Impacts of vitrification on the transcriptome of human ovarian tissue in patients with gynecological cancer.

Objective: This study investigated the effects of a vitrification/warming procedure on the mRNA transcriptome of human ovarian tissues. Design: Human ovarian tissues were collected and processed through vitrification (T-group) and then subjected to RNA sequencing (RNA-seq) analysis, HE, TdT-mediated dUTP nick-end labeling (TUNEL), and real-time quantitative PCR, and the results were compared to those of the fresh group (CK). Results: A total of 12 patients, aged 15-36 years old, with a mean anti-Müllerian hormone level of 4.57 ± 3.31 ng/mL were enrolled in this study. According to the HE and TUNEL results, vitrification effectively preserved human ovarian tissue. A total of 452 significantly dysregulated genes (|log2FoldChange| > 1 and p < 0.05) were identified between the CK and T groups. Among these, 329 were upregulated and 123 were downregulated. A total of 372 genes were highly enriched for 43 pathways (p < 0.05), which were mainly related to systemic lupus erythematous, cytokine-cytokine receptor interaction, the TNF signaling pathway, and the MAPK signaling pathway. IL10, AQP7, CCL2, FSTL3, and IRF7 were significantly upregulated (p < 0.01), while IL1RN, FCGBP, VEGFA, ACTA2, and ASPN were significantly downregulated in the T-group (p < 0.05) compared to the CK group, which agreed with the results of the RNA-seq analysis. Conclusion: These results showed (for the first time to the authors' knowledge) that vitrification can induce changes in mRNA expression in human ovarian tissues. Further molecular studies on human ovarian tissues are required to determine whether altered gene expression could result in any downstream consequences.

Hypoxia and its possible relationship with endometrial receptivity in adenomyosis: a preliminary study.

BACKGROUND: Adenomyosis (AM) is an important cause of female infertility. However, the underlying mechanism remains unclear. This report describes a preliminary study of hypoxia and its possible association with endometrial receptivity in AM. METHODS: The study was divided into in vitro and in vivo experiments. In vitro, expression levels of the endometrial receptivity markers HOXA10 and HOXA11 in the implantation period were examined using real-time PCR and western blotting. Endometrial expression of hypoxia-inducible factor (HIF)-1α, HIF-2α, and HIF-3α was determined using immunohistochemistry. In vivo, using an AM mouse model established by oral administration of tamoxifen, we inhibited expression of HIF-2α using an HIF-2α antagonist (PT2399; 30 mg/kg body weight, twice daily by oral gavage for 2 days) and then examined expression levels of Hoxa10 and Hoxa11 using real-time PCR and western blotting. RESULTS: Endometrial mRNA and protein expression levels of HOXA10 and HOXA11 were significantly lower in patients with AM than in control patients. Expression of HIF-2α was significantly higher in the AM group than in the control group, whereas that of HIF-1α and HIF-3α was equivalent in both groups. In vivo analysis showed that administration of the HIF-2α antagonist resulted in increased expression of Hoxa10 and Hoxa11 at both the mRNA and protein levels in AM model mice. CONCLUSIONS: HIF-2α overexpression may be one reason for decreased endometrial receptivity in AM. The current findings provide insight into HIF-2α-mediated AM-related infertility and suggest that PT2399 has potential as a treatment for AM. TRIAL REGISTRATION: This trial was retrospectively registered.

Preincubation with glutathione ethyl ester improves the developmental competence of vitrified mouse oocytes.

PURPOSE: Oocyte vitrification is currently used for human fertility preservation. However, vitrification damage is a problem caused by decreasing ooplasmic levels of glutathione (GSH). The GSH donor glutathione ethyl ester (GSH-OEt) can significantly increase the GSH content in oocytes. However, it is difficult to obtain oocyte from woman. To overcome this, we used mouse oocytes to replace human oocytes as a model of study. METHODS: Oocytes from B6D2F1 mice were preincubated for 30 min with 2.5 mmol/L GSH-OEt (GSH-OEt group), without GSH-OEt preincubation before vitrification (control vitrification group) or in nonvitrified oocytes (fresh group). After thawing, oocytes were fertilized for evaluating the developmental competence of embryos in vitro and in vivo. Immunofluorescence, Polscope equipment and quantitative reverse transcription polymerase chain reaction (RT-qPCR) were used to analyze damage, including mitochondrial distribution, reactive oxygen species (ROS) levels, spindle morphology, and gene expression levels (Bcl-2, BAX, and MnSOD). RESULTS: The rates of fertilization, 3-4 cell, blastocyst formation and expanded blastocysts were significantly higher (p < 0.05) in the GSH-OEt group (90.4%; 91.1%; 88.9% and 63.0%) than in the control (80.0%; 81.4%; 77.7% and 50.5%). Provided embryos overcame the 2-cell block and developed to the blastocyst stage, birth rates of all groups were similar. Vitrification altered mitochondrial distribution, increased ROS levels, and caused abnormal spindle morphology; GSH-OEt preincubation could improve such damage. RT-qPCR showed that the expression of Bcl-2 was lower in the control group compared with the GSH-OEt group; BAX and MnSoD expression levels were higher in the control group than in the GSH-OEt group (p < 0.05). CONCLUSIONS: The beneficial effect of GSH-OEt preincubation occurred before the 2-cell stage.

