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Yu Tian Institute of Reproductive Health, Center for Reproductive Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China

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Li-quan Zhou Institute of Reproductive Health, Center for Reproductive Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China

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Invasion or damage of the male reproductive system is one of the reported outcomes of viral infection. Current studies have documented that SARS-CoV-2, which causes COVID-19, can damage the male reproductive system in large part by inflammatory damage caused by a cytokine storm. However, whether SARS-CoV-2 can infect the human testis directly and enter semen is controversial. Other adverse effects of SARS-CoV-2 on male reproduction are also of concern and require comprehensive evaluation. Here, we analyze the invasiveness of SARS-CoV-2 in the testis and examine reported mechanisms by which SARS-CoV-2 interferes with male reproduction. Long-term implications of SARS-CoV-2 infection on male reproduction are also discussed. It should be emphasized that although COVID-19 may induce testicular damage, a substantial decrease in male reproductive capacity awaits clinical evidence. We propose that there is an urgent need to track male COVID-19 patients during their recovery. The development of suitable experimental models, including human reproductive organoids, will be valuable to further investigate the viral impact on reproduction for current and future pandemics.

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Hui Li Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, Michigan, USA

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Qianhui Huang Department of Biostatistics, School of Public Health, University of Michigan, Ann Arbor, Michigan, USA

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Yu Liu Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, Michigan, USA

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Lana X Garmire Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, Michigan, USA

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Human placenta is a complex and heterogeneous organ interfacing between the mother and the fetus that supports fetal development. Alterations to placental structural components are associated with various pregnancy complications. To reveal the heterogeneity among various placenta cell types in normal and diseased placentas, as well as elucidate molecular interactions within a population of placental cells, a new genomics technology called single cell RNA-seq (or scRNA-seq) has been employed in the last couple of years. Here we review the principles of scRNA-seq technology, and summarize the recent human placenta studies at scRNA-seq level across gestational ages as well as in pregnancy complications, such as preterm birth and preeclampsia. We list the computational analysis platforms and resources available for the public use. Lastly, we discuss the future areas of interest for placenta single cell studies, as well as the data analytics needed to accomplish them.

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Na Li Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China

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Ling Zhang Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China

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Qi Li Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China

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Yu Du Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China

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Hengwei Liu Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China

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Yi Liu Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China

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Wenqian Xiong Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China

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Oestrogen has been reported to control the invasiveness of endometrial stromal cells in endometriosis. Notch signalling, a master regulator of cell invasion in tumours, is regulated by oestrogen in other diseases and hyperactivated in endometriotic stromal cells. Therefore, we hypothesized that an interaction between Notch signalling and oestrogen may exist in the regulation of endometrial stromal cell invasion, which is essential for the development of endometriosis. Western blot analysis of tissues showed that the expression levels of Notch components (JAG1 and NOTCH1) and Notch activity were markedly higher in ectopic endometria than in their eutopic and normal counterparts. Primary stromal cells obtained from normal endometria cultured with oestrogen presented significant increases in the expression of Notch components and Notch activity, the cytoplasmic and nuclear accumulation of NOTCH1 intracellular domain, the expression of matrix metallopeptidase 9 and vascular endothelial growth factor and cell invasiveness. Knockdown of NOTCH1 markedly alleviated oestrogen-induced matrix metallopeptidase 9 and vascular endothelial growth factor expression and cell invasion. ICI (an oestrogen receptor α antagonist) also blocked these oestrogenic effects. Oestrogen-responsive elements were found in the promoters of NOTCH1 and JAG1. A luciferase reporter analysis revealed that oestrogen regulated the expression of Notch components via oestrogen receptor alpha, which is bound to oestrogen-responsive elements in the JAG1 and NOTCH1 promoters. Collectively, our findings indicate that oestrogen engages in crosstalk with Notch signalling to regulate cell invasion in endometriosis via the activation of oestrogen receptor alpha and the enhancement of Notch activity. Notch signalling blockade may therefore be a novel therapeutic target for endometriosis.

