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Hang Qi Department of Obstetrics and Gynecology, International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China
Institute of Embryo-Fetal Original Adult Disease, Affiliated to School of Medicine, Shanghai Jiaotong University, Shanghai, China

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Guiling Liang Department of Obstetrics and Gynecology, International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China
Institute of Embryo-Fetal Original Adult Disease, Affiliated to School of Medicine, Shanghai Jiaotong University, Shanghai, China

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Jin Yu Department of Obstetrics and Gynecology, International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China
Institute of Embryo-Fetal Original Adult Disease, Affiliated to School of Medicine, Shanghai Jiaotong University, Shanghai, China

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Xiaofeng Wang Department of Obstetrics and Gynecology, International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China
Institute of Embryo-Fetal Original Adult Disease, Affiliated to School of Medicine, Shanghai Jiaotong University, Shanghai, China

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Yan Liang Department of Obstetrics and Gynecology, International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China
Institute of Embryo-Fetal Original Adult Disease, Affiliated to School of Medicine, Shanghai Jiaotong University, Shanghai, China

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Xiaoqing He Department of Obstetrics and Gynecology, International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China
Institute of Embryo-Fetal Original Adult Disease, Affiliated to School of Medicine, Shanghai Jiaotong University, Shanghai, China

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Tienan Feng Clinical Research Center, Shanghai Jiao Tong University School of Medicine, Shanghai, China

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Jian Zhang Department of Obstetrics and Gynecology, International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China
Institute of Embryo-Fetal Original Adult Disease, Affiliated to School of Medicine, Shanghai Jiaotong University, Shanghai, China

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MicroRNA (miRNA) expression profiles in tubal endometriosis (EM) are still poorly understood. In this study, we analyzed the differential expression of miRNAs and the related gene networks and signaling pathways in tubal EM. Four tubal epithelium samples from tubal EM patients and five normal tubal epithelium samples from uterine leiomyoma patients were collected for miRNA microarray. Bioinformatics analyses, including Ingenuity Pathway Analysis (IPA), Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, were performed. Quantitative real-time polymerase chain reaction (qRT-PCR) validation of five miRNAs was performed in six tubal epithelium samples from tubal EM and six from control. A total of 17 significantly differentially expressed miRNAs and 4343 potential miRNA-target genes involved in tubal EM were identified (fold change >1.5 and FDR-adjusted P value <0.05). IPA indicated connections between miRNAs, target genes and other gynecological diseases like endometrial carcinoma. GO and KEGG analysis revealed that most of the identified genes were involved in the mTOR signaling pathway, SNARE interactions in vesicular transport and endocytosis. We constructed an miRNA-gene-disease network using target gene prediction. Functional analysis showed that the mTOR pathway was connected closely to tubal EM. Our results demonstrate for the first time the differentially expressed miRNAs and the related signal pathways involved in the pathogenesis of tubal EM which contribute to elucidating the pathogenic mechanism of tubal EM-related infertility.

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Xiaokui Yang
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Ying Zhou
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Sha Peng
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Liang Wu Department of Human Reproductive Medicine, State Key Laboratory of Reproductive Biology, Graduate School of the Chinese Academy of Sciences, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing 100006, China
Department of Human Reproductive Medicine, State Key Laboratory of Reproductive Biology, Graduate School of the Chinese Academy of Sciences, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing 100006, China

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Hai-Yan Lin Department of Human Reproductive Medicine, State Key Laboratory of Reproductive Biology, Graduate School of the Chinese Academy of Sciences, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing 100006, China

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Shuyu Wang
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Hongmei Wang Department of Human Reproductive Medicine, State Key Laboratory of Reproductive Biology, Graduate School of the Chinese Academy of Sciences, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing 100006, China

