NAT10-mediated N4-acetylcytidine modification is required for meiosis entry and progression in male germ cells.

Post-transcriptional RNA modifications critically regulate various biological processes. N4-acetylcytidine (ac4C) is an epi-transcriptome, which is highly conserved in all species. However, the in vivo physiological functions and regulatory mechanisms of ac4C remain poorly understood, particularly in mammals. In this study, we demonstrate that the only known ac4C writer, N-acetyltransferase 10 (NAT10), plays an essential role in male reproduction. We identified the occurrence of ac4C in the mRNAs of mouse tissues and showed that ac4C undergoes dynamic changes during spermatogenesis. Germ cell-specific ablation of Nat10 severely inhibits meiotic entry and leads to defects in homologous chromosome synapsis, meiotic recombination and repair of DNA double-strand breaks during meiosis. Transcriptomic profiling revealed dysregulation of functional genes in meiotic prophase I after Nat10 deletion. These findings highlight the crucial physiological functions of ac4C modifications in male spermatogenesis and expand our understanding of its role in the regulation of specific physiological processes in vivo.

[Genetic analysis of carriers of complex chromosome rearrangement].

OBJECTIVE: To comprehensively analyze the numbers of involved chromosomes and breakpoints and the clinical phenotypes of the patients with complex chromosome rearrangement (CCR). METHODS: We selected 23 745 patients with abnormal fertility seeking medical care in the Center of Reproductive Medicine of Peking University Third Hospital from 2011 to 2015, and analyzed their peripheral blood chromosomal karyotypes using G-banding, C-banding and fluorescence in situ hybridization (FISH). RESULTS: A total of 28 CCR carriers (0.118%) were detected among the 23 745 patients with abnormal fertility, including 18 males mainly with azoospermia or oligoasthenospermia and 10 females mainly with infertility, recurrent abortion, embryo termination and abnormal birth. Of the 28 cases of CCR, tripartite rearrangement was found in 9 (32.14%), double translocation in 7 (25%) and special translocation in 12 (42.86%). CCR carrier-related chromosomes were all involved but chromosomes 12 and 19, while 2 and 5 were involved most frequently. CONCLUSION: At present, the incidence of CCR is low. CCR carriers with normal phenotypes are often found because of reproductive problems, and their low chance of having a normal baby necessitates the use of preimplantation genetic test to improve the rate of live birth. Due to the diversity of the chromosomes and breakpoints involved in CCR, it is crucial to give each CCR carrier precise genetic counseling.

[Six-sequence-tagged site (STS) versus eight-STS scheme for detection of Y chromosome microdeletions].

OBJECTIVE: To compare the six-sequence-tagged site (STS) with the eight-STS scheme in the detection of Y chromosome microdeletions. METHODS: Using real-time quantitative PCR, we compared the results of the six-STS (sY84, sY86, sY127, sY134, sY254, sY255) scheme with those of the eight-STS (sY84, sY86, sY127, sY134, sY254, sY255, sY145, sY152) scheme in detecting Y chromosome microdeletions. RESULTS: No statistically significant difference was found in the detection rate of the deletion of the azoospermia factor (AZF) regions between the six-STS and eight-STS methods (9.34% ï¼»575/6177ï¼½ vs 8.85% ï¼»542/6122ï¼½, P > 0.05). CONCLUSION: Though the eight-STS scheme increased the detection of AZFd, its detection rate of the AZF region deletion was not significantly different from that of the six-STS method. From the perspectives of experimental operation, economic cost and clinical strategy guidance, the six-STS is better than the eight-STS scheme for the detection of Y chromosome microdeletions.

Single-cell RNA sequencing reveals abnormal fluctuations in human eight-cell embryos associated with blastocyst formation failure.

Infertility has become a global health issue, with the number of people suffering from the disease increasing year by year, and ART offering great promise for infertility treatment. However, the regulation of early embryonic development is complicated and a series of processes takes place, including the maternal-to-zygotic transition. In addition, developmental arrest is frequently observed during human early embryonic development. In this study, we performed single-cell RNA sequencing on a biopsied blastomere from human eight-cell embryos and tracked the developmental potential of the remaining cells. To compare the sequencing results between different eight-cell embryos, we have combined the research data of this project with the data previously shared in the database and found that cells from the same embryo showed a higher correlation. Additionally, the transcriptome of embryos with blastocyst formation failure was significantly different from developed embryos, and the gene expression as well as cell signaling pathways related to embryonic development were also altered. In particular, the expression of some maternal and zygotic genes in the failed blastocyst formation group was significantly altered: the overall expression level of maternal genes was significantly higher in the failed blastocyst than the developed blastocyst group. In general, these findings provide clues for the causes of human embryonic arrest after the eight-cell stage, and they also provide new ideas for improving the success rate of ART in clinical practice.

