• Investigating the effects of increasing ROS during Cryopreservation technique on sperm quality
  • Zahra Sadeghi,1,* Sayed Mehrdad Azimi,2 Ali Akbari,3
    1. Department of Anatomical and Molecular Biology Sciences, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
    2. Department of Anatomical and Molecular Biology Sciences, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
    3. Department of Anatomical and Molecular Biology Sciences, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran


  • Introduction: Today, infertility is a global problem that, in addition to economic costs, also has destructive effects on the mental health of couples. According to the World Health Organization (WHO), about half of infertility cases are caused by male factors. Due to the importance of the issue of infertility, various techniques have been proposed to help in the treatment of it. Sperm cryopreservation (SC) is one of the techniques used in infertility treatment centers and sperm banks, however, this method has destructive effects on sperm. One of the most important damages caused to sperm in this method is the increase in the production of reactive oxygen species (ROS). ROS include different types that through different mechanisms such as DNA damage, cell membrane, and other membrane organelles damage lead to a decrease in the chance of successful fertilization. Due to the importance of the infertility issue, in this review, first, the damage caused by ROS on the male reproductive system is discussed. Below is some information on the effect of antioxidants to help improve ROS levels in sperm.
  • Methods: An exhaustive literature review was conducted to assess original investigations. Relevant studies published between 1996 and 2023 were identified by searching the PubMed, Web of Science, and Google Scholar databases using keyword combinations related to male infertility, sperm, cryopreservation, oxidative stress, apoptosis, DNA damage, and signaling pathways. Inclusion criteria stipulated original research articles published in English that directly examined indicators of cryopreservation-induced damage and antioxidant efficacy in sperm samples. Additional applicable papers were identified by hand-searching the reference lists of reviews and included articles. Overall, articles met all inclusion criteria and were compiled to qualitatively summarize the evidence on mechanisms of cryopreservation damage, antioxidant mechanisms of action, and future directions to optimize sperm integrity during freeze-thaw procedures. This comprehensive review synthesizes current knowledge on pathways of sperm damage during cryopreservation and the protective effects of antioxidant supplementation based on the past 3 decades of articles.
  • Results: After skimming and fully understanding the articles, this review found that the increase in ROS levels leads to increased oxysterol production and membrane permeability to cytochrome C. Additionally, it can elevate the production of procaspase 9, apoptotic bodies, caspase 3, and DNA fragmentation. Also, by activating the SIRT1/Nrf2 pathway and releasing cytochrome C from mitochondria, it decreased the expression of Bax and reversely increased the expression of Bcl-2. On the other hand, elevation of ROS can reduce the PI3K/AKT/mTOR pathway, induce NOX and CHAC1, formation of TNF, and induction of NF-kB, which has harmful results (e.g., cell damage, apoptosis, mitochondrial activity impairment, DNA damage, inhibition of sperm motility, sperm morphology alteration) on spermatogenesis. As an attachment for the review, various antioxidants including vitamins C and E were traced. Many studies have shown the role of various antioxidants and have confirmed the effective removal of ROS through the reduction of malondialdehyde production and lipid peroxidation.
  • Conclusion: This literature review demonstrates that SC leads to elevated ROS levels, which induces pathological changes through diverse molecular pathways. The increased ROS prompts oxysterol production and membrane permeabilization, enabling the release of apoptogenic factors like cytochrome C from the mitochondria. ROS activates caspase cascades (caspase 9, caspase 3) and nuclear fragmentation, culminating in apoptotic sperm death. However, the stimulation of cytoprotective mechanisms like the SIRT1/Nrf2 pathway and enhanced Bcl-2 expression can counteract apoptotic signaling. Conversely, ROS impairs sperm function by reducing PI3K/AKT/mTOR activity, inducing expression of pro-oxidant enzymes (NOX), and damaging DNA integrity. Additional deleterious effects include impaired sperm motility, aberrant morphology, and inhibition of mitochondrial activity. These pathological effects are mediated through ROS-induced signaling molecules like CHAC1, TNF, and NF-kB. Numerous antioxidants have proven effective at mitigating ROS-induced damage by reducing lipid peroxidation and oxidative stress. For instance, supplementation with vitamin C and vitamin E decreased malondialdehyde levels, confirming their role as ROS scavengers. In summary, this review elucidates the diverse molecular cascades through which accumulated ROS during cryopreservation adversely impact sperm viability and function. It also verifies the protective capacity of antioxidant molecules to preserve sperm from oxidative damage. Further research should explore combinatorial antioxidant therapies to optimize sperm integrity and mitochondrial health during cryopreservation procedures.
  • Keywords: Male infertility, DNA, Oxidative stress, Sperm Cryopreservation