle both influence breast cancer pathophysiology.

Is cervical dilatation during parturition at term associated with apoptosis?

AIMS: Cellular turnover may be involved in remodeling of the cervix during parturition. Therefore, the number and localization of apoptotic and proliferating cells during cervical dilatation at term were determined. METHODS: Biopsy specimens from the lower uterine segment of 36 women undergoing cesarean section with a cervical dilatation of < 2 cm (n = 10), 2- < 4 cm (n = 9), 4-6 cm (n = 8), and > 6 cm (n = 9) were examined for nuclear fragmentation by the TUNEL assay, and for cell survival by the apoptosis-blocking bcl-2. Proliferation was marked by Ki-67, epithelial cells by cytokeratin and leukocytes by CD 45. For quantification of apoptotic and proliferating cells, eight random fields of each specimen stained for TUNEL or Ki-67 were blindly counted by two investigators. For statistical evaluation, 90% confidence intervals based on a Poisson distribution were used; groups with non-overlapping intervals were considered significantly different. RESULTS: Apoptotic cells were found exclusively within the stromal compartment, while bcl-2 was expressed in epithelial cells and leukocytes. Proliferating cells were of stromal and epithelial origin. The number of apoptotic as well as proliferating cells ranged from 0 to 2 cells per high-power field (median number 0) in all groups. The confidence intervals were overlapping for all groups, showing no statistical difference between them. CONCLUSION: Apoptosis does not seem to play a decisive role in the process of cervical dilatation during parturition at term.

Interleukin-11 expression: its significance in eutopic and ectopic human implantation.

Embryo implantation and subsequent decidualization, trophoblast invasion and formation of a functional placenta are crucial for establishment and maintenance of pregnancy. Interleukin-11 signalling has been shown to be obligatory for adequate decidualization and trophoblast invasion in mice. Defects in IL-11 signalling in mice result in trophoblast over-invasion and fetal loss. The pathological situation of human tubal pregnancy resembles that of IL-11Ralpha(-/-) mice concerning these symptoms. As our interest is focused on the human early pregnancy, we compared IL-11 expression at the implantation site of ectopic tubal pregnancy (EP) to 1st and 2nd trimester of normal intrauterine pregnancies (IP), and to the normal cycling endometrium. The mRNA expression of IL-11 and IL-11Ralpha was analysed by semiquantitative RT-PCR. Protein expression was detected by western blotting and immunohistochemistry. IL-11Ralpha is expressed constitutively in all tissue specimens analysed. IL-11 is expressed predominantly during follicular and early luteal phase of the menstrual cycle. In IP, IL-11 expression peaks during the 1st trimester and declines from the beginning of the 2nd trimester onwards. In tubal abortions, IL-11 expression is reduced in comparison to vital EP and IP. Cultured primary endometrial and decidual epithelial cells were analysed for hormonal regulation of IL-11 by enzyme-linked immunosorbent assay and RT-PCR. IL-11 is up-regulated by estrogen and down-regulated by progesterone. Overall, our results indicate that in humans, IL-11 signalling is significantly involved in regulation of trophoblast invasion. In the case of tubal abortion, inadequate IL-11 signalling may therefore result in dysregulation of trophoblast invasion.

Haptoglobin expression and release by rabbit oviduct and endometrium, its localization in blastocyst extra-embryonic matrix and fluid during preimplantation time.

BACKGROUND: Evidence is emerging that haptoglobin, an acute phase protein with immunomodulatory properties, is expressed by the endometrium of various species. The present study describes an in-depth investigation of haptoglobin expression and release in the rabbit reproductive tract and in preimplantation embryos. METHODS: The full-length cDNA sequence of rabbit haptoglobin was determined by rapid amplification of cDNA ends PCR. Haptoglobin expression was studied in the oviductal ampull, and isthmus, endometrium and embryos from the time of ovulation up to adhesion. These results were completed by western blot analysis of reproductive tract secretions and embryonic tissues. RESULTS: cDNA sequencing showed a high homology between rabbit and human haptoglobin (84.1%). In oviductal tissues haptoglobin mRNA is clearly expressed from 6 h post-conception (p.c.) to day 3, and in the uterus on days 5 and 6. In the oviductal fluid highest haptoglobin protein content was found between 6 h p.c and day 2, and in the uterine fluid on days 5 and 6 p.c. Embryos do not express haptoglobin mRNA during preimplantation development. However, considerable amounts of maternal haptoglobin protein were detected in the blastocyst coverings and in blastocyst fluid. CONCLUSIONS: Already during periovulatory time and oviductal passage, high amounts of haptoglobin are present in the microenvironment surrounding the oocyte/embryo. Two days before implantation, again, high haptoglobin levels are detectable in the embryo's environment. The incorporation of haptoglobin into the extra-embryonic matrix may be of particular functional significance.