Meiotic chromatid recombination and segregation assessed with human single cell genome sequencing data.

BACKGROUND: The human oocyte transmits one set of haploid genome into female pronucleus (FPN) while discards the remaining genome into the first polar body (PB1) and the second polar body (PB2). The FPN genome carries an assembly of maternal and paternal genome that resulted from homologous recombination during the prophase of the first meiosis. However, how parental genome has been shuffled and transmitted is difficult to assess by analysing only the progeny's genome. OBJECTIVE: To assess meiotic chromatid recombination and segregation in human oocytes. METHODS: Single cell genome sequencing data of PB1, PB2 and FPN that originated from the same oocyte were used to analyse the human oocyte homologous chromosome interaction and segregation. To analyse whether chromosomes were non-randomly segregated into polar bodies or pronucleus, we analysed the ratio of crossover in PB2 and FPN, and constructed a model to detect the randomness of oocyte chromosome segregation. RESULTS: We found that during oocyte meiosis, in addition to homologous chromosome recombination, there was also a genome conversion phenomenon which generated a non-reciprocal genetic information transmission between homologous chromosomes. We also inferred that during meiosis, DNA breaks and repairs frequently occurred at centromere-adjacent regions. From our data we did not find obvious evidence supporting the crossover number-based or SNP-based meiotic drive in oocytes. CONCLUSION: In addition to the crossover-based recombination, during human oocyte meiosis, a direct genome conversion between homologous chromosomes is used in some oocytes. Our findings are helpful in understanding the specific features of meiotic chromatid recombination and segregation in human oocytes.

The 'normal' range of FMR1 triple CGG repeats may be associated with primary ovarian insufficiency in China.

The aim of this study was to investigate the relationship between normal Fragile X mental retardation gene 1 (FMR1) CGG repeat numbers and primary ovarian insufficiency (POI) occurrence or subsequent resumption of ovarian function. A total of 122 women with POI and 105 controls were followed up and analysed in our centre. The prevalence of premutation and intermediate range of FMR1 CGG repeats in Han Chinese women with POI was only 0.81% (1/122) and 1.64% (2/122), respectively. The risk of POI occurrence for less than 26 CGG repeats and 29 or more CGG repeats in allele1 (smaller allele) was significantly higher than that for 26-28 CGG repeats (odds ratio 13.50, 95% confidence interval: 3.21 to 56.77 and 6.32, 95% confidence interval: 2.49 to 16.09 respectively; both P < 0.001). No significant difference was found in the CGG repeat distribution (<26, 26-28, or ≥29) in FMR1 allele1 between POI cases whose ovarian function resumed and those whose ovarian function did not. It is suggested that the CGG repeat number in allele1, but not that in allele2 (longer allele), was significantly associated with POI occurrence (P < 0.001). Fewer than 26 or more than 28 CGG repeats in FMR1 allele1 were both risk factors of POI occurrence.

Human single follicle growth in vitro from cryopreserved ovarian tissue after slow freezing or vitrification.

