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Kaiyue Zhang Center for Reproductive Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai, China

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Wanxia Zhong Center for Reproductive Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai, China

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Wei-Ping Li Center for Reproductive Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai, China

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Zi-Jiang Chen Center for Reproductive Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai, China

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Cong Zhang Center for Reproductive Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai, China
Key Laboratory of Animal Resistance Biology of Shandong Province, College of Life Science, Shandong Normal University, Ji’nan, Shandong, China

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Poor ovarian response is a significant problem encountered during in vitro fertilization and embryo transfer procedures. Many infertile women may suffer from poor ovarian response and its incidence tends to be increasing in young patients nowadays. It is a major cause of maternal infertility because it is associated with low pregnancy and live birth rates. However, the cause of poor ovarian response is not clear. In this study, we extracted microRNAs from human follicular fluid and performed miRNA sequencing to investigate a potential posttranscriptional mechanism underlying poor ovarian response. The results showed that many miRNAs were obviously different between the poor ovarian response and non-poor ovarian response groups. We then performed quantitative polymerase chain reaction, Western blot analysis and used an in vitro culture system to verify the sequencing results and to study the mechanism. Notably, we found that miRNA-15a-5p was significantly elevated in the young poor ovarian response group. Furthermore, we demonstrated that high levels of miR-15a-5p in the young poor ovarian response group repressed granulosa cell proliferation by regulating the PI3K-AKT-mTOR signaling pathway and promoted apoptosis through BCL2 and BAD. This could explain the reduced oocyte retrieval number seen in poor ovarian response patients.

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Ting Zhang
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Pengyuan Dai Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Key Laboratory of Animal Resistance Research, Shandong Center for Disease Control and Prevention, Renji hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200135, China

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Dong Cheng Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Key Laboratory of Animal Resistance Research, Shandong Center for Disease Control and Prevention, Renji hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200135, China

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Liang Zhang Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Key Laboratory of Animal Resistance Research, Shandong Center for Disease Control and Prevention, Renji hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200135, China

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Zijiang Chen
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Xiaoqian Meng Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Key Laboratory of Animal Resistance Research, Shandong Center for Disease Control and Prevention, Renji hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200135, China

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Fumiao Zhang Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Key Laboratory of Animal Resistance Research, Shandong Center for Disease Control and Prevention, Renji hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200135, China

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Xiaoying Han Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Key Laboratory of Animal Resistance Research, Shandong Center for Disease Control and Prevention, Renji hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200135, China

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Jianwei Liu Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Key Laboratory of Animal Resistance Research, Shandong Center for Disease Control and Prevention, Renji hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200135, China

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Jie Pan Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Key Laboratory of Animal Resistance Research, Shandong Center for Disease Control and Prevention, Renji hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200135, China

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Guiwen Yang Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Key Laboratory of Animal Resistance Research, Shandong Center for Disease Control and Prevention, Renji hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200135, China

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Cong Zhang
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The Apolipoprotein (Apo) family is implicated in lipid metabolism. There are five types of Apo: Apoa, Apob, Apoc, Apod, and Apoe. Apoe has been demonstrated to play a central role in lipoprotein metabolism and to be essential for efficient receptor-mediated plasma clearance of chylomicron remnants and VLDL remnant particles by the liver. Apo e-deficient (Apoe −/− ) mice develop atherosclerotic plaques spontaneously, followed by obesity. In this study, we investigated whether lipid deposition caused by Apo e knockout affects reproduction in female mice. The results demonstrated that Apoe −/− mice were severely hypercholesterolemic, with their cholesterol metabolism disordered, and lipid accumulating in the ovaries causing the ovaries to be heavier compared with the WT counterparts. In addition, estrogen and progesterone decreased significantly at D 100. Quantitative PCR analysis demonstrated that at D 100 the expression of cytochromeP450 aromatase (Cyp19a1), 3β-hydroxysteroid dehydrogenase (Hsd3b), mechanistic target of rapamycin (Mtor), and nuclear factor-κB (Nfkb) decreased significantly, while that of BCL2-associated agonist of cell death (Bad) and tuberous sclerosis complex 2 (Tsc2) increased significantly in the Apoe −/− mice. However, there was no difference in the fertility rates of the Apoe −/− and WT mice; that is, obesity induced by Apoe knockout has no significant effect on reproduction. However, the deletion of Apoe increased the number of ovarian follicles and the ratio of ovarian follicle atresia and apoptosis. We believe that this work will augment our understanding of the role of Apoe in reproduction.

