title: Minimal volume vitrification of epididymal spermatozoa results in successfulin vitrofertilization and embryo development in mice.
abstract: This study compared three cryopreservation protocols on sperm functions, IVF outcomes, and embryo development. Epididymal spermatozoa cryopreserved using slow-cooling (18% w/v raffinose, RS-C) were compared with spermatozoa vitrified using 0.25 M sucrose (SV) or 18% w/v raffinose (RV). The motility, vitality, and DNA damage (TUNEL assay) of fresh control (FC) spermatozoa were compared with post-thawed or warmed RS-C, RV, and SV samples. Mouse oocytes (n = 267) were randomly assigned into three groups for insemination: RV (n = 102), RS-C (n = 86), and FC (n = 79). The number and the proportion of two-cell embryos and blastocysts from each treatment were assessed. Sperm motility (P < 0.01) and vitality (P < 0.05) were significantly reduced after vitrification compared with slow-cooled spermatozoa. However, DNA fragmentation was significantly reduced in spermatozoa vitrified using sucrose (15 ± 1.8% [SV] vs 26 ± 2.8% [RV] and 27 ± 1.2% [RS-C]; P < 0.01). Although the number of two-cell embryos produced by RS-C, RV, and FC spermatozoa was not significantly different, the number of blastocysts produced from two-cell embryos using RV spermatozoa was significantly higher than FC spermatozoa (P = 0.0053). This simple, small volume vitrification protocol and standard insemination method allows successful embryo production from small numbers of epididymal spermatozoa and may be applied clinically to circumvent the need for ICSI, which has the disadvantage of bypassing sperm selection.
authors.affiliation: Education Program in Reproduction and Development, Department Obstetrics and Gynaecology, Monash University, Clayton VIC 3168, Australia.|Department of Obstetrics and Gynaecology, Unit of Reproductive Medicine, Clínica Las Condes, Lo Fontecilla 441, Las Condes, Santiago, Region Metropolitana, 7550000, Chile.
authors.forename: Fabrizzio
authors.initials: F
authors.lastname: Horta
authors.affiliation: Education Program in Reproduction and Development, Department Obstetrics and Gynaecology, Monash University, Clayton VIC 3168, Australia.
authors.forename: Hamida
authors.initials: H
authors.lastname: Alzobi
authors.affiliation: Education Program in Reproduction and Development, Department Obstetrics and Gynaecology, Monash University, Clayton VIC 3168, Australia.
authors.forename: Sutthipat
authors.initials: S
authors.lastname: Jitanantawittaya
authors.affiliation: Education Program in Reproduction and Development, Department Obstetrics and Gynaecology, Monash University, Clayton VIC 3168, Australia.
authors.forename: Sally
authors.initials: S
authors.lastname: Catt
authors.affiliation: Education Program in Reproduction and Development, Department Obstetrics and Gynaecology, Monash University, Clayton VIC 3168, Australia.
authors.forename: Penny
authors.initials: P
authors.lastname: Chen
authors.affiliation: Education Program in Reproduction and Development, Department Obstetrics and Gynaecology, Monash University, Clayton VIC 3168, Australia.
authors.forename: Mulyoto
authors.initials: M
authors.lastname: Pangestu
authors.affiliation: Education Program in Reproduction and Development, Department Obstetrics and Gynaecology, Monash University, Clayton VIC 3168, Australia.
authors.forename: Peter
authors.initials: P
authors.lastname: Temple-Smith
mesh_terms: D000818:Animals; D001755:Blastocyst; D002470:Cell Survival; D015925:Cryopreservation / Q000379:methods*; D053938:DNA Fragmentation; D047108:Embryonic Development; D005260:Female; D005307:Fertilization in Vitro / Q000379:methods; D008297:Male; D051379:Mice; D009865:Oocytes; D012662:Semen Preservation / Q000379:methods*; D013081:Sperm Motility; D053625:Sperm Retrieval; D013094:Spermatozoa / Q000378:metabolism*; D058989:Vitrification