STUDY QUESTION: What is the effect of human ovarian tissue cryopreservation on single follicular development in vitro? SUMMARY ANSWER: Vitrification had a greater negative effect on growth and gene expression of human ovarian follicles when compared with fresh follicles. WHAT IS KNOWN ALREADY: For human ovarian cortex cryopreservation, the conventional option is slow freezing while more recently vitrification has been demonstrated to maintain good quality and function of ovarian tissues. STUDY DESIGN, SIZE, DURATION: Ovarian tissues were collected from 11 patients. For every patient, the ovarian cortex was divided into three samples: Fresh, slow-rate freezing (Slow) and vitrification (Vit). Tissue histology was performed and follicles were isolated for single-cell mRNA analysis and in vitro culture (IVC) in 1% alginate for 8 days. PARTICIPANTS/MATERIALS, SETTING, METHODS: Follicle morphology was assessed with hematoxylin-eosin analysis. Follicles were individually embedded in alginate (1% w/v) and cultured in vitro for 8 days. Follicle survival and growth were assessed by microscopy. Follicle viability was observed after Calcein-AM and ethidium homodimer-I (Ca-AM/EthD-I) staining. Expression of genes, including GDF9 (growth differentiation factor 9), BMP15 (bone morphogenetic protein 15) and ZP3 (zona pellucida glycoprotein 3) in oocytes and AMH (anti-Mullerian hormone), FSHR (FSH receptor), CYP11A (cholesterol side-chain cleavage cytochrome P450) and STAR (steroidogenic acute regulatory protein) in GCs, was evaluated by single-cell mRNA analysis. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 129 follicles were separated from ovarian cortex (Fresh n = 44; Slow n = 40; Vit n = 45). The percentage of damaged oocytes and granulosa cells was significantly higher in both the Slow and Vit groups, as compared with Fresh control (P< 0.05). The growth of follicles in vitro was significantly delayed in the Vit group compared with the Fresh group (P< 0.05). Both slow freezing (P< 0.05) and vitrification (P< 0.05) down-regulated the mRNA levels of ZP3 and CYP11A compared with Fresh group, while there was no significant difference between the Slow and Vit groups (P> 0.05). Vitrification also down-regulates AMH mRNA levels compared with Fresh group (P< 0.05). LIMITATIONS, REASONS FOR CAUTION: Only short-term IVC studies (8 days) are reported. Further study should be performed to examine and improve follicular development in a long-term culture system after cryopreservation. WIDER IMPLICATIONS OF THE FINDINGS: This is the first comparison of gene expression and growth of single human ovarian follicles in vitro after either slow freezing or vitrification. With the decreased gene expression and growth during IVC, damage by cryopreservation still exists and needs to be minimized during the long-term IVC of follicles in the future for eventual clinical application. STUDY FUNDING/COMPETING INTERESTS: This work was supported by the National Natural Science Foundation of China (31230047, 81571386, 81471508, 31429004 and 81501247), National Natural Science Foundation of Beijing (7142166) and Mega-projects of Science Research for the 12th five-year plan (2012ba132b05). There are no conflicts of interest to declare.

Gonadotropin-mediated dynamic alterations during bovine oocyte maturation in vitro.

Gonadotropins have been widely used in human-assisted reproduction and animal science for the past four decades. However, the effects of gonadotropins on oocyte maturation at the molecular and biochemical levels are poorly understood. To determine the effects of gonadotropins (recombinant follicle stimulating hormone and urinary human menopausal gonadotropin) on oocyte maturation, we used the bovine oocyte in vitro maturation model. First, we studied the effects of increasing gonadotropin concentrations on nuclear maturation and mitochondrial function in oocytes. Gonadotropins at concentrations of 0.075 and 0.75 IU/ml improved nuclear maturation and increased inner mitochondrial membrane potential and ATP levels; however, there were no beneficial effects at concentrations of 7.5 and 75 IU/ml. Second, we studied the effects of increasing gonadotropin concentrations on the status of methylation in matured (MII) oocytes. Aberrant methylation and demethylation of H19, SNRPN, and PEG3 genes were observed in MII oocytes at all concentrations except 0.075 IU/ml. The expression of genes that function in spindle formation, cell cycle control, and methylation was also downregulated by high gonadotropin concentrations. In conclusion, we established the optimal gonadotropin concentration (i.e., 0.075 IU/ml) to be used for bovine oocyte in vitro maturation studies. These results may provide a guide for clinical stimulation protocols and help to reduce the risks associated with gonadotropin administration during in vitro fertilization treatment.

Basic fibroblast growth factor promotes the development of human ovarian early follicles during growth in vitro.

STUDY QUESTION: What is the effect of basic fibroblast growth factor (bFGF) on the growth of individual early human follicles in a three-dimensional (3D) culture system in vitro? SUMMARY ANSWER: The addition of 200 ng bFGF/ml improves human early follicle growth, survival and viability during growth in vitro. WHAT IS KNOWN ALREADY: It has been demonstrated that bFGF enhances primordial follicle development in human ovarian tissue culture. However, the growth and survival of individual early follicles in encapsulated 3D culture have not been reported. STUDY DESIGN, SIZE, DURATION: The maturation in vitro of human ovarian follicles was investigated. Ovarian tissue (n= 11) was obtained from 11 women during laparoscopic surgery for gynecological disease, after obtaining written informed consent. One hundred and fifty-four early follicles were isolated by enzymic digestion and mechanical disruption. They were individually encapsulated into alginate (1% w/v) and randomly assigned to be cultured with 0, 100, 200 or 300 ng bFGF/ml for 8 days. PARTICIPANTS/MATERIALS, SETTING, METHODS: Individual follicles were cultured in minimum essential medium α (αMEM) supplemented with bFGF. Follicle survival and growth were assessed by microscopy. Follicle viability was evaluated under confocal laser scanning microscope following Calcein-AM and Ethidium homodimer-I (Ca-AM/EthD-I) staining. MAIN RESULTS AND THE ROLE OF CHANCE: After 8 days in culture, all 154 follicles had increased in size. The diameter and survival rate of the follicles and the percentage with good viability were significantly higher in the group cultured with 200 ng bFGF/ml than in the group without bFGF (P < 0.05). The percentage of follicles in the pre-antral stage was significantly higher in the 200 ng bFGF/ml group than in the group without bFGF (P < 0.05), while the percentages of primordial and primary follicles were significantly lower (P < 0.05). LIMITATIONS, REASONS FOR CAUTION: The study focuses on the effect of bFGF on the development of individual human early follicles in 3D culture in vitro and has limited ability to reveal the specific effect of bFGF at each different stage. The findings highlight the need to improve the acquisition and isolation of human ovarian follicles. WIDER IMPLICATIONS OF THE FINDINGS: The in vitro 3D culture of human follicles with appropriate dosage of bFGF offers an effective method to investigate their development. Moreover, it allows early follicles to be cultured to an advanced stage and therefore has the potential to become an important source of mature oocytes for assisted reproductive technology; particularly as an option for fertility preservation in women, including patients with cancer. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Basic Research Program of China (2011|CB944504, 2011CB944503) and the National Natural Science Foundation of China (81200470, 81000275, 31230047, 8110197). There are no conflicts of interest to declare.