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Recent studies implicate the regulatory function of microRNAs (miRNAs) in oocyte maturation and ovarian follicular development. Differentially expressed miRNAs are found in the plasma of premature ovarian failure (POF) patients and normal cycling women. In this study, miRNA-regulated signaling pathways and related genes were described using Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes pathway analysis. The effect of mir-23a on granulosa cell apoptosis was also studied by examining the protein expression of X-linked inhibitor of apoptosis protein (XIAP) and caspase-3, followed by subsequent counting of apoptotic cells after Hoechst 33258 staining. Both GO analysis and pathway analysis suggested that many signaling pathways, including the AKT signaling pathway, steroid hormone receptor signaling pathways, and others, were regulated by this group of differentially expressed miRNAs. A decrease in XIAP expression (mRNA and protein level) and caspase-3 protein levels and an increase in cleaved caspase-3 protein were observed in human ovarian granulosa cells transfected with pre-mir-23a, along with an increased occurrence of apoptosis. In conclusion, differentially expressed miRNAs in the plasma of POF patients may have regulatory effects on proliferation and apoptosis of granulosa cells by affecting different signaling pathways. Mir-23a may play important roles in regulating apoptosis via decreasing XIAP expression in human ovarian granulosa cells.

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Keqin Yan Department of Obstetrics and Gynecology, China–Japan Friendship Hospital, Beijing, China

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Dingqing Feng Department of Obstetrics and Gynecology, China–Japan Friendship Hospital, Beijing, China

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Jing Liang Department of Obstetrics and Gynecology, China–Japan Friendship Hospital, Beijing, China

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Qing Wang Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, Beijing, China

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Lin Deng Department of Obstetrics and Gynecology, China–Japan Friendship Hospital, Beijing, China

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Xiao Zhang Department of Obstetrics and Gynecology, China–Japan Friendship Hospital, Beijing, China

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Bin Ling Department of Obstetrics and Gynecology, China–Japan Friendship Hospital, Beijing, China

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Daishu Han Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, Beijing, China

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Viral infections of the ovary may perturb ovarian functions. However, the mechanisms underlying innate immune responses in the ovary are poorly understood. The present study demonstrates that cytosolic viral DNA sensor signaling initiates the innate immune response in mouse ovarian granulosa cells and affects endocrine function. The cytosolic DNA sensors p204 and cGAS and their common signaling adaptor stimulator of interferon (IFN) genes (STING) were constitutively expressed in granulosa cells. Transfection with VACV70, a synthetic vaccinia virus (VACV) DNA analog, induced the expression of type I interferons (IFNA/B) and major inflammatory cytokines (TNFA and IL6) through IRF3 and NF-κB activation respectively. Moreover, several IFN-inducible antiviral proteins, including 2′,5′-oligoadenylate synthetase, IFN-stimulating gene 15 and Mx GTPase 1, were also induced by VACV70 transfection. The innate immune responses in granulosa cells were significantly reduced by the transfection of specific small-interfering RNAs targeting p204, cGas or Sting. Notably, the VACV70-triggered innate immune responses affected steroidogenesis in vivo and in vitro. The data presented in this study describe the mechanism underlying ovarian immune responses to viral infection.

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Yu-Xiang Liang Shanxi Key Laboratory of Birth Defect and Cell Regeneration, Shanxi Medical University, Taiyuan, China
College of Veterinary Medicine, South China Agricultural University, Guangzhou, China

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Wei Hu College of Veterinary Medicine, South China Agricultural University, Guangzhou, China
College of Life Science and Resources and Environment, Yichun University, Yichun, China

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Zhi-Yong Jin College of Veterinary Medicine, South China Agricultural University, Guangzhou, China

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Hong-Lu Diao Reproductive Medicine Center, Renmin Hospital, Hubei University of Medicine, Shiyan, Hubei, China

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Li Liu College of Veterinary Medicine, South China Agricultural University, Guangzhou, China

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Yan Yang College of Veterinary Medicine, South China Agricultural University, Guangzhou, China

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Tao Fu College of Veterinary Medicine, South China Agricultural University, Guangzhou, China

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Zeng-Ming Yang College of Veterinary Medicine, South China Agricultural University, Guangzhou, China

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Embryo implantation and decidualization are crucial steps during early pregnancy. We recently showed that nucleolar stress is involved in embryo implantation. This study was to explore whether nucleolar stress participates in mouse and human decidualization. Our data demonstrated that a low dose of actinomycin D (ActD) could induce nucleolar stress in stroma cells. Nucleolar stress promotes the stromal-epithelial transition during mouse in vitro decidualization through nucleophosmin1 (NPM1). Under nucleolar stress, Wnt family member 4 (Wnt4), a decidualization marker, is significantly increased, but decidua/trophoblast prolactin-related protein (Dtprp/Prl8a2) expression remains unchanged. For translational significance, we also examined the effects of nucleolar stress on human decidualization. Nucleolar stress stimulated by a low dose of ActD enhances human stromal–epithelial transition during human decidualization, but has no effects on the expression of insulin-like growth factor-binding protein 1 (IGFBP1). Our study indicates that nucleolar stress may promote only the mesenchymal–epithelial transition (MET), but not for all the molecular changes during decidualization.

