Follicular fluid progesterone downregulated HPGD and COX2 in granulosa cells via suppressing NF-ĸB in endometriosis†.

Increasing evidences showed that ovulatory dysfunction, possibly caused by luteinized unruptured follicular follicle syndrome (LUFS), is one of the reasons for endometriosis-related infertility. The present study was conducted to explore the potential effect of elevated progesterone in follicular fluid (FF) on ovulation in endometriosis. A prospective study including 50 ovarian endometriosis patients and 50 control patients with matched pairs design was conducted with alterations in FF and peritoneal fluid (PF) components identified by metabolomics analyses and differentially expressed genes in granulosa cells (GCs) identified by transcriptome analysis. Patients with endometriosis exhibited a significantly higher progesterone level in serum, FF, and PF. Granulosa cells from endometriosis patients revealed decreased expression of HPGD, COX-2, and suppressed NF-ĸB signaling. Similarly, progesterone treatment in vitro downregulated HPGD and COX2 expression and suppressed NF-ĸB signaling in granulosa tumor-like cell line KGN (Bena Culture Collection, China) and primarily cultured GCs, as manifested by decreased expressions of IL1R1, IRAK3, reduced pIĸBα/IĸBα ratio, and nucleus translocation of p65. On the contrary, TNF-α treatment increased expression of IL1R1, IRAK3, pIĸBα, p65, and HPGD in GCs. One potential p65 binding site was identified in the promoter region of HPGD by chromatin immunoprecipitation. In conclusion, we found that intrafollicular progesterone might downregulate HPGD and COX-2 in GCs via suppressing the NF-ĸB signaling pathway, shedding light on the mechanism underlying the endometriosis-related ovulatory dysfunction.

High expression of L-selectin ligand in secretory endometrium is associated with better endometrial receptivity and facilitates embryo implantation in human being.

PROBLEM: L-selectin ligand has displayed mediating adhesion at the maternal-fetal interface. Therefore, we investigated the impact of L-selectin ligand on establishing pregnancy in women undergoing in vitro fertilization and embryo transfer (IVF-ET). METHOD OF STUDY: Endometrium between cycle days LH +6 to +9 was obtained from 56 Chinese women referred for IVF and tested for L-selectin ligand by immunohistochemistry and Western blot. The standard gonadotropin-releasing hormone agonist long protocol was used for ovarian stimulation. RESULTS: L-selectin ligand was localized in the endometrial gland and luminal epithelial cells. Western blot analysis of endometrium identified four bands and levels of component 1, 2 and 4 were significantly higher in the pregnancy group than in the non-pregnancy group (P < 0.05). Clinical pregnancy and implantation rates were higher in patients with high level L-selectin ligand compared with those with low level (53.6%versus 25.0%, and 27.1%versus 12.1%, respectively, P < 0.05). CONCLUSION: The presence of higher level L-selectin ligand was associated with a better pregnancy outcome.

[Expression of aquaporin 9 in granulosa cells of patients with polycystic ovary syndrome in IVF-cycles].

OBJECTIVE: To investigate aquaporin 9 (AQP9) mRNA and protein expression in antrum follicle and luteinizing granulosa cells of polycystic ovarian syndrome (PCOS) ovary, and its relation to follicular fluid steroids hormone levels during IVF cycles. METHODS: AQP9 mRNA expression on luteinizing granulosa cells in IVF cycles was detected by RT-PCR. AQP9 protein expression in antrum follicles of PCOS ovary and luteinizing granulosa cells was measured by immunohistochemistry. The concentrations of estradiol (E2), progesterone (P) and testerone (T) in follicular fluid were measured by radioimmunoassay (RIA). RESULT: The expression of AQP9 mRNA in luteinizing granulosa cells during IVF cycles was positive by RT-PCR. No significant differences in AQP9 mRNA levels in granulosa cells between PCOS and control group were found during IVF cycles. The expression level of AQP9 mRNA in large follicles was higher than that in small follicles, but not significantly. The immunoreactivity for AQP9 was localized in membrane and cytoplast of granulosa cells in antrum follicles from PCOS ovary and luteinizing granulosa cells during IVF cycles. Multiple regression analysis showed that AQP9 mRNA levels on granulosa cells were not correlated with E2, P and T levels in follicular fluid during IVF cycles. CONCLUSION: AQP9 may play an important role in the follicle development and antrum formation through water transport and AQP9 may be involved in the mechanism of follicle development in PCOS.

50-Hertz electromagnetic fields induce gammaH2AX foci formation in mouse preimplantation embryos in vitro.