ART do not increase the risk of Y-chromosome microdeletion in 19 candidate genes at AZF regions.

Y-chromosome microdeletions (YCMs) have been found at a much higher rate in infertile men than fertile controls. A specific deletion in the azoospermia factor locus (AZF) at Yq11 is significantly associated with male infertility. Whether assisted reproductive technology (ART) increases the risk of YCM in ART-derived offspring remains unclear. In this study the occurrence of YCM in 199 fathers and their 228 sons (Chinese, Han ethnicity), including 85 offspring conceived by IVF, 73 by intra-cytoplasmic sperm injection (ICSI) and 70 by natural conception, was investigated. Nineteen candidate genes related to YCM were analysed by multiplex ligation-dependent probe amplification. We identified one de novo YCM from 70 naturally-conceived offspring and none from 158 ART-conceived offspring and found no statistical significance between these two groups. There was no statistically-significant difference in the detection rate of the father's Y-chromosome microdeletion group: IVF 10.7% (8/75), ICSI 3.2% (2/63), natural conception 8.2% (5/61). These results suggest that ART does not increase the risk of YCM in male offspring.

Metabolic heterogeneity of follicular amino acids in polycystic ovary syndrome is affected by obesity and related to pregnancy outcome.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a heterogeneous endocrine disorder frequently accompanied by obesity and by insulin resistance, and patients with this syndrome suffer from infertility and poor pregnancy outcome. Disturbances in plasma amino acid (AA) metabolism have been implicated in women with PCOS. However, direct evidence on follicular AA metabolic profiles in PCOS patients and their relationship with pregnancy outcome is sparse. METHODS: We conducted a prospective study in 63 PCOS patients and 48 controls in the Division of Reproductive Center, Peking University Third Hospital. Follicular AA levels were measured by the liquid chromatography-tandem mass spectrometric method, and the results were analyzed based on different grouping criteria. RESULTS: The levels of aromatic amino acid (AAA) increased in PCOS patients independent of obesity (P < 0.05), whereas the levels of branched-chain amino acid (BCAA), glutamic acid, phenylalanine, alanine, and arginine increased with body mass index irrespective of the PCOS status (all P < 0.05). In addition, compared with non insulin resistant-PCOS patients and controls, insulin resistant-PCOS group had higher levels of leucine, valine and glutamic acid (all P < 0.05). In PCOS group, aspartic acid and serine levels were elevated in pregnant patients compared with the non-pregnant subjects (both P < 0.05). Moreover, the levels of BCAA and valine were higher in the non-pregnant group than in the pregnant group (both P < 0.05). The pregnancy rate (45.00%) of subjects with elevated BCAA level was significantly lower than that (66.67%) in control subjects (P = 0.036) at a BCAA cutoff value of 239.10 µM, while the abortion rate was much higher (33.33% versus 2.78%, P = 0.004). CONCLUSIONS: Both PCOS and obesity were accompanied by follicular AA metabolic disturbances, with obesity exerting a more pronounced effect on AA metabolic profiles. The disruptions in specific AAs in the follicular fluid might account for the inferior pregnancy outcome in obese patients and increased risk of abortion in PCOS patients.

Improvement in the quality of heterotopic allotransplanted mouse ovarian tissues with basic fibroblast growth factor and fibrin hydrogel.