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Hui Li Laboratory for Reproductive Immunology, Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, NPFPC Key Laboratory of Contraceptive Drugs & Devices, Hospital of Obstetrics and Gynecology, Fudan University, Zhao Zhou Road 413, Shanghai 200011, China

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Yu-Han Meng Laboratory for Reproductive Immunology, Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, NPFPC Key Laboratory of Contraceptive Drugs & Devices, Hospital of Obstetrics and Gynecology, Fudan University, Zhao Zhou Road 413, Shanghai 200011, China

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Wen-Qing Shang Laboratory for Reproductive Immunology, Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, NPFPC Key Laboratory of Contraceptive Drugs & Devices, Hospital of Obstetrics and Gynecology, Fudan University, Zhao Zhou Road 413, Shanghai 200011, China

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Li-Bing Liu Laboratory for Reproductive Immunology, Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, NPFPC Key Laboratory of Contraceptive Drugs & Devices, Hospital of Obstetrics and Gynecology, Fudan University, Zhao Zhou Road 413, Shanghai 200011, China

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Xuan Chen Laboratory for Reproductive Immunology, Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, NPFPC Key Laboratory of Contraceptive Drugs & Devices, Hospital of Obstetrics and Gynecology, Fudan University, Zhao Zhou Road 413, Shanghai 200011, China

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Min-Min Yuan Laboratory for Reproductive Immunology, Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, NPFPC Key Laboratory of Contraceptive Drugs & Devices, Hospital of Obstetrics and Gynecology, Fudan University, Zhao Zhou Road 413, Shanghai 200011, China

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Li-Ping Jin Laboratory for Reproductive Immunology, Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, NPFPC Key Laboratory of Contraceptive Drugs & Devices, Hospital of Obstetrics and Gynecology, Fudan University, Zhao Zhou Road 413, Shanghai 200011, China
Laboratory for Reproductive Immunology, Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, NPFPC Key Laboratory of Contraceptive Drugs & Devices, Hospital of Obstetrics and Gynecology, Fudan University, Zhao Zhou Road 413, Shanghai 200011, China

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Ming-Qing Li Laboratory for Reproductive Immunology, Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, NPFPC Key Laboratory of Contraceptive Drugs & Devices, Hospital of Obstetrics and Gynecology, Fudan University, Zhao Zhou Road 413, Shanghai 200011, China
Laboratory for Reproductive Immunology, Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, NPFPC Key Laboratory of Contraceptive Drugs & Devices, Hospital of Obstetrics and Gynecology, Fudan University, Zhao Zhou Road 413, Shanghai 200011, China
Laboratory for Reproductive Immunology, Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, NPFPC Key Laboratory of Contraceptive Drugs & Devices, Hospital of Obstetrics and Gynecology, Fudan University, Zhao Zhou Road 413, Shanghai 200011, China

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Da-Jin Li Laboratory for Reproductive Immunology, Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, NPFPC Key Laboratory of Contraceptive Drugs & Devices, Hospital of Obstetrics and Gynecology, Fudan University, Zhao Zhou Road 413, Shanghai 200011, China
Laboratory for Reproductive Immunology, Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, NPFPC Key Laboratory of Contraceptive Drugs & Devices, Hospital of Obstetrics and Gynecology, Fudan University, Zhao Zhou Road 413, Shanghai 200011, China

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Chemokine CCL24, acting through receptor CCR3, is a potent chemoattractant for eosinophil in allergic diseases and parasitic infections. We recently reported that CCL24 and CCR3 are co-expressed by trophoblasts in human early pregnant uterus. Here we prove with evidence that steroid hormones estradiol (E), progesterone (P), and human chorionic gonadotropin (hCG), as well as decidual stromal cells (DSCs) could regulate the expression of CCL24 and CCR3 of trophoblasts. We further investigate how trophoblast-derived CCL24 mediates the function of trophoblasts in vitro, and conclude that CCL24/CCR3 promotes the proliferation, viability and invasiveness of trophoblasts. In addition, analysis of the downstream signaling pathways of CCL24/CCR3 show that extracellular signal-regulated kinases (ERK1/2) and phosphoinositide 3-kinase (PI3K) pathways may contribute to the proliferation, viability and invasiveness of trophoblasts by activating intracellular molecules Ki67 and matrix metallopeptidase 9 (MMP9). However, we did not observe any inhibitory effect on trophoblasts when blocking c-Jun N-terminal kinase (JNK) or p38 pathways. In conclusion, our data suggests that trophoblast-derived CCL24 at the maternal-fetal interface promotes trophoblasts cell growth and invasiveness by ERK1/2 and PI3K pathways. Meanwhile, pregnancy-related hormones (P and hCG), as well as DSCs could up-regulate CCL24/CCR3 expression in trophoblasts, which may indirectly influence the biological functions of trophoblasts. Thus, our results provide a possible explanation for the growth and invasion of trophoblasts in human embryo implantation.