Transcriptome analysis of endometrial tissues following GnRH agonist treatment in a mouse adenomyosis model.

PURPOSE: Adenomyosis is a common, benign gynecological condition of the female reproductive tract characterized by heavy menstrual bleeding and dysmenorrhea. Gonadotropin-releasing hormone (GnRH) agonists are one of the medications used in adenomyosis treatment; however, their underlying mechanisms are poorly understood. Moreover, it is difficult to obtain endometrial samples from women undergoing such treatment. To overcome this, we generated an adenomyosis mouse model, which we treated with an GnRH agonist to determine its effect on pregnancy outcomes. We also analyzed endometrial gene expression following GnRH agonist treatment to determine the mechanisms that may affect pregnancy outcome in individuals with adenomyosis. METHODS: Neonatal female mice were divided into a control group, an untreated adenomyosis group, and an adenomyosis group treated with a GnRH agonist (n=6 each). The pregnancy outcome was observed and compared among the groups. Then, three randomly chosen transcriptomes from endometrial tissues from day 4 of pregnancy were analyzed between the adenomyosis group and the GnRH agonist treatment group by RNA sequencing and quantitative reverse transcription polymerase chain reaction (PCR). RESULTS: The litter size was significantly smaller in the adenomyosis group than in the control group (7±0.28 vs 11±0.26; P<0.05). However, the average live litter size was increased (10±0.28 vs 7±0.28; P<0.05) after GnRH agonist treatment. Three hundred and fifty-nine genes were differentially expressed in the GnRH agonist-treated group compared with the untreated group (218 were downregulated and 141 were upregulated). Differentially expressed genes were related to diverse biological processes, including estrogen metabolism, cell cycle, and metabolite biosynthesis. CONCLUSION: GnRH agonist treatment appears to improve the pregnancy outcome of adenomyosis in a mouse model. Besides pituitary down-regulation, other possible mechanisms such as the regulation of cell proliferation may play a role in this. These new insights into GnRH agonist mechanisms will be useful for future adenomyosis treatment.

Associations between insulin resistance, free fatty acids, and oocyte quality in polycystic ovary syndrome during in vitro fertilization.

CONTEXT: Both polycystic ovary syndrome (PCOS) and obesity are associated with specific reproductive health complications, including lower oocyte quality and clinical pregnancy rates in assisted conception cycles, which may be a result of metabolism-induced changes in the oocyte through the microenvironment of follicular fluid. Free fatty acids (FFAs) are important biomedical indicators of abnormal lipid metabolism and have pronounced effects on cells, leading to changes in metabolism, cell growth, and differentiation. OBJECTIVE: Our objective was to determine the effect of FFA metabolism in plasma and follicular fluid on oocyte quality in the women with PCOS undergoing in vitro fertilization. DESIGN AND SETTING: Ninety-three women undergoing in vitro fertilization treatment, including 55 with PCOS and 38 age-matched controls, were recruited. PCOS patients were divided into obese and nonobese subgroups on the basis of their body mass index. MAIN OUTCOME MEASURES: Embryo quality was morphologically assessed, and serum sex hormone and insulin levels were measured. FFAs in plasma and follicular fluid were measured using gas chromatography-mass spectrometry. RESULTS: PCOS was found to be associated with significantly higher LH/FSH, total T, free androgen index (FAI), and lower SHBG levels, independent of obesity(P < .05). Obese women with PCOS had a significantly higher total T level, FAI, fasting insulin, insulin resistance index as determined by homeostasis model assessment for insulin resistance, and lower SHBG levels than the nonobese women with PCOS (P < .05). The embryo fragmentation score was significantly positively correlated with the oleic acid concentration in all PCOS patients (r = 0.22, P = .04, for nonobese patients and r = 0.25, P = .03, for obese patients). CONCLUSIONS: Our findings clearly demonstrated that PCOS is associated with significantly higher FAI and insulin resistance levels and decreased plasma SHBG levels, independent of body mass index. Obese PCOS patients had higher palmitoleic acid and oleic acid levels in both the plasma and follicular fluid than did the control subject and nonobese PCOS patients. Our results indicated that developmental competence is associated with oleic and stearic acid concentrations, which may contribute to the poor pregnancy outcomes in patients with PCOS.