Kristof Chwalisz

Luminal and Glandular Endometrial Epithelium Express Integrins Differentially Throughout the Menstrual Cycle: Implications for Implantation, Contraception, and Infertility

PROBLEM: Integrins belong to a family of cell adhesion molecules that are present on virtually all cells. The temporal and spatial expression of these important proteins on the human endometrium suggests that certain integrins may participate in the cascade of molecular events leading to successful implantation.

Characterization of the functional progesterone receptor in an endometrial adenocarcinoma cell line (Ishikawa): Progesterone-induced expression of the α1 integrin

Endometrial progesterone receptors (PR) are regulated by both estrogen (E2) and progesterone (P) and mediate the expression of specific endometrial proteins. Ishikawa cells are a well-differentiated human endometrial adenocarcinoma cell line, with both estrogen receptors (ER) and PR, regulated in a manner similar to that of normal endometrium. Immunohistochemical and biochemical analyses demonstrate that the concentration of PR is increased by E2 priming and decreased by subsequent treatment with P. Scatchard plot analysis showed a K(d) of 1 nM. On the basis of biochemical analysis, PR concentrations reached approximately 1400 fmol/mg cytosol protein in cells after treatment with E2 (10(-8) M) for 4 days. Immunoprecipitation and Western blot studies revealed the presence of both the 116 kDa and 81 kDa proteins with multiple isoforms of the high molecular weight (MW) protein. Northern blot analysis demonstrated transcriptional control of PR by steroid treatment. These studies demonstrate the coordinate regulation of all PR mRNA species. The functionality of Ishikawa PR was demonstrated by the expression of alpha1beta1 integrin in response to E2 plus P, at the level of transcription and translation. This effect was blocked by the addition of the anti-progestin, RU-486. These studies reconfirm that the Ishikawa cell line is an excellent model for the study of hormonally regulated events in the human endometrial epithelium.

Physiological dilation of uteroplacental arteries in the guinea pig depends on nitric oxide synthase activity of extravillous trophoblast.

The trophoblast invasion of uteroplacental arteries in the guinea pig has been studied by means of electron microscopy and immunohistochemisty. To identify trophoblast cells, smooth muscle cells, and endothelial cells, antibodies against cytokeratins, smooth muscle myosin, desmin, and vimentin were employed. Furthermore, the immunohistochemical expression patterns of nitric oxide synthase isoforms (eNOS, mNOS and bNOS) were studied and were compared with the enzyme histochemical staining for NADPH-diaphorase. Dilation of uteroplacental arteries begins prior to day 30, when trophoblast cells that coexpress endothelial and macrophage nitric oxide synthase can be found in the vicinity of the vessels and replace the surrounding peritoneal mesothelium. Trophoblast invasion of the arterial walls and the subsequent wall destruction are only secondary effects. Starting around day 50, the final steps of pregnancy-dependent vessel modifications involve intraarterial trophoblast adhesion to the endothelium and subsequent replacement of the endothelium by the trophoblast cells. These may centrifugally invade the vessel media eventually forming intraluminal plugs. These findings led us to the conclusion that in the guinea pig pregnancy-induced physiological dilation of the uteroplacental arteries is due to the effect of nitric oxide rather than being caused by trophoblast-induced media destruction.

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.

Modulation of the Action of Chorionic Gonadotropin in the Baboon (Papio anubis) Uterus by a Progesterone Receptor Antagonist (ZK 137.316)

Abstract Signals from the developing mammalian blastocyst rescue the corpus luteum (CL) and modulate the uterine environment in preparation for implantation and early pregnancy. Our previous studies demonstrated that both short- and long-term administration of chorionic gonadotropin (CG) markedly alters the morphology and the biochemical activity of the receptive endometrium. Because the effects of CG were superimposed on a progesterone-primed endometrium, this study was undertaken to determine if the inhibition of progesterone action by progesterone receptor antagonists (PRa) in intact and ovariectomized baboons would alter the action of CG on the endometrium at the time of uterine receptivity. In the short-term hCG-treated baboons, the PRa reduced the epithelial plaque reaction, completely inhibited α-smooth muscle actin (αSMA) expression in stromal fibroblasts, and induced the reappearance of the progesterone (PR) and estrogen (ERα) receptors in epithelial cells. However, this treatment protocol had no effect on the expression of glycodelin in the glandular epithelium. In contrast, glycodelin expression in addition to αSMA was suppressed in the ovariectomized animals. In the long-term hCG-treated baboons, the PRa had a similar effect on both αSMA, PR, and ER. In addition, this treatment also resulted in an inhibition of glycodelin expression in the glandular epithelium. These results indicate that blocking the action of progesterone on the endometrium even for a short period of time has a profound effect on the hCG-induced response in stromal fibroblasts. In contrast, for the diminution of glandular epithelial function in the presence of an ovary requires prolonged inhibition of progesterone action, suggesting a potential paracrine effect on the endometrium from the CL in response to hCG.