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Baoheng Gui The State Key Laboratory of Medical Genetics of China, The Reproductive Medical Center of Xiangya Hospital, Yikon Genomics Co. Ltd, Central South University, No. 110 Xiangya Road, Changsha, Hunan 410008, China

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Zhongyuan Yao The State Key Laboratory of Medical Genetics of China, The Reproductive Medical Center of Xiangya Hospital, Yikon Genomics Co. Ltd, Central South University, No. 110 Xiangya Road, Changsha, Hunan 410008, China

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Yanping Li The State Key Laboratory of Medical Genetics of China, The Reproductive Medical Center of Xiangya Hospital, Yikon Genomics Co. Ltd, Central South University, No. 110 Xiangya Road, Changsha, Hunan 410008, China

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Donge Liu The State Key Laboratory of Medical Genetics of China, The Reproductive Medical Center of Xiangya Hospital, Yikon Genomics Co. Ltd, Central South University, No. 110 Xiangya Road, Changsha, Hunan 410008, China

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Nenghui Liu The State Key Laboratory of Medical Genetics of China, The Reproductive Medical Center of Xiangya Hospital, Yikon Genomics Co. Ltd, Central South University, No. 110 Xiangya Road, Changsha, Hunan 410008, China

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Yan Xia The State Key Laboratory of Medical Genetics of China, The Reproductive Medical Center of Xiangya Hospital, Yikon Genomics Co. Ltd, Central South University, No. 110 Xiangya Road, Changsha, Hunan 410008, China

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Yanru Huang The State Key Laboratory of Medical Genetics of China, The Reproductive Medical Center of Xiangya Hospital, Yikon Genomics Co. Ltd, Central South University, No. 110 Xiangya Road, Changsha, Hunan 410008, China

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Libin Mei The State Key Laboratory of Medical Genetics of China, The Reproductive Medical Center of Xiangya Hospital, Yikon Genomics Co. Ltd, Central South University, No. 110 Xiangya Road, Changsha, Hunan 410008, China

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Ruiyu Ma The State Key Laboratory of Medical Genetics of China, The Reproductive Medical Center of Xiangya Hospital, Yikon Genomics Co. Ltd, Central South University, No. 110 Xiangya Road, Changsha, Hunan 410008, China

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Sijia Lu The State Key Laboratory of Medical Genetics of China, The Reproductive Medical Center of Xiangya Hospital, Yikon Genomics Co. Ltd, Central South University, No. 110 Xiangya Road, Changsha, Hunan 410008, China

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Desheng Liang The State Key Laboratory of Medical Genetics of China, The Reproductive Medical Center of Xiangya Hospital, Yikon Genomics Co. Ltd, Central South University, No. 110 Xiangya Road, Changsha, Hunan 410008, China

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Lingqian Wu The State Key Laboratory of Medical Genetics of China, The Reproductive Medical Center of Xiangya Hospital, Yikon Genomics Co. Ltd, Central South University, No. 110 Xiangya Road, Changsha, Hunan 410008, China

