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Ovarian aging refers to the gradual decline of ovarian function with increasing physiological age, manifested as decreased ovarian reserve, elevated aging-related markers, and reduced oocyte quality. With a declining female fertility and a growing aging population, it is urgent to delay ovarian aging to maintain fertility and improve the life quality of women. Theaflavin 3, 3 ′ -digallate (TF3) is a naturally bioactive polyphenol compound extracted from black tea, and its antioxidant properties play an important role in maintaining human health and delaying aging; however, the effects of TF3 on female reproduction and ovarian function are not yet clear. Here, we show that TF3 can preserve primordial follicle pool, partially restore the estrous cycle, and increase the offspring number of aged mice. Meanwhile, TF3 gavage increased the number of oocytes retrieved, decreased the level of reactive oxygen species, increased the level of glutathione, and decreased the abnormal rate of oocyte spindle after ovulation induction. Moreover, TF3 inhibited human granulosa cell apoptosis and improved their antioxidative stress ability. High-throughput sequencing and small-molecule-targeted pharmacological prediction show that TF3 affects multiple pathways and gene expression levels, mainly involved in reproductive and developmental processes. It may also affect cellular function by targeting mTOR to regulate the autophagic pathway, thereby delaying the process of ovarian aging. This study shows that TF3 can be used as a potential dietary supplement to protect ovary function from aging and thereby improving the life quality of advanced-age women.

From the collection of human follicular fluid to the isolation of primary granulosa cells, the duration of these two time points is variable. And there is no specific experiment to explore which temperature will make the cell state better in subsequent experiments. In this study, the collected follicular fluid was divided into 7 groups: one fresh control group, three in vitro storage temperatures (4℃, Room Temperature, and 37℃) and two storage duration times (4h and 8h). After isolating the cells, they were cultured in vitro to evaluate their state and function. The results showed that granulosa cells of the fresh control group had better cell viability and steroid hormone secretion ability than the other treatment groups; with the prolongation of in vitro storage time, the cell viability and steroid hormone secretion ability have decreased to varying degrees; low-temperature in vitro storage is conducive to the maintenance of cell survival and steroid hormone secretion ability, which is also confirmed by the detection of ROS, which can reflect the oxidative stress state of cells. This study showed that after obtaining follicular fluid, the in vitro storage time should be shortened as much as possible, and stored at low temperature, which can keep cells maintain good activity and steroid hormone secretion ability, and reduce oxidative stress and aging. It is of great significance for improving the state of human primary granulosa cells after isolation and subsequent experiment, and it also has certain guiding significance for standardization experimental research programs.

Loss of follicles together with decreased oocyte quality and quantity contribute to age-associated ovarian senescence and infertility. Although underlying mechanisms for ovarian senescence are still unknown, mitochondrial dysfunctions have been reported. Here, we showed age-dependent decreases in ovarian Nicotinamide Adenine Dinucleotide (NAD+) levels in mice whereas supplementing aging mice with nicotinamide riboside (NR), an NAD+ precursor, increased ovarian NAD+ content. We found that increases in ovarian NAD+ levels in aging mice led to increased number of ovarian follicles and ovulatory potential as well as increased live birth rate. NR supplementation also reduced levels of reactive oxygen species and decreased spindle anomalies in aging oocytes, together with increased mitochondrial membrane potential (ΔΨm) and decreased mitochondrial clustering. In addition, NR supplementation improved ovarian mitochondrial energy metabolism. Our data suggested that supplementation with NAD+ precursors in vivo and in vitro could be potential therapeutic approaches for treating age-related ovarian infertility.

Background The blastocyst morphology provided valuable roles for predicting pregnancy and live birth, but was still not fully understood for evaluating miscarriage. The aim of this study was to explore the association between blastocyst morphologic evaluation and first trimester miscarriage combined with karyotype of miscarried conceptus. Methods This retrospective cohort study included a total of 2873 clinical pregnancy cycles with single blastocyst transfer performed from January 2013 to April 2019. Chromosome karyotype of miscarried conceptus was analyzed via single nucleotide polymorphism array analysis. Miscarriage and karyotype of miscarried conceptus associated with blastocyst morphology were analyzed by chi-square and logistic regression analysis. Results A total of 354 (12.3%) cycles resulted in first trimester miscarriage. Miscarriage rates increased with trophectoderm (TE) grade from A to C (P = 0.012), while three morphologic parameters (blastocoele expansion degree, inner cell mass (ICM) and TE) showed no statistical significance with miscarriage after multivariable analysis. The rate of aneuploidy was 47.7% (83 of 174) in total miscarried conceptuses. For euploid miscarriages, the grade B of ICM occupied a higher proportion compared with aneuploidy, with OR of 2.474, (95% CI, 1.311–4.699), P = 0.005. Conclusions Chromosomal aberration of embryo is an important genetic factor for first trimester miscarriage, and the quality of ICM is a potential indicator for euploid miscarriage. Blastocysts with grade A of ICM should be given priority during single blastocyst transfer to reduce potential miscarriage.

To some extent, the use of metformin may improve endometrial receptivity and pregnancy outcomes of women with polycystic ovarian syndrome (PCOS) undergoing in vitro fertilization/intracytoplasmic sperm injection. However, the mechanism is not well-known. The endometrium of metformin-treated group (metformin-treated patients with PCOS) and the control group (non-metformin-treated patients with PCOS) were analyzed for the expression of homeobox A10 (HOXA10) and integrin beta-3 (ITGB3) and differential micro RNA (miRNA) expression profiles. On this basis, miRDB and Target Scan databases were used to predict and screen out that miR-491-3p and miR-1910-3p may target HOXA10 and ITGB3. Furthermore, we verified the effects of metformin on the expression of HOXA10 and ITGB3, and regulatory effects of miR-1910-3p and miR-491-3p on HOXA10 and ITGB3 using Ishikawa cell line. Metformin induced a significant dose-dependent upregulation of HOXA10 and ITGB3. The results from the microarray analyses showed there were 40 differentially expressed miRNAs between the 2 groups. Among them, miR-1910-3p and miR-491-3p were the 2 significantly downregulated miRNAs. Bioinformatics prediction indicated that HOXA10 and ITGB3 are potential target genes for miR-1910-3p and miR-491-3p. In Ishikawa cells transfected with miR-491-3p mimics, the expression of HOXA10 and ITGB3 on both messenger RNA (mRNA) and protein level were lower than those in control group (P .05). Metformin likely improves endometrial receptivity through downregulating the expression of miR-491-3p and miR-1910-3p, thereby increasing the expression of HOXA10 and ITGB3 in the endometrium of PCOS women.

