The objective of these studies was to evaluate the effect of cryopreservation-thawing of human spermatozoa on DNA fragmentation and membrane integrity. This was a prospective, controlled cohort study, performed at a university-based infertility center. Ejaculates were examined from 5 donors and 16 men undergoing infertility evaluation. Purified sperm populations were prepared by gradient centrifugation, cryopreserved using a manual method and TEST-yolk buffer and glycerol (TYB-G), followed by quick-thaw. Annexin V binding was used for assessing membrane translocation of phosphatidylserine, and terminal deoxynucleotidyl transferase-me-diated dUTP nick end labeling (TUNEL) was utilized for the evaluation of DNA fragmentation. The results were as follows: the percentage of live cells with intact membranes (annexin V−, live) was significantly reduced after cryopreservation-thawing. On the other hand, the percentages of live cells with phosphatidylserine translocation (annexin V+, live) and of necrotic (dead) cells increased significantly after thawing. TUNEL revealed percentages of cells with DNA fragmentation in the prefreeze and postthaw samples that were not significantly different. In a further attempt to examine differences in response to various cryoprotection protocols, experiments were carried out using no cryoprotection, glycerol alone, or TYB-G. Samples frozen with TYB-G demonstrated significantly higher percentages of live cells without phosphatidylserine translocation than the other conditions. We concluded that cryopreservation-thawing of human sperm from patients and donors was associated with membrane change, as revealed by membrane translocation of phosphatidylserine, while having no major impact on DNA fragmentation.

Cryopreservation-Thawing of fractionated human spermatozoa is associated with membrane phosphatidylserine externalization and not DNA fragmentation.

https://www.fertstert.org/article/S0015-0282(00)01694-0/pdf

Cryopreservation-thawing of fractionated human spermatozoa and plasma membrane translocation of phosphatidylserine

The aim of this study was to develop and to test the standardized aseptic technology of permeable cryoprotectant-free vitrification of human spermatozoa in capillaries (for intracytoplasmic sperm injection [ICSI] or in vitro fertilization [IVF]). To test the effect of vitrification on basic sperm parameters, each of 68 swim-up–prepared ejaculates from oligo-astheno-terato-zoospermic patients were aliquoted and distributed into 3 groups: 1) nontreated control, 2) 10 μL of spermatozoa cryopreserved by slow conventional freezing with glycerol-contented medium, and 3) 10 μL of spermatozoa vitrified in 50-μL plastic capillaries in culture medium with 0.25 M sucrose. Spermatozoa motility (1, 24, and 48 hours after warming), plasma membrane integrity, acrosomal integrity, and spontaneous capacitation-like changes were determined after warming. Aseptic cryoprotectant-free vitrification showed a significantly stronger cryoprotective effect compared with conventional freezing. One hour after warming, motility, plasma membrane integrity, and acrosomal integrity were significantly higher than is observed for conventionally frozen spermatozoa (28% vs 18%, 56% vs 22%, and 55% vs 21%, respectively; P .1) (2% in fresh spermatozoa). The newly developed technology of aseptic vitrification of human spermatozoa in capillaries can effectively preserve these cells from cryo-injures. Spermatozoa, vitrified by this technology, are free from seminal plasma owing to swim-up preceding vitrification and are free from permeable cryoprotectants. They are ready for further use immediately after warming without any additional treatment. Therefore, the reported technology has a great potential for use in ICSI/IVF.

https://onlinelibrary.wiley.com/doi/pdf/10.2164/jandrol.111.013789

Vitrification of human ICSI/IVF spermatozoa without cryoprotectants: new capillary technology.

Assessment of sperm cryodamage and strategies to improve outcome

This article focuses on a range of issues that are of current concern to those working in human sperm cryobanking and considers them within a general framework of risk analysis and management, taking into account the available evidence and perceptions of 'best practice' from both the medical and legal perspectives In particular, issues arising from concerns over the risk of cross-contamination between samples during storage immersed in liquid nitrogen have been analysed in detail Even though such an occurrence has never been reported for spermatozoa or embryos, and the risk is generally accepted to be vanishingly small, it does represent a finite risk and all reasonable measures should be taken to reduce the chance of its occurring It is concluded that all methods used to collect, cryopreserve, store, thaw and use cryobanked human spermatozoa must address the risk of contamination from any source throughout the entire process To assist workers in this area, a series of recommendations as to current best practice are made, based upon conclusions from risk analyses using currently available information

Current and future concepts and practices in human sperm cryobanking

An intrauterine insemination-ready cryopreservation method compared with sperm recovery after conventional freezing and post-thaw processing.

https://www.fertstert.org/article/S0015-0282(99)00094-1/pdf

Fecundability trends among sperm donors as a measure of donor performance.

Sperm penetration assay (SPA) scores obtained from cryobanked semen were correlated with therapeutic insemination (TI) fecundity in a group of established sperm donors, thereby evaluating the efficacy of the SPA in screening donors for sperm banking. While the SPA has been used to separate fertile from infertile males, we altered assay conditions to use frozen semen and to distinguish performance among fertile donors. Three frozen ejaculates from 11 pregnancy-proven donors were analysed. Of 905 TI cycles, 275 recipients achieved 95 pregnancies. There were no significant relationships between fecundity and donor semen, washed sperm parameters, sperm recoveries or recipient age. A significant relationship was revealed between mean SPA scores (range 8.7-66.6 penetrations/ ovum) and donor fecundity (range 0.04-0.16, P < 0.03). Sperm concentration was varied in an effort to establish the most sensitive test condition. Using 0.25 × 10 6 motile spermatozoa/ml, a highly significant relationship was observed (P < 0.002). The four donors with the lowest SPA scores achieved the four lowest fecundities. It is concluded that a modified SPA can be used on frozen donor semen to estimate donor fertility potential. If applied routinely in donor semen banking, poor quality applicants could be excluded, thereby increasing pregnancy rates while decreasing donor screening costs.

/pdf/the-relationship-between-fertility-potential-measurements-on-4mhfoh82lu.pdf

Barbara A. Mixon

Papers

An intrauterine insemination-ready cryopreservation method compared with sperm recovery after conventional freezing and post-thaw processing.

Fecundability trends among sperm donors as a measure of donor performance.

The relationship between fertility potential measurements on cryobanked semen and fecundity of sperm donors