Antiprogestins in the induction of labor.

There are maternal or fetal indications for the induction of labor and delivery in approximately 10-15% of term and near-term pregnancies. It is well recognized that the successful induction of labor is largely dependent on the condition of the cervix. If labor induction is performed in the presence of an “unripe” cervix, the duration of labor is prolonged resulting in a high failure rate of induction. As a consequence, there is a significant increase in the overall incidence of instrumental deliveries and cesarean sections as well as a variety of other complications.’ The introduction of prostaglandins, in particular, vaginally or endocervically administered PGEz, into the clinical routine to soften the cervix before labor induction with oxytocin or intravenous PGE, has to be considered as a major advance in obstetrics during the last decade. However, prostaglandin use is often associated with side effects and some studies have also reported an increased risk of hyperstimulation of the uterus and higher fetal heart rate abnormalities after local PGE, application.’ Moreover, in about one third of women prostaglandins are not effective in inducing cervical ripening. Therefore, there is a need for further advances in cervical ripening and labor induction techniques, especially in terms of increased efficacy and reduced risk in uterine hyperstimulation. The ideal method for the induction of labor should replicate the mechanisms governing spontaneous parturition as closely as possible, i . e . , by sequential use of apriming agent with oxytocin. Such a priming treatment should prepare the uterus for labor induction by increasing cervical scores and myometrial responsiveness without stimulating myometrial contractions. During pregnancy, which is characterized by uterine quiescence, the myometrium is highly unresponsive to uterotonic stimuli and the uterine cervix which is firm and rigid remains closed. Toward the end of pregnancy the myometrium becomes more responsive to uterotonic agents, and cervical ripeness, characterized by changes in the shape and consistency of the cervix, improves. The start

Onapristone (ZK 98.299): A potential antiprogestin for endometrial contraception

OBJECTIVESnThe effects of the antiprogestin onapristone (ZK 98.299) on fertility; menstrual cycle length; duration of menses; serum estradiol, progesterone, and cortisol concentrations; and endometrial morphologic features were studied in adult bonnet monkeys.nnnSTUDY DESIGNnFive animals were treated subcutaneously with the vehicle and another nine with either 2.5 (n = 4) or 5 mg of onapristone per animal (n = 5). Treatment was initiated on day 5 of the first treatment cycle, and thereafter onapristone was administered every third day for four to seven consecutive cycles. The females were placed with adult males during the periovulatory period, which was assessed by frequent analysis of serum estradiol concentrations. In the final treatment cycle an endometrial biopsy was performed on day 8 after a midcycle estradiol peak in the ovulatory cycle, or around day 20 if the cycle was anovulatory. Blood samples for estradiol, progesterone, and cortisol measurement were collected every third day, except for the periovulatory period when sampling was more frequent.nnnRESULTSnEach of the five animals treated with the vehicle became pregnant: one in the first, three in the second, and one in the third mated cycle, whereas only one of nine treated with onapristone became pregnant. Four animals treated with 2.5 mg of onapristone for 17 cycles and another four treated with a 5 mg dose for 21 cycles did not conceive. In eight animals that did not conceive the first three treatment cycles of six were ovulatory, and in the remaining two animals two cycles of each were ovulatory. During treatment the mean menstrual cycle length was not altered significantly; however, in one it was shortened and in another two it was prolonged. Similarly, the mean duration of menses was not significantly affected, but in some cycles it was reduced. Moreover, there was only slight bleeding in some treatment cycles. Ovulation occurred in 30 of 45 treatment cycles, including the final treatment cycle during which the biopsy was taken, as indicated by serum estradiol and progesterone concentrations. In some of the ovulatory cycles prolonged treatment suppressed luteal activity; however, in the ovulatory cycles the duration of follicular and luteal phases was not significantly affected. In the anovulatory cycles there was a delayed increase in serum estradiol concentrations, suggesting a partial inhibition of folliculogenesis. In treated animals endometrial growth and development was retarded and rendered out of phase. In animals treated with the higher (5 mg) onapristone does the endometrial glands had partially regressed, the secretory activity was blocked, and stromal compaction was evident. The treatment had no significant effect on serum cortisol levels.nnnCONCLUSIONSnThis study demonstrates that low-dose onapristone treatment throughout the menstrual cycle prevents pregnancy without disturbing the menstrual cycle and ovulation in the majority of cycles. However, anovulation and luteal insufficiency occurred in some animals during prolonged treatment. The contraceptive effect in the ovulatory cycles seems primarily related to the retardation of endometrial development resulting in the inhibition of endometrial receptivity. It appears likely that a dose or treatment regimen of onapristone that will inhibit endometrial receptivity and prevent implantation without affecting the menstrual cycle even on prolonged treatment could be identified.