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Xiaohui Deng
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Hua Zheng
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Xuan Yu
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Hongling Yu
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Chengmei Zhang Center for Reproductive Medicine, Laboratory Animal Center of Shandong University, Department of Pathology, Qilu Hospital of Shandong University, Jinan 250012, Shandong, People's Republic of China

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Lan Chao
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Ruichang Li
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Wenjun Liu Center for Reproductive Medicine, Laboratory Animal Center of Shandong University, Department of Pathology, Qilu Hospital of Shandong University, Jinan 250012, Shandong, People's Republic of China

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The functional longevity of cryopreserved ovarian grafts is one of the most challenging questions regarding ovarian transplantation at present. This study used a rat ovarian grafting model to investigate whether ovarian tissues from adult rats, which had been cryopreserved by vitrification and followed by heterotopic transplantation, could establish long-term hormone secretion and follicle development. Fresh and cryopreserved ovarian tissues were autologously transplanted under the kidney capsule. One-third of the animals in each group (sham-operated, fresh autografts, cryopreserved autografts, or castrated) were killed 5, 8, or 10 months after transplantation. Vaginal cytology, serum estradiol (E2), progesterone, and the morphology of the reproductive tract were used to assess ovarian function. Both fresh and cryopreserved ovarian grafts survived well in all the animal models with comparable proportion of follicles at each stage of folliculogenesis at all three time points. The serum E2 and progesterone concentrations in the groups with fresh or cryopreserved grafts remained comparable with those in sham-operated controls at all investigated time points. However, a loss of grafts and primordial follicles following heterotopic transplantation was noted. In conclusion, the heterotopic autotransplantation of vitrified ovarian tissues from adult rat without vascular anastomosis can maintain long-term ovarian function and exert endocrine function in target organs, in spite of the reduction in follicle pool.

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Tengteng Li Department of Obstetrics and Gynecology, The First Affiliated Hospital of Anhui Medical University, Hefei, China

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Jiajia Fei Department of Obstetrics and Gynecology, The First Affiliated Hospital of Anhui Medical University, Hefei, China

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Huihui Yu Department of Obstetrics and Gynecology, The First Affiliated Hospital of Anhui Medical University, Hefei, China

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Xingxing Wang Department of Obstetrics and Gynecology, The First Affiliated Hospital of Anhui Medical University, Hefei, China

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Dan Li Department of Scientific Research, The Second Affiliated Hospital of Anhui Medical University, Hefei, China

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Zongzhi Yin Department of Obstetrics and Gynecology, The First Affiliated Hospital of Anhui Medical University, Hefei, China
NHC Key Laboratory of the Study of Abnormal Gametes and the Reproductive Tract, Anhui Medical University, Hefei, China

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In brief

During pregnancy, uterine kept quiescence along with uterine overdistention before labor. Prolonged stretching induced uterus myometrial hypoxia, increased TREK1 expression, and relaxed the myometrium, which may contribute to uterine quiescence and atony during pregnancy.

Abstract

The mechanisms underlying pre-labor uterine quiescence and uterine atony during overdistention are unclear. TREK1 (a two-pore domain potassium channel) and hypoxia-inducible factor-1α (HIF-1α) are activated by mechanical stretch, and their expression is upregulated by decreased uterine contractility. HIF-1α is a nuclear factor which regulates numerous target proteins, but whether it regulates TREK1 during the uterine stretch to cause uterine quiescence and/or atony is unclear. We investigated uterine contractility at different gestational stages in rats, as well as in non-pregnant uteri, which were induced by prolonged stretching and hypoxia. We also assessed the effects of incubating the uteri with or without echinomycin or l-methionine. Moreover, we analyzed HIF-1α and TREK1 expression levels in each group, as well as at various gestational stages of pregnant human uteri. We found that contractility was significantly decreased in pregnant uteri when compared with non-pregnant uteri, and this decrease was associated with increases in HIF-1α and TREK1 expression levels. HIF-1α and TREK1 expression levels in human uteri increased with the gestational length. Decreased uterine contractility and increased HIF-1α and TREK1 expression levels were also observed in non-pregnant rat uteri under 8 g of stretching tension or hypoxia. Inhibition of hypoxia with echinomycin restored normal uterine contractility, while HIF-1α and TREK1 protein expression remained reduced. TREK1 inhibition with l-methionine also restored uterine contractility under tension or hypoxia. In conclusion, we demonstrated that prolonged stretching induces myometrial hypoxia, increases TREK1 expression, and relaxes the myometrium, which may contribute to uterine quiescence and atony.

