A novel functional variant in Wilms' Tumor 1 (WT1) is associated with idiopathic non-obstructive azoospermia.

Idiopathic nonobstructive azoospermia (INOA) is one of the most severe forms of male infertility, yet its pathophysiology remains unclear. WT1 (Wilms' tumor 1) regulates the polarity of Sertoli cells, thereby playing a critical, indirect role in spermatogenesis. Here, we evaluated WT1 gene variation associates with INOA by assessing its promoter and coding regions in 200 patients diagnosed with INOA and 200 proven-fertile men. Three novel variants in the WT1 coding region were detected only in INOA patients, including two synonymous variants and one missense variant, p.Phe435Leu (p.F435L), which was predicted to be deleterious to protein function. The results of dual luciferase reporter showed that the WT1 p.F435L variant decreases transcription of COL4A1 and WNT4 promoters through a dominant-negative effect. Furthermore, chromatin immunoprecipitation assays revealed that COL4A1 and WNT4 promoter is directly bound by wild-type WT1 protein, but not the p.F435L WT1 variant. Thus, we identified a novel functional variant of WT1 functionally associated with INOA. Mol. Reprod. Dev. 84: 222-228, 2017. © 2017 Wiley Periodicals, Inc.

Significantly lengthened telomere in granulosa cells from women with polycystic ovarian syndrome (PCOS).

PURPOSE: Polycystic ovary syndrome (PCOS) is the most common endocrinopathy among women at reproductive age. However, its etiology remains poorly understood. Recent studies indicated that telomere length was related to PCOS. However, the association between telomere length and PCOS has only been shown in leucocytes and remained controversial across different studies. To clarify the association between telomere length and PCOS, the current study interrogated telomere length not only in leucocytes, but also in follicular granulosa cells, which is essential for folliculogenesis and steroidogenesis. METHODS: Seventy-five patients with PCOS and 81 controls with mechanical infertility undergoing their first in vitro fertilization cycle were enrolled. Their peripheral blood and granulosa cells were collected on the oocyte retrieval day. Telomere length of both leucocytes in the blood and granulosa cells was assayed by quantitative polymerase chain reaction. RESULTS: No significant difference was found in the leucocyte telomere length between controls and PCOS patients (0.99 ± 0.44 vs. 1.00 ± 0.38, p = 0.93). Interestingly, when comparing telomere length in granulosa cells between controls and PCOS subjects, significantly lengthened telomere length was found in PCOS subjects (1.00 ± 0.37 vs. 1.57±0.67, p < 0.0001). After adjustments for age and body mass index, the p value remained significant (p < 0.0001). CONCLUSIONS: This finding reinforced the association between telomere abnormalities and PCOS. Given the importance of telomere length in cellular proliferation, our findings provided novel insights into the pathophysiology of PCOS that abnormalities in telomere length possibly disturb folliculogenesis and subsequently result in PCOS.

The effect of gonadotropin-releasing hormone agonist downregulation in conjunction with hormone replacement therapy on endometrial preparation in patients for frozen-thawed embryo transfer.

Objective: To investigate the effects of combining gonadotropin-releasing hormone agonist (GnRHa) downregulation with hormone replacement therapy (HRT, GnRHa-HRT) on the clinical outcomes of patients undergoing frozen-thawed embryo transfer (FET). Methods: In this retrospective study, we included patients who had FET between January 2018 and December 2022. They were categorized into HRT and GnRHa-HRT groups based on the endometrial preparation protocol. The study compared the clinical outcomes of patients in two groups. Possible factors affecting clinical outcomes were analyzed using univariate analysis. To analyze the impact of two endometrial preparation methods on clinical outcomes, multifactorial logistic regression was performed. Results: The rates of clinical pregnancy (47.31% vs. 59.60%), embryo implantation (37.58% vs. 49.65%), biochemical pregnancy (52.36% vs. 64.31%), and early abortion (7.07% vs. 10.77%) were statistically different between the two groups (p < 0.05). Analysis using multifactorial logistic regression showed that there was a 1.65-fold increase in clinical pregnancy rates (OR = 1.65, 95% CI: 1.29-2.12, p < 0.001) and a 1.55-fold increase in embryo implantation rates (OR = 1.55, 95% CI: 1.27-1.90, p < 0.001) in the GnRHa-HRT group when compared to the HRT group. For blastocyst transfer, the clinical pregnancy and implantation rates of the GnRHa-HRT group were significantly higher than those of the HRT group (OR = 1.75, 95% CI: 1.30-2.37, p < 0.001; OR = 1.73, 95% CI: 1.35-2.21, p < 0.001). Conclusion: In FET cycles, leuprorelin (as a GnRHa) downregulation combined with HRT may improve the clinical outcome of patients compared to the HRT cycle, especially for the clinical pregnancy and embryo implantation rates of patients with blastocyst transfer.

