title: Human single follicle growth in vitro from cryopreserved ovarian tissue after slow freezing or vitrification.
abstract: OBJECTIVE
  What is the effect of human ovarian tissue cryopreservation on single follicular development in vitro?
  
  
  CONCLUSIONS
  Vitrification had a greater negative effect on growth and gene expression of human ovarian follicles when compared with fresh follicles.
  
  
  BACKGROUND
  For human ovarian cortex cryopreservation, the conventional option is slow freezing while more recently vitrification has been demonstrated to maintain good quality and function of ovarian tissues.
  
  
  METHODS
  Ovarian tissues were collected from 11 patients. For every patient, the ovarian cortex was divided into three samples: Fresh, slow-rate freezing (Slow) and vitrification (Vit). Tissue histology was performed and follicles were isolated for single-cell mRNA analysis and in vitro culture (IVC) in 1% alginate for 8 days.
  
  
  METHODS
  Follicle morphology was assessed with hematoxylin-eosin analysis. Follicles were individually embedded in alginate (1% w/v) and cultured in vitro for 8 days. Follicle survival and growth were assessed by microscopy. Follicle viability was observed after Calcein-AM and ethidium homodimer-I (Ca-AM/EthD-I) staining. Expression of genes, including GDF9 (growth differentiation factor 9), BMP15 (bone morphogenetic protein 15) and ZP3 (zona pellucida glycoprotein 3) in oocytes and AMH (anti-Mullerian hormone), FSHR (FSH receptor), CYP11A (cholesterol side-chain cleavage cytochrome P450) and STAR (steroidogenic acute regulatory protein) in GCs, was evaluated by single-cell mRNA analysis.
  
  
  RESULTS
  A total of 129 follicles were separated from ovarian cortex (Fresh n = 44; Slow n = 40; Vit n = 45). The percentage of damaged oocytes and granulosa cells was significantly higher in both the Slow and Vit groups, as compared with Fresh control (P< 0.05). The growth of follicles in vitro was significantly delayed in the Vit group compared with the Fresh group (P< 0.05). Both slow freezing (P< 0.05) and vitrification (P< 0.05) down-regulated the mRNA levels of ZP3 and CYP11A compared with Fresh group, while there was no significant difference between the Slow and Vit groups (P> 0.05). Vitrification also down-regulates AMH mRNA levels compared with Fresh group (P< 0.05).
  
  
  CONCLUSIONS
  Only short-term IVC studies (8 days) are reported. Further study should be performed to examine and improve follicular development in a long-term culture system after cryopreservation.
  
  
  CONCLUSIONS
  This is the first comparison of gene expression and growth of single human ovarian follicles in vitro after either slow freezing or vitrification. With the decreased gene expression and growth during IVC, damage by cryopreservation still exists and needs to be minimized during the long-term IVC of follicles in the future for eventual clinical application.
  
  
  BACKGROUND
  This work was supported by the National Natural Science Foundation of China (31230047, 81571386, 81471508, 31429004 and 81501247), National Natural Science Foundation of Beijing (7142166) and Mega-projects of Science Research for the 12th five-year plan (2012ba132b05). There are no conflicts of interest to declare.
authors: Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, No.49 North HuaYuan Road, HaiDian District, Beijing 100191, China Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Shengjing Hospital of China Medical University, Shenyang 100004, China.
authors: Tian-ren
authors: TR
authors: Wang
authors: Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, No.49 North HuaYuan Road, HaiDian District, Beijing 100191, China Key Laboratory of Assisted Reproduction, Ministry of Education, Beijing 100191, China.
authors: Jie
authors: J
authors: Yan
authors: Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, No.49 North HuaYuan Road, HaiDian District, Beijing 100191, China Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproduction, Beijing 100191, China.
authors: Cui-ling
authors: CL
authors: Lu
authors: Center for Reproductive Medicine, Peking University Shenzhen Hospital, FuTian District, Shenzhen, Guangdong 518000, China.
authors: Xi
authors: X
authors: Xia
authors: Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, No.49 North HuaYuan Road, HaiDian District, Beijing 100191, China Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproduction, Beijing 100191, China.
authors: Tai-lang
authors: TL
authors: Yin
authors: Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, No.49 North HuaYuan Road, HaiDian District, Beijing 100191, China Key Laboratory of Assisted Reproduction, Ministry of Education, Beijing 100191, China.
authors: Xu
authors: X
authors: Zhi
authors: Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, No.49 North HuaYuan Road, HaiDian District, Beijing 100191, China Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproduction, Beijing 100191, China.
authors: Xiao-hui
authors: XH
authors: Zhu
authors: Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, No.49 North HuaYuan Road, HaiDian District, Beijing 100191, China Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproduction, Beijing 100191, China.
authors: Ting
authors: T
authors: Ding
authors: Department of Obstetrics and Gynecology, General Hospital of Chinese People's Armed Police Forces, Beijing 100191, China.
authors: Wei-hong
authors: WH
authors: Hu
authors: Department of Obstetrics and Gynecology, Peking University Third Hospital, Beijing 100191, China.
authors: Hong-yan
authors: HY
authors: Guo
authors: Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, No.49 North HuaYuan Road, HaiDian District, Beijing 100191, China Key Laboratory of Assisted Reproduction, Ministry of Education, Beijing 100191, China.
authors: Rong
authors: R
authors: Li
authors: Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, No.49 North HuaYuan Road, HaiDian District, Beijing 100191, China Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproduction, Beijing 100191, China yanliyingkind@aliyun.com jie.qiao@263.net.
authors: Li-ying
authors: LY
authors: Yan
authors: Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, No.49 North HuaYuan Road, HaiDian District, Beijing 100191, China Key Laboratory of Assisted Reproduction, Ministry of Education, Beijing 100191, China Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproduction, Beijing 100191, China yanliyingkind@aliyun.com jie.qiao@263.net.
authors: Jie
authors: J
authors: Qiao
mesh_terms: D000328:Adult; D054304:Anti-Mullerian Hormone / Q000235:genetics / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D002470:Cell Survival; D002681:China; D002786:Cholesterol Side-Chain Cleavage Enzyme; D015925:Cryopreservation; D005260:Female; D005615:Freezing; D018507:Gene Expression Regulation, Developmental; D006107:Granulosa Cells / Q000502:physiology; D006801:Humans; D059471:In Vitro Oocyte Maturation Techniques; D006080:Ovarian Follicle / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D059010:Single-Cell Analysis; D013997:Time Factors; D046509:Tissue Culture Techniques; D058989:Vitrification; D000072216:Zona Pellucida Glycoproteins / Q000235:genetics / Q000378:metabolism
chemical_list: D015415:Biomarkers; D012333:RNA, Messenger; C000606517:ZP3 protein, human; D000072216:Zona Pellucida Glycoproteins; D054304:Anti-Mullerian Hormone; D002786:Cholesterol Side-Chain Cleavage Enzyme
keywords: human follicle; in vitro 3D culture; ovarian tissue cryopreservation; single-cell mRNA analysis; slow freezing; vitrification