Expression of Wilms' tumor suppressor gene (WT1) in term human trophoblast: regulation by cyclic adenosine 3',5'-monophosphate.

The Wilms' tumor suppressor gene (WT1), which is deleted in some Wilms' tumors, encodes a zinc finger transcription factor. We studied WT1 messenger ribonucleic acid (mRNA) in human term placenta and cytotrophoblasts differentiating into syncytiotrophoblasts in vitro by RT-PCR. The results suggest that WT1 mRNA is expressed in the trophoblasts in a cell-specific fashion. WT1 mRNA expression has been observed to decline remarkably in trophoblast cells after 72 h, when these cells are morphologically differentiated into multinucleated syncytiotrophoblasts. As it is well known that cAMP as a second messenger plays a significant role in cellular proliferation and differentiation of placental cells, we examined the effect of 8-bromo-cAMP on WT1 mRNA expression in undifferentiated cytotrophoblasts and differentiated syncytiotrophoblasts. We observed that cAMP enhanced WT1 mRNA expression in cytotrophoblasts, but remained ineffective in altering WT1 mRNA in syncytiotrophoblasts. In summary, the results of this investigation demonstrate that the WT1 gene is developmentally regulated during trophoblast differentiation. An involvement of the cAMP-mediated system in regulating the WT1 gene in the trophoblast is suggested.

Human endometrial leukemia inhibitor factor (LIF) secretion and its relationship to sonographic endometrial appearance.

PROBLEM: LIF is believed to be involved in human reproduction. Because little is known about the function of this cytokine in proliferative phase of cycle and because LIF is found to regulate the cellular growth we evaluated the possible relationship between endometrial LIF secretion and endometrial growth. METHOD: The present is prospective, blinded study with clinical and immunobiochemical correlation between endometrial LIF concentration and endometrial ultrasound pattern. Twenty-four patients who were candidates for IVF and oocyte donation are included in this study. At day 10 of their cycle the endometrial biopsy was performed, followed by vaginal sonographic measurements of endometrial thickness and pattern. Endometrial LIF concentration was measured by ELISA in supernatants taken from cultured explants. RESULTS: Endometrial LIF production significantly negatively correlated with endometrial thicknesses (P < 0.05). There was a 4-fold elevation in LIF production when the endometrial thickness was below 5 mm. Strong correlation was found also between endometrial LIF production and sonographic endometrial pattern (P < 0.05). The most significant amount of LIF was found in nonfavorable endometria. CONCLUSION: In the proliferative phase of cycle there is a dynamic relationship between endometrial sonographic appearance and local LIF secretion. Specifically LIF production is negatively correlated with endometrial thickness and pattern. The low amount of cytokine is a normal uteral environment for endometrial development, whereas deregulation of cytokine production toward its overexpression may lead to a strong inhibitory effect on endometrial growth. This finding might have an important clinical implication.

Endometrial leukemia inhibitory factor (LIF) as a possible cause of unexplained infertility and multiple failures of implantation.

PROBLEM: The possible role of leukemia inhibiting factor (LIF) in unexplained infertility and multiple failures of implantation (MFI) was evaluated. METHOD OF STUDY: By a specific and sensitive enzyme-linked immunosorbent assay (ELISA), the in vitro endometrial LIF secretion by explant cultures from women with unexplained infertility (n = 32) was examined. Endometrial samples were obtained on either days 8-11 of the proliferative phase or days 18-21 of the secretory phase. In some subjects (n = 11) an endometrial biopsy was performed twice, both in the proliferative and in the secretory phase of the cycle. The control group consisted of fertile women (n = 17). RESULTS: In fertile women the endometrial LIF secretion was 2.2 times higher in the secretory phase samples than in the proliferative phase samples (mean +/- SEM, 3259 +/- 314 pg in the proliferative phase vs. 7726 +/- 1192 in the secretory phase; P < 0.05). In contrast, infertile women exhibited no such elevation of cytokine production. Moreover, in infertile women with MFI the level of LIF in the secretory phase demonstrated the tendency to decrease (mean +/- SEM, 4953 +/- 1125 pg vs. 2162 +/- 541 pg; P > 0.05). When the amount of cytokine secretion was compared on the same day of the cycle between the two groups of women, the LIF production in fertile women on days 18-21 of the menstrual cycle was 3.5 times greater than in the infertile women with MFI and 2.2 times greater than in women without MFI (P < 0.01 and P < 0.05, respectively). On days 8-11 of the cycle, the level of LIF in these groups did not differ significantly, however, in infertile women the range of distribution of cytokine was largely varied, demonstrating the highest amplitude of variations in subjects with MFI. Analyses of the data for women to whom LIF was examined in both phases of the cycle showed that some subjects (27%) exhibited an elevated amount of LIF in the secretory phase of the cycle. This suggests that in these cases the endometrial factor(s) may be irrelevant to infertility. CONCLUSION: In the majority of infertile women there is a deregulation of LIF production in the endometrium during both the proliferative and the secretory phases of the cycle. The dysfunction of cytokine production is more profound in patients with MFI. The deregulation of endometrial LIF secretion throughout the menstrual cycle may be a possible cause of unexplained infertility and repetitive failures of implantation.