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Bo Zheng Center for Reproduction and Genetics, Suzhou Municipal Hospital, The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou, China
State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing, China

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Jun Yu State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing, China
Department of Obstetrics and Gynecology, Affiliated Hospital of Jiangsu University, Jiangsu University, Zhenjiang, China

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Yueshuai Guo State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing, China
The Affiliated Wuxi Matemity and Child Health Care Hospital of Nanjing Medical University, Wuxi, China

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Tingting Gao State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing, China
Center of Clinical Reproductive Medicine, The Affiliated Changzhou Matemity and Child Health Care Hospital of Nanjing Medical University, Changzhou, China

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Cong Shen Center for Reproduction and Genetics, Suzhou Municipal Hospital, The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou, China
State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing, China

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Xi Zhang State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing, China

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Hong Li Center for Reproduction and Genetics, Suzhou Municipal Hospital, The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou, China

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Xiaoyan Huang State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing, China

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The cellular nucleic acid-binding protein (CNBP), also known as zinc finger protein 9, is a highly conserved zinc finger protein that is strikingly conserved among vertebrates. Data collected from lower vertebrates showed that CNBP is expressed at high levels and distributed in the testes during spermatogenesis. However, the location and function of CNBP in mammalian testes are not well known. Here, by neonatal mouse testis culture and spermatogonial stem cells (SSC) culture methods, we studied the effect of CNBP knockdown on neonatal testicular development. Our results revealed that CNBP was mainly located in the early germ cells and Sertoli cells. Knockdown of CNBP using morpholino in neonatal testis culture caused disruption of seminiferous tubules, mislocation of Sertoli cells and loss of germ cells, which were associated with the aberrant Wnt/β-catenin pathway activation. However, knockdown of CNBP in SSC culture did not affect the survival of germ cells. In conclusion, our study suggests that CNBP could maintain testicular development by inhibiting the Wnt/β-catenin pathway, particularly by influencing Sertoli cells.

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Jing Tong Center for Reproductive Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai, China

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Shile Sheng Department of Nuclear Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China

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Yun Sun Center for Reproductive Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai, China

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Huihui Li Center for Reproductive Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai, China
Department of Obstetrics and Gynecology, Center for Reproductive Medicine, Qilu Hospital of Shandong University, Jinan, China

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Wei-Ping Li Center for Reproductive Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai, China

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Cong Zhang Center for Reproductive Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai, China
Key Laboratory of Animal Resistance Research, College of Life Science, Shandong Normal University, Ji’nan, Shandong, China

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Zi-Jiang Chen Center for Reproductive Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai, China
Center for Reproductive Medicine, Shandong Provincial Hospital, Shandong University, Jinan, China
National Research Center for Assisted Reproductive Technology and Reproductive Genetics, Jinan, China
The Key Laboratory for Reproductive Endocrinology of Ministry of Education, Jinan, China
Shandong Provincial Key Laboratory of Reproductive Medicine, Jinan, China