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Yu-Yin Liu Laboratory for Reproductive Immunology, Hospital of Obstetrics and Gynecology, Fudan University, Shanghai, People’s Republic of China

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Yu-Kai Liu Laboratory for Reproductive Immunology, Hospital of Obstetrics and Gynecology, Fudan University, Shanghai, People’s Republic of China

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Wen-Ting Hu Laboratory for Reproductive Immunology, Hospital of Obstetrics and Gynecology, Fudan University, Shanghai, People’s Republic of China

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Ling-Li Tang 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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Chun-Yan Wei Laboratory for Reproductive Immunology, Hospital of Obstetrics and Gynecology, 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
NHC Key Lab of Reproduction Regulation (Shanghai Institute of Planned Parenthood Research), Hospital of Obstetrics and Gynecology, Fudan University, Shanghai, People’s Republic of China
Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Shanghai, People’s Republic of China

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Xiao-Yong Zhu Laboratory for Reproductive Immunology, Hospital of Obstetrics and Gynecology, Fudan University, Shanghai, People’s Republic of China
NHC Key Lab of Reproduction Regulation (Shanghai Institute of Planned Parenthood Research), Hospital of Obstetrics and Gynecology, Fudan University, Shanghai, People’s Republic of China
Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Shanghai, People’s Republic of China

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Endometriosis (EMS) is a chronic inflammatory disease characterized by the presence of extrauterine endometrial tissues. It has been previously reported that the refluxed blood containing viable endometrial tissues and the defective elimination of peritoneal macrophages in the pelvic cavity may involve in EMS pathogenesis. However, the mechanism by which macrophages exhibit attenuated phagocytic capability in EMS remains undetermined. Herein, we found that heme, the byproduct of lysed erythrocytes, accumulated abnormally in the peritoneal fluid (PF) of patients with EMS (14.22 μmol/L, 95% confidence interval (CI): 12.54–16.71), compared with the EMS-free group (9.517 μmol/L, 95% CI: 8.891–10.1053). This abnormal accumulation was not associated with the color of PF, phase of the menstrual cycle or severity of the disease. The reduced phagocytic ability of peritoneal macrophages (pMφs) was observed in the EMS group. Consistently, a high-concentration (30 μmol/L) heme treatment impaired EMS-pMφs phagocytosis more than a low-concentration (10 μmol/L) heme treatment. A similar phenomenon was observed in the EMS-free control pMφs (Ctrl-pMφs) and the CD14+ peripheral monocytes (CD14+ Mos). These results indicated that a high heme concentration exhibits a negative effect on macrophage phagocytosis, which supplements the mechanism of impaired scavenger function of pMφs in EMS.

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Chao Wei Anhui Provincial Laboratory for Local Livestock and Poultry Genetic Resource Conservation and Breeding, College of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui 230036, China

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Xia Li Anhui Provincial Laboratory for Local Livestock and Poultry Genetic Resource Conservation and Breeding, College of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui 230036, China

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Pengfei Zhang Anhui Provincial Laboratory for Local Livestock and Poultry Genetic Resource Conservation and Breeding, College of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui 230036, China

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Yu Zhang Anhui Provincial Laboratory for Local Livestock and Poultry Genetic Resource Conservation and Breeding, College of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui 230036, China

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Tong Liu Anhui Provincial Laboratory for Local Livestock and Poultry Genetic Resource Conservation and Breeding, College of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui 230036, China

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Shaoshuai Jiang Anhui Provincial Laboratory for Local Livestock and Poultry Genetic Resource Conservation and Breeding, College of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui 230036, China

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Fei Han Anhui Provincial Laboratory for Local Livestock and Poultry Genetic Resource Conservation and Breeding, College of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui 230036, China

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Yunhai Zhang Anhui Provincial Laboratory for Local Livestock and Poultry Genetic Resource Conservation and Breeding, College of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui 230036, China