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Balanced chromosomal rearrangements (CRs) are among the most common genetic abnormalities in humans. In the present study, we have investigated the degree of consistency between the chromosomal composition of the blastocyst inner cell mass (ICM) and trophectoderm (TE) in carriers with balanced CR, which has not been previously addressed. As a secondary aim, we have also evaluated the validity of cleavage-stage preimplantation genetic diagnosis (PGD) based on fluorescence in situ hybridization (FISH) of blastocysts from CR carriers. Blastocyst ICM and TE were screened for chromosomal aneuploidy and imbalance of CR-associated chromosomes based on whole-genome copy number variation analysis by low-coverage next-generation sequencing (NGS) following single-cell whole-genome amplification by multiple annealing and looping-based amplification cycling. The NGS results were analyzed without knowledge of cleavage-stage FISH results. NGS results for blastocyst ICM and TE from CR carriers were 86.49% (32/37) consistent. Of the 1702 (37×46) chromosomes examined, 99.47% (1693/1702) showed consistency. However, only 40.0% (18/45) of all embryos had consistent results for chromosomes involved in CR, as determined by blastocyst NGS and cleavage-stage FISH. Of the 85 CR-affected chromosomes analyzed by FISH, 37.65% (32/85) were incongruous with NGS results, with 87.5% (28/32) showing imbalanced composition by FISH but balanced composition by NGS. These results indicate that chromosomal composition of blastocyst ICM and TE in balanced CR carriers is highly consistent, and that PGD based on cleavage-stage FISH is inaccurate; therefore, using blastocyst TE biopsies for NGS-based PGD is recommended for identifying chromosomal imbalance in embryos from balanced CR carriers.

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Rui-Qi Chang The Center for Reproductive Medicine, Department of Obstetrics and Gynecology, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, People’s Republic of China
Joint International Research Lab for Reproduction and Development, Ministry of Education, Chongqing, People’s Republic of China
Reproduction and Stem Cell Therapy Research Center of Chongqing, Chongqing, People’s Republic of China

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Jing-Cong Dai The Center for Reproductive Medicine, Department of Obstetrics and Gynecology, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, People’s Republic of China

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Yu-Han Qiu The Center for Reproductive Medicine, Department of Obstetrics and Gynecology, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, People’s Republic of China

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Yan Liang The Center for Reproductive Medicine, Department of Obstetrics and Gynecology, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, People’s Republic of China

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Xiao-Yu Hu The Center for Reproductive Medicine, Department of Obstetrics and Gynecology, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, People’s Republic of China

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Ming-Qing Li Laboratory for Reproductive Immunology, Hospital of Obstetrics and Gynecology, Fudan University, Shanghai, People’s Republic of China

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Fan He The Center for Reproductive Medicine, Department of Obstetrics and Gynecology, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, People’s Republic of China
Joint International Research Lab for Reproduction and Development, Ministry of Education, Chongqing, People’s Republic of China
Reproduction and Stem Cell Therapy Research Center of Chongqing, Chongqing, People’s Republic of China

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In brief

The mechanism underlying the accumulation of γδT cells in the decidua, which helps maintain maternal–fetal immunotolerance in early pregnancy, is unknown. This study reveals that DSC-derived RANKL upregulates ICAM-1 expression via the NF-κB pathway to enable γδT cell accumulation in the early decidua.

Abstract

Decidual γδT (dγδT) cells help maintain maternal–fetal immunotolerance in early pregnancy. However, the mechanism underlying the accumulation of γδT cells in the decidua is unknown. Previous work showed that RANKL upregulated intercellular adhesion molecule 1 (ICAM-1) in decidual stromal cells (DSCs), and Rankl knockout mice had limited dγδT cell populations. In this study, we measured the expression levels of RANKL/RANK and ICAM-1 in DSCs, in addition to the integrins of ICAM-1 on dγδT cells, and the number of dγδT cells from patients with recurrent spontaneous abortion (RSA) and normal pregnant women in the first trimester. RSA patients showed significantly decreased RANKL/RANK and ICAM-1/CD11a signaling in decidua, and a decreased percentage of dγδT cells, which was positively correlated with DSC-derived RANKL and ICAM-1. Next, an in vitro adhesion experiment showed that the enhanced attraction of human DSCs to dγδT cells after RANKL overexpression was almost completely aborted by anti-ICAM-1. Furthermore, Rankl knockout mice showed a significant reduction in NF-κB activity compared with wild-type controls. Finally, we applied a selective NF-κB inhibitor named PDTC to validate the role of NF-κB in RANKL-mediated ICAM-1 upregulation. Taken together, our data show that DSC-derived RANKL upregulates ICAM-1 expression via the NF-κB pathway to enable γδT cell accumulation in the early decidua. A reduction in RANKL/ICAM-1 signaling in DSCs may result in insufficient accumulation of γδT cells in decidua and, in turn, RSA.