Background: Studies have found that ATG7 knockout mice have defects in acrosome development, and the sperm formed by them are similar to human round-headed sperm. miR-188-3p expression is alleviated in the testis tissues of NOA samples. It is unclear whether ATG7 gene is related to non-obstructive azoospermia and whether miR-188-3p and ATG7 have direct target regulation relationship to affect spermatogenesis.Methods: The testicular tissues of 14 non-obstructive azoospermia (NOA) patients and 13 healthy patients were collected for basic scientific research from October 2017 to June 2018. High-throughput sequencing, qRT-PCR, Western blot, immunohistochemical staining, immunofluorescence, electron microscope, and luciferase experiment were performed to confirm the correlation between miR-188-3p and ATG7 in non-obstructive azoospermia.Results: ATG7 was highly expressed in NOA group. ATG7 protein was localized in the cytoplasm of spermatogenic cells at various levels, and the expression in NOA group was dramatically higher than that of normal individuals. After overexpression of miR-188-3p, the ATG7 3'UTR-WT luciferase activity was impeded; while the ATG7 3'UTR-MUT luciferase activity had no significant difference. LC3 and Beclin-1 gene and protein expression in the NOA samples were elevated in comparison with the normal samples; LC3 was mainly located in the cytoplasm of spermatogenic cells at all levels. LC3 and Beclin-1 genes expression after miR-188-3p overexpression were prominently in comparison with the normal samples; after miR-188-3p was inhibited, LC3 and Beclin-1 expressions were dramatically higher. The degree of LC3 punctate aggregation in the miR-188-3p mimic NC samples was greater, and that in the miR-188-3p inhibitor group was greater. The number of autophagosomes of the miR-188-3p mimic samples was less compared with the mimic NC group.Conclusion: We demonstrated here that the ATG7 gene was located in all levels of spermatogenic cells and was highly expressed in the NOA group. The expression of ATG7 gene was negatively correlated with miR-188-3p that may regulate the target gene ATG7 to participate in autophagy and affect the occurrence of NOA.

Background: A retrospectively cohort study was performed to compare the birth weight of different blastocyst grades in fresh transplantation cycle and explore the related factors affecting the birth weight. Methods: The 1301 fresh cycles of single blastocyst transplantation and single live birth profile were analyzed,four groups were divided according to the grade of transplanted blastocyst. There are 170 cycles in group A with AA blastocyst grade, 312 cycles in group B with AB/BA blastocyst grade, 559 cycles in group C with BB/CA/AC blastocyst grade and 260 cycles in group D with BC/CB blastocyst grade. Comparison were made among four groups of birth weight, general conditions, fertilization rate, embryo rate, cleavage rate and D5,D6 blastocyst formation rate and other laboratory indicators. And then comparison were performed among the birth weight of different groups which were divided by the degree of blastocyst expansion, and the classification of inner cell mass(ICM) and the trophectoderm(TE). Results: The study shows that the birth weight of group A is significantly higher than that of the other three groups (P < 0.05). And the high quality embryo rate and blastocyst rate of group A are significantly higher than those of the other three groups (P < 0.05). What’s more, the clinical pregnancy rate, implantation rate and live birth rate of high grade blastocyst are higher, but there are no significant difference in abortion rate. The birth weight of the degree of blastocyst expansion in grade 3 and below is significantly lower than that of those with grade 3 and above (P < 0.05). The birth weight of grade A of ICM is significantly higher than that of grade B (P < 0.05). The birth weight of grade B of TE is significantly heavier than that of grade C (P < 0.05). Conclusions: Our results indicate that high grade blastocyst transplantation can achieve better pregnancy outcome. Different blastocyst grades affect birth weight, and low grade blastocyst transplantation is associated with a single birth weight loss.

At present, embryo morphology assessment based on the observation of the embryonic morphological characteristics at several specific time points has been mainly used for selecting the high-quality embryo. However, we all know that embryo development is a dynamic process. Many research results on the correlation between the embryo morphokinetic parameters and embryo quality and development potential were inconsistent. With the help of time-lapse imaging, the development processes and outcomes of a total of 365 embryos were cultured and analyzed in this study. The results showed that the mean tPNf and t2 of the high-quality embryo were significantly shorter than the low-quality embryo; the mean t2PBe and tPNa of the high-quality embryo from the implantation group were significantly shorter than those from the non-implantation group. In addition, based on the quartile grouping of each morphokinetic parameter, the embryos that had 21.15≤tPNf≤25.30 value were significantly higher in embryo quality when compared with the embryos that had the tPNf values outside the range on Days 3. Similarly, the embryos that had values of t2≤25.60 were significantly higher in embryo quality than those with outside the range values on Days 3. Thus, we demonstrated that the morphokinetic parameter evaluated using a time-lapse embryo monitoring system can predict the embryo quality, and be benefit for the selection of the high-quality embryos and improvement for the implantation success of the patient in assisted reproductive technologies.

Epigenetics of human embryonic development Substantial epigenetic reprogramming occurs in mammalian early development. Xia et al. investigated the reprogramming dynamics of three key histone modifications in human early embryonic development. The reprogramming in humans is highly species-specific and different than that in mice. A globally permissive chromatin state connects parental and zygotic epigenomes during maternal-to-zygotic transition. Inner-cell mass-specific, but not trophectoderm-specific, genes are asymmetrically patterned by a histone mark during early lineage segregation. Science , this issue p. 353

Backgrounds Long non-coding RNA is a novel group of non-protein coding transcripts over 200 nt in length. Recent studies have found that they are widely involved in many pathological and physiological processes. In our previous study, we found that lnc-GULP1–2:1 was significantly down-regulated in the ovarian cortical tissue of patients with primary ovarian insufficiency and predicted that lnc-GULP1–2:1 has a regulatory effect on COL3A1. Results In this study, we found that lnc-GULP1–2:1 was mainly localized in the cytoplasm of luteinized granulosa cells. The expression of lnc-GULP1–2:1 was lower in patients with diminished ovarian reserve but substantially elevated in patients with polycystic ovary syndrome. Overexpression of lnc-GULP1–2:1 in KGN cells significantly inhibited cell proliferation, likely through cell cycle related genes CCND2 and p16. Moreover, lnc-GULP1–2:1 expression was positively correlated with the level of COL3A in luteinized granulosa cells from patients with different ovarian functions as well as in multiple cell lines. Overexpression of lnc-GULP1–2:1 in KGN cells promoted the expression of COL3A1 and its translocation into the nucleus. Consistently, silencing COL3A1 in KGN cells also significantly inhibited cell proliferation. Conclusions Lnc-GULP1–2:1 affects the proliferation of granulosa cells by regulating the expression and localization of COL3A1 protein, and may participate in the regulation of ovarian follicle development. This study will provide new insight into molecular mechanisms underlying ovarian follicular development, which will help generate novel diagnostic and therapeutic strategies for diseases related to ovarian follicular development disorders.

A disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1) is an extracellular matrix metalloproteinase that plays an important role in the process of ovulation. According to previous studies, the expression level of ADAMTS1 in the granulosa cells of polycystic ovarian syndrome (PCOS) patients and the mechanism for regulating oocyte quality and embryonic development potential are still unclear. Our research clarified that ADAMTS1 was significantly increased in granulosa cells of PCOS patients as compared to ovulatory controls. After silencing ADAMTS1 in granulosa cells, cell proliferation and E2 secretion were significantly inhibited, which may be related to the down-regulation of B-cell lymphoma 2 (Bcl2) family genes and key genes involved in E2 synthesis. Through retrospective analysis of the clinical data, it was found that the expression level of ADAMTS1 was significantly positively correlated to the oocyte maturation rate and good-quality embryo rate in PCOS patients. The downregulation of ADAMTS1 in primary granulosa cells lead to the changes in the expression of marker genes for oocyte and embryonic quality. By using immunofluorescence staining, it was found ADAMTS1 was expressed in various stages of pre-implantation embryo but its expression level gradually decreases with the development of the embryo. In addition, the silence of ADAMTS1 in 3PN zygotes significantly prolonged the development time of the zygote to the morula stage. This is, to our knowledge, the first time to explored the mechanism by which ADAMST1 is involved in affecting the quality of oocytes and embryonic development potential, which will provide new evidence for further understanding of the follicular microenvironment and embryo development.

Uniparental embryos derived from only the mother (gynogenetic [GG]) or the father (androgenetic [AG]) are unique models for studying genomic imprinting and parental contributions to embryonic development. Human parthenogenetic embryos can be obtained following artificial activation of unfertilized oocytes, but the production of AG embryos by injection of two sperm into one denucleated oocyte leads to an extra centriole, resulting in multipolar spindles, abnormal cell division, and developmental defects. Here, we improved androgenote production by transferring the male pronucleus from one zygote into another haploid androgenote to prevent extra centrioles and successfully generated human diploid AG embryos capable of developing into blastocysts with an identifiable inner cell mass (ICM) and trophectoderm (TE). The GG embryos were also generated. The zygotic genome was successfully activated in both the AG and GG embryos. DNA methylome analysis showed that the GG blastocysts partially retain the oocyte transcription-dependent methylation pattern, whereas the AG blastocyst methylome showed more extensive demethylation. The methylation states of most known imprinted differentially methylated regions (DMRs) were recapitulated in the AG and GG blastocysts. Novel candidate imprinted DMRs were also identified. The production of uniparental human embryos followed by transcriptome and methylome analysis is valuable for identifying parental contributions and epigenome memory transitions during early human development.

FBN1 encodes asprosin, a glucogenic hormone, following furin cleavage of the C-terminus of profibrillin 1. Based on evolutionary conservation between FBN1 and FBN2, together with conserved furin cleavage sites, we identified a peptide hormone placensin encoded by FBN2 based on its high expression in trophoblasts of human placenta. In primary and immortalized murine hepatocytes, placensin stimulates cAMP production, protein kinase A (PKA) activity, and glucose secretion, accompanied by increased expression of gluconeogenesis enzymes. In situ perfusion of liver and in vivo injection with placensin also stimulate glucose secretion. Placensin is secreted by immortalized human trophoblastic HTR-8/SVneo cells, whereas placensin treatment stimulates cAMP-PKA signaling in these cells, accompanied by increases in MMP9 transcripts and activities, thereby promoting cell invasion. In pregnant women, levels of serum placensin increase in a stage-dependent manner. During third trimester, serum placensin levels of patients with gestational diabetes mellitus are increased to a bigger extent compared to healthy pregnant women. Thus, placensin represents a placenta-derived hormone, capable of stimulating glucose secretion and trophoblast invasion.

Objective To investigate the expression of aquaporin 7 (AQP7) and aquaporin 9 (AQP9) in the granulosa cells of patients with polycystic ovary syndrome (PCOS) and healthy women and detect their localization in oocytes at the germinal vesicle (GV), metaphase I (MI), MII, embryo, and blastocyst stages and the in vitro response to insulin stimulation. Design Randomized, assessor-blinded study. Setting Reproductive medical center. Patient(s) A total of 40 women (aged 20–38 years) comprising 29 cases of primary infertility and 11 cases of secondary infertility, of whom 17 had an initial diagnosis of PCOS and three received a PCOS diagnosis after an infertility examination. Intervention(s) Controlling different concentrations of insulin and different treatment times in cultures of normal human granulosa cells in vitro. Main Outcome Measure(s) Expression of AQP7 and AQP9 genes and proteins in granulosa cells detected by real-time quantitative polymerase chain reaction, and localization in oocytes at the GV, MI, MII, embryo, and blastocyst stages by Western blot, immunohistochemical, and immunofluorescence assays, and concentrations of insulin in follicular fluid by enzyme-linked immunosorbent assay. Result(s) The expression levels of the AQP7 mRNA and protein in the granulosa cells of patients with PCOS were higher than found in healthy controls. We found AQP7 protein expressed in human oocytes at GV, MI, MII, embryo, and blastocyst stages; it was mainly located in the nucleoplasm. In the PCOS group, the expression level of AQP9 mRNA and protein in granulosa cells was lower, and AQP9 protein was expressed in oocytes at the GV, MI, MII, embryo, and blastocyst stages; it was localized on the nuclear membrane. Compared with healthy women, the insulin expression in patients with PCOS was higher. In cultures of normal human granulosa cells in vitro, the expression of AQP7 and AQP9 mRNA and protein decreased with the increase in insulin concentration; expression statistically significantly decreased when the insulin concentration was 100 nmol/L, and after 6 to 24 hours of exposure the lowest expression levels were found at 12 hours. Conclusion(s) The different localization and expression of AQP7 and AQP9 between the two groups suggests that they might be involved in oocyte maturation and embryonic development through different regulatory pathways. The expression levels of AQP7 and AQP9 were negatively correlated with insulin regulation, suggesting that insulin might affect the maturation of PCOS follicles by changing AQP7 and AQP9 expression.

