New Technique for Human Sperm Cryopreservation Using Emptied Sheep’s Ovarian Follicles

This study aimed to investigate the efficacy of emptied ovarian follicles of sheep as a container for cryopreservation of human spermatozoa to preserve the presence of low concentrations of spermatozoa at the post-thawing stage. This research was performed on 30 semen samples from oligozoospermic patients and 10 samples from normozoospermic males. They were diagnosed according to the standard criteria of the World Health Organization 2010. Semen samples were classified into four groups of G1-G4 according to sperm concentration: 3-5 million/mL, 6-10 million/mL, 11-15 million/mL, and 16-20 million/mL, respectively. Each sample was divided into two equal parts. One part was cryopreserved without cryoprotectant, while the other was diluted 1:1 with 10% glycerol-based cryosolution. The ovarian follicles of sheep were obtained from a local slaughterhouse and prepared by slicing the ovaries and evacuating the follicular fluid and oocyte. The emptied follicles were injected with the prepared semen samples. After cryopreservation and thawing, the semen mixture aspired outside the follicles, and sperm parameters were measured, namely concentration, progressive motility, total motility, and normal morphology. Sperm concentration and progressive and total sperm motility were significantly (P<0.01) decreased in all groups at the post-thawing stage, compared to the pre-freezing stage. The sperm concentration was significantly higher (P<0.01) in samples cryopreserved without cryoprotectant, compared to that in those cryopreserved with glycerol. However, progressive and total motility were significantly (P<0.01) higher in samples cryopreserved with glycerol, compared to that in the samples cryopreserved without cryoprotectant in all groups. Moreover, no significant difference was found between the pre-freezing and post-thawing stages in terms of normal morphology. Emptied ovarian follicles are an appropriate carrier for cryopreservation of human sperms, especially for patients with oligozoospermia. The best sperm survival rate in this technique was observed when using glycerol-based cryosolution.