Human spermatozoa vitrified in the absence of permeable cryoprotectants: birth of two healthy babies.

Herein, we report the birth of two healthy babies to a woman following intracytoplasmic sperm injection (ICSI) using motile spermatozoa vitrified without permeable cryoprotectants. Spermatozoa (in a case of oligoasthenoteratozoospermia) were cooled in cut standard straws in human tubal fluid supplemented with 0.5% human serum albumin and 0.25 M sucrose. Sperm motility, capacitation-like changes, acrosome reaction and mitochondrial membrane potential (MMP) were compared in fresh and vitrified spermatozoa. Eight mature (MII) oocytes were microinjected with the vitrified-warmed motile spermatozoa. Although the motility of vitrified-warmed spermatozoa was markedly lower than that of fresh spermatozoa (60% v. 90%, respectively), there were no immediate visible differences in the percentages of capacitated and acrosome-reacted vitrified and fresh spermatozoa (10% v. 8% and 5% v. 8%, respectively). However, the MMP in vitrified spermatozoa was apparently adversely affected in the ejaculate used for ICSI compared with fresh spermatozoa (63% v. 96% spermatozoa with high MMP). Eighteen hours later, six oocytes showed signs of normal fertilisation. Two-pronuclear oocytes were cultured in vitro for 24h and two four-blastomere embryos were transferred. Two healthy girls were born at term. Our findings suggest that permeable cryoprotectant-free vitrification can be applied successfully for some procedures in assisted reproduction, in particular in ICSI with motile vitrified spermatozoa, to achieve normal pregnancy and birth.