Loss of follicles together with decreased oocyte quality and quantity contribute to age-associated ovarian senescence and infertility. Although underlying mechanisms for ovarian senescence are still unknown, mitochondrial dysfunctions have been reported. Here, we showed age-dependent decreases in ovarian Nicotinamide Adenine Dinucleotide (NAD

Mammalian ovarian follicular development is an intricate, elaborate, and well-organized phenomenon regulated by various signaling pathways; however, the underlying mechanism remains unclear. Mammalian sirtuins (sirtuin 1 to sirtuin 7) are a group of NAD+ -dependent deacetylases implicated in various physiological processes including cell proliferation, apoptosis, cell cycle progression, and insulin signaling. Mammalian ovarian sirtuins have been studied using adult and aged bovine, porcine, and murine models. However, limited information is available regarding their precise expression patterns and the localization of follicle development in mice. This study aimed to assess the dynamic expression and localization of all seven sirtuins in early postnatal mouse ovaries through real-time polymerase chain reaction analysis and immunohistochemistry, respectively. During postnatal ovarian follicle development, sirtuin 1, sirtuin 4, and sirtuin 6 were downregulated compared with those in 1-day postnatal mouse ovaries (p < .05), indicating that these three sirtuin genes may be markers of follicular development. Combining their localization in granulosa cells through immunohistochemical studies, sirtuin 1, sirtuin 4, and sirtuin 6 are suggested to play negative regulatory roles in mammal ovarian follicular granulosa cell development. Furthermore, we found that sirtuin 2 (p < .05) and sirtuin 7 (p < .05) mRNA were constantly upregulated relative to sirtuin 1, although limited information is available regarding sirtuin 7. Among all sirtuins in mouse ovaries, sirtuin 1 was relatively and steadily downregulated. Upon sirtuin 1 overexpression in 1-day postnatal mouse ovaries via sirtuin 1-harboring adenoviruses in vitro, the emergence of primary follicles was delayed, as was the emergence of secondary follicles in 4-day postnatal ovaries. Further studies on KGN cell lines reported that interfering with sirtuin 1 expression in granulosa cell significantly affected granulosa cell proliferation and the expression of mitochondrial genes. This study presents the first systemic analysis of dynamic patterns of sirtuin family expression in early postnatal mice ovaries, laying the foundation for further studies on less discussed sirtuin subtypes, such as sirtuin 5 and sirtuin 7.

Upon fertilization, drastic chromatin reorganization occurs during preimplantation development 1 . However, the global chromatin landscape and its molecular dynamics in this period remain largely unexplored in humans. Here we investigate chromatin states in human preimplantation development using an improved assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) 2 . We find widespread accessible chromatin regions in early human embryos that overlap extensively with putative cis-regulatory sequences and transposable elements. Integrative analyses show both conservation and divergence in regulatory circuitry between human and mouse early development, and between human pluripotency in vivo and human embryonic stem cells. In addition, we find widespread open chromatin regions before zygotic genome activation (ZGA). The accessible chromatin loci are readily found at CpG-rich promoters. Unexpectedly, many others reside in distal regions that overlap with DNA hypomethylated domains in human oocytes and are enriched for transcription factor-binding sites. A large portion of these regions then become inaccessible after ZGA in a transcription-dependent manner. Notably, such extensive chromatin reorganization during ZGA is conserved in mice and correlates with the reprogramming of the non-canonical histone mark H3K4me3, which is uniquely linked to genome silencing3–5. Taken together, these data not only reveal a conserved principle that underlies the chromatin transition during mammalian ZGA, but also help to advance our understanding of epigenetic reprogramming during human early development and in vitro fertilization.

// Tingting Cheng 1 , Kui Zhai 2 , Yan Chang 2 , Guidong Yao 1 , Jiahuan He 1 , Fang Wang 1 , Huijuan Kong 1 , Hang Xin 1 , Huiwen Wang 2 , Meng Jin 2 , Bing Gong 3 , Lei Gu 2 , Zhiguang Yang 2 , Yanyun Wu 2 , Guangju Ji 2 , Yingpu Sun 1 1 Center for Reproductive Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China 2 National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China 3 Department of Cardiac Surgery, State Key Laboratory of Cardiovascular Disease, Fuwai Hospital, National Center for Cardiovascular Disease, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China Correspondence to: Guangju Ji, email: gj28@ibp.ac.cn Yingpu Sun, email: syp2008@vip.sina.com Keywords: CHIR99021, retinoic acid, primordial germ cells, human embryonic stem cells, β-catenin Received: September 24, 2016 Accepted: December 01, 2016 Published: December 15, 2016 ABSTRACT Primordial germ cells (PGCs) derived from human embryonic stem cells (hESCs) represent as a desirable experimental model as well as a potential strategy for treating male infertility. Here, we developed a simple and feasible method for differentiation of PGCs from hESCs by using CHIR99021 (an inhibitor of glycogen synthase kinase 3) and retinoic acid (RA). We firstly found that the deleted in azoospermia-like (DAZL) protein can be detected in 3 d CHIR99021 plus 9 d retinoic acid treated cultures and 12 d CHIR99021 plus retinoic acid co-treated cultures, but not expressed in single CHIR99021 treated cultures, single retinoic acid treated cultures, as well as 3 d retinoic acid plus 9 d CHIR99021 treated cultures. Next, we showed that several PGCs’ markers were expressed in the 12 d CHIR99021 and retinoic acid co-treated cultures or 3 d CHIR99021 plus 9 d retinoic acid treated cultures. Moreover, meiosis was initiated in CHIR99021 and retinoic acid co-treated cultures as evidenced by a significant expression of the punctate synaptonemal complex protein 3 (SCP3). Fluorescent in situ hybridization (FISH) analysis indicated that a small percentage of putative 1N populations were formed. Mechanically, we found that β-catenin relocated into nucleus after the treatment of 3 d CHIR99021 suggesting that Wnt signaling pathway was activated. Furthermore, blockade of Wnt signaling pathway by IWR-1 can reverse CHIR99021 and retinoic acid mediated-effects. Taken together, our results indicate that CHIR99021 combined with retinoic acid can effectively differentiate hESCs into PGCs via activating Wnt signaling pathway.

Context: Recently, two patients with primary ovarian insufficiency (POI) delivered healthy babies after in vitro activation (IVA) treatment followed by auto-transplantation of frozen-thawed ovarian tissues. Objective: This study sought to report the first case of live birth after IVA treatment following fresh ovarian tissue grafting in patients with POI, together with monitoring of follicle development and serum hormonal changes. Design: This was a prospective observational cohort study. Setting: We performed IVA treatment in 14 patients with POI with mean age of 29 years, mean duration since last menses of 3.8 years, and average basal FSH level of 94.5 mIU/mL. Interventions: Prior to IVA treatment, all patients received routine hormonal treatments with no follicle development. We removed one ovary from patients with POI and treated them with Akt stimulators. We improved upon early procedures by grafting back fresh tissues using a simplified protocol. Main Outcome Measures: In six of the 14 patients (43%), a total of 15 follicle development waves were detected, and four patients had successful oocyte retrieval to yield six oocytes. For two patients showing no spontaneous follicle growth, human menopausal gonadotropin treatment induced follicle growth at 6–8 months after grafting. After vitro fertilization of oocyte retrieved, four early embryos were derived. Following embryo transfer, one patient became pregnant and delivered a healthy baby boy, with three other embryos under cryopreservation. Conclusion: IVA technology can effectively activate residual follicles in some patients with POI and allow them to conceive their own genetic offspring. IVA may also be useful for treating patients with ovarian dysfunction including aging women and cancer survivors., We performed in vitro activation treatment following fresh ovarian tissue grafting in primary ovarian insufficiency patients, and achieved the first case of live birth with this method.