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Hui-Hui Shen Laboratory for Reproductive Immunology, Hospital of Obstetrics and Gynecology, Fudan University, Shanghai, People’s Republic of China

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Cheng-Jie Wang Laboratory for Reproductive Immunology, Hospital of Obstetrics and Gynecology, Fudan University, Shanghai, People’s Republic of China

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Xin-Yan Zhang Laboratory for Reproductive Immunology, Hospital of Obstetrics and Gynecology, Fudan University, Shanghai, People’s Republic of China

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Yan-Ran Sheng Laboratory for Reproductive Immunology, Hospital of Obstetrics and Gynecology, Fudan University, Shanghai, People’s Republic of China

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Shao-Liang Yang Laboratory for Reproductive Immunology, Hospital of Obstetrics and Gynecology, Fudan University, Shanghai, People’s Republic of China

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Zi-Meng Zheng Laboratory for Reproductive Immunology, Hospital of Obstetrics and Gynecology, Fudan University, Shanghai, People’s Republic of China

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Jia-Lu Shi Laboratory for Reproductive Immunology, Hospital of Obstetrics and Gynecology, Fudan University, Shanghai, People’s Republic of China

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Xue-Min Qiu Laboratory for Reproductive Immunology, Hospital of Obstetrics and Gynecology, Fudan University, Shanghai, People’s Republic of China
Clinical Research Center, Obstetrics and Gynecology Hospital of Fudan University, Shanghai, People’s Republic of China

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Feng Xie Medical Center of Diagnosis and Treatment for Cervical and Intrauterine Diseases, Obstetrics and Gynecology Hospital of Fudan University, Shanghai, People’s Republic of China

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Ming-Qing Li Laboratory for Reproductive Immunology, Hospital of Obstetrics and Gynecology, Fudan University, Shanghai, People’s Republic of China
Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Hospital of Obstetrics and Gynecology, Fudan University, Shanghai, People’s Republic of China
Department of Obstetrics and Gynecology, Maternal and Child Health Hospital of Longgang District, Shenzhen, Guangdong Province, People’s Republic of China

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Heme oxygenase 1 (HO-1, encoded by the HMOX1 gene) is the rate-limiting enzyme that catalyzes heme degradation, and it has been reported to exert antioxidative effects. Recently, decidualization has been reported to confer resistance to environmental stress signals, protecting against oxidative stress. However, the effects and regulatory mechanism of HO-1 in decidual stromal cells (DSCs) during early pregnancy remain unknown. Here, we verified that the levels of HO-1 and heme in DSCs are increased compared with those in endometrial stromal cells. Additionally, the upregulation of HIF1A expression led to increased HMOX1 expression in DSCs possibly via nuclear factor erythroid 2-related factor (encoded by the NFE2L2 gene). However, addition of the competitive HO-1 inhibitor zinc protoporphyrin IX resulted in an increase in HIF1A expression. Hydrogen peroxide (H2O2) induced the production of reactive oxygen species (ROS), decreased the cell viability of DSCs in vitro, and upregulated the level of heme. As an HO-1 inducer, cobalt protoporphyrin IX decreased ROS production and significantly reversed the inhibitory effect of H2O2 on cell viability. More importantly, patients with unexplained spontaneous abortion had low levels of HO-1 that were insufficient to protect against oxidative stress. This study suggests that the upregulation of HO-1 expression via HIF1A protects DSCs against excessive heme-mediated oxidative stress. Furthermore, the excessive oxidative stress injury and impaired viability of DSCs associated with decreased HO-1 expression should be associated with the occurrence and/or development of spontaneous abortion.