Chronic low-dose antiprogestin impairs preimplantation embryogenesis, but not oocyte nuclear maturation or fertilization in rhesus monkeys

Continual administration of low doses of the antiprogestin ZK137316 permits ovarian/menstrual cyclicity, but prevents pregnancy in female rhesus monkeys. The sites of contraceptive action remain unknown. This study determined whether chronic, low-dose antiprogestin exposure during follicular development impairs oocyte maturation in vivo, as well as fertilization and preimplantation embryogenesis in vitro. Adult, female rhesus monkeys exhibiting normal menstrual cycles received vehicle (n=9) or 0.03 mg ZK137316 (n=8)/kg body weight i.m. daily for 3 months. Controlled ovarian stimulation with recombinant gonadotropins was initiated in the 3rd month. Oocytes collected from preovulatory follicles were evaluated for nuclear maturity and inseminated in vitro. Preimplantation embryonic development was monitored in vitro. The total number of oocytes and percentage collected at each nuclear stage were similar in both groups. More (P<0.05) atretic oocytes were recovered following antiprogestin relative to vehicle treatment. Fertilization rates and percentages of embryos that progressed to the morula stage were similar between groups, but antiprogestin-treated females exhibited less (P<0.05) normal cleavage. Embryonic development was accelerated by 1 day (P<0.05) from the 16-cell to the morula stage in the antiprogestin group relative to vehicle. Despite this, the majority of embryos became blastocysts within 6 days in vitro in the antiprogestin group, but fewer expanded (P=0.09) and hatched (P<0.05) compared to vehicle. During in vivo treatment with chronic, low-dose antiprogestin, oocytes retained their ability to resume and complete meiosis as well as fertilize following insemination in vitro. However, preimplantation embryogenesis in vitro was impaired, particularly during the later stages of blastocyst development. Thus, antiprogestin exposure during follicular development altered oocyte functions that are critical for normal preimplantation embryogenesis; this may contribute to pregnancy prevention.

Effects of the Progesterone Antagonists Onapristone (ZK 98 299) and ZK 136 799 on Surgically Induced Endometriosis in Intact Rats

The effects of the progesterone antagonists (antiprogestins) onapristone (ZK 98 299) and ZK 136 799 on surgically induced endometriosis were studied in intact female rats. Endometriosis was induced by transplanting homologous endometrium to the parietal peritoneum of the abdominal wall (location A) and to the mesentery of the small intestine (location B). The animals were treated daily for 4 weeks at doses of 0.4 and 2.0 mg onapristone or ZK 136 799. The growth of the endometriosis-like foci was measured with a calliper during both pre- and post-treatment laparotomy. Both antiprogestins exerted inhibitory effects on the growth of the endometriosis-like foci in terms of complete remission. A 40 and 50% remission of endometriosis was observed at each location after the administration of 2.0 mg onapristone, whereas 50 and 63% (location A) and 50 and 75% (location B) remissions were found after the administration of 0.4 and 2.0 mg of ZK 136 799 respectively. ZK 136 799 was also more potent than onapristone in growth inhibition (85 versus 48% for location B) in animals with persistent endometriosis. Growth inhibition of the endometriosis-like foci was confirmed by histology and immunohistochemical staining of the proliferating cell nuclear antigen. The antiprogestins caused a reduction in glandular and luminal epithelial cells in the ectopic endometrium. Both antiprogestins tended to cause a decrease in uterine weight. Unlike the inhibitory effects in the ectopic endometrium, both onapristone and ZK 136 799 exhibited some stimulatory effects on the epithelial cells within the eutopic endometrium. Serum 17 beta-oestradiol concentrations did not vary significantly among the different treatment groups. No antiglucocorticoid effect of the antiprogestins was observed at either dose. This study indicates that the antiprogestins onapristone and ZK 136 799 exhibit antiproliferative effects in the ectopic but not the eutopic endometrium via mechanisms which remain to be established. The better efficacy of ZK 136 799 is more likely caused by its higher antiprogestagenic activity than its partial androgenic activity. These findings may be a further indication of the future potential of antiprogestins such as onapristone and ZK 136 799 in the treatment of endometriosis.