STUDY QUESTION: Does basic fibroblast growth factor (bFGF) in combination with fibrin hydrogel improve follicle development and revascularization of heterotopically transplanted mouse ovarian tissues? SUMMARY ANSWER: Treatment of transplanted ovarian tissues with higher concentrations (75, 100 and 150 µg/ml), but not lower concentrations (25 and 50 µg/ml), of bFGF significantly improved primordial follicle survival and angiogenesis, while apoptosis of follicles and stromal cells was significantly decreased. WHAT IS KNOWN ALREADY: Use of transplanted ovarian tissues in female fertility preservation is limited by the massive loss of follicles and ischemia-reperfusion injury due to the expected delay in revascularization. STUDY DESIGN, SIZE AND DURATION: Ovarian tissues from 18-day-old ICR mice were encapsulated in fibrin hydrogel mixed with different concentrations of bFGF, then transplanted under the skin of adult female mice for 1 week. The ovarian tissues treated without fibrin hydrogels and bFGF were designated as Control group I, and the ovarian tissues treated with fibrin hydrogels but without bFGF were designated as Control group II. The ovarian tissues treated with 25 and 50 µg/ml bFGF were designated as the lower concentration group, and the ovarian tissues treated with 75, 100 and 150 µg/ml bFGF were designated as the higher concentration group. MATERIALS, SETTING AND METHODS: Assessment of follicular quantity and follicle classification was carried out by histologic analysis. Follicle proliferation was evidenced by immunostaining with proliferating cell nuclear antigen and apoptosis was verified by anti-active caspase-3 staining. Epithelial cells of new blood vessels were stained using CD31 antibody to evaluate neoangiogenesis, and the blood vessel density was analyzed by immunohistochemistry. MAIN RESULTS AND THE ROLE OF CHANCE: The ovarian tissues were recovered 1 week post-transplantation. Compared with the control group, the survival and proliferation of the follicles was significantly increased, the apoptosis of follicles and stromal cells was significantly decreased, and angiogenesis was significantly enhanced when the transplanted ovarian tissues were treated with a higher concentration of bFGF. Treatment with a lower concentration of bFGF did not improve follicle survival and blood revascularization. LIMITATIONS, REASONS FOR CAUTION: The results obtained may not be fully extrapolated to humans because of the physiologic differences between mice and humans. WIDER IMPLICATIONS OF THE FINDINGS: For the first time, the present study investigated the role of bFGF in transplanted ovarian tissues and demonstrated that bFGF might significantly improve the quality of transplanted ovarian tissues by increasing follicle quantity and promoting neoangiogenesis. This study sets the stage for further study and application of ovarian tissue transplantation in clinics, and may eventually benefit females for fertility preservation. STUDY FUNDING/COMPETING INTEREST(S): This work was partially supported by the Ministry of Science and Technology of China Grants (973 Program; 2011CB944503 to Q.J.), the Program for Changjiang Scholars and Innovative Research Team in University of Ministry of Education of China (30825038 to Q.J.), and the National Natural Science Funds for Young Scholar (81200470 to Y.J. and 81000275 to Y.L.Y.). None of the authors have any conflicts of interest.

Y chromosome AZFc microdeletion may not affect the outcomes of ICSI for infertile males with fresh ejaculated sperm.

PURPOSE: To explore whether the presence of a Y chromosome AZFc microdeletion confers any adverse effect on the outcomes of intracytoplasmic sperm injection (ICSI) with fresh ejaculated sperm. METHODS: A total of 143 oligozoospermia patients with Y chromosome AZFc microdeletion in ICSI cycles in a five-year period were studied. Infertile men with normal Y chromosome in ICSI at the same time-frame were used as controls matched to the study group for age of female, female's body mass index, male's age, infertility duration and number of oocytes retrieved. Retrospective case-control study was used. RESULTS: There were no significant differences between groups in clinical outcomes of endometrial thickness, transferred embryos, good embryo rates, implantation rates, biochemical pregnancy rates, clinical pregnancy rates, ectopic pregnancy rates, miscarriage rates, preterm birth rates, the ratio of male and female babies, newborn body height, newborn weight, low birth weight and birth defects (P > 0.05). Patients with Y chromosome AZFc microdeletion had a lower fertilization rate (61.8 % vs. 67.8 %, P < 0.05) and higher cleaved embryo rate (94.0 % vs. 88.1 %, P < 0.05). CONCLUSIONS: ICSI clinical outcomes for oligozoospermic patients with Y chromosome AZFc microdeletion are basically comparable to that of infertile patients with normal Y chromosomes. The results of ICSI were not affected by the AZFc deletion. Preimplantation genetic diagnosis (PGD) before ICSI for Y chromosome AZFc microdeletion may not be a justifiable regular procedure if the couples didn't care the vertical transmission of Y chromosome deletion.