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Rui-Song Ye
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Meng Li
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Chao-Yun Li Chinese National Engineering Research Center for Breeding Swine Industry, SCAU-Alltech Research Joint Alliance, Guandong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, China

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Qi-En Qi Chinese National Engineering Research Center for Breeding Swine Industry, SCAU-Alltech Research Joint Alliance, Guandong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, China

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Ting Chen Chinese National Engineering Research Center for Breeding Swine Industry, SCAU-Alltech Research Joint Alliance, Guandong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, China

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Xiao Cheng Chinese National Engineering Research Center for Breeding Swine Industry, SCAU-Alltech Research Joint Alliance, Guandong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, China

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Song-Bo Wang Chinese National Engineering Research Center for Breeding Swine Industry, SCAU-Alltech Research Joint Alliance, Guandong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, China

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Gang Shu Chinese National Engineering Research Center for Breeding Swine Industry, SCAU-Alltech Research Joint Alliance, Guandong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, China

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Li-Na Wang Chinese National Engineering Research Center for Breeding Swine Industry, SCAU-Alltech Research Joint Alliance, Guandong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, China

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Xiao-Tong Zhu Chinese National Engineering Research Center for Breeding Swine Industry, SCAU-Alltech Research Joint Alliance, Guandong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, China

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Qing-Yan Jiang Chinese National Engineering Research Center for Breeding Swine Industry, SCAU-Alltech Research Joint Alliance, Guandong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, China

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Qian-Yun Xi Chinese National Engineering Research Center for Breeding Swine Industry, SCAU-Alltech Research Joint Alliance, Guandong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, China

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Yong-Liang Zhang Chinese National Engineering Research Center for Breeding Swine Industry, SCAU-Alltech Research Joint Alliance, Guandong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, China

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FSH plays an essential role in processes involved in human reproduction, including spermatogenesis and the ovarian cycle. While the transcriptional regulatory mechanisms underlying its synthesis and secretion have been extensively studied, little is known about its posttranscriptional regulation. A bioinformatics analysis from our group indicated that a microRNA (miRNA; miR-361-3p) could regulate FSH secretion by potentially targeting the FSHB subunit. Herein, we sought to confirm these findings by investigating the miR-361-3p-mediated regulation of FSH production in primary pig anterior pituitary cells. Gonadotropin-releasing hormone (GnRH) treatment resulted in an increase in FSHB synthesis at both the mRNA, protein/hormone level, along with a significant decrease in miR-361-3p and its precursor (pre-miR-361) levels in time- and dose-dependent manner. Using the Dual-Luciferase Assay, we confirmed that miR-361-3p directly targets FSHB. Additionally, overexpression of miR-361-3p using mimics significantly decreased the FSHB production at both the mRNA and protein levels, with a reduction in both protein synthesis and secretion. Conversely, both synthesis and secretion were significantly increased following miR-361-3p blockade. To confirm that miR-361-3p targets FSHB, we designed FSH-targeted siRNAs, and co-transfected anterior pituitary cells with both the siRNA and miR-361-3p inhibitors. Our results indicated that the siRNA blocked the miR-361-3p inhibitor-mediated upregulation of FSH, while no significant effect on non-target expression. Taken together, our results demonstrate that miR-361-3p negatively regulates FSH synthesis and secretion by targeting FSHB, which provides more functional evidence that a miRNA is involved in the direct regulation of FSH.

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Shu-Zhen Liu State Key Laboratory of Reproductive Biology, Institute of Zoology, Graduate School, Chinese Academy of Sciences, Beijing 100080, China and College of Life Science, Shandong Normal University, Jinan 250014, China

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Li-Juan Yao State Key Laboratory of Reproductive Biology, Institute of Zoology, Graduate School, Chinese Academy of Sciences, Beijing 100080, China and College of Life Science, Shandong Normal University, Jinan 250014, China

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Man-Xi Jiang State Key Laboratory of Reproductive Biology, Institute of Zoology, Graduate School, Chinese Academy of Sciences, Beijing 100080, China and College of Life Science, Shandong Normal University, Jinan 250014, China

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Zi-Li Lei State Key Laboratory of Reproductive Biology, Institute of Zoology, Graduate School, Chinese Academy of Sciences, Beijing 100080, China and College of Life Science, Shandong Normal University, Jinan 250014, China

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Li-Sheng Zhang State Key Laboratory of Reproductive Biology, Institute of Zoology, Graduate School, Chinese Academy of Sciences, Beijing 100080, China and College of Life Science, Shandong Normal University, Jinan 250014, China