The cytokine receptor gp130 and its soluble form are under hormonal control in human endometrium and decidua.

The transmembrane protein gp130 plays a central role in cytokine action as a signal transducing receptor subunit common to all interleukin-6 type cytokines. Endometrial tissue obtained from women with a normal menstrual cycle and decidua obtained from women in the first or second trimester of pregnancy were assessed for gp130 by western blotting, immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) analysis. By immunoblotting, two forms of gp130 were detected: one-the soluble form-of approximately 100 kDa and a larger membrane-bound form of approximately 150 kDa. The latter became clearly visible in the mid to late secretory phase and was more pronounced in decidual tissue of second trimester compared to first trimester. Immunohistochemically, gp130 was located in glandular epithelial cells during the mid to late secretory phase, whereas staining in the proliferative phase was rather weak. In first and second trimester decidua, glandular cells were also positively stained. In addition, the invading trophoblast cells were gp130 positive. Soluble gp130 release was measured in the supernatants from primary endometrial and decidual cell cultures by ELISA and reached maximum values in cell cultures without addition of hormones. In cultured endometrial epithelial cells obtained during the proliferative phase of the cycle, the soluble gp130 release increased significantly under combined estradiol/progesterone supplementation which mimics the secretory phase conditions compared to estradiol supplementation alone. In cultured epithelial cells derived from decidual tissue of first trimester of pregnancy, similar effects of hormonal regulation were observed. Our results suggest that the balance between soluble gp130 and its membrane-bound form may play an important role in regulating cytokine action necessary for blastocyst implantation and for further interaction between the decidualized endometrium and the invading trophoblast.

Apoptosis of extravillous trophoblast cells limits the trophoblast invasion in uterine but not in tubal pregnancy during first trimester.

During the first trimester of pregnancy extravillous trophoblast cells (EVT) invade the maternal decidua. Invasion normally is reduced from the second trimester onwards and stops in the inner third of the myometrium. By contrast, in extrauterine tubal pregnancy, trophoblast invasion may even penetrate the tubal wall, which ultimately leads to the rupture of the fallopian tube. Induction of apoptosis of EVT cells, by maternal immune competent cells, may be an important mechanism to limit EVT invasion in uterine pregnancy. Tissue specimens from first and second trimester uterine pregnancy and first trimester tubal pregnancy were analyzed for apoptosis by TUNEL- and M30-staining. By immunohistochemical double labelling, maternal leukocyte subtypes were co-localized to apoptotic cells and in this context, the number of CD56(+)NK cells was analyzed. Our data show that apoptosis is confined to the decidua basalis. Most apoptotic cells are single cytokeratin-positive epithelial cells residing in the stromal compartment. Consequently these cells can only be EVT cells. Maternal leukocytes are not apoptotic. They are located in close contact to apoptotic cells. The number of apoptotic cells in the second trimester (1.8+/-0.7 per cent) is reduced compared to first trimester (5.6+/-0.7 per cent) of uterine pregnancy. In parallel, the number of NK cells declines from first (24.4+/-2.9) to second (12.4+/-1.8) trimester. Furthermore, apoptosis is significantly reduced in ectopic (0.9+/-0.3 per cent) compared to eutopic first trimester pregnancies. Consequently, we suggest that in first trimester uterine pregnancy, induction of EVT cell apoptosis by the maternal immune system is one mechanism to limit EVT invasion. During the second trimester, in parallel to declining numbers of NK cells, the mechanism changes. However, in tubal pregnancy due to differing immunological microenvironments at the ectopic implantation site, apoptosis induction fails, which deleteriously may result in uncontrolled invasion and penetration of the tubal wall.

Endometrial secretion aspiration prior to embryo transfer does not reduce implantation rates.