Effects of electromagnetic fields (EMFs) on DNA damage in mammals are still controversial. In the present study, the effects of EMFs on DNA damage in preimplantation mouse embryos in vitro were investigated by using gammaH2AX foci formation, a new sensitive indicator for detecting DNA double-strand breaks (DSBs). The data obtained demonstrated that EMFs decreased the cleavage rate of preimplantation mouse embryos. This decreasing effect of EMFs was related to the DNA-damaging effect indicated by the induction of gammaH2AX foci formation in preimplantation mouse embryos. The inducing effects of EMFs on gammaH2AX foci formation could be inhibited by the treatment of noise MFs or wortmannin, a phosphatidylinositol 3-kinase (PI3K) family inhibitor. Furthermore, the data obtained also showed that EMFs could activate the DNA damage-repair mechanism by recruiting repair factor Rad50 to the damaged DNA sites to repair the corresponding DNA damage. These findings suggest that EMFs could cause DNA damage in preimplantation embryos in vitro and that the adverse effects of EMFs on development might at least partly act through DNA damage. The DNA damage induced by EMFs could be at least partly repaired by the natural activation of DNA damage-repair mechanism or prevented by the simultaneous treatment of noise magnetic fields.

Ovarian stimulation with GnRH agonist, but not GnRH antagonist, partially restores the expression of endometrial integrin beta3 and leukaemia-inhibitory factor and improves uterine receptivity in mice.

BACKGROUND: The impact of different ovarian stimulation (OS) protocols on endometrial receptivity remains controversial. In this study, the effects of different OS on the expression of endometrial integrin beta3 subunit and leukaemia-inhibitory factor (LIF) during the implantation window and the implantation rate in mice were investigated. METHODS: Three OS protocols were used, involving either pregnant mare's serum gonadotrophin (PMSG) alone, PMSG plus GnRH agonist or PMSG plus GnRH antagonist. Uterus samples were collected at 48 h after OS or ovulation and were detected with immunohistochemistry, Western blot and RT-PCR analyses. Normal embryos at gestation day 4 were transferred into the uteri of mice in the control and OS groups. RESULTS: All OS groups showed a significant decrease in the expression of both the endometrial integrin beta3 subunit and LIF during the implantation window and the implantation rate. Among the three OS groups, GnRH agonist-treated mice showed a higher endometrial integrin beta3 subunit and LIF expression and a higher implantation rate. No significant difference was found in the measured indices between the GnRH antagonist and PMSG groups. CONCLUSIONS: OS may inhibit the expression of endometrial integrin beta3 subunit and LIF and impair endometrial receptivity in mice. OS with GnRH agonist, but not GnRH antagonist, may partially restore the endometrial physiological secretion and improve uterine receptivity.

Aquaporin-2 expression in human endometrium correlates with serum ovarian steroid hormones.

The aim of the present study was to examine the expression of aquaporin-2 (AQP2), a member of the water channel family aquaporins (AQPs), in human uterine endometrium and its modulation of ovarian steroid hormone at the proliferative and secretory phases. Western blot, immunohistochemistry, and RT-PCR were employed in the present study. Western blot revealed a 29-kDa band that represented AQP2 in human endometrium. The expression of AQP2 in endometrium was confirmed by RT-PCR and immunohistochemical results. The immunohistochemical analysis demonstrated that AQP2 was prominent in luminal and glandular epithelial cells of endometrium. The levels of endometrial AQP2 expression changed during the menstrual cycle and were higher in the secretory endometrium than in the proliferative endometrium. A significantly high level of AQP2 was detected at the mid-secretory phase. There was a positive correlation between the levels of the endometrial AQP2 expression and the concentrations of the serum 17beta-estradiol (E2) or/and progesterone (P4). These data for the first time corroborate that AQP2 is expressed in human endometrium and that the expression of AQP2 in human endometrium might be regulated by E2 or/and P4. The changed expression of AQP2 at different phases of the menstrual cycle may be essential to reproductive physiology in human. The high level of endometrial AQP2 expression was observed at the mid-secretory phase, the time of embryo implantation, suggesting that AQP2 might play physiological roles in the uterine receptivity.

[Protein and mRNA expression of aquaporin-1 in epithelial ovarian tumors and its clinic significance].

OBJECTIVE: To investigate protein and mRNA expression of aquaporin-1 (AQP1) in epithelial ovarian tumors and its clinic significance. METHODS: The protein and mRNA expressions of AQP1 were measured by immunohistochemical technique, western blot and RT-PCR in 65 cases of epithelial ovarian tumors and 13 cases of normal ovary tissue. RESULTS: AQP1 located in microvascular and small vessel epithelial cells. The protein and mRNA expressions of AQP1 in ovarian cancer (0.39 +/- 0.12, 0.93 +/- 0.51, respectively) and ovarian borderline tumors (0.43 +/- 0.21, 0.95 +/- 0.34, respectively) were significantly higher than that of ovarian benign tumors (0.27 +/- 0.13, 0.51 +/- 0.41, respectively; P < 0.05) and normal ovary tissue (0.24 +/- 0.13, 0.34 +/- 0.29, respectively; P < 0.05). Of all ovarian cancers, expression of AQP1 in cases with ascites more than 1000 ml (0.46 +/- 0.13, 1.25 +/- 0.57, respectively) was higher than that of ascites less than 1 approximately 499 ml (0.35 +/- 0.11, 0.75 +/- 0.45, respectively; P < 0.05). CONCLUSION: Over-expression of AQP1 plays an important role in development of epithelial ovarian tumors, and may be related with formation of ascites of ovarian carcinoma.

Effects of preimplantation embryos on expressions of DNA methyltransferase 1 in mouse oviduct epithelial cells.

OBJECTIVE: To investigate the effects of mouse preimplantation embryos on the expressions of DNA methyltransferase 1(Dnmt1) of mouse oviduct epithelial cells. METHODS: The histological location of Dnmt1 protein was detected by immunohistochemical staining and the expression levels of Dnmt1 mRNA and protein in mouse oviduct were assayed by real-time reverse transcription-PCR(RT-PCR) and Western blotting in both pregnant and pseudopregnant mice at the 2-cell, 4-cell and 8-cell stages. RESULTS: The expressions of Dnmt1 protein were mainly located in the epithelial cells of mouse oviduct. It was found that during all three stages, the expression levels of Dnmt1 mRNA in the epithelial cells of the pregnant mice were significantly lower than those in the pseudopregnant mice (P< 0.05), and the level of Dnmt1 protein expression in the pregnant mice was significantly decreased as compared with that in pseudopregnant mice at the 4-cell stage. CONCLUSION: Expressions of both Dnmt1 mRNA and protein in the epithelial cells of mouse oviduct could be regulated by mouse preimplantation embryos, which might play an important role in the expression changes of some genes in oviduct epithelial cells during the preimplantation period.

[Polymorphism in insulin receptor gene exon 17 in women with polycystic ovary syndrome].

OBJECTIVE: To investigate insulin receptor (INSR) genotype exon 17 frequencies in women with polycystic ovary syndrome (PCOS) and to elucidate its role in the pathogenesis of PCOS. METHODS: The study involved 33 women with PCOS and 28 healthy control women who were genotyped for polymorphism of INSR gene exon 17 by single strand conformation polymorphism (SSCP) analysis. Body mass index (BMI), insulin sensitive index (ISI), the expression of INSR beta subunit, and serum concentration of luteinizing hormone (LH), total testosterone between the genotypes were compared. RESULTS: (1) The T-to-C mutation was observed in the INSR gene exon 17 (1008 bp). The frequency of the C/C genotype was significantly higher in patients (39%) than in the controls (11%) (P < 0.05). There was no significant difference in the distribution of genotypes between obese PCOS and non-obese PCOS, and between PCOS with insulin resistance (IR) and PCOS without IR. (2) In comparison of mutation genotype groups with wild genotype (T/T), ISI was significantly decreased (C/C genotype, P < 0.01; C/T genotype, P < 0.05), and no significant difference was observed in the other indices. CONCLUSIONS: There is polymorphism in INSR gene exon 17 in patients with PCOS. This variant leads to increased risk of IR in women with PCOS. It does not influence the expression of INSR beta subunit.

[Preimplantation genetic diagnosis of chromosome abnormality by fluorescence in-situ hybridization].

OBJECTIVE: To perform preimplantation genetic diagnosis (PGD) of chromosome abnormality using fluorescence in-situ hybridization (FISH). METHODS: Ten couples were presented for preimplantation genetic diagnosis. They had a total of 10 oocyte pick-up cycles. The collected oocytes were inseminated by intracytoplasmic sperm injection. PGD was carried out using cleavage-stage (day 3) embryo biopsy, fluorescence in-situ hybridization, and day 4 embryo transfer. RESULTS: Ten oocyte pick-up cycles yielded 158 oocytes. Among the 94 embryos obtained, 54 embryos were biopsied and FISH analyses were performed for 51 blastomeres. Twenty-four embryos were transferred on the fourth day. There were 4 clinical pregnancies: 3 infants have been born, and 1 couple had ectopic pregnancy. CONCLUSION: PGD is a valuable method to prevent the high risk of spontaneous miscarriages and conceiving chromosomally unbalanced offspring.

[Preimplantation genetic diagnosis: two successful cases]

OBJECTIVE: To establish a technology of preimplantation genetic diagnosis. METHODS: Intracytoplasm sperm injection and blastomere biopsy were performed on two women at the advanced age with the fallopian tube obstruction. Normal embryos were selected for embryo transfer after fluorescence in-situ hybridziation in biopsied blastomere. RESULTS: The levels of serum HCG were increased 20 days after embryo transfer and ultrasonography in 16 gestation weeks showed the fetal growth and structure are normal. CONCLUSION: Two successful clinical pregnancies achieved after preimplantation genetic diagnosis.