Novel animal protein-free sperm cryopreservation media results in post-thaw survival equivalent to standard sperm freezing media

Abstract

ObjectiveOptimal sperm survival following cryopreservation and thawing is essential, especially in patients with low sperm counts and donor samples. Sperm cryopreservation media has historically been supplemented with proteins sourced from human and/or animal. This study tested a novel media free of human and animal proteins eliminating potential sources of contamination while maintaining cryoprotective properties.DesignProspective comparison of sperm cryopreservation mediaMaterials and Methods15 sperm samples designated for disposal were pooled and washed in Quinn's Fertilization media (Sage) then split for 5 runs with two treatments: standard Freezing media (Test Yolk Buffer, Irvine) or the experimental Cryolink Freezing media (Biogenetics). Freeze media was added drop-wise and gently mixed for 10 minutes. Samples were placed in cryotubes (1ml per tube) and plunged into LN2. On a later date, samples were thawed in a 37° water bath for 5 minutes, mixed with wash media drop-wise over 6 minutes. Samples were washed, pelleted and resuspended in 0.5ml of wash media. Recovery and survival was assessed by count, motility, progression and morphology post thaw and at 0, 3, and 24 hrs after washing. Data was analyzed by Wilcoxen ranked signs test.ResultsTabled 1CRYO-PROTECTANTPRE-FREEZE MOTILITYPOST THAW MOTILITY (0 hr)CELLS RECOVERED (0 hr)POST THAW MOTILITY (3 hr)CELLS RECOVERED (3 hr)POST THAW MOTILITY (24 hr)CELLS RECOVERED (24 hr)TEST YOLK63%45%71%32%51%15%24%CRYOLINK59%47%80%31%63%17%29% Open table in a new tab ConclusionAnimal and/or human proteins are potential sources of bacterial, viral or prion contamination. We tested a novel sperm cryopreservation media prepared free of animal or human protein. No difference in sperm survival post-thaw was observed between Test Yolk buffer and Cryolink media. With further validation, Cryolink media may provide a safer and simplified means of cryopreserving sperm for reproductive use. ObjectiveOptimal sperm survival following cryopreservation and thawing is essential, especially in patients with low sperm counts and donor samples. Sperm cryopreservation media has historically been supplemented with proteins sourced from human and/or animal. This study tested a novel media free of human and animal proteins eliminating potential sources of contamination while maintaining cryoprotective properties. Optimal sperm survival following cryopreservation and thawing is essential, especially in patients with low sperm counts and donor samples. Sperm cryopreservation media has historically been supplemented with proteins sourced from human and/or animal. This study tested a novel media free of human and animal proteins eliminating potential sources of contamination while maintaining cryoprotective properties. DesignProspective comparison of sperm cryopreservation media Prospective comparison of sperm cryopreservation media Materials and Methods15 sperm samples designated for disposal were pooled and washed in Quinn's Fertilization media (Sage) then split for 5 runs with two treatments: standard Freezing media (Test Yolk Buffer, Irvine) or the experimental Cryolink Freezing media (Biogenetics). Freeze media was added drop-wise and gently mixed for 10 minutes. Samples were placed in cryotubes (1ml per tube) and plunged into LN2. On a later date, samples were thawed in a 37° water bath for 5 minutes, mixed with wash media drop-wise over 6 minutes. Samples were washed, pelleted and resuspended in 0.5ml of wash media. Recovery and survival was assessed by count, motility, progression and morphology post thaw and at 0, 3, and 24 hrs after washing. Data was analyzed by Wilcoxen ranked signs test. 15 sperm samples designated for disposal were pooled and washed in Quinn's Fertilization media (Sage) then split for 5 runs with two treatments: standard Freezing media (Test Yolk Buffer, Irvine) or the experimental Cryolink Freezing media (Biogenetics). Freeze media was added drop-wise and gently mixed for 10 minutes. Samples were placed in cryotubes (1ml per tube) and plunged into LN2. On a later date, samples were thawed in a 37° water bath for 5 minutes, mixed with wash media drop-wise over 6 minutes. Samples were washed, pelleted and resuspended in 0.5ml of wash media. Recovery and survival was assessed by count, motility, progression and morphology post thaw and at 0, 3, and 24 hrs after washing. Data was analyzed by Wilcoxen ranked signs test. ResultsTabled 1CRYO-PROTECTANTPRE-FREEZE MOTILITYPOST THAW MOTILITY (0 hr)CELLS RECOVERED (0 hr)POST THAW MOTILITY (3 hr)CELLS RECOVERED (3 hr)POST THAW MOTILITY (24 hr)CELLS RECOVERED (24 hr)TEST YOLK63%45%71%32%51%15%24%CRYOLINK59%47%80%31%63%17%29% Open table in a new tab ConclusionAnimal and/or human proteins are potential sources of bacterial, viral or prion contamination. We tested a novel sperm cryopreservation media prepared free of animal or human protein. No difference in sperm survival post-thaw was observed between Test Yolk buffer and Cryolink media. With further validation, Cryolink media may provide a safer and simplified means of cryopreserving sperm for reproductive use. Animal and/or human proteins are potential sources of bacterial, viral or prion contamination. We tested a novel sperm cryopreservation media prepared free of animal or human protein. No difference in sperm survival post-thaw was observed between Test Yolk buffer and Cryolink media. With further validation, Cryolink media may provide a safer and simplified means of cryopreserving sperm for reproductive use.