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.
Highlights
Embryo cryopreservation is one of the most widely used techniques in infertility management
We evaluated the effects of vitrification and revitrification on alterations of microRNA expressions involved in apoptosis, implantation and target genes (BCL2, BAX, ITGβ3) in human blastocysts
The results in this study showed that the expression of Bcl2 and BAX genes in the re-vitrification group was significantly reduced compared to the vitrification group and the expression of ITGβ3 gene in the re-vitrification group was reduced compared to the vitrification group, this decrease was not significant
Summary
Embryo cryopreservation is one of the most widely used techniques in infertility management This technique plays a key role in preserving fertility potential of those suffering from premature ovarian failure and provides an embryo resource to be thawed/warmed for. Few studies have been performed on re-verification of mouse embryos, indicating re-verification did not have any effects on developmental potential and gene expression [5, 6]. Re-verification of embryos seems to be a successful and useful method for preserving extra embryos, but so far, its safety and genetic and epigenetic effects of re-verification/warming of human embryos have not been studied. MicroRNAs are innate, small noncoding RNAs with 20–23 nucleotides in length, having an important role in controlling posttranscriptional gene expression and are regulated by genetic and environmental conditions. To date the importance of microRNAs has been recognized in primary developmental steps including embryo implantation, development, cell growth, and differentiation in many species from C.elegans to mammals [7, 8]
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