Comparative Epigenetic Analysis of Imprinting Genes Involved in Fertility, in Cryopreserved Human Sperms with Rapid Freezing versus Vitrification Methods
Authors: Nahid Khosronezhad; Vahideh Hassanzadeh; Maryam Hezavehei; Abdolhossein Shahverdi; Maryam Shahhoseini
Number of views: 16
Objective: Choosing the optimal method for human sperm cryopreservation seems necessary to reduce cryoinjury.
The aim of this study is to compare two cryopreservation methods including rapid-freezing and vitrification, in terms
of cellular parameters, epigenetic patterns and expression of paternally imprinted genes (PAX8, PEG3 and RTL1) in
human sperm which play a role in male fertility.
Materials and Methods: In this experimental study, semen samples were collected from 20 normozoospermic men.
After washing the sperms, cellular parameters were investigated. DNA methylation and expression of genes were
investigated using methylation-specific polymerase chain reaction (PCR) and real-time PCR methods, respectively.
Results: The results showed a significant decrease in sperm motility and viability, while a significant increase was
observed in DNA fragmentation index of cryopreserved groups in comparison with the fresh group. Moreover, a significant
reduction in sperm total motility (TM, P<0.01) and viability (P<0.01) was determined, whereas a significant increase
was observed in DNA fragmentation index (P<0.05) of the vitrification group compared to the rapid-freezing group.
Our results also showed a significant decrease in expression of PAX8, PEG3 and RTL1 genes in the cryopreserved
groups compared to the fresh group. However, expression of PEG3 (P<0.01) and RTL1 (P<0.05) genes were reduced
in the vitrification compared to the rapid-freezing group. Moreover, a significant increase in the percentage of PAX8,
PEG3 and RTL1 methylation was detected in the rapid-freezing group (P<0.01, P<0.0001 and P<0.001, respectively)
and vitrification group (P<0.01, P<0.0001 and P<0.0001, respectively) compared to the fresh group. Additionally,
percentage of PEG3 and RTL1 methylation in the vitrification group was significantly increased (P<0.05 and P<0.05,
respectively) compared to the rapid-freezing group.
Conclusion: Our findings showed that rapid-freezing is more suitable method for maintaining sperm cell quality. In
addition, due to the role of these genes in fertility, changes in their expression and epigenetic modification may affect