Analysis of protein patterns in endometrial secretion fluid may offer a relatively non-invasive means of assessing endometrial receptivity during fertility treatment cycles. In order to study the impact of the removal of endometrial secretions on embryo implantation, a prospective matched controlled study was performed. In 66 women undergoing IVF, endometrial fluid was obtained transcervically by aspiration just prior to embryo transfer (study group). Biochemical and ongoing pregnancy rates were compared with 66 control patients matched for stimulation treatment protocol, age, number of collected oocytes and number of high quality embryos. The protein content and uterine fluid protein profile in each sample was determined. Respective biochemical and ongoing pregnancy rates per embryo transfer were 36 and 33% in patients who underwent aspiration of endometrial secretion, compared with 33 and 30% respectively in matched control patients (P = 0.84 and P = 0.85). The protein content in endometrial fluid was sufficient for protein pattern analysis. Uterine fluid aspiration prior to IVF embryo transfer is a safe method for obtaining sufficient material for uterine secretion electrophoresis, thus allowing analysis of protein patterns serving as receptivity markers during treatment cycles. This technique may offer a novel tool for assessing endometrial receptivity during treatment cycles without affecting implantation rates.

Embryo-maternal communication in bovine - strategies for deciphering a complex cross-talk.

Early embryonic development, implantation and maintenance of a pregnancy are critically dependent on an intact embryo-maternal communication. So far, only few signals involved in this dialogue have been identified. In bovine and other ruminants, interferon tau is the predominant embryonic pregnancy recognition signal, exhibiting antiluteolytic activity. However, this is just one aspect of the complex process of embryo-maternal signalling, and a number of other systems are more likely to be involved. To gain a more comprehensive understanding of these important mechanisms, integrated projects involving specialists in embryology, reproductive biotechnology and functional genome research are necessary to perform a systematic analysis of interactions between pre-implantation stage embryos and oviduct or uterine epithelial cells, respectively. State-of-the-art transcriptomic and proteomic technologies will identify reciprocal signals between embryos and their maternal environment and the respective downstream reaction cascades. For in vivo studies, the use of monozygotic twins as recipient animals provides elegant model systems, thus eliminating genetic variability as a cause of differential gene expression. In addition, suitable systems for the co-culture of oviduct epithelial or endometrium cells with the respective embryonic stages need to be established for functional validation of candidate genes potentially involved in the dialogue between embryos and their maternal environment. The knowledge of these mechanisms should help to increase the pregnancy rate following embryo transfer and to avoid embryonic losses. Candidate genes involved in embryo-maternal communication will also be used to define new quality criteria for the selection of embryos for transfer to recipients. Another application is the supplementation of embryotrophic factors or components of embryo-maternal signalling in optimized formulations, such as bioartificial matrices. As a long-term goal, signalling mechanisms identified in bovine will also be functionally evaluated in other species, including the human.

Proteins in the extraembryonic matrix of preimplantation rabbit embryos.

Preimplantation embryos of several species are surrounded by an extraembryonic matrix (often simply named zona pellucida) until briefly before implantation. All signals of the early embryo-maternal dialogue have to pass this matrix and therefore are detectable inside. We investigated the protein pattern of the extraembryonic matrices of 3-6-day-old rabbit embryos by two-dimensional gel-electrophoresis. Using (35)S-methionine incorporation, embryonic proteins were labelled and could be distinguished from maternal proteins. Furthermore, the presence of three different proteins (insulin-like growth factor-binding protein-3, uteroglobin, haptoglobin) within the matrices of day-6 embryos was investigated by Western blot analysis. The pattern and numbers of protein spots detected was clearly dependent on the time of embryonic development. Of all proteins detected, 19.3% and 33% are of embryonic origin (day 5 and day 6, respectively). At day 4 the zona proteins are no longer detectable, reflecting the degradation of the zona pellucida. From day 4 to day 5 proteins detectable within the extraembryonic matrices increase enormously. This demonstrates that embryo-maternal signalling accelerates at least 2 days before implantation. Insulin-like growth factor-binding protein-3, uteroglobin and haptoglobin are part of the early signalling as shown by Western blot analysis. Insulin-like growth factor-binding protein-3 could be detected as one spot at 38 kDa pI 6.1, uteroglobin at 8 kDa pI 6.0 and haptoglobin as two spots/isoforms at 36/38 kDa pI 5.8 and pI 6.0. These results demonstrate that extraembryonic matrices serving as a mailbox are a valuable tool for investigating early embryo-maternal signalling.

