Cryopreservation of Low Number of Human Spermatozoa; Which is Better: Vapor Phase or Direct Submerging in Liquid Nitrogen?

Abstract

Cryopreservation and subsequent survival of semen samples with low numbers of human spermatozoa in assisted reproductive technology (ART) facilities can be challenging. The aim of this study was to compare the quality of warmed human spermatozoa following vitrification using direct submerging (DS) in liquid nitrogen (LN) or with LN vapour (V). Normozoospermic ejaculates were prepared by the swim-up technique and the motile sperm fraction was divided into three groups: (i) fresh (control); (ii) DS methods in LN (DS Group); or (iii) cryopreserved in V (Group V). Cryopreservation was performed in a small volume using a Cryotech device. After warming, sperm parameters (motility, viability, acrosome, DNA fragmentation and chromatin integrity) were assessed using cytochemical assays. Progressive motility, viability, chromatin integrity and DNA fragmentation differed significantly after warming when compared with the control. Sperm progressive motility and total motility rates were significantly higher in the DS Group compared to Group V. However, normal morphology, acrosome integrity and DNA damage were not significantly different between two groups. Also, sperm chromatin condensation using chromomycin-A3 (CMA3) and Aniline Blue (AB) staining showed fewer alterations in the DS Group compared to Group V. The rates of spermatozoa with an intact acrosome decreased significantly after thawing from 81.30 ± 6.76% to 68.84 ± 14.70% in the DS Group and to 60.92 ± 8.06% in Group V. Cryopreservation of a small number of spermatozoa with the DS method showed superior outcomes with regard to motility, viability and chromatin configuration.

Does conventional freezing affect sperm DNA fragmentation?

“…However, the degree to which these parameters decrease has shown variation according to the use of various cryopreservation/thawing methods or CPAs. In many previous studies, motility after thawing decreased by 20% to 50% following different cryopreservation methods [25-27]. The decrease in sperm motility that we found may have been due to cryopreservation-induced damage to the mitochondrial membrane.…”

“…This hypothesis has been controversial for many years. Some authors have reported that DNA fragmentation after cryopreservation increased [27,30]. According to Spano et al [31], sperm quality (including sperm DNA integrity assessed by SCSA) worsens after thawing.…”

Human sperm vitrification: the state of the art

“…They concluded that the sperm ultra-rapid freezing was superior to slow freezing [30]. More recently, Hosseini et al [46] prepared human samples from normozoospermic ejaculates by swim-up, and low number of human spermatozoa were frozen by directly submerging in LN 2 or its vapor. They found that direct submerging generated higher sperm progressive motility and total motility rates, lower alterations in sperm chromatin indicated by chromomycin-A3 and Aniline Blue staining, but did not affect morphology, acrosome integrity or DNA damage [46].…”

Sperm Cryopreservation: Principles and Biology

“…Although the detrimental effects of freeze-thaw cycles on various sperm parameters and fertilization capacity is well elucidated, there are two opposite thoughts regarding the DNA damage imposed by such procedures. A set of studies suggest that cryopreservation of sperm exert a negative impact on DNA integrity and causes its fragmentation due to oxidative stress caused by ROS [33][34][35][36]. The second line of thought believes that sperm DNA integrity is not affected by freezing and thawing process [37][38][39].…”