Supplementation with low concentrations of melatonin improves nuclear maturation of human oocytes in vitro.

PURPOSE: Studies in bovine and porcine have indicated that melatonin (MT) could induce meiotic maturation of immature oocytes in vitro. The object of the current study was to investigate if MT could ameliorate human oocytes maturation during rescue in vitro maturation (IVM). METHODS: Two hundred seventy eight germinal vesicle (GV) oocytes and 451 (MI) metaphase I oocytes were vitrified, thawed and then matured in vitro. All the oocytes were randomly allocated into six groups in which the oocytes were cultured in medium supplemented with different concentrations of MT (0, 10(-2), 1, 10(2), 10(4), 10(6) nM) and nuclear maturation was evaluated at 6 h, 12 h, 18 h, 24 h and 48 h of culture. RESULTS: The optimal MT concentration for both GV and MI oocytes was 1 nM. At 24 h of culture, nuclear maturation rate of MI oocytes cultured in 1 nM MT medium was significantly higher than other groups (P < 0.05); Nuclear maturation rate of GV oocytes cultured in 1 nM MT medium was also significantly higher than the control group (P < 0.05). On the other hand, decreased nuclear maturation rate was observed in the high MT concentration group (10(6) nM). CONCLUSIONS: The current study demonstrated that low concentration of exogenous MT could ameliorate nuclear maturation of human oocyte during rescue IVM, while high concentration of MT presented negative effects.

Blastocyst quality and perinatal outcomes of frozen-thawed single blastocyst transfer cycles.

Objective: To investigate the effects of blastocyst quality and morphological grade on the perinatal outcomes in patients undergoing frozen-thawed single blastocyst transfer cycles. Methods: This single-center retrospective cohort study included 2648 singleton neonates resulting from frozen-thawed single blastocyst transfers performed between January 2017 and September 2021. Multivariate logistic regression was performed to evaluate perinatal outcomes for their association with blastocyst quality and morphological parameters. Result: Transfer of a good-quality blastocyst in a frozen-thawed cycle was associated with a lower rate of preterm delivery (PTD, adjusted OR =0.7, 95% CI 0.5-0.9; P=0.020) and a higher likelihood of a male neonate (adjusted OR =1.2, 95%CI 1.0-1.5; P=0.048). Compared with grade C inner cell mass (ICM) blastocyst transfer, grade B ICM (adjusted OR =0.5, 95%CI 0.2-0.9; P=0.027) and grade A ICM (adjusted OR =0.6, 95%CI 0.3-1.5; P=0.290) blastocyst transfers were associated with a lower rate of PTD, which was more evident for grade B ICM. After adjusting for confounders, the likelihood of a male neonate (grade B TE, OR =1.2, 95%CI 1.0-1.5, P=0.037; grade A TE OR =1.9, 95%CI 1.3-28, P=0.002) increased with increasing trophectoderm (TE) quality. Compared with expansion stage 4, the likelihood of a male neonate was 1.5 times greater with transfer of a stage 6 blastocyst (OR =1.5, 95%CI 1.0-2.3; P=0.06), and the risk of small for gestational age (SGA) was greater with transfer of a stage 5 blastocyst (adjusted OR =3.5, 95%CI 1.5-8.0; P=0.004). The overall grading of the blastocyst, expansion stage, ICM grade, and TE grade were not associated with length at birth, birthweight, large for gestational age (LGA), or birth defects (all P>0.05). Conclusions: In frozen-thawed single blastocyst transfer cycles, transfer of a good-quality blastocyst was associated with a lower rate of PTD and a greater likelihood of a male neonate. Transfer of grade B ICM blastocysts decreased the rate of PTD, and TE quality was positively correlated with the likelihood of a male neonate.