Endometrial expression and secretion of interleukin-6 throughout the menstrual cycle.

Previous findings revealing reduced endometrial interleukin-6 (IL-6) mRNA expression in patients with recurrent early abortions gave rise to the analysis of IL-6 synthesis in the human endometrium, and how it relates to concentrations in uterine secretions. Endometrial tissues and uterine secretions were collected from patients undergoing hysterectomy. Expression of IL-6 receptor and gp130 mRNA (n = 28) in total endometrium, of IL-6 mRNA in endometrial epithelial and stromal cells, and CD45-positive leukocytes were investigated by RNAase protection assay throughout the cycle. IL-6 protein was assessed in endometrial tissue by immunohistochemistry (n = 32) and in uterine secretions by enzyme-linked immunosorbent assay (ELISA) (n = 33). IL-6 mRNA was expressed at low levels in the proliferative phase, and expression increased progressively in the secretory phase. The increase was attributed to epithelial and stromal cells and leukocytes. Concentrations of IL-6 protein in endometrial glands and in uterine secretions were low in the proliferative phase, and increased 5-10-fold in the mid- to late secretory phase. mRNA expression of IL-6 receptor and gp130 remained constant in total endometrium throughout the menstrual cycle. High concentrations of IL-6 in the mid-secretory phase, the putative implantation window, and a further increase in the late secretory phase, the premenstrual period, support a role of IL-6 in the regulation of endometrial functions.

Ovarian stimulation for IVF and endometrial receptivity--the missing link.

The contemporary approach to ovarian stimulation for IVF treatment results in supraphysiological concentrations of steroids during the follicular and luteal phases of the menstrual cycle. These sex steroids act directly and indirectly to mature the endometrium, influencing receptivity for implantation. Corpus luteum function is distinctly abnormal in IVF cycles, and therefore luteal support is widely used. Various reasons may underlie the defective luteal phase, including (i) ovarian hyperstimulation per se, (ii) gonadotrophin-releasing hormone (GnRH) analogue co-treatment and (iii) the use of human chorionic gonadotrophin (HCG) to induce final oocyte maturation. The recent introduction of GnRH antagonist co-treatment for the prevention of a premature LH rise during the late follicular phase allows for different approaches to ovarian stimulation for IVF. However, a recent meta-analysis showed that implantation rates may be compromised by using GnRH antagonists in currently employed regimens. The development of endometrium receptive to embryo implantation is a complex process and may be altered by inappropriate exposure to sex steroids in terms of timing, duration and magnitude. New approaches to the assessment of endometrial receptivity are now required. Novel approaches to ovarian stimulation aimed at adjusted GnRH antagonist regimens and achieving a more physiological luteal phase endocrinology are now appearing in the literature and may represent an important step in the improvement of the overall health economics of IVF.

[The implantation receptive luteal phase of the endometrium. On the current status of molecular and cell biology research]. / Die implantationsgerechte Lutealphase des Endometriums. Zum Stand der molekularen und zellbiologischen Forschung.

The biological aim of the differentiation and maturation of endometrial tissue compartments during any menstrual cycle is the achievement of suitable conditions for blastocyst implantation and the establishment of pregnancy. Infertility and early embryonic loss are frequently caused by insufficient endometrial differentiation. Even any incomplete receptivity stage of the luteal phase endometrium will prevent attachment and implantation. We have studied the physiological changes throughout an endometrial cycle to elucidate causes of endometrial insufficiency leading to subfertility or infertility. Up to now, the histological changes described by Noyes et al. are understood as classical diagnostic approaches. However, evidence is accumulating that molecular deficits of endometrial differentiation are by no means detectable histologically, and consequently ask for the research on new diagnostic methods and parameters. There are histochemical localizations of specific protein molecules, adhesion molecules and cytokines, which permit by far more detailed and significant molecular analyses than any classical morphological means could yield. Moreover, there are convincing arguments to use further biochemical assessments on proteins of the uterine secretions as specific diagnostic parameters. The electrophoretical resolution presents typical protein patterns, which in turn can be interpreted as characteristic reflexions of the functional phases of the endometrial cycle. What is demonstrated as the so-called adequate luteal phase protein pattern clearly is the product of the receptive endometrium, reflecting the "implantation window". This is established already two days after ovulation and persists usually eight further days, if the endometrial cycle is undisturbed (15th to 24th day of the cycle).

