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 24 h 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.