Early cumulus cell removal could reduce the available embryo rate in human IVF.

PURPOSE: This study was designed to compare cumulus cell removal at different times and to evaluate their effects on embryo developmental potential and the outcomes of in vitro fertilization (IVF). METHODS: We retrieved 606 IVF cycles with standard long down regulation protocol from January 2010 to December 2010. These cycles were divided into two groups: group A, 364 cycles with high risk of fertilization failure, whose cumulus cells were removed 4 h after short gamete coincubation; group B, 242 cycles as control, whose cumulus cells removal were performed 17 to 18 h after insemination. The epidemiological, clinical, laboratory factors and the outcomes of these cycles were analyzed. RESULTS: The available embryo rate of group A was significantly lower (P = 0.002). There were no significant differences in other laboratory parameters and outcomes between the two groups (P > 0.05). CONCLUSIONS: For patients with high risk of fertilization failure, there could be a risk of compromising the rate of available embryos, if fertilization is judged by the presence of 2 PB by cumulus cell removal only 4 h post-insemination. Therefore, this strategy is not recommended to all IVF cycles and future studies are needed to confirm its reliability.

Good quality blastocyst from non-/mono-pronuclear zygote may be used for transfer during IVF.

Although healthy infants have developed from non- and mono-pronuclear zygotes, the transfer of embryos from non- and mono-pronuclear zygotes is not recommended because there are no proper selection criteria. In the present study, we discuss how to select non- and mono-pronuclear embryos with the highest developmental potential at 19-20 hours post-insemination. We found that the percentage of blastocysts with normal chromosome constitution in non-pronuclear zygotes was slightly higher than in mono-pronuclear zygotes. Non- and mono-pronuclear embryos that were at the 4-cell stage on D2 and/or at the 6- to 8-cell stage on D3 had higher incidence rates of blastocysts with normal chromosome constitutions. We also found higher incidences of blastocysts with normal chromosome constitution on D6 than on D5. The results suggest that if high quality non- and mono-pronuclear zygotes develop to the 4-cell stage on D2 and the 6-to 8- cell stages on D3, along with high quality D6 blastocysts, the incidence of blastocysts with normal chromosome constitution is higher.

Patients with polycystic ovary syndrome have successful embryo arrest.

In this retrospective study, we investigate the relationship between embryo arrest and polycystic ovary syndrome (PCOS) during in vitro fertilization-embryo transfer (IVF-ET). In this study, 667 subjects were enrolled, including 330 patients with PCOS and 337 subjects without PCOS. The subjects underwent in vitro fertilization/intracytoplasmic sperm injection and embryo transfer (IVF/ICSI-ET) cycles at the Reproductive Medical Centre of Henan Provincial Hospital from January 2009 to December 2012. Four protocols were used to stimulate the ovaries, including long protocol, super-long down-regulation protocol, short protocol and antagonist protocol. Oocytes were retrieved using transvaginal ultrasound guidance. Pronuclei were checked on the next morning after IVF/ICSI. Cleavage stage embryo was assessed after 62-66 hours. Women with PCOS had significantly elevated body mass index, basal luteinizing hormone, estradiol and testosterone compared with normal women. Basal Follicle stimulating hormone level in PCOS patients was lower compared with that in control group. After IVF-ET, PCOS patients had more available oocytes than subjects in control group. PCOS patients had slightly lower fertilization rate than the controls in IVF cycles, but in ICSI cycles, fertilization rate in PCOS patients was significantly higher than that in controls. For either IVF or ICSI, the embryo arrest rate was not changed by PCOS. Moreover, there was no significant difference in embryo arrest rate between both groups adopting different stimulation protocols. Interestingly, embryo arrest rate was not correlated with testosterone for patients in PCOS group. The data indicated that patients with PCOS had successful early embryo arrest during IVF-ET.