Effects of Slow Freezing and Vitrification of Human Semen on Post-Thaw Semen Quality and miRNA Expression

Abstract

Semen cryopreservation has played an important role in medically assisted reproduction for decades. In addition to preserving male fertility, it is sometimes used for overcoming logistical issues. Despite its proven clinical usability and safety, there is a lack of knowledge of how it affects spermatozoa at the molecular level, especially in terms of non-coding RNAs. Therefore, we conducted this study, where we compared slow freezing and vitrification of good- and poor-quality human semen samples by analyzing conventional sperm quality parameters, performing functional tests and analyzing the expression of miRNAs. The results revealed that cryopreservation of normozoospermic samples does not alter the maturity of spermatozoa (protamine staining, hyaluronan binding), although cryopreservation can increase sperm DNA fragmentation and lower motility. On a molecular level, we revealed that in both types of cryopreservation, miRNAs from spermatozoa are significantly overexpressed compared to those in the native semen of normozoospermic patients, but in oligozoospermic samples, this effect is observed only after vitrification. Moreover, we show that expression of selected miRNAs is mostly overexpressed in native oligozoospermic samples compared to normozoospermic samples. Conversely, when vitrified normozoospermic and oligozoospermic samples were compared, we determined that only miR-99b-5p was significantly overexpressed in oligozoospermic sperm samples, and when comparing slow freezing, only miR-15b-5p and miR-34b-3p were significantly under-expressed in oligozoospermic sperm samples. Therefore, our results imply that cryopreservation of normozoospermic sperm samples can modulate miRNA expression profiles in spermatozoa to become comparable to those in oligozoospermic samples.

Keywords: assisted reproduction; cryopreservation; infertility; microRNA; semen; slow freezing; spermatozoa; vitrification.

Figure 1

MicroRNA (miRNA) expression in spermatozoa from normozoospermic individuals under different sperm cryopreservation conditions. Expression profiles of 12 selected miRNAs in spermatozoa from 30 individuals, which were partitioned and subjected to vitrification (group VF; n = 30) and to a slow freezing (group SF; n = 30) and compared to native spermatozoa (group Native; n = 30). The horizontal line within the boxplot denotes the median, and the horizontal border lines denote the interquartile range. Each dot represents the log₂ miRNA fold change of an individual sample. The symbol × denotes the average log₂-fold change value. The data were evaluated in a pairwise manner using paired Student’s t tests. A p value of <0.05 was considered to indicate statistical significance.

Figure 2

MicroRNA (miRNA) expression in spermatozoa from patients with oligozoospermia under different sperm cryopreservation conditions. Expression profiles of 12 selected miRNAs in spermatozoa subjected to vitrification (group VF; n = 13) and a slow freezing technique (group SF; n = 14) compared to native spermatozoa (group Native; n = 13). The horizontal line within the boxplot denotes the median, and the horizontal border lines denote the interquartile range. Each dot represents the log² miRNA fold change of an individual sample. The symbol × denotes the average log²-fold change value. The data were evaluated using the unpaired Student’s t test. A p value of <0.05 was considered to indicate statistical significance.

Figure 3

MicroRNA (miRNA) expression in spermatozoa from patients with oligozoospermia compared to normal spermatozoa under each sperm cryopreservation condition. Expression profiles of 12 selected miRNAs in spermatozoa from patients with oligozoospermia compared to spermatozoa from normozoospermic individuals following vitrification (group VF) (A) or slow freezing (group SF) (B) and in native spermatozoa (group Native) (C). Analysis was performed on 13 oligozoospermic and 30 normozoospermic samples in the VF and Native groups (A,C) and on 14 oligozoospermic and 30 normozoospermic samples in the SF group (B). The horizontal line within the boxplot denotes the median, and the horizontal border lines denote the interquartile range. Each dot represents the log₂ miRNA fold change of an individual sample. The symbol × denotes the average log₂-fold change value. The data were evaluated using the unpaired Student’s t test. For miR-34b-3p and miR-92a-3p, the Mann-Whitney U test was used due to non-normal data distribution. An asterisk indicates a significant difference compared with the control normozoospermic group (* p < 0.05; ** p < 0.01; *** p < 0.001). A p value of <0.05 was considered to indicate statistical significance.

Figure 4

Overview of the study design for normozoospermic patients.