Leukemia inhibitory factor (LIF) production by human decidua and its relationship with pregnancy hormones.

The expression of leukemia inhibitory factor (LIF) by murine uterus was shown to be regulated by maternal hormones and was not dependent on the presence of an embryo. The objective of this study was to investigate whether in humans the secretion of LIF during early pregnancy is under maternal control and whether its production is correlated with pregnancy hormones, progesterone and beta-human chorionic gonadotropin (beta hCG). To exclude the possibility of paracrine interaction of decidua with trophoblast, we examined the secretion of LIF in women with extrauterine pregnancy. The present study was designed as a prospective, blinded, clinical and immunobiochemical study. The endometrial biopsies were performed on 12 women during surgery for ectopic pregnancy. On the same day, the level of progesterone and beta hCG in maternal plasma was examined. LIF concentration was determined in supernatants taken from cultured decidual explants. LIF production by decidual culture explants was found in all women with an ectopic pregnancy (Median 5015 pg, range 1389-19,304 pg). There was no correlation between the LIF production and the term of pregnancy, or with the level of circulated beta hCG (p > 0.05). However, when the concentration of progesterone in circulating plasma was less than 5 ng/ml, the secretion of LIF was 2.3-fold higher as compared to women who had progesterone levels of more than 5 ng/ml (p < 0.01). Therefore, we conclude that LIF is actively produced by human decidua and that the production of this cytokine does not depend on the presence of fetotrophoblast. This study demonstrates for the first time that progesterone downregulates the secretion of LIF in the decidua during early pregnancy.

Differential expression and regulation of inducible nitric oxide synthase (iNOS) mRNA in human trophoblasts in vitro.

OBJECTIVES: To determine the expression of inducible nitric oxide synthase (iNOS) in human trophoblast and to examine the possible regulation of iNOS gene by cytokines. MATERIALS AND METHODS: Total RNA was isolated from: 1) homogenized placental tissue; from 2) isolated and purified cytotrophoblast cells; and 3) cytotrophoblast and syncytiotrophoblast cells treated with cytokines in vitro. RNA was reverse transcribed and amplified by polymerase chain reaction, using specific primers for iNOS. Trophoblast cells were treated in vitro by interferon-gamma (IFN-gamma) in a dose of 10 ng/mL, Interleukin 1beta (IL-1beta) (4 ng/mL) and leukemia inhibitory factor (LIF) (1 ng/mL). Trophoblasts were also subjected to immunocytochemistry using iNOS-specific antibody to detect iNOS protein expression in these cells. RESULTS: The expression of iNOS mRNA was found both in placental tissue and isolated cytotrophoblast cells. In culture, the highly differentiated syncytiotrophoblast expressed more mRNA than cytotrophoblast cells. IFN-gamma and LIF, but not IL-1beta, induced iNOS mRNA expression in trophoblast cells in vitro. The effects of these cytokines on iNOS mRNA were only observed in syncytiotrophoblast cells, but not in cytotrophoblast cells. Immunocytochemical staining confirmed the trophoblast cells as a major source of the iNOS synthase production. CONCLUSIONS: 1) Human trophoblast cells are able to express the iNOS mRNA, hence suggesting a role for NO in placental growth and function. 2) LIF and IFN-gamma, but not IL-1beta, induce the iNOS mRNA expression in syncytiotrophoblast cells in vitro, suggesting possible similar regulatory mechanisms in vivo. 3) This study, for the first time, demonstrates the stimulating effect of LIF on iNOS gene expression in human tissue.