Effects of trophoblast invasion on the distribution of leukocytes in uterine and tubal implantation sites.

OBJECTIVE: To distinguish endocrine and paracrine influences on leukocyte subpopulations at uterine and tubal implantation sites. DESIGN: Retrospective immunohistochemical study. SETTING: Departments of Anatomy, and Obstetrics and Gynecology, School of Medicine, RWTH University of Aachen, Aachen, Germany. PATIENT(S): Ten women with a viable ectopic pregnancy (EP), 25 women who had undergone elective first-trimester termination of pregnancy, and 4 women who had undergone hysterectomy with adnexectomy. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Quantitative analysis of leukocyte subpopulations at the implantation sites and their corresponding noninvaded tissues, decidual tissue from patients with EP, and tubal mucosa from normal menstrual cycle. RESULT(S): Similar numbers and characteristic distribution patterns of macrophages, T cells, and B cells were found at both normal intrauterine and tubal implantation sites. Natural killer (NK) cells were always absent from tubal mucosa. The number and distribution of leukocytes within decidual tissue from women with EP corresponded to those in the noninvaded decidual compartment in intrauterine pregnancy (IUP). CONCLUSION(S): Leukocyte populations present in the tubal and uterine mucosa are an intrinsic characteristic of these tissues. The distinct leukocyte distribution pattern at the implantation sites suggests that the invading trophoblast exerts a paracrine influence on endometrial and endosalpingeal leukocytes. The absence of natural killer cells from the tubal wall may be one reason for the higher degree of invasiveness of the trophoblast at the tubal implantation site.

Expression and regulation of 17beta-hydroxysteroid dehydrogenase 7 in the rabbit.

We cloned partial cDNA sequences of rabbit 17beta-hydroxysteroid dehydrogenase 1 (17beta-HSD1) and 17beta-HSD7. We analyzed the tissue distribution of 17beta-HSD7 as well as the expression in corpus luteum and endometrium during pseudopregnancy. The obtained cDNA sequence of 17beta-HSD7 coded for all functional regions of the protein and showed 86 and 81% similarity to human and rodent sequences, respectively. The partial sequence of rabbit 17beta-HSD1 was 76 and 82% similar to rodent and human sequences. By Northern analysis 17beta-HSD7 expression was predominantly found in reproductive organs like ovary, oviduct, endometrium, placenta and mammary gland. Substantial expression was also apparent in the heart, stomach and cerebellum. The 17beta-HSD1 could be detected in placenta by reverse transcriptase-polymerase chain reaction (RT-PCR), so far. During rabbit pseudopregnancy 17beta-HSD7 expression was found to be regulated in corpus luteum as well as in endometrium. In the corpus luteum, strongest expression occurred from d10 to d14 of pseudopregnancy (p. hCG) and was downregulated on d16 p. hCG. In endometrium strongest expression of 17beta-HSD7 was found on d6 p. hCG, when the endometrium was differentiated to its implantation permissive status.

Progestins, progesterone receptor modulators, and progesterone antagonists change VEGF release of endometrial cells in culture.

The influences of the synthetic progestin, medroxyprogesterone acetate (MPA), the progesterone receptor modulator J867, and the antagonist ZK137316 were studied in vitro on isolated endometrial epithelial cells, as well as endometrial fibroblasts. We evaluated the expression of estrogen receptor alpha (ER) and the progesterone receptor (PR) by RT-PCR. ER and PR were strongly expressed in the fibroblasts and epithelial cells under treatment with 10(-8) M 17beta-estradiol (E(2)). Treatment with 10(-6) M J867 or ZK137316 upregulated the PR expression as did E(2), in contrast to treatment with 10(-6) M MPA, which caused a downregulation of PR in epithelial cells, but not in fibroblasts. In addition, the vascular endothelial growth factor (VEGF) release into the cell culture medium was analyzed by a VEGF-ELISA. VEGF which plays an important role in angiogenesis, is regulated by steroid hormones as well as hypoxia. E(2) stimulates VEGF release into the medium in both cell types. MPA reduces VEGF release significantly in the fibroblast cell culture, but increases it in the epithelial cell culture. ZK137316, in the presence or absence of E(2), reduces VEGF release in fibroblast cell culture. J867 increases the VEGF production in fibroblasts only in the presence of E(2). Both compounds show no significant effects, compared to E(2), in epithelial cell culture. The different results for the epithelial cells and fibroblasts indicate that the pharmacological effects of PR modulators (PRMs) and progesterone antagonists (PAs) may be cell specific and depend on the presence or absence of partial progestagenic agonistic activities. This observation opens up new perspectives for various clinical applications.

