Molecular analysis of magnetic activated sperm sorting in level and expression level of apoptotic micro RN in oligospermia patients

Background and Aim: MicroRNAs are family of small non-coding RNAs composed of ۲۲ nucleotide sequences that regulate gene silencing after transcription by binding to the complement bases of the target mRNAs in untranslated region. Recent evidences suggest that these fragments are not aberrant and play an important role in controlling gene expression in the cell. Micro RNAs play an important role in spermatogenesis. Some of these microRNAs can affect sperms survival by targeting pro and anti-apoptotic genes expression. In this study, we investigated the relationship between Mir ۱۸۱, Mir ۱۲۲, Mir ۳۴c-۵ and their role in apoptosis in sperm cells and the effect of the MACS method on separation of apoptotic cells from normal cells.Methods: In this study, the expression levels of microRNAs inclouding: Mir ۱۸۱, Mir ۱۲۲ and Mir ۳۴c-۵ were investigated on the sperm samples of ۱۵ oligospermic patients. At the first, sperm samples were obtained from patients by ejaculation. After liquefying and initial washing of semen samples, primary investigation were done like confirmation of oligospermia and examination of basic features such as sperm motility and morphology. Isolation of normal cells from apoptotic cells were done using negative selection of MACS technique by targeting annexin v as an apoptotic cell surface marker. After isolation of RNAs from sperm samples, the expression levels of Mir ۱۸۱, Mir ۱۲۲ and Mir ۳۴c-۵ were measured before and after sperm separation by MACS using quantitative real time-PCR.Results: Our results showed that the expression levels of Mir ۱۸۱, Mir ۱۲۲ and Mir ۳۴c-۵ were decreased in sperm samples which separated by MACS compared to other samples (P value <۰.۰۵).Conclusion: Currently, most of the sperm selection criteria's for Assisted Reproductive Technology (ART) are based on sperm count, motility and morphology. Several factors affect IVF successfulness and one of the most important of them is selection of healthy and motile sperm. Our results indicate that separation of sperms by MACS based on their apoptosis status decreased apoptotic sperms and therefore increased healthy sperm selection chance. Further studies may select sperms by deleting abnormal sperms that are not detectable by morphological methods, including mitochondrial wall degradation, caspase-۳ activity, chromatin degradation, and loss of genome integrity.

Keywords:

micro RNA , apoptosis , oligospermia , MACS