Identification of risk genes in Chinese nonobstructive azoospermia patients based on whole-exome sequencing.

Nonobstructive azoospermia (NOA) is a severe condition in infertile men, and increasing numbers of causative genes have been identified during the last few decades. Although certain causative genes can explain the presence of NOA in some patients, a proportion of NOA patients remain to be addressed. This study aimed to investigate potential high-risk genes associated with spermatogenesis in idiopathic NOA patients by whole-exome sequencing. Whole-exome sequencing was performed in 46 male patients diagnosed with NOA. First, screening was performed for 119 genes known to be related to male infertility. Next, further screening was performed to determine potential high-risk causative genes for NOA by comparisons with 68 healthy male controls. Finally, risk genes with high/specific expression in the testes were selected and their expression fluctuations during spermatogenesis were graphed. The frequency of cystic fibrosis transmembrane conductance regulator (CFTR) gene pathogenic variant carriers was higher in the NOA patients compared with the healthy controls. Potential risk genes that may be causes of NOA were identified, including seven genes that were highly/specifically expressed in the testes. Four risk genes previously reported to be involved in spermatogenesis (MutS homolog 5 [MSH5], cilia- and flagella-associated protein 54 [CFAP54], MAP7 domain containing 3 [MAP7D3], and coiled-coil domain containing 33 [CCDC33]) and three novel risk genes (coiled-coil domain containing 168 [CCDC168], chromosome 16 open reading frame 96 [C16orf96], and serine protease 48 [PRSS48]) were identified to be highly or specifically expressed in the testes and significantly different in the 46 NOA patients compared with 68 healthy controls. This study on clinical NOA patients provides further evidence for the four previously reported risk genes. The present findings pave the way for further functional investigations and provide candidate risk genes for genetic diagnosis of NOA.

Association between maternal MTHFR C677T/A1298C combination polymorphisms and IVF/ICSI outcomes: a retrospective cohort study.

STUDY QUESTION: What are the roles of maternal 5,10-methylenetetrahydrofolate reductase (MTHFR) C677T/A1298C combination polymorphisms on the embryological and clinical outcomes of IVF/ICSI? SUMMARY ANSWER: Our study reveals for the first time that the oocyte maturation potential gradually decreases with a reduction of maternal MTHFR activity determined by combined C677T/A1298C polymorphisms, while embryo quality was worse in women with intermediate MTHFR activity. WHAT IS KNOWN ALREADY: Although many previous studies have explored the association between MTHFR polymorphisms and IVF/ICSI outcomes, the results remain contradictory due to inadequate samples, no adjustment for potential confounders and/or the study of C677T and A1298C separately. Few studies have systematically investigated the exact role of MTHFR activity determined by combined C677T/A1298C polymorphisms on the embryological and clinical outcomes of IVF/ICSI. STUDY DESIGN SIZE DURATION: This is a retrospective cohort study investigating 1160 women who were referred for MTHFR genotyping and IVF/ICSI treatment at Peking University Third Hospital from May 2017 to May 2020. PARTICIPANTS/MATERIALS SETTING METHODS: Women who were referred for MTHFR genotyping and their first IVF/ICSI treatment at our hospital were included and those undergoing preimplantation genetic testing cycles were excluded. The included women were divided into different cohorts according to their C677T, A1298C and combined C677T/A1298C genotypes. The embryological outcomes, including oocytes retrieved, metaphase II oocytes, oocyte maturation rate, normal fertilization rate and transplantable embryo rate, were evaluated by generalized linear regression models. The clinical outcomes, including biochemical pregnancy rate, clinical pregnancy rate and live birth rate, were evaluated by log-binomial regression models. All outcomes were adjusted for potential confounders. MAIN RESULTS AND THE ROLE OF CHANCE: Women with the combined 677TT/1298AA genotype (hereafter abbreviated as TT/AA, as with other combined genotypes), whose enzyme activity was the lowest, had a lower oocyte maturation rate compared with those with the wild-type genotype (P = 0.007). Moreover, the oocyte maturation rate decreased linearly with the decline in MTHFR enzyme activity determined by combined C677T/A1298C genotypes (P-trend = 0.001). The combined CC/AC, CC/CC&CT/AA and CT/AC genotypes with intermediate enzyme activity were associated with a lower transplantable embryo rate (P = 0.013, 0.030 and 0.039, respectively). The differences in clinical outcomes between women with wild-type genotype and combined C677T/A1298C variant genotypes were not significant. LIMITATIONS REASONS FOR CAUTION: Our study population had comparable embryological outcomes but worse clinical outcomes than other women undergoing IVF/ICSI treatment at our hospital. Therefore, the results related to the clinical outcomes should be generalized with caution. In addition, we did not detect the folate concentration of each patient during pregnancy. However, this might not have much influence on our results because almost all of our study participants took sufficient folic acid around pregnancy. WIDER IMPLICATIONS OF THE FINDINGS: We provide a holistic view of the effect of MTHFR C677T and A1298C polymorphisms on the IVF/ICSI outcomes, which can contribute to providing reasonable folic acid supplementation suggestions for women with different MTHFR genotypes, especially for those with a low oocyte maturation rate and/or low embryo quality. STUDY FUNDING/COMPETING INTERESTS: This work was funded by the National Natural Science Foundation of China (31871447, and 82101677), the National Key Research and Development Program (2019YFA0801400) and the Natural Science Foundation of Beijing Municipality (7202226). The authors declare that they have no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Gonadotrophin-induced paracrine regulation of human oocyte maturation by BDNF and GDNF secreted by granulosa cells.