PURPOSE: The preimplantation genetic testing for monogenic defects (PGT-M) is a beneficial strategy for the patients suffering from a Mendelian disease, which could protect their offspring from inheriting the disease. The purpose of this study is to report the effectiveness of PGT-M based on karyomapping for three cases of dynamic mutation diseases with trinucleotide repeat expansion. METHODS: PGT-M was carried out on three couples, whose family members were diagnosed with Huntington’s disease or spinocerebellar ataxias 2 or 12. The whole genome amplification was obtained using the multiple displacement amplification (MDA) method. Then, karyomapping was performed to detect the allele that is carrying the trinucleotide repeat expansion using single nucleotide polymorphism (SNP) linkage analyses, and the copy number variations (CNVs) of the embryos were also identified. Prenatal diagnosis was performed to validate the accuracy of PGT-M. RESULTS: PGT-M was successfully performed on the three couples, and they accepted the transfers of euploid blastocysts without the relevant pathogenic allele. The clinical pregnancies were acquired and the prenatal diagnosis of the three families confirmed the effectiveness of karyomapping. The three born babies were healthy and free of the pathogenic alleles HTT, ATXN2, or PPP2R2B corresponding to Huntington’s disease, spinocerebellar ataxias 2 or 12, respectively. CONCLUSION: This study shows that karyomapping is a highly powerful and efficient approach for dynamic mutation detection in preimplantation embryos. In this work, we first report the birth of healthy babies that are free of the pathogenic gene for dynamic mutation diseases in patients receiving PGT-M by karyomapping. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10815-020-01718-5) contains supplementary material, which is available to authorized users.

Previous studies have shown that long noncoding RNAs (lncRNAs) show a highly tissue- and disease-specific expression pattern and that they regulate the expression of neighboring genes. Because lncRNAs have been shown to be secreted into the general circulation, they may be used as diagnostic tools for some diseases. Primary ovarian insufficiency (POI) is a disease in which women have menstrual cessation before the age of 40, accompanied by elevated follicle stimulating hormone and decreased estrogen levels. In this study, ovarian cortical tissues from five women with normal menstrual cycles and from five POI patients were used for next-generation RNA sequencing. We found 20 differentially expressed lncRNAs with 12 upregulated and eight downregulated lncRNAs in cortical tissues of POI ovaries, compared with normal controls (fold change ≥ 2 and false discovery rate[FDR] ≤ 0.05). We also found 52 differentially expressed messenger RNA transcripts, with 33 upregulated and 19 downregulated ones (foldchange ≥ 2 and FDR ≤ 0.05). Functional annotation showed that these differentially expressed transcripts were associated with follicular development and granulosa cell function. Thirteen differentially expressed lncRNAs and their targeted neighboring transcripts were coregulated in ovarian cortical tissues, including lnc-ADAMTS1-1:1/ADAMTS1, lnc-PHLDA3-3:2/CSRP1, lnc-COL1A1-5:1/COL1A1, lnc-SAMD14-5:3/COL1A1, and lnc-GULP1-2:1/COL3A1. Furthermore, serum levels of these lncRNAs in POI patients were significantly different from those in normal patients ( p < 0.05), and expression differences were consistent with those in ovarian cortical tissues. This study showed that key lncRNAs were differentially expressed in both ovarian cortical tissues and serum samples between women with normal menstrual cycles and POI patients. Further studies on the regulation of ovarian lncRNAs during follicular development are critical in understanding the etiologies of POI. Analyses of lncRNA expression in serum samples might provide a basis for early diagnosis and treatment of POI.

OBJECTIVE To evaluate the safety of rapid cryopreservation for human testicular tissues by comparing the meiotic recombination in the fresh and thawed testis after rapid freezing. METHODS Twelve male patients with prostate cancer (PC) who had given birth to healthy children at youth and need to receive surgical removal of testicular tissue at present were selected in this study. Testicular tissues from 4 cases of PC patients were randomly divided into two parts, one part as fresh tissue and the other to receive rapid freezing treatment. Fidelity analysis for homologous genetic recombination and synapsis were performed by immunofluorescence after prepared by a micro-spreading technique. RESULTS The average number of MLH1 foci per cell, autosomal synaptonemal complex (SC) containing 0∼5 MLH1 foci and percent of cells with one MLH1 focus on XY chromosome showed no difference between the fresh and frozen thawed testicular tissues from the same case (P >0.05). And, no significant difference in the frequency of gaps and splits on SCs was observed in fresh and thawed spermatocytes (P > 0.05). CONCLUSION Rapid cryopreservation showed little effect on the frequency of meiotic recombination and fidelity of synapsis in human spermatocytes from PC patients, and acted as an effective method for preserving male fertility.

Background/Aims: To observe the effects of vitrification on spindle, zona pellucida, embryonic aneuploidy and DNA injury in in vivo-maruted, in vitro-mature and immature human oocytes. Methods: Between January 2009 and February 2015, 223 immature oocytes from 450 infertile patients, and 31 in vivo-mature oocytes from 3 infertile couples were collected. Of the 223 immature oocytes, 113 were used for in vitro culture before vitrification. Some oocytes were randomly divided into in vivo-mature group (group A, n = 15), in vitro-mature group (group B, n = 88) and immature group (group C, n = 85), and then the oocytes with spindle in these three groups after freezing-thawing were selected to use for Polscope imaging, embryonic aneuploidy screening and embryo development evaluation. Other oocytes were randomly divided into group A (n = 16), group B (n = 25) and group C (n = 25) for detecting DNA injury. Results: After thawing, spindle occurrence rate, spindle Retardance value, and cleavage rate were significantly higher in groups A and B than in group C (all P < 0.05), but there were no statistical differences in fertility rate, high-quality embryo rate, blastulation rate and aneuploidy rate amongst the three groups (all P > 0.05). Zona pellucida density (ZPD) was significantly lower in group A than in groups B and C both before and after vitrification (all P < 0.05). ZPD was significantly higher after thawing than before vitrification (all P < 0.05), but zona pellucida thickness (ZPT) was not significantly changed in all the three groups (all P > 0.05). Rate of comet cells was significantly lower in group A than in groups B and C (all P < 0.01). Comet tail was significantly longer in group C than in groups B and A (all P < 0.05). Conclusion: In vivo- and in vitro-mature human oocytes are more suitable to vitrification than immature human oocytes. Spindle Retardance value has more predictive value for embryonic development potential than ZPD and ZPT.

