It has been widely acknowledged that anti-Müllerian hormone (AMH) is a golden marker of ovarian reserve. Declined ovarian reserve (DOR), based on experience from reproductive-aged women, refers to both the quantitative and qualitative reduction in oocytes. This view is challenged by a recent study clearly showing that the quality of oocytes is similar in young women undergoing IVF cycles irrespective of the level of AMH. However, it remains elusive whether AMH indicates oocyte quality in women with advanced age (WAA). The aim of this study was to investigate this issue. In the present study, we retrospectively analysed the data generated from a total of 492 IVF/ICSI cycles (from January 2017 to July 2020), and these IVF/ICSI cycles contributed 292 embryo transfer (ET) cycles (from June 2017 to September 2019, data of day 3 ET were included for analysis) in our reproductive centre. Based on the level of AMH, all patients (= > 37 years old) were divided into 2 groups: the AMH high (H) group and the AMH low (L) group. The parameters of in vitro embryo development and clinical outcomes were compared between the two groups. The results showed that women in the L group experienced severe DOR, as demonstrated by a higher rate of primary diagnosis of DOR, lower antral follicle count (AFC), higher level of basal follicle stimulating hormone (FSH) and cancelation cycles, lower level of E2 production on the day of surge, and fewer oocytes and MII oocytes retrieved. Compared with women in the H group, women in the L group showed slightly reduced top embryo formation rate but a similar normal fertilization rate and blastocyst formation rate. More importantly, we found that the rates of implantation, spontaneous miscarriage and livebirth were similar between the two groups, while the pregnancy rate was significantly reduced in the L group compared with the H group. Further analysis indicated that the higher pregnancy rate of women in the H group may be due to more top embryos transferred per cycle. Due to an extremely low implantation potential for transfer of non-top embryos from WAA (= > 37 years old) in our reproductive centre, we assumed that all the embryos that implanted may result from the transfer of top embryos. Based on this observation, we found that the ratio of embryos that successfully implanted or eventually led to a livebirth to top embryos transferred was similar between the H and the L groups. Furthermore, women with clinical pregnancy or livebirth in the H or L group did not show a higher level of serum AMH but were younger than women with non-pregnancy or non-livebirth. Taken together, this study showed that AMH had a limited role in predicting in vitro embryo developmental potential and had no role in predicting the in vivo embryo developmental potential, suggesting that in WAA, AMH should not be used as a marker of oocyte quality. This study supports the view that the accumulation of top embryos via multiple oocyte retrieval times is a good strategy for the treatment of WAA.

Alkylating agents (AAs) that are commonly used for cancer therapy cause great damage to the ovary. Pyrroloquinoline-quinine (PQQ), which was initially identified as a redox cofactor for bacterial dehydrogenases, has been demonstrated to benefit the fertility of females. The aim of this study was to investigate whether PQQ dietary supplementation plays a protective role against alkylating agent-induced ovarian dysfunction. A single dose of busulphan (20 mg/kg) and cyclophosphamide (CTX, 120 mg/kg) were used to establish a mouse model of ovarian dysfunction. Feed containing PQQNa₂ (5 mg/kg) was provided starting 1 week before the establishment of the mouse model until the date of sacrifice. One month later, estrous cycle period of mice were examined and recorded for consecutive 30 days. Three months later, some mice were mated with fertile male mice for fertility test. The remaining mice were sacrificed to collect serum samples and ovaries. One day before sacrifice, some mice received a single injection of BrdU to label proliferating cells. Serum samples were used for test hormonal levels. Ovaries were weighted and used to detect follicle counts, cell proliferation, cell apoptosis and cell senescence. In addition, the levels of inflammation, oxidative damage and Pgc1α expression were detected in ovaries. Results showed that PQQ treatment increased the ovarian weight and size, partially normalized the disrupted estrous cycle period and prevented the loss of follicles of mice treated with AAs. More importantly, we found that PQQ treatment significantly increased the pregnancy rate and litter size per delivery of mice treated with AAs. The protective effects of PQQ appeared to be directly mediated by promoting cell proliferation of granulosa, and inhibiting cell apoptosis of granulosa and cell senescence of ovarian stromal cells. The underlying mechanisms may attribute to the anti-oxidative stress, anti-inflammation and pro-mitochondria biogenesis effects of PQQ.Our study highlights the therapeutic potential of PQQ against ovarian dysfunction caused by alkylating agents.

