Alterations of miR-16, miR-let-7a and their target genes expression in human blastocysts following vitrification and re-vitrification

Abstract

Embryo cryopreservation is a widely used technique in infertility management and today is an essential part of assisted reproductive technology (ART). In some cases, re-vitrification can be applied to good quality supernumerary warmed embryos that have not been transferred in the present cycle. However, there is no study about re-vitrification impact on microRNA and gene expression in human embryos. The purpose of this study is to evaluate miR-16, miR-let7a and target genes expression in in vitro produced human blastocysts following re-vitrification.Day3 embryos obtained from ICSI cycles of fertile couples referring for family balancing program were biopsied and cultured individually. On the fourth day (post-ICSI) male ones (choices of their parents) were transferred and the females (good quality embryos) were donated for research. Donated embryos were cultured to blastocyst stage and assigned to three groups: fresh, vitrified and re-vitrification. Embryos were vitrified on Cryotech carriers. Then blastocysts of three groups were individually assessed for expression of miR-16, miR-let7a and target genes.The results showed that re-vitrification of human blastocysts did not affect the ability to re-expand in culture. In addition, significant decrease was observed in miR-16 and miR-let7a expression in re-vitrified group compared to fresh (p < 0.05). A significant upregulation of the target genes ITGβ3 and BCL-2 in re-vitrified and vitrified embryos was observed compared to the fresh group (p < 0.05). The expression of BAX as a pro-apoptotic gene showed a significant decrease in re-vitrification group comparing with the fresh one (P < 0.05).The results of this research indicated that re-vitrification of embryos changes the expression of miR-16, miR-let-7a and their target genes. These alterations include increased expression of BCl-2 and ITGβ3 genes which play important roles in embryo survival and implantation, respectively. Clinical proof of these effects requires further research.

Double vitrification and warming does not compromise the chance of live birth after single unbiopsied blastocyst transfer

“…In an attempt to explain an underlying mechanism, the authors argued that the cryopreservation procedures could have elicited transcriptional changes in the embryos enough to hinder their developmental competence in utero. With this regard, only one study has assessed and shown that re-vitrification can alter the RNA of human blastocysts ( Daneshvar et al , 2021 ). Similarly, Farhi and colleagues reported lower pregnancy rates after double cryopreservation (n = 25) with slow freezing at the cleavage and blastocyst stages compared with after single cryopreservation (n = 50) at blastocyst stage only ( Farhi et al , 2019 ).…”

Feline microRNAome in ovary and testis: Exploration of in-silico miRNA-mRNA networks involved in gonadal function and cellular stress response

“…In addition, a family of miRNAs termed CryoMiRs have been shown to be associated with freezing tolerance, and regulate metabolism in mammalian hibernators and insects ( Lyons et al, 2013 ; Singh and Storey, 2021 ). Finally, changes in miRNA expressions have been identified in cryopreserved oocytes, embryos and spermatozoa compared to fresh counterparts in human ( Daneshvar et al, 2021 ), mouse ( Heidari et al, 2019 ; Li et al, 2019 ; Movahed et al, 2019 ; Daneshvar et al, 2021 ; Xu et al, 2021 ), cow ( Shangguan et al, 2020 ) and pig ( Zhang et al, 2017 ; Dai et al, 2019 ).…”