In this Letter, the ‘Open chromatin’ label in Fig. 4a should have been centred above the first three columns, and the black horizontal line underneath the label should have been removed. In addition, there should have been a vertical black line between the last two sets of panels for consistency. Minor changes have also been made to Fig. 1 and to the legend of Fig. 3. These errrors have been corrected online, and see Supplementary Information to the accompanying Amendment for the original Fig. 4.

The quality of postovulatory metaphase II oocytes undergoes a time-dependent deterioration as a result of the aging process. Melatonin is considered to be an anti-aging agent. However, the underlying mechanisms of how melatonin improves the quality of postovulatory aged oocytes remain largely unclear. In this study, by using mouse model, we found that there were elevated reactive oxygen species levels and impaired mitochondrial function demonstrated by reduced mitochondrial membrane potential and increased mitochondrial aggregation in oocytes aged 24 h, accompanied by an increased number of meiotic errors, unregulated autophagy-related proteins and early apoptosis, which led to decreased oocyte quality and disrupted developmental competence. However, all of these events can be largely prevented by supplementing the oocyte culture medium with 10−3 M melatonin. Additionally, we found that the expression of sirtuin family members (SIRT1, 2 and 3) was dramatically reduced in aged oocytes. In addition,in vitrosupplementation with melatonin significantly upregulated the expression of SIRT1 and antioxidant enzyme MnSOD, but this action was not observed for SIRT2 and SIRT3. Furthermore, the protective effect of melatonin on the delay of oocyte aging vanished when the SIRT1 inhibitor EX527 was used to simultaneously treat the oocytes with melatonin. Consistent with this finding, we found that the postovulatory oocyte aging process was markedly attenuated when the oocytes were treated with the SIRT1 activator SRT1720. In conclusion, our data strongly indicate that melatonin delays postovulatory mouse oocyte aging via a SIRT1–MnSOD-dependent pathway, which may provide a molecular mechanism support for the further application of melatonin in the assisted reproductive technology field.

Background Excessive nerve growth factor (NGF) is commonly found in the follicular fluid of patients with polycystic ovary syndrome (PCOS). Furthermore, oocytes from PCOS patients exhibit lower developmental competence. The purpose of this study was to explore the association between excessive NGF and low oocyte competence in vitro. Methods Excessive NGF was added to mouse cumulus oocyte complexes (COCs) cultured in vitro to investigate meiotic maturation of the oocyte. After culture, mRNA expression levels of Pfkp and Ldha genes in cumulus cells (CCs) and Gdf9, Bmp15 and Fgf8 genes in oocytes, were determined by real-time quantitative polymerase chain reaction (qPCR). We also investigated the mRNA content of Pfkp and Ldha in CCs from PCOS and non-PCOS patients. Results Excessive NGF significantly inhibited oocyte meiotic maturation. The inhibitory effect was mediated by the NGF high-affinity receptor, NTRK1. mRNA content of Pfkp and Ldha genes in CCs was significantly reduced by excessive NGF stimulation. Moreover, the expression levels of Gdf9, Bmp15 and Fgf8 were also decreased in oocytes, and was induced by excessive NGF-stimulated CCs. In addition, lower expression levels of Pfkp and Ldha in CCs were identified in Chinese PCOS patients with excessive NGF (PCOS, 22 ± 2.63 ng/ml, n = 13; non-PCOS, 7.18 ± 2.42 ng/ml, n = 9; p

Nerve growth factor (NGF) is known to play key roles in ovarian follicular development, such as the assembly of early follicles and follicular ovulation through its high-affinity receptor, tyrosine kinase receptor A (trkA). Herein, the molecular mechanism controlling NGF-induced granulosa cell (GC) proliferation was not clear. In this study, we found that NGF is abundant in preantral GCs and knockdown of trkA in GCs attenuated NGF-induced GC proliferation and further decreased the levels of phosphorylated extracellular regulated protein kinases 1/2 (ERK1/2). Cyclin-dependent kinase inhibitor 1A (CDKN1A), also named p21, a factor which could be either a negative or a positive regulator via transformation related protein 53 (TRP53, also named p53)-dependent or independent pathways in cell proliferation, was up-regulated during the process of NGF-induced GC proliferation. Blockade of trkA (K252α) and ERK1/2 (U0126) in GCs decreased NGF-induced expression of CDKN1A and did not alter the expression of TRP53, indicating that NGF stimulates CDKN1A expression via the trkA-ERK1/2 pathway in a TRP53-independent manner. Meanwhile, ESR2, a tumor suppressor which is exclusively expressed in GCs, was suppressed in NGF-induced GC proliferation, and this effect was abrogated by U0126. Blockade of ESR2 (ICI182,780) caused the promotion of GC proliferation and CDKN1A expression, indicating that ESR2 may be downstream of the ERK1/2 pathway in mediating the effect of CDKN1A on NGF-induced GC proliferation. Therefore, ESR2 may be involved in the integration of intracellular signal cascades and cell cycle proteins in affecting GC proliferation. Here, we provide mechanistic insights into the roles of CDKN1A in NGF-induced GC proliferation. Understanding potential cross-points between CDKN1A and ESR2 affecting GC proliferation will help in the discovery of new therapeutic targets in some female infertility disorders.

Reciprocal translocations (RecT) and Robertsonian translocations (RobT) are among the most common chromosomal abnormalities that cause infertility and birth defects. Preimplantation genetic testing for aneuploidy using comprehensive chromosome screening for in vitro fertilization enables embryo selection with balanced chromosomal ploidy; however, it is normally unable to determine whether an embryo is a translocation carrier. Here we report a method named “Mapping Allele with Resolved Carrier Status” (MaReCs), which enables chromosomal ploidy screening and resolution of the translocation carrier status of the same embryo. We performed MaReCs on 108 embryos, of which 96 were from 13 RecT carriers and 12 were from three RobT carriers. Thirteen of the sixteen patients had at least one diploid embryo. We have confirmed the accuracy of our carrier status determination in amniotic fluid karyotyping of seven cases as well as in the live birth we have thus far. Therefore, MaReCs accurately enables the selection of translocation-free embryos from patients carrying chromosomal translocations. We expect MaReCs will help reduce the propagation of RecT/RobT in the human population.

