The influence of slow freezing and vitrification on expression of miRNA in human spermatozoa

Cryopreservation of sperm is an extremely important approach in maintaining male fertility. There are several reasons why a man's fertility is at risk. One of the common ones is because of cancer or some other serious illness that requires therapy, which could have a negative effect on sperm production or have a negative effect on sperm quality, despite seemingly normal spermatogenesis. Therefore, it is important that appropriate care is taken before gonadotoxic therapy to maintain patient fertility. Semen cryopreservation is also an effective method of addressing other problems that arise during the infertility treatment process or may also be a partial cause of infertility. Sperm cryopreservation has been used successfully in clinical practice for many years, but it is also known that the freezing and thawing process itself can partially affect the quality of spermatozoa. The negative effect is usually observed as lowered viability and motility of the spermatozoa, but usually enough spermatozoa of sufficient quality survive to carry out the ICSI procedure. In addition to standard semen quality parameters, a number of other parameters can be determined or various functional tests performed, which are not yet widely used in clinical practice or are mostly still in the research phase. Such parameters are e.g. DNA fragmentation, determining the maturity of chromatin in terms of ratio bewteen histones and protamins, a functional test for determining the percentage of bound spermatozoa to hyaluronan (HBA), or various genetic/epigenetic tests. The advances in genetic/epigenetic testing have recently provided additional possibilities for determining sperm quality, but most of these studies focused mostly on fresh semen. From the genetic/epigenetic aspect (e.g. DNA methylation, expression of non-coding RNAs, e.g. miRNAs), the impact of freezing/thawing of the semen is rarely studied, and most of the performed studies were conducted on animal models. Since epigenetic changes are of the utmost importance as they can also be transmitted to offspring and even across generations, it is very important to determine whether the freezing/thawing process affects these traits as well. It has already been shown in animal models that cryopreservation can alter miRNA expression, but in humans, no such study has been performed. Therefore, our group will conduct this study and determine if the candidate miRNAs are expressed differently in the frozen/thawed semen as in the fresh samples. The semen cryopreservation method that is currently used in clinical practice is often referred to as the slow freezing procedure, since the semen sample, after the addition of cryoprotectants, is exposed to a slow drop in temperature from room temperature to -196 ° C. So far, there is no standard clinical protocol for human semen vitrification yet. Currently published studies comparing different types of semen cryopreservation focus only on assessment of the basic parameters of semen quality, and there are practically no studies to examine how this affects genetic/epigenetic traits. Therefore, in our study, we will test whether the results of functional sperm tests the expression of the candidate miRNAs differ regarding to sperm cryopreservation protocol. The results of such studies can be greatly influenced by the quality of the sperm used. Normally, sperm of normal quality are most commonly used in the initial stages of similar studies, but this method is questionable, since in clinical practice, patients who need these procedures for treatment are most likely to have poor semen quality. Therefore, in our study, we will also check whether the results of functional tests and epigenetic assay (miRNA expression) differ with respect to semen quality and with respect to the sperm cryopreservation protocol. We will compare two groups, namely the group with normal semen quality and the group with poor semen quality (diagnosed as oligoasthenoteratozoospermia).