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Partially reprogrammed induced pluripotent stem cells (PiPSCs) have great potential for investigating reprogramming mechanisms and represent an alternative potential material for making genetically modified animals and regenerative medicine. To date, PiPSCs have scarcely been reported in detail when compared with mice and humans. In this study, we obtained PiPSCs from porcine adipose-derived stem cells (pADSCs) by ectopic expression of human transcription factors (OCT4, SOX2, c-MYC, and KLF4) in feeder-free condition. The morphology and proliferation activity of porcine PiPSCs (pPiPSCs) were similar to those of porcine fully reprogrammed iPSCs (pFiPSCs); furthermore, pPiPSCs expressed higher levels of the typical surface molecules (CD29) found in pADSCs. However, pPiPSCs were negative for key proteins (NANOG) connected with stemness and possessed lower differentiation ability in vivo and in vitro. When differentiation-inhibiting factors were withdrawn, pPiPSCs-derived cells (pPiPSC-DCs) showed similar features to pADSCs in many aspects, including proliferation, differentiation, and immunosuppression. When both types of cells were used to produce cloned embryos, we found that the blastocyst formation rate of 19DC (one of the pPiPSC-DC cell lines)-derived cloned embryos was obviously higher than that of others. The total cell number of 19DC-derived blastocysts was significantly higher than the 30DC (one pFiPSC-DC cell line)-derived blastocysts. In all, through limited differentiation ability, the proliferation activity of pPiPSCs is similar to that of pFiPSCs, and pPiPSCs can retain several of the features of pADSCs, which are beneficial to cell therapy. Furthermore, the differentiation of pPiPSCs is more favorable for producing high-quality reconstructed embryos.

Free Chinese abstract: A Chinese translation of this abstract is freely available at http://www.reproduction-online.org/content/149/5/485/suppl/DC2.

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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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Wei Wang College of Animal Science and Technology, Nanjing Agricultural University, Weigang 1, Nanjing 210095, People's Republic of China

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Xia Chen College of Animal Science and Technology, Nanjing Agricultural University, Weigang 1, Nanjing 210095, People's Republic of China

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Xinxiu Li College of Animal Science and Technology, Nanjing Agricultural University, Weigang 1, Nanjing 210095, People's Republic of China

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Li Wang College of Animal Science and Technology, Nanjing Agricultural University, Weigang 1, Nanjing 210095, People's Republic of China

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Haiyan Zhang College of Animal Science and Technology, Nanjing Agricultural University, Weigang 1, Nanjing 210095, People's Republic of China

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Yu He College of Animal Science and Technology, Nanjing Agricultural University, Weigang 1, Nanjing 210095, People's Republic of China

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Jingjing Wang College of Animal Science and Technology, Nanjing Agricultural University, Weigang 1, Nanjing 210095, People's Republic of China

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Yongyan Zhao College of Animal Science and Technology, Nanjing Agricultural University, Weigang 1, Nanjing 210095, People's Republic of China

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Baole Zhang College of Animal Science and Technology, Nanjing Agricultural University, Weigang 1, Nanjing 210095, People's Republic of China

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Yinxue Xu College of Animal Science and Technology, Nanjing Agricultural University, Weigang 1, Nanjing 210095, People's Republic of China

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FSH plays a critical role in granulosa cell (GC) proliferation and steroidogenesis through modulation by factors including bone morphogenetic proteins family, which belongs to transforming growth factor β (TGFB) superfamily. TGFBs are the key factors in maintaining cell growth and differentiation in ovaries. However, the interaction of FSH and TGFB on the GCs' proliferation and steroidogenesis remains to be elucidated. In this study, we have investigated the role of SMAD4, a core molecule mediating the intracellular TGFB/SMAD signal transduction pathway, in FSH-mediated proliferation and steroidogenesis of porcine GCs. In this study, SMAD4 was knocked down using interference RNA in porcine GCs. Our results showed that SMAD4-siRNA causes specific inhibition of SMAD4 mRNA and protein expression after transfection. Knockdown of SMAD4 significantly inhibited FSH-induced porcine GC proliferation and estradiol production and changed the expression of cyclin D2, CDK2, CDK4, CYP19a1, and CYP11a1. Thus, these observations establish an important role of SMAD4 in the regulation of the response of porcine GCs to FSH.

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