Permeable cryoprotectants-free vitrification of human TESE, PESA and OAT spermatozoa: clinical outcomes

Cryopreservation of testicular and epididymal spermatozoa is more challenging in comparison to ejaculated spermatozoa due to lower sperm concentration and motility, and higher sperm sensitivity to cryoprotectants. Sperm vitrification without the use of potentially toxic permeable cryoprotectants is an attractive freezing alternative for testicular and epididymal spermatozoa, as well as oligoasthenoteratozoospermia (OAT) samples. Our study is a retrospective analysis of outcomes in IVF cycles involving a total of 70 testicular, 77 epididymal and 69 ejaculated OAT samples vitrified in a closed double-straw device using mHTF medium supplemented with protein and sucrose, without any permeable cryoprotectant. In total, 71 frozen samples were used for intracytoplasmic sperm injection (ICSI). Results were compared to fresh samples (26 testicular, 53 epididymal and 63 ejaculated OAT samples) that served as controls. Elective single frozen embryo transfers of euploid or unknown-ploidy blastocysts were performed. While sperm motility is expected to diminish following slow sperm freezing and thawing, our data demonstrated that vitrification of testicular, epididymal and OAT samples had a mean motility rate comparable to fresh samples. No statistically significant differences (p > 0.05) were observed between vitrified versus fresh TESE in fertilization (64.1% vs. 59.5%), blastocyst development (54.9% vs. 56.7%), blastocyst euploidy (36.4% vs. 33.3%), clinical pregnancy (47.8% vs. 36.4%) and live birth rates (43.5% vs. 24.2%). Similarly, vitrified versus fresh PESA showed no statistically significant differences (p > 0.05) in the analyzed results respectively: (69.4% vs.74.9%; 62.6% vs. 59.7%; 40.5% vs. 48.1%; 36.0% vs.37.7%; and 32.0% vs. 27.5%). For vitrified OAT samples, there was a significant difference in blastocyst development and euploidy rates when compared to the control group. Our results demonstrate that human testicular, epididymal spermatozoa and samples with OAT can be successfully vitrified in small volumes in a closed system without using any permeable cryoprotectants, allowing utilization of this technique in clinical settings.