BACKGROUND: In mammalian ovaries, diverse paracrine factors have been identified to mediate or modulate LH-induced changes during ovulation. Due to the difficulty in obtaining non-stimulated granulosa cells during IVF, little is known about the LH-induced paracrine factors in the human ovary. Based on earlier studies using murine ovarian cells showing the paracrine roles of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) in promoting oocyte maturation, we investigated the expression of these ligands in human granulosa cells and their regulation of human oocyte development. METHODS: Non-stimulated granulosa cells were obtained from non-stimulated IVM (in vitro maturation) patients after oocyte retrieval. Women undergoing non-stimulated IVM treatment at a mean age of 30.8 ± 1.3 (n = 10) were recruited for this study. Immature oocytes and granulosa cells were collected from IVF patients undergoing gonadotrophin stimulation and ICSI. Immunocytochemical analyses of granulosa cells were carried out to investigate expression profiles of BDNF and GDNF, together with real-time RT-PCR to analyze the gonadotrophin regulation of BDNF and GDNF transcript levels. In addition, immature oocytes were cultured to analyze the regulation of oocyte maturation by BDNF and GDNF. RESULTS: BDNF and GDNF were found to be expressed in non-stimulated granulosa cells. After gonadotrophin (FSH and/or hCG) treatment, transcripts levels for BDNF and GDNF were significantly increased (P < 0.05). In cultured immature oocytes, treatment with BDNF or GDNF promoted total yields of metaphase II oocytes. CONCLUSIONS: These findings demonstrate that FSH and hCG treatments augment the expression of BDNF and GDNF by granulosa cells and that these granulosa-cell-derived factors are candidate paracrine factors capable of promoting oocyte maturation.

ER-α36, a novel variant of estrogen receptor α, is involved in EGFR-related carcinogenesis in endometrial cancer.

OBJECTIVE: To explore the role of estrogen receptor-α36 (ER-α36) in epidermal growth factor receptor (EGFR)-related carcinogenesis in endometrial cancer. STUDY DESIGN: The expression of ER-α36, EGFR, and phospho-extracellular signal-regulated kinase was analyzed using immunohistochemistry in endometrial cancer samples. The cellular localization of ER-α36 and EGFR was determined using immunofluorescence in the endometrial cancer Hec1A cells. The level of phospho-extracellular signal-regulated kinase of Hec1A cells was determined using Western blotting after treatment with epidermal growth factor. RESULTS: Positive rate of ER-α36 was increased in high-stage (P = .03) and high-grade (P = .224) endometrial cancer; expression of ER-α36 and EGFR exhibited a significant positive correlation (r = 0.334, P = .025) and they showed substantial colocalization on the plasma membrane of glandular cells; phospho-extracellular signal-regulated kinase positive rate in ER-α36 positive group and EGFR positive group was higher than that of ER-α36 negative group (P = .014) and EGFR negative group (P = .016); finally, ER-α36 mediated epidermal growth factor-stimulated extracellular signal-regulated kinase activation in Hec1A cells. CONCLUSION: ER-α36 mediates EGFR-related extracellular signal-regulated kinase activation in endometrial cancer.

Poor intracytoplasmic sperm injection outcome in infertile males with azoospermia factor c microdeletions.