In-vivo administration of progesterone inhibits the secretion of endometrial leukaemia inhibitory factor in vitro.

Human interleukin for DA cells, also called leukaemia inhibitory factor (LIF), is of cardinal importance for successful murine embryo implantation. Recent studies suggest it may also play an important role in human embryo implantation. Our objective was to study the hormonal regulation of the production/secretion of LIF by the human endometrium. Endometrial LIF secretion in specimens obtained from women without ovarian function (n+/-14) at day 10 (4 mg of oestradiol regimen) or day 20 (oestradiol plus 300 mg of progesterone) of a simulated menstrual cycle was examined. LIF was detected in all cultured explants obtained both in the proliferative and secretory phase of the stimulated cycles. The levels of cytokine production by day 10 endometrial culture explants were 5-fold higher than by day 20 endometrial samples (mean+/-SEM, 24.3+/-8.6 versus 4.5+/-2.1 pg/mg, P < 0.01). This suggests that progesterone significantly down-regulates the endometrial LIF secretion. The effect of progesterone on LIF secretion by the endometrium in vitro was also examined. Explants of endometrium obtained from the same patients on day 10 of cycle were treated with 0.5 ng/ml of progesterone in vitro. This progesterone treatment significantly reduced LIF secretion by endometrial explants in vitro (mean+/-SEM, 20.3+/-4.8 versus 10.7+/-2.3 pg/mg, P < 0.05). These results suggest that LIF endometrial production is regulated by progesterone both in-vivo and in-vitro. The possible mechanisms of LIF regulation are discussed briefly.

[In vitro modulation of the production of the cytokine HILDA/LIF, secreted by human endometrial explants: preliminary results]. / Modulation in vitro de la production de la cytokine HILDA/LIF sécrétée par des explants d'endomètre humain: résultats préliminaires.

Human endometrium from fertile women secrete HILDA/LIF in vitro. In endometrial explants from women suffering from an unexplained sterility or women whose fertilized eggs do not implant the in vitro secretion is significantly reduced. HILDA/LIF secretion is increased by IL-1 and decreased by IL-4. CSF-1 has little effects on secretion from fertile women but clearly inhibits secretion from some infertile women. Altogether, the results suggest that HILDA/LIF might be involved in human implantation.

In-vitro endometrial secretion of human interleukin for DA cells/leukaemia inhibitory factor by explant cultures from fertile and infertile women.

Human interleukin for DA cells/leukaemia inhibitory factor (HILDA/LIF) is a cytokine with pleiotropic effects involved in successful murine implantation. We evaluated human uterine HILDA/LIF production by monitoring its in-vitro secretion by endometrial explant cultures obtained from individuals in either normal or pathological conditions. The cytokine secretion was standardized using the day 5:day 1 ratio of HILDA/LIF concentration in supernatants of such cultures, hereby termed HILDA/LIF production index (HLPI). Our results confirmed that HILDA/LIF is secreted by the human endometrium as assessed by secretion at every phase of the cycle in either normal fertile women, or women bearing intrauterine devices. This was also the case for samples obtained from infertile women presenting repeated failures of embryonic implantation or unexplained primary sterility. However, the HLPI were significantly lower in those latter two groups when compared to fertile women. These results suggest an abnormal regulation of HILDA/LIF secretion in such circumstances, and the clinical implication of those data is discussed.

[Abnormal endometrial reactivity to colony stimulating factor 1 and leukemia inhibitory factor dependent female infertility]. / Réactivité anormale de l'endomètre au CSF-1 et stérilité féminine dépendante du LIF.

Maternal LIF is essential for embryo implantation in mice and it may also be the case in humans. We recently reported that endometrial LIF secretion from infertile women presenting repeated failures of embryonic implantation or unexplained sterility was significantly lower than the secretion of explants from fertile women. We now report on the modulation of the endometrial LIF secretion according to the obstetrical status. CSF-1 has little effect or increases LIF secretion from fertile women whereas it inhibits secretion from infertile women with repeated failures of embryonic implantation.