The progesterone antagonist onapristone retards the advanced endometrial transformation after gonadotropin stimulation in rabbits.

Ovarian stimulation with gonadotropins (GN) during human in vitro fertilization and embryo transfer (IVF/ET) therapy alters the ovarian steroid output, especially that of progesterone. As a consequence, endometrial transformation is advanced, and embryo implantation is hampered. This study used the rabbit model to determine if the application of the progesterone antagonist (PA) onapristone (ONA) could retard endometrial development after GN-stimulation. Rabbits were GN-stimulated twice daily with 5 IU FSH and 5 IU LH on 3 consecutive days with a) hMG (n = 10) or b) with a mixture of recombinant FSH and LH (n = 10). The animals were then mated, and hCG was injected i.v. to ensure ovulation. This day is designated as day 0 post coitum (d 0 p.c.). On day 2 p.c., five animals of each group were treated with 20 mg ONA/kg body weight and five with vehicle for control. On d 5 p.c. endometrial transformation was analyzed by morphology, uteroglobin (Ugl)-mRNA expression, and proliferation. Embryos were flushed from the uteri. Their number and morphology was evaluated. The endometrium of GN-stimulated control animals demonstrated very long endometrial glands and narrow stromal septa. Ugl-mRNA expression was restricted to the cells at the bottom of the gland. 17.0 +/- 4.6% (mean +/- SD) of glandular cells and 6.0 +/- 5.3% of luminal epithelial cells proliferated. In ONA-treated animals, endometrial glands were significantly shorter, and the pattern of arborization was less pronounced. Endometrial gland cells and luminal epithelial cells expressed Ugl-mRNA. Furthermore, glandular and luminal cells proliferated with high intensity (38.6 +/- 6.8% and 36.4 +/- 9.3%, respectively). These results indicate that the status of endometrial differentiation was retarded after ONA-treatment. Nevertheless, the embryos of these ONA-treated animals were well developed. In conclusion, after GN-stimulation, ONA treatment retarded the advanced endometrial transformation in rabbits. Therefore, postovulatory administration of a PA might be a possible strategy to modulate the advanced endometrial development in IVF-cycles.

Modulation of endometrial transformation in gonadotrophin-stimulated and unstimulated pseudo-pregnant rabbits: studies with the progesterone receptor antagonist, onapristone.

Advanced endometrial transformation often occurs in IVF and embryo transfer therapy after ovarian stimulation with gonadotrophins. One reason is probably the early rise in peripheral progesterone concentration after ovulation induction. Consequently, we studied in a rabbit model, whether the post-ovulatory application of the progesterone receptor antagonist, onapristone, could prevent such an advancement of endometrial transformation after stimulation with different gonadotrophin preparations. The inhibitory effect of onapristone on the endometrium is dependent upon the strength of ovarian stimulation. In unstimulated animals or animals treated with recombinant LH (nine corpora lutea/animal in both groups), secretory differentiation and proliferation of the endometrium was strongly inhibited by onapristone. After weak ovarian stimulation with a 3:1 mixture of FSH and LH (22 corpora lutea/animal), secretory differentiation was strongly inhibited, while proliferation was enhanced. After strong stimulation with either a 1:1 mixture of FSH and LH, or human menopausal gonadotrophin (HMG; >40 corpora lutea/animal), only limited inhibitory effects of onapristone on secretory transformation or proliferation could be detected. In conclusion, these graded effects of onapristone after stimulation with gonadotrophins, resemble the basic observations from which a therapeutic strategy emerges, to modulate the advanced endometrial transformation which occurs in many IVF patients after ovarian stimulation.