Background Polycaprolactone (PCL) has been regarded as a promising synthetic material for bone tissue engineering application. Owing to its unique biochemical properties and great compatibility, PCL fibers have come to be explored as a potential delivering scaffold for stem cells to support bone regeneration during clinical application. Methods The human derived mesenchymal stem cells (MSCs) were obtained from umbilical cord (UC), bone marrow (BM), and adipose tissue (AD), respectively. The osteogenic differentiation potency of various human MSCs on this novel synthetic biomaterial was also investigated in vitro. Results Here, we illustrated that those human UC-, BM-, and AD-derived MSCs exhibited fibroblast-like morphology and expressed characteristic markers. Impressively, PCL nanofiber scaffold could support those MSC adhesion and proliferation. Long-term culture on PCL nanofiber scaffold maintained the viability as well as accelerated the proliferation of those three different kinds of human MSCs. More importantly, the osteogenic differentiation potency of those human MSCs was increased significantly by culturing on PCL nanofiber scaffold. Of note, BM-derived MSCs demonstrated greater differentiation potency among the three kinds of MSCs. The Wnt/β-catenin and Smad3 signaling pathways contributed to the enhanced osteogenesis of human MSCs, which was activated consistently by PCL nanofiber scaffold. Conclusions The utilization of PCL nanofiber scaffold would provide a great application potential for MSC-based bone tissue repair by enhancing the osteogenic differentiation of human MSCs.

Tyrosine kinase receptor A (TrkA), the high-affinity receptor of nerve growth factor (NGF), is known to play key roles in ovarian follicular development, such as assembly of early follicles and follicular ovulation. However, little is known about the roles of TrkA in cumulus oocyte complex (COC) expansion. In this study, we found that TrkA was abundant in large antral follicles and knockdown of TrkA in COCs attenuated epidermal growth factor (EGF)-induced COC expansion and further decreased the ovulation rate. The effect of TrkA on COC expansion was not mediated through downstream EGF effectors, phosphorylation of extracellular regulated protein kinases 1/2 (ERK1/2) or drosophila mothers against decapentaplegic protein (SMAD), or through up-regulation of COC expansion-related transcripts such as prostaglandin-endoperoxide synthase 2 (Ptgs2), hyaluronan synthase 2 (Has2), TNF-induced protein 6 (Tnfaip6) or pentraxin 3 (Ptx3). However, pharmacological blockade of TrkA transducing activity (K252α) in COCs decreased the mRNA expression and protein secretion of interleukin-6 (IL-6), identified from mRNA microarray of K252α-treated COCs. Meanwhile, knockdown of IL-6 attenuated EGF-induced COC expansion. In addition, IL-6 rescued the inhibitory effect of K252α on EGF-induced cumulus expansion. Therefore, IL-6 may act as a new potential cumulus expansion-related transcript, which may be involved in the integration of TrkA and EGF signaling in affecting COC expansion. Here, we provide mechanistic insights into the roles of TrkA in EGF-induced cumulus expansion. Understanding potential cross-points between TrkA and EGF affecting cumulus expansion will help in the discovery of new therapeutic targets in ovulation-related diseases.

Introduction Reciprocal translocations (RecT) and Robertsonian translocations (RobT) are among the most common chromosomal abnormalities that cause infertility and birth defects. Preimplantation genetic testing for aneuploidy using comprehensive chromosome screening for in vitro fertilization enables embryo selection with balanced chromosomal ploidy. However, it is normally unable to determine whether an embryo is a translocation carrier. We have reported a method named “Mapping Allele with Resolved Carrier Status” (MaReCs), which enables chromosomal ploidy screening and resolution of the translocation carrier status of the same embryo ( J. Xu et al. 2017 ). Here we introduce MaReCs and update the clinical study results. Material & Methods From September, 2017 to now, we have performed MaReCs on 69 patients, among them 55 were RecT carriers and 14 were RobT carriers. Result Totally 176 embryos from patients were identified ploidy-balanced, of which embryos from 41 patients were successfully identified as normal or balanced translocation. Other embryos’ translocation status could not be identified because of lacking reference embryos and these patients have accepted further identification in next cycles. And we have confirmed the accuracy of our carrier status determination in amniotic fluid karyotyping of seven cases as well as in the live birth we have thus far. Conclusions MaReCs accurately enables the selection of translocation-free embryos from patients carrying chromosomal translocations. It can work as an effective technology to help reduce the propagation of RecT/RobT in the human population.

MicroRNAs (miRNAs) have been indicated to play key roles in ovarian follicular development. However, little is known about how the miRNA gene expression itself is regulated in the mammalian ovary. We previously reported that miR-224 is involved in TGF-β1-mediated follicular granulosa cell (GC) growth and estradiol (E2) production by targeting Smad4. Here, the transcriptional regulation of miR-224 expression in GCs was further investigated. Our results showed that both the tumor suppressor gene p53 and NF-κB p65 subunit suppressed the TGF-β1-induced increase in pri-miR-224 expression in GCs. ChIP assays demonstrated that TGF-β1 enhanced the binding of p53 and p65 to the proximal promoter region of GABAA receptor ε subunit (miR-224 host gene). p53 and p65 transcriptionally cooperated to inactivate the GABAA receptor ε subunit promoter. In addition, p53/p65 could up-regulate Smad4 expression by inhibiting its target miR-224 in GCs which contributed, at least partially, to the effects of miR-224 and Smad4 on GC proliferation and E2 release. Our results provide new data about the interplay between transcription factors involved in GC proliferation and function by cooperatively regulating miRNA expression.

Clinically discarded human embryos, which are generated from both normal and abnormal fertilizations, have the potential of developing into blastocysts. A total of 1,649 discarded human embryos, including zygotes containing normal (2PN) and abnormal (0PN, 1PN, 3PN and ≥4PN) pronuclei and prematurely cleaved embryos (2Cell), were collected for in vitro culture to investigate their developmental potential and chromosomal constitution using an SNP array-based chromosomal analysis. We found that blastocyst formation rates were 63.8% (for 2Cell embryos), 22.6% (2PN), 16.7% (0PN), 11.2% (3PN) and 3.6% (1PN). SNP array-based chromosomal analysis of the resultant blastocysts revealed that the percentages of normal chromosomes were 55.2% (2Cell), 60.7% (2PN), 44.4% (0PN) and 47.4% (0PN). Compared with clinical preimplantation genetic diagnosis (PGD) data generated with clinically acceptable embryos, results of the SNP array-based chromosome analysis on blastocysts from clinically discarded embryos showed similar values for the frequency of abnormal chromosome occurrence, aberrant signal classification and chromosomal distribution. The present study is perhaps the first systematic analysis of the developmental potential of clinically discarded embryos and provides a basis for future studies.

