title: A novel method for cryopreservation of individual human spermatozoa.
abstract: The purpose of this study is to develop a novel method for the cryopreservation and efficient post-thaw recovery of individual or small numbers of human spermatozoa. Spermatozoa equilibrated in cryoprotectant buffer were injected with an intracytoplasmic sperm injection (ICSI) needle into a droplet of cryoprotectant on a homemade cryoleaf. The droplet was of cryoprotectant and seminal plasma at a ratio of 1:1. The sperm-loaded cryoleaf was slowly lowered over and stored in liquid nitrogen. Spermatozoa were thawed in a 37°C oil bath without dilution and centrifugation. To test the fertilizing ability of these spermatozoa, the recovered spermatozoa were injected by ICSI into 1-d-old or in vitro-matured human oocytes. Fresh spermatozoa from the same semen samples served as controls. The trials were performed in two separate experiments. In the first set of experiments, 92 spermatozoa were thawed and carefully investigated. The spermatozoa from percutaneous epididymal sperm aspiration had a motility recovery of 92.9% (13/14); ejaculated spermatozoa had a motility recovery of 61.5% (48/78), and only 1.3% (1/78) was lost. Together in the first and second set of experiments, the fertilization rates for the fresh and frozen-thawed spermatozoa were 67.6% (25/37) and