Background: Recurrent implantation failure (RIF) is a major limitation of assisted reproductive technology, which is associated with impaired endometrial receptivity. Although N⁶-methyladenosine (m⁶A) has been demonstrated to be involved in various biological processes, its potential role in the endometrium of women with RIF has been poorly studied. Methods: Global m⁶A levels and major m⁶A methyltransferases/demethylases mRNA levels in mid-secretory endometrium from normal and RIF women were examined by colorimetric m⁶A quantification strategy and quantitative real-time PCR, respectively. The effects of METTL3-mediated m⁶A modification on embryo attachment were evaluated by an vitro model of a confluent monolayer of Ishikawa cells co-cultured with BeWo spheroids, and the expression levels of homeo box A10 (HOXA10, a well-characterized marker of endometrial receptivity) and its downstream targets were evaluated by quantitative real-time PCR and Western blotting in METTL3-overexpressing Ishikawa cells. The molecular mechanism for METTL3 regulating HOXA10 expression was determined by methylated RNA immunoprecipitation assay and transcription inhibition assay. Results: Global m⁶A methylation and METTL3 expression were significantly increased in the endometrial tissues from women with RIF compared with the controls. Overexpression of METTL3 in Ishikawa cells significantly decreased the ration of BeWo spheroid attachment, and inhibited HOXA10 expression with downstream decreased β3-integrin and increased empty spiracles homeobox 2 expression. METTL3 catalyzed the m⁶A methylation of HOXA10 mRNA and contributed to its decay with shortened half-life. Enforced expression of HOXA10 in Ishikawa cells effectively rescued the impairment of METTL3 on the embryo attachment in vitro. Conclusion: Increased METTL3-mediated m⁶A modification represents an adverse impact on embryo implantation by inhibiting HOXA10 expression, contributing to the pathogenesis of RIF.

In males, aging is accompanied by decline in serum testosterone levels due to impairment of testicular Leydig cells. The polycomb protein BMI1 has recently been identified as an anti-aging factor. In our previous study, BMI1 null mice showed decreased serum testosterone and Leydig cell population, excessive oxidative stress and p16/p19 signaling activation. However, a cause-and-effect relationship between phenotypes and pathways was not investigated. Here, we used the rescue approach to study the role of oxidative stress or p16/p19 in BMI1-mediated steroidogenesis. Our results revealed that treatment with antioxidant NAC, but not down-regulation of p16/p19, largely rescued cell senescence, DNA damage and steroidogenesis in BMI1-deficient mouse MLTC-1 and primary Leydig cells. Collectively, our study demonstrates that BMI1 orchestrates steroidogenesis mainly through maintaining redox homeostasis, and thus, BMI1 may be a novel and potential therapeutic target for treatment of hypogonadism.

Sperm preparation in IVF cycles using density gradient centrifugation (DGC) in combination with swim-up (SU) has been widely adopted in reproductive centres worldwide. It is a fact that the sperm recovery rate following one DGC from poor semen samples (showing liquefaction defects/containing too many unresolvable clots or rare sperm) is relatively low. Our results showed that double DGC (DDGC) is effective at increasing the sperm recovery rate from poor semen samples. However, DDGC may increase the mechanical stress of sperm, thereby potentially impairing embryo development. Therefore, it is necessary to evaluate the safety of using sperm prepared by DDGC/SU for IVF cycles. In this study, we retrospectively analysed the data generated from a total of 529 IVF cycles (from June 2017 to June 2018), and these IVF cycles contributed 622 transfer cycles (from June 2017 to December 2018) in Changzhou Maternal and Child Health Care Hospital. Of them, 306 IVF cycles and the related 355 transfer cycles (normal semen samples prepared by DGC/SU) were set as the normal group, while 223 IVF cycles and the related 267 transfer cycles (poor semen prepared by DDGC/SU) were set as the observation group. The main outcome measures, including the normal fertilization rate, top D3 embryo formation rate, blastocyte formation rate, clinical pregnancy rate and live birth rate, birth weight and duration of pregnancy, were compared between the two groups. Compared to semen in the DGC/SU group, semen in the DDGC/SU group showed increased levels of the DNA fragmentation index (DFI) and reduced sperm concentration, percentage of progressive motility (PR) sperm, and percentage of normal morphology sperm. The indicators reflecting in vitro embryo development and clinical outcomes were similar in the DGC/SU group and DDGC/SU group, including the normal fertilization rate, top D3 embryo formation rate, blastocyte formation rate, pregnancy rate, implantation rate, spontaneous abortion rate, live birth rate, birth weight and duration of pregnancy. Furthermore, we found that the 1PN zygote formation rate was significantly lower in the DDGC/SU group than that in the DGC/SU group. We concluded that oocytes fertilized by sperm from poor semen samples separated by DDGC/SU achieved the same outcomes as oocytes fertilized by sperm from normal semen separated by DGC/SU, suggesting that DDGC/SU is an effective and safe method of sperm enrichment for poor semen samples in IVF. The main contribution of the present study is the verification of the effectiveness of DDGC/SU in improving sperm recovery from poor semen samples and the safety of using sperm prepared by DDGC/SU for IVF.