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The effect of cryopreservation on DNA methylation patterns of the chromosome 15q11–q13 region in human spermatozoa

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Abstract

Human sperm cryopreservation is a common technique which is used in assisted reproductive technologies. Despite the existence of evidence supporting the production of ROS and DNA fragmentation during sperm cryopreservation, there is little and equivocal information about the cryopreservation effects on methylation of imprinted genes and imprinting control regions. In this study, we have investigated the effects of cryopreservation on DNA methylation in promoter regions of SNURF-SNRPN and UBE3A imprinted genes, PWS-ICR and AS-ICR in the chromosome 15q11–q13 region. Semen samples from 10 healthy normozoospermic men were collected and each sample was divided into four equal aliquots: fresh, cryoprotectant, cryopreservation, and H2O2. We measured the ROS levels and DNA fragmentation using DCFH-DA and TUNEL assay respectively by flow cytometry. DNA methylation in promoter regions of SNURF-SNRPN and UBE3A imprinted genes, PWS-ICR and AS-ICR in the chromosome 15q11–q13 region were evaluated by quantitative methylation-specific PCR technique. Intracellular levels of ROS and percentage of TUNEL-positive spermatozoa significantly increased in cryopreservation group compared to fresh group. Exposure to cryoprotectant had no significant effect on ROS levels and DNA fragmentation. Neither cryopreservation nor exposure to cryoprotectant significantly affected DNA methylation of the selected gene regions. However, DNA fragmentation had positive correlation with DNA methylation of AS-ICR. In conclusion, based on our study, clinical use of sperm cryopreservation for fertility treatments appear to be safe in regard to DNA methylation in the chromosome 15q11–q13 region.

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Acknowledgements

The authors would like to thank the Tehran University of Medical Sciences for the financial support (Grant No: 32185). Also, the authors would like to express their profound gratitude to Dr. Nancy L. Brackett (Professor of Neurological Surgery and Urology, University of Miami Miller School of Medicine) for her constructive suggestions on revising. Her willingness to give her time so generously is very much appreciated.

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Authors and Affiliations

  1. Faculty of Anatomy, School of Medicine, Tehran University of Medical Science, 16 Azar Avenue, Poor Sina Street, Tehran, Iran

    Zahra Khosravizadeh, Gholamreza Hassanzadeh, Kajal Khodamoradi, Maryam Khanehzad & Farid Abolhassani

  2. Faculty of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, 16 Azar Avenue, Poor Sina Street, Tehran, Iran

    Javad Tavakkoly Bazzaz

  3. Faculty of Genomic Psychiatry and Behavioral Genomics (DGPBG), Roozbeh Hospital, School of Medicine, Tehran University of Medical Science, South Kargar Avenue, Tehran, Iran

    Fatemeh Alizadeh

  4. Faculty of Stem Cells and Developmental Biology, Royan Institute, Tehran, Iran

    Mehdi Totonchi

  5. Faculty of Gynecology, School of Medicine, Fertility and Infertility Research Center, Bandar Abbas, Iran

    Ensieh Salehi

  6. Dr. Ali Shariati Hospital, Shahid Mohammadi Hospital, Hormozgan University of Medical Sciences, Bandar Abbas, Iran

    Ensieh Salehi

  7. Faculty of Urology, Sina Hospital, Tehran University of Medical Science, Imam Khomeini Street, Tehran, Iran

    Seyed Reza Hosseini

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  1. Zahra Khosravizadeh
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Correspondence to Seyed Reza Hosseini or Farid Abolhassani.

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Human semen samples were collected in accordance with Research Ethical Committee (IR.TUMS.MEDICINE.REC.1395.860) of Tehran University of Medical Sciences (Tehran, Iran) and all participants have given the written informed consent for participation in this study.

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Khosravizadeh, Z., Hassanzadeh, G., Tavakkoly Bazzaz, J. et al. The effect of cryopreservation on DNA methylation patterns of the chromosome 15q11–q13 region in human spermatozoa. Cell Tissue Bank 21, 433–445 (2020). https://doi.org/10.1007/s10561-020-09828-1

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