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Original article
Biallelic mutations in CFAP65 cause male infertility with multiple morphological abnormalities of the sperm flagella in humans and mice
  1. Weiyu Li1,2,3,4,
  2. Huan Wu4,5,6,
  3. Fuping Li7,8,
  4. Shixiong Tian1,2,
  5. Zine-Eddine Kherraf9,10,
  6. Jintao Zhang11,
  7. Xiaoqing Ni4,5,6,
  8. Mingrong Lv4,5,6,
  9. Chunyu Liu1,2,3,4,
  10. Qing Tan4,5,6,
  11. Ying Shen12,
  12. Amir Amiri-Yekta9,10,13,
  13. Caroline Cazin9,10,
  14. Jingjing Zhang4,5,6,
  15. Wangjie Liu1,2,3,
  16. Yan Zheng7,8,
  17. Huiru Cheng4,5,6,
  18. Yingbi Wu7,8,
  19. Jiajia Wang4,5,6,
  20. Yang Gao4,5,6,
  21. Yujie Chen4,5,6,
  22. Xiaomin Zha4,5,6,
  23. Li Jin1,
  24. Mingxi Liu11,
  25. Xiaojin He4,5,6,
  26. Pierre F Ray9,10,
  27. Yunxia Cao4,5,6,
  28. Feng Zhang1,2,3,4
  1. 1 Obstetrics and Gynecology Hospital, NHC Key Laboratory of Reproduction Regulation (Shanghai Institute of Planned Parenthood Research), State Key Laboratory of Genetic Engineering at School of Life Sciences, Fudan University, Shanghai, China
  2. 2 Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Shanghai, China
  3. 3 State Key Laboratory of Reproductive Medicine, Center for Global Health, School of Public Health, Nanjing Medical University, Nanjing, China
  4. 4 Reproductive Medicine Center, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Anhui Medical University, Hefei, China
  5. 5 NHC Key Laboratory of Study on Abnormal Gametes and Reproductive Tract, Anhui Medical University, Hefei, China
  6. 6 Key Laboratory of Population Health Across Life Cycle (Anhui Medical University), Ministry of Education of the People’s Republic of China, Hefei, China
  7. 7 Human Sperm Bank, West China Second University Hospital, Sichuan University, Chengdu, China
  8. 8 Key Laboratory of Birth Defects and Related Disease of Women and Children (Sichuan University), Ministry of Education, Chengdu, China
  9. 9 Genetic Epigenetic and Therapies of Infertility, Institute for Advanced Biosciences, INSERM U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France
  10. 10 Centre Hospitalier Universitaire de Grenoble, UM GI-DPI, Grenoble, France
  11. 11 State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing, China
  12. 12 Department of Obstetrics/Gynecology, Joint Laboratory of Reproductive Medicine (SCU-CUHK), Key Laboratory of Obstetric, Gynecologic and Pediatric Diseases and Birth Defects of Ministry of Education, West China Second University Hospital, Sichuan University, Chengdu, China
  13. 13 Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, Academic Center for Education, Culture, and Research, Tehran, Iran
  1. Correspondence to Professor Feng Zhang, Fudan University, Shanghai 200011, China; zhangfeng{at}; Professor Yunxia Cao, Anhui Medical University, Hefei 230022, China; caoyunxia6{at}


Background Male infertility is a prevalent issue worldwide, mostly due to the impaired sperm motility. Multiple morphological abnormalities of the sperm flagella (MMAF) present aberrant spermatozoa with absent, short, coiled, bent and irregular-calibre flagella resulting in severely decreased motility. Previous studies reported several MMAF-associated genes accounting for approximately half of MMAF cases.

Methods and result We conducted genetic analysis using whole-exome sequencing in 88 Han Chinese MMAF probands. CFAP65 homozygous mutations were identified in four unrelated consanguineous families, and CFAP65 compound heterozygous mutations were found in two unrelated cases with MMAF. All these CFAP65 mutations were null, including four frameshift mutations (c.1775delC [p.Pro592Leufs*8], c.3072_3079dup [p.Arg1027Profs*41], c.1946delC [p.Pro649Argfs*5] and c.1580delT [p.Leu527Argfs*31]) and three stop-gain mutations (c.4855C>T [p.Arg1619*], c.5270T>A [p.Leu1757*] and c.5341G>T [p.Glu1781*]). Additionally, two homozygous CFAP65 variants likely affecting splicing were identified in two MMAF-affected men of Tunisian and Iranian ancestries, respectively. These biallelic variants of CFAP65 were verified by Sanger sequencing and were absent or very rare in large data sets aggregating sequence information from various human populations. CFAP65, encoding the cilia and flagella associated protein 65, is highly and preferentially expressed in the testis. Here we also generated a frameshift mutation in mouse orthologue Cfap65 using CRISPR-Cas9 technology. Remarkably, the phenotypes of Cfap65-mutated male mice were consistent with human MMAF.

Conclusions Our experimental observations performed on both human subjects and on Cfap65-mutated mice demonstrate that the presence of biallelic mutations in CFAP65 causes the MMAF phenotype and impairs sperm motility.

  • male infertility
  • exome sequencing
  • mouse model
  • sperm flagella
  • CFAP65

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  • WeL, HW and FL contributed equally.

  • Contributors WeL, XH, YunC and FZ designed the study. HW, FL, XN, MingrL, QT, YS, AA-Y, JingZ, YZ, HC, YW, JW, YG, YCh, XZ, XH and YunC provided patients’ data and performed clinical assessments. WeL, ST, Z-EK, JintZ, CL, CC and WaL conducted experiments. WeL, HW, FL, LJ, MingxL, XH, PFR, YunC and FZ analysed data. WeL, HW, XH, PFR, YunC and FZ wrote the manuscript. PFR, YunC and FZ supervised the study.

  • Funding This work was supported by National Natural Science Foundation of China (31625015, 31521003 and 81601340), Special Foundation for Development of Science and Technology of Anhui Province (2017070802D150 and YDZX20183400004194), Foundation of the Education Department of Anhui Province (KJ2016A370), Natural Science Foundation of Anhui Province (1708085QC59 and 1908085QH313), Shanghai Medical Center of Key Programs for Female Reproductive Diseases (2017ZZ01016) and Shanghai Municipal Science and Technology Major Project (2017SHZDZX01).

  • Competing interests None declared.

  • Patient consent for publication Obtained.

  • Ethics approval This study was approved by the ethical committees of the centers participating in this study and the animal ethics committee at the School of Life Science, Fudan University.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Data availability statement All data relevant to the study are included in the article or uploaded as supplementary information.