Human semen cryopreservation: a sperm DNA fragmentation study with alkaline and neutral Comet assay

Abstract

SUMMARYSperm cryopreservation is widely used for both research and reproduction purposes, but its effect on sperm DNA damage remains controversial. Sperm DNA fragmentation (SDF) has become an important biomarker to assess male infertility. In particular, the differentiation between single-and double-stranded DNA fragmentation (ssSDF and dsSDF) has clinical implications for male infertility where ssSDF is associated with reduced fertility, whereas dsSDF is associated with increased risk of miscarriage. In this study, semen samples from 30 human males have been analysed in both fresh and cryopreserved using the alkaline and neutral Comet assays. Results show an increase of about 10% of ssSDF, assessed by the alkaline Comet assay, regardless of the male fertility status. Neutral Comet analysis of dsSDF does not show any statistical increase when comparing fresh and cryopreserved samples in any of the patient groups. Results support previous reports that oxidative stress is the major effector in DNA damage during sample cryopreservation, as, on one hand, ssSDF has previously been related to oxidative damage and, on the other hand, we have not found any effect on dsSDF. Therefore, there might be a slight risk of decreased fertility after using a freezed sample, but no evidence for increased miscarriage risk from cryopreserved spermatozoa should be expected.

Sperm preparation before freezing improves sperm motility and reduces apoptosis in post-freezing-thawing sperm compared with post-thawing sperm preparation

“…The negative effects relating to rapid decreases in temperature, such as osmotic injury, cellular dehydration, intra-cellular ice crystal formation, and oxidative stress, also lead to damage to sperm DNA, chromatin instability and DNA denaturation [4][5][6][7][8]. Therefore, the reproductive outcome following fertilization with the sperm containing the damaged genetic material may be poor [8][9][10]. In cases where pregnancy is achieved using this sperm, the risk of abortion is increased because of the damaged genetic material in the sperm [11].…”

Effect of Seminal Transforming Growth Factorß1 (TGFß1) and Glutathione on Apoptosis in Spermatozoa from Tunisian Infertile Men

“…In this study, we examined the relationship between apoptosis, the rate of seminal TGFß1, the rate of the different forms of glutathione (GSHt, GSSG, GSHr) and fragmentation of the sperm DNA. As far as we know, this is the first analysis that examines all these parameters simultaneously [41][42][43][44][45][46][47].…”

“…For cell per meabilization, slides were incubated in phosphate buffer saline (PBS) with a solution of 1% Triton X100 (Sigma) [45][46][47].…”

Oxidative stress level in fresh ejaculate is not related to semen parameters or to pregnancy rates in cycles with donor oocytes

“…DNA repair in spermatozoa occurs during spermatogenesis and stops during nuclear condensation in the epididymis [11,12]. However, spermatozoa can be exposed to oxidative damage in the vas deferens [13]; from this point onward, spermatozoa DNA repair can only occur after entry into the oocyte; ssDNA seems to be more readily repaired than double-strand DNA (dsDNA) by the oocyte, and in the Capsule Semen oxidative stress was measured in 132 patients' ejaculates by nitro blue tetrazolium testing before performing ICSI with donor oocytes. The measured level did not correlate with either seminal parameters or reproductive outcomes.…”