OBJECTIVE: To explore whether the presence of azoospermia factor c (AZFc) microdeletions adversely affects intracytoplasmic sperm injection (ICSI) outcome. DESIGN: Retrospective cohort. SETTING: University hospital. PATIENT(S): A total of 293 patients with azoospermia or severe oligozoospermia AZFc deletions underwent 345 ICSI cycles, and 363 idiopathic patients with normal Y chromosome underwent 462 ICSI cycles. INTERVENTION(S): Testicular sperm aspiration, microdissection testicular sperm extraction. MAIN OUTCOME MEASURE(S): The main clinical outcome parameters were cumulative clinical pregnancy rate, cumulative live birth delivery rate, and no embryo suitable for transfer cycle rate. RESULT(S): Compared with the control group, the AZFc deletion group exhibited poorer ICSI outcome, with significant differences between the 2 groups for cumulative clinical pregnancy rate (45.39% vs. 67.49%; odds ratio [OR], 2.843; 95% confidence interval [CI]), cumulative live birth delivery rate (35.15% vs. 53.44%; OR, 2.234; 95% CI), no embryo suitable for transfer cycle rate (15.07% vs. 8.23%; OR, 0.565; 95% CI), fertilization rate (46.80% vs. 53.37%; adjusted ß, -0.074; 95% CI), implantation rate (28.63% vs. 31.26%; adjusted ß, -0.075; 95% CI) separately. The poor ICSI outcome of the AZFc deletion group was related to AZFc microdeletions by linear and logistic regression analyses. CONCLUSION(S): AZFc microdeletions adversely affect ICSI outcome; patients with AZFc deletion should be informed that they have reduced opportunities to be biological fathers.

Effects of oocyte vitrification on histone modifications.

Vitrification has been widely used as an assisted reproductive technology in animals and humans, yet the impact of oocyte vitrification and warming on survival and histone modifications has to be evaluated. In the present study, the survival of mouse MII oocytes was assessed after freezing, as were changes in histone 3 lysine 9 (H3K9) dimethylation, histone 4 lysine 5 (H4K5) acetylation and histone 3 lysine 14 (H3K14) acetylation. The results show that, in oocytes subjected to vitrification, H3K9 methylation and H4K5 acetylation were increased. H3K14 acetylation could not be detected in either non-vitrified or vitrified oocytes. Oocytes are very sensitive to changes in H3K9 and H4K5 following vitrification. Both these histone modifications could be useful markers to monitor epigenetic perturbations induced by various experimental vitrification protocols and eventually for optimising the cryopreservation of human oocytes.

Single-cell DNA methylation sequencing reveals epigenetic alterations in mouse oocytes superovulated with different dosages of gonadotropins.

BACKGROUND: Epigenetic abnormalities caused by superovulation have recently attracted increasing attention. Superovulation with exogenous hormones may prevent oocytes from establishing an appropriate epigenetic state, and this effect may extend to the methylation programming in preimplantation embryos, as de novo DNA methylation is a function of developmental stage of follicles and oocyte size. Follicle-stimulating hormone (FSH) and human menopausal gonadotropin (hMG) are common gonadotropins used for superovulation, and appropriate concentrations of these gonadotropins might be necessary. However, no systematic study on the effects of DNA methylation alterations in oocytes associated with superovulation with different dosages of FSH/hMG at the single-cell level has yet been reported. In the current study, different dosages of FSH/hMG combined with human chorionic gonadotropin (hCG) were used in female mice to generate experimental groups, while naturally matured oocytes and oocytes superovulated with only hCG were respectively used as controls. Single-cell level DNA methylation sequencing was carried out on all these matured oocytes. RESULTS: In this study, we revealed that the genome-wide methylation pattern and CG methylation level of the maternal imprinting control regions of all mature oocytes were globally conserved and stable. However, methylation alterations associated with superovulation were found at a specific set of loci, and the differentially methylated regions (DMRs) mainly occurred in regions other than promoters. Furthermore, some of the annotated genes in the DMRs were involved in biological processes such as glucose metabolism, nervous system development, cell cycle, cell proliferation, and embryo implantation and were altered in all dosages of FSH/hMG group (for example, Gfod2 and SYF2). Other genes were impaired only after high gonadotropin dosages (for instance, Sox17 and Phactr4). CONCLUSIONS: In conclusion, the current study addressed the effects of superovulation on DNA methylation from the perspective of different dosages of gonadotropins at the single-cell level. We found that the genome-wide DNA methylation landscape was globally preserved irrespective of superovulation or of the kind and dosage of gonadotropins used, whereas the methylation alterations associated with superovulation occurred at a specific set of loci. These observed effects reflect that superovulation recruits oocytes that would not normally be ovulated or that have not undergone complete epigenetic maturation. Our results provide an important reference for the safety assessment of superovulation with different dosages of gonadotropins. However, it should be noted that this study has some limitations, as the sample number and library coverage of analyzed oocytes were relatively low. Future studies with larger sample sizes and high-coverage libraries that examine the effects of superovulation on embryo development and offspring health as well as the underlying mechanisms are still needed.