To evaluate the safety of rapid cryopreservation for human testicular tissues by comparing the meiotic recombination in the fresh and thawed testis after rapid freezing.Twelve male patients with prostate cancer (PC) who had given birth to healthy children at youth and need to receive surgical removal of testicular tissue at present were selected in this study. Testicular tissues from 4 cases of PC patients were randomly divided into two parts, one part as fresh tissue and the other to receive rapid freezing treatment. Fidelity analysis for homologous genetic recombination and synapsis were performed by immunofluorescence after prepared by a micro-spreading technique.The average number of MLH1 foci per cell, autosomal synaptonemal complex (SC) containing 0∼5 MLH1 foci and percent of cells with one MLH1 focus on XY chromosome showed no difference between the fresh and frozen thawed testicular tissues from the same case (P0.05). And, no significant difference in the frequency of gaps and splits on SCs was observed in fresh and thawed spermatocytes (P 0.05).Rapid cryopreservation showed little effect on the frequency of meiotic recombination and fidelity of synapsis in human spermatocytes from PC patients, and acted as an effective method for preserving male fertility.

Leukocyte telomere lengths (LTLs) are shorter in obese compared with normal weight people. However, it is not known whether sperm telomere length (STL) is related to obesity. The aim of the study was to evaluate the impact of men’s body mass index (BMI) on STL, embryo quality and clinical outcomes in couples undergoing IVF. In total, 651 couples were recruited, including 345 men with a normal BMI and 306 men with an overweight BMI (normal BMI group: 20–25 kg/m2; overweight BMI group: >28 kg/m2). We found that couples with male’s BMI over 28 kg/m2 exhibited a significantly lower fertilization rate, good-quality embryo rate and clinical pregnancy rate compared to their normal BMI counterparts. The mean STL in the overweight BMI group was also significantly shorter than that of the normal BMI group. The results also showed that individuals with higher BMI had higher ROS (Reactive oxygen species) content and sperm DNA fragmentation rate when compared with normal BMI individuals. Mitochondrial activity was also lower in the overweight BMI group than in the normal BMI group. This is the first report to find that STL is shorter in overweight/obese men, which may account for their poorer treatment outcomes in IVF cycles.

Whether there are differences in the pregnancy outcomes of blastocysts cryopreserved during different developmental stages remains under debate because the results among studies are inconsistent. We analyzed blastocyst quality and pregnancy outcomes by considering blastocyst euploidy and investigated the differences in the development potential between blastocysts of different developmental stages (frozen-thawed day 5 [D5] and day 6 [D6] cycles) and their relationship with clinical pregnancy outcomes. In total, 1374 D5 and 255 D6 frozen-thawed blastocyst transfer cycles were retrospectively analyzed. Additionally, the chromosome euploidy and clinical pregnancy rates of 237 blastocysts from 50 pre-implantation genetic diagnosis (PGS) cycles were statistically analyzed. The corresponding euploidy rate and pregnancy outcomes of the D5 and D6 blastocyst transfers were also compared. The clinical pregnancy rate (47.2 vs 40.0 %; P = 0.04) and implantation rate (34.2 vs 28.8 %; P = 0.03) of the D5 blastocysts were higher than were those of the D6 blastocysts. However, the clinical pregnancy rate (52.4 vs 52.6 %; P = 0.97) and implantation rate (38.9 vs 35.6 %; P = 0.39) of the high-quality D5 blastocysts did not significantly differ from those of the high-quality D6 blastocysts. Analysis of blastocyst euploidy in 237 blastocysts examined in 50 PGS cycles showed that the euploidy rates of the D5 and D6 blastocysts were both 48.1 % (P = 0.99). The clinical pregnancy rate of the D5 blastocysts (48.5 vs 17.6 %; P = 0.03) was higher than that of the D6 blastocysts. The euploidy rates (55.2 vs 55.3 %; P = 0.99) and clinical pregnancy rates (60.0 vs 42.9 %; P = 0.77) of the high-quality D5 and D6 blastocysts did not differ. The euploidy rate (55.3 vs 41.5 %, P = 0.03) and clinical pregnancy rate (54.5 vs 25.0 %, P = 0.03) of the high-quality blastocysts were higher than were those of the poor-quality blastocysts. The euploidy rates between the D5 and D6 blastocysts did not differ. High-quality D6 blastocysts in frozen-thawed cycles had similar developmental potential and pregnancy outcomes compared to those of high-quality D5 blastocysts. The quality of the blastocysts was an important factor that affected the pregnancy outcomes of the frozen-thawed cycles.

Our previous studies have shown that microRNA-383 (miR-383) is one of the most down-regulated miRNA in TGF-β1-treated mouse ovarian granulosa cells (GC). However, the roles and mechanisms of miR-383 in GC function during follicular development remain unknown. In this study, we found that miR-383 was mainly expressed in GC and oocytes of mouse ovarian follicles. Overexpression of miR-383 enhanced estradiol release from GC through targeting RNA binding motif, single stranded interacting protein 1 (RBMS1). miR-383 inhibited RBMS1 by affecting its mRNA stability, which subsequently suppressed the level of c-Myc (a downstream target of RBMS1). Forced expression of RBMS1 or c-Myc attenuated miR-383-mediated steroidogenesis-promoting effects. Knockdown of the transcription factor steroidogenic factor-1 (SF-1) significantly suppressed the expression of Sarcoglycan zeta (SGCZ) (miR-383 host gene), primary and mature miR-383 in GC, indicating that miR-383 was transcriptionally regulated by SF-1. Luciferase and chromatin immunoprecipitation assays revealed that SF-1 specifically bound to the promoter region of SGCZ and directly transactivated miR-383 in parallel with SGCZ. In addition, SF-1 was involved in regulation of miR-383- and RBMS1/c-Myc-mediated estradiol release from GC. These results suggest that miR-383 functions to promote steroidogenesis by targeting RBMS1, at least in part, through inactivation of c-Myc. SF-1 acts as a positive regulator of miR-383 processing and function in GC. Understanding of regulation of miRNA biogenesis and function in estrogen production will potentiate the usefulness of miRNA in the control of reproduction and treatment of some steroid-related disorders.

We compared the vitrified outcomes between early and expanded blastocysts with or without laser drilling. The grade III embryos from the patients undergoing in vitro fertilization-embryo transfer (IVF-ET) in our reproductive center from September 2009 to February 2015 were incubated into early blastocysts and expanded blastocysts. The early blastocysts and expanded blastocysts were, respectively, divided into laser group (vitrification after laser drilling), non-laser group (direct vitrification), and control group (fresh non-vitrified blastocysts). After thawing, the blastular anabiosis rate, expansion rate, hatching rate, and apoptosis were observed in each group and then were compared amongst groups. This study indicated that the blastular expansion rate (all P