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Good-quality oocytes are critical for the success of in vitro fertilization (IVF), but, to date, there is no marker of ovarian reserve available that can accurately predict oocyte quality. Melatonin exerts its antioxidant actions as a strong radical scavenger that might affect oocyte quality directly as it is the most potent antioxidant in follicular fluid. To investigate the precise role of endogenous melatonin in IVF outcomes, we recruited 61 women undergoing treatment cycles of IVF or intracytoplasmic sperm injection (ICSI) procedures and classified them into three groups according to their response to ovarian stimulation. Follicular fluid was collected to assess melatonin levels using a direct RIA method. We found good correlations between melatonin levels in follicular fluid with age, anti-Müllerian hormone (AMH) and baseline follicle-stimulating hormone (bFSH), all of which have been used to predict ovarian reserve. Furthermore, as melatonin levels correlated to IVF outcomes, higher numbers of oocytes were collected from patients with higher melatonin levels and consequently the number of oocytes fertilized, zygotes cleaved, top quality embryos on D3, blastocysts obtained and embryos suitable for transplantation was higher. The blastocyst rate increased in concert with the melatonin levels across the gradient between the poor response group and the high response group. These results demonstrated that the melatonin levels in follicular fluid is associated with both the quantity and quality of oocytes and can predict IVF outcomes as well making them highly relevant biochemical markers of ovarian reserve.

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Xiao Han State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, People’s Republic of China
College of Life Sciences, Qingdao Agricultural University, Qingdao, People’s Republic of China

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Cong Zhang State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, People’s Republic of China
College of Life Sciences, Qingdao Agricultural University, Qingdao, People’s Republic of China

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Xiangping Ma State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, People’s Republic of China
College of Life Sciences, Qingdao Agricultural University, Qingdao, People’s Republic of China

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Xiaowei Yan State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, People’s Republic of China
College of Life Sciences, Qingdao Agricultural University, Qingdao, People’s Republic of China

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Bohui Xiong State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, People’s Republic of China
College of Life Sciences, Qingdao Agricultural University, Qingdao, People’s Republic of China

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Wei Shen College of Life Sciences, Qingdao Agricultural University, Qingdao, People’s Republic of China

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Shen Yin College of Life Sciences, Qingdao Agricultural University, Qingdao, People’s Republic of China

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Hongfu Zhang State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, People’s Republic of China

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Qingyuan Sun College of Life Sciences, Qingdao Agricultural University, Qingdao, People’s Republic of China
Fertility Preservation Lab, Reproductive Medicine Center, Guangdong Second Provincial General Hospital, Guangzhou, People’s Republic of China

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Yong Zhao State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, People’s Republic of China
College of Life Sciences, Qingdao Agricultural University, Qingdao, People’s Republic of China

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Muscarinic acetylcholine receptor (mAChR) antagonists have been reported to decrease male fertility; however, the roles of mAChRs in spermatogenesis and the underlying mechanisms are not understood yet. During spermatogenesis, extensive remodeling between Sertoli cells and/or germ cells interfaces takes place to accommodate the transport of developing germ cells across the blood-testis barrier (BTB) and adluminal compartment. The cell–cell junctions play a vital role in the spermatogenesis process. This study used ICR male mice and spermatogonial cells (C18-4) and Sertoli cells (TM-4). shRNA of control or M5 gene was injected into 5-week-old ICR mice testes. Ten days post-viral grafting, mice were deeply anesthetized with pentobarbital and the testes were collected. One testicle was fresh frozen for RNA-seq analysis or Western blotting (WB). The second testicle was fixed for immunofluorescence staining (IHF). C18-4 or TM-4 cells were treated with shRNA of control or M5 gene. Then, the cells were collected for RNA-seq analysis, WB, or IHF. Knockdown of mAChR M5 disrupted mouse spermatogenesis and damaged the actin-based cytoskeleton and many types of junction proteins in both Sertoli cells and germ cells. M5 knockdown decreased Phldb2 expression in both germ cells and Sertoli cells which suggested that Phldb2 may be involved in cytoskeleton and cell–cell junction formation to regulate spermatogenesis. Our investigation has elucidated a novel role for mAChR M5 in the regulation of spermatogenesis through the interactions of Phldb2 and cell–cell junctions. M5 may be an attractive future therapeutic target in the treatment of male reproductive disorders.

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