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Yan-Ling Zhang State Key Laboratory of Reproductive Biology, Institute of Zoology, Graduate School, Chinese Academy of Sciences, Beijing 100080, China and College of Life Science, Shandong Normal University, Jinan 250014, China

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Qing-Yuan Sun State Key Laboratory of Reproductive Biology, Institute of Zoology, Graduate School, Chinese Academy of Sciences, Beijing 100080, China and College of Life Science, Shandong Normal University, Jinan 250014, China

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Yue-Liang Zheng State Key Laboratory of Reproductive Biology, Institute of Zoology, Graduate School, Chinese Academy of Sciences, Beijing 100080, China and College of Life Science, Shandong Normal University, Jinan 250014, China

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Xiang-Fen Song State Key Laboratory of Reproductive Biology, Institute of Zoology, Graduate School, Chinese Academy of Sciences, Beijing 100080, China and College of Life Science, Shandong Normal University, Jinan 250014, China

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Da-Yuan Chen State Key Laboratory of Reproductive Biology, Institute of Zoology, Graduate School, Chinese Academy of Sciences, Beijing 100080, China and College of Life Science, Shandong Normal University, Jinan 250014, China

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In this study, we investigated the development, the cell number of the blastocyst, and apoptosis in rabbit nuclear transfer (NT) embryos derived from adult fibroblasts and cumulus cells as compared with embryos derived from in vivo fertilization and in vitro culture. The developmental rate and the total cell number of the blastocyst were significantly lower in NT embryos than in fertilized embryos (FEs). The type of donor cells did not affect the embryonic developmental rate and the total cell number of blastocysts in NT groups. The present study investigated the onset and the frequency of apoptosis in NT embryos and FEs by using a terminal deoxynucleotidyl transferase-mediated dUTP nick and labeling (TUNEL) assay. The earliest positive TUNEL signals were detected at the eight-cell stage in NT embryos and at the morula stage in FEs. The apoptotic index of the total blastocysts, the inner cell mass and the trophoderm was greatly higher in the NT embryos than in FEs. Moreover, the apoptotic index of the blastocyst from fibroblasts was significantly higher than that of the blastocyst from cumulus cells.

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Y-F Liu College of Veterinary Medicine, South China Agricultural University, Guangzhou, China

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M-Y Li College of Veterinary Medicine, South China Agricultural University, Guangzhou, China

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Y-P Yan College of Veterinary Medicine, South China Agricultural University, Guangzhou, China

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W Wei College of Veterinary Medicine, South China Agricultural University, Guangzhou, China

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B Li College of Veterinary Medicine, South China Agricultural University, Guangzhou, China

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H-Y Pan College of Veterinary Medicine, South China Agricultural University, Guangzhou, China

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Z-M Yang College of Veterinary Medicine, South China Agricultural University, Guangzhou, China

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X-H Liang College of Veterinary Medicine, South China Agricultural University, Guangzhou, China

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Maintenance of a suitable uterine milieu is important for embryo development and subsequent implantation during early pregnancy. High estrogen level in proestrous and estrous stages is essential for uterine anti-bacterial activity during preimplantation period. Lipocalin-2 is an essential molecule which prevents bacterial infection by sequestering iron. In this study, the highest expression of lipocalin-2 is observed in the endometrial epithelium on day 1 of normal pregnancy and pseudopregnancy, which exhibit a similar hormone scenario. By injecting the agonists for estrogen receptor α and estrogen receptor β in ovariectomized mice, we found estrogen receptor α is the dominant member for estrogen regulation on lipocalin-2 expression. Estrogen treatment in estrogen receptor α-knockout mice further confirmed the role of estrogen receptor α. Using published data from whole-genome estrogen receptor α binding site assay, significant estrogen receptor α recruitment peaks are found at the downstream of lipocalin-2 gene after estrogen treatment. Furthermore, to study the anti-bacterial activity of lipocalin-2 in uterus, Escherichia coli is injected to mimic bacterial infection. Our results showed an obvious induction of lipocalin-2 in Escherichia coli-treated group. Taken together, this study indicates estrogen regulation of lipocalin-2 in uterine epithelium is mediated by estrogen receptor α, and lipocalin-2 may have anti-bacterial activity during early pregnancy.

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