The Risk Factors Analysis And The WT1 And DMC1 Gene In Premature Ovarian Failure

Part ⅠRisks associated with premature ovarian failure in Han Chinese womenContext:Premature ovarian failure (POF) is defined as primary or secondary amenorrhea, infertility, estrogen deficiency and elevated gonadotropin concentration (FSH> 40IU/L) in women younger than 40 years. Women with POF are now recognized to be at an increased risk for premature morbidity and mortality owing to experiencing estrogen deficiency before the median age of natural menopause. The physical consequences of premature hypoestrogen in women with POF include infertility, menopausal symptoms and higher risk of osteoporosis and cardiovascular disorders. In addition, POF also affects psychological health. Multiple causes of POF have been defined, such as genetic, autoimmune, infectious, environmental factors and iatrogenic agents (e.g. pelvic surgery, chemotherapy and radiations). The underlying causes, however, remain unexplained in most cases. The complexity and heterogeneity of POF make diagnosis difficult before irreversible damage to the ovarian reserve. Therefore, identification of risk factors for ovarian damage is of great importance.Objective:The aim of the present study was to analyze the relationship between POF and demographic characteristics, past history, general lifestyle behavior and environmental factors to determine possible risk factors for POF in Han Chinese.Methods:In this case-control study,553 patients with POF and 400 controls were recruited from the Center for Reproductive Medicine, Shandong Provincial Hospital Affiliated to Shandong University between January 2008 and October 2013. Information about participants was recorded on a predefined questionnaire, including demographic characteristics, past history, reproduction, general lifestyle behavior and environmental factors. The binary and categorical logistic regression was used to calculate odds ratios (OR).95% confidence intervals (95% CI). and P-values for each risk factor.Results:A total of 65 (65/544.11.95%) women presented with primary amenorrhea. and 479 (479/544.88.05%) with secondary amenorrhea. The demographic characteristics such as occupation and education showed no significant difference between patients with POF and controls. Housing renovation, exposure to agricultural chemicals and chemical agents were associated with an increased risk of POF (OR= 4.76 [2.98 to 7.61]:OR= 11.56 [5.51 to 24.25]; OR= 4.47 [2.09 to 9.58]). Women who had relatives with abnormal menstrual cycles, a history of mumps and pelvic surgery, including ovarian cystectomy (e.g. endometrial, simple serous and teratoma cysts), unilateral oophorectomy and tubal ectopic pregnancy surgery (e.g. salpingectomy and salpingostomy) had an increased risk for POF (OR= 28.12 [8.84 to 89.46]; OR= 3.26 [2.38 to 4.47]; OR= 5.53 [2.15 to 14.23]). Tea consumption, vegetarian diet and mineral water were associated with a decreased risk of POF (OR= 0.04 [0.03 to 0.07]; OR= 0.27 [0.19 to 0.37]; OR= 0.63 [0.47 to 0.85]).Conclusion:Heredity, pelvic surgery, mumps and exposure to chemical agents were identified as risk factors for POF, whereas vegetarian diet, tea consumption and mineral water drinking were protective. Therefore, genetic consultation could help those women whose relatives manifested an early or premature menopause to avoid the consequences of possible premature ovarian function cessation. Avoidance of exposure to endocrine disrupters and flavonoids intake should be considered.Part ⅡNovel WT1 Missense Mutations in Han Chinese Women with Premature Ovarian FailureContext:Premature ovarian failure (POF) is a heterogeneous disease. Heritability exists but the causes of most cases remain unknown. The Wilms’tumors gene WTl encodes an essential transcription factor, which functions in mammalian urogenital development. In rat, the expression of WT1 in GCs decreased along with the follicular development. WT1 was speculated as a repressor of genes responsible for GCs differentiation. In a recent study, the Wt1+ R394W mice has been shown to be subfertile due to defect in follicle development. Compared to controls, the Wt1+ R394W mice have smaller ovaries and significantly fewer follicles, which is similar to the characteristics of patients with POF. These findings indicate that the WTl gene play a critical role in follicle development and might be a plausible candidate for POF.Objective:To explore the contribution of WTl involved in the etiology of POF. the coding region of WTl gene was screened in patients with POF and vitro experiments were done to explore the effect of novel missense mutants on transcription.Methods:Mutation screening in the WT1 gene in 384 Han Chinese women with non-syndromic POF were performed with direct sequencing. Subsequently, function of missense mutations were predicted by Mutation Taster, Polyphen-2 and SIFT. The wild type and mutant Wt1 was over-expressed in mouse granulosa cells and the expressions of downstream gens (Amh. Fshr, Lhr. Cypl9. Par6h and Cdh1) were detected by quantity real time PCR (qRT-PCR) to determine the causative role of the novel variants identified in patients with POF.Results:By screening the coding region of WT1 gene,6 novel variations were identified, including two missense mutations (p. Pro126Ser in exonl and p. Arg370His in exon7) and four intronic variants (C.647-26C>T, c.647-13G>C, c.647-13G>A in intron1 and c.950+14T>C in intron 4). In vitro experiments showed that in the Wtl-overexpressing differentiated GCs, the expression of Amh, Cdhl, and Par6b was up-regulated while Fshr, Lhr, and Cyp19 down-regulated by Wtl.Both mutations p. Prol26Ser and p. Arg370His repressed the expression of Amh and Cdhl, and induced the expression of Fshr and Cypl9 (P<0.05).Conclusion:The downstream genes(AMH, FSHR, CYP19 and CDH1) are required for granulosa cells (GCs) differentiation and oocyte-GCs interaction. The novel mutations p. P126S and p. R370H found in patients with POF potentially impaired GCs differentiation and oocyte-GCs interaction, which might result in loss of follicles prematurely. Therefore, WT1 is a plausible causal gene for POF.Part IIIMutations in DMC1 are not responsible for premature ovarian failure in Han Chinese womenContext:DNA meiotic recombinase 1 gene, DMC1 is a meiosis-specific gene, encoding a protein essential for meiotic homologous recombination. It participates in the formation of synaptonemal complexes and repair of double-strand breaks at recombination hotspots. Dmc1-/- mice are infertile due to decreased germ cells, failing to form synaptonemal complexes and arresting at the leptotene or zygotene stage in late meiosis I. The phenotypes of Dmc1-/- ovaries are similar to clinical characteristics found in women with POF, which suggest it may be a plausible candidate gene for POF.Objective:This study investigated whether mutations in DMC1 contribute to POF’in Han Chinese women.Methods:A total of 192 Chinese women with idiopathic POF were recruited from the Center for Reproductive Medicine, Shandong Provincial Hospital Affiliated to Shandong University. The coding region of DMC1 gene was screened by direct sequencing. The HapMap-CHB database was applied to compare with the POF population.Results:Two known singlenucleotide polymorphisms (SNPs), rs11570392 in intron 4 and rs4987164 in intron 10, were identified. Comparisons of genotype and allelic frequencies between POF cases and the general population showed no significant differences of the two polymorphisms (P>0.05).Conclusion:These results indicate that mutations in the coding exons of DMC1 are not common in Han Chinese women with POF. Future studies in larger cohorts from different ethnic populations are necessary to determine the role of DMC1 played in POF.Part IVEffects of dehydroepiandrosterone on ovarian reaction and pregnancy outcome in women with poor ovarian responseContext:In assisted reproduction technique (ART) cycles, women with poor ovarian response (POR) produce few oocytes and yield few normal embryos when exposed to maximal gonadotrophin stimulation. The number of oocytes and normal embryos was associated with pregnancy outcome. Dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulphate (DHEA-S) are ubiquitous steroids of primarily adrenocortical reticularis zonal origin. These hormones circulate in high amounts in female reproductive life; however, concentrations fall progressively with age, leading to speculation that replacement of DHEA and DHEA-S in the elderly may have age-retardant effects.Objective:This study investigated the effects of DHEA on ovarian reaction and pregnancy outcome in in vitro fertilization and embryo transfer (1VF-ET) or intra cytoplasmic sperm injection and embryo transfer (ICIS-ET) cycles.Methods:A total 170 women with POR were recruited from the Center for Reproductive Medicine, Shandong Provincial Hospital Affiliated to Shandong University. Patients who agreed to participate in the study began taking micronized DHEA by prescription. 25 mg orally, two times a day. If clinically possible, patients received approximately 12 weeks of DHEA treatment before any post-treatment IVF-ET/ICS1-ET cycles. The patients experienced both their pre-and post-DHEA treatment IVF cycles at our hospital. IVF cycle characteristics of the 170 patients, before and after DHEA treatment, were compared by paired-samples ttests, nonparametric tests and chi-square tests.Results:The average age of the 170 patients was 36. A total of 85 (85/170,50%) women presented with primary fertility, and 85 (85/170,50%) with secondary fertility. After DHEA treatment, the number of> 14mm antral follicles, oocytes,2PN oocytes and normal embryos (5.59±4.09,6.42±5.33,0[0-3],3[1-5] and 0[0-2]) were significantly increased (P<0.05). Compared to before DHEA treatment, the rate of clinical pregnancy was significantly increased and biochemical pregnancy were significantly decreased (P<0.01).Conclusion:After DHEA treatment, more oocytes were produced and the oocytes and embryos were improved. The DHEA therapy could increase the clinical pregnancy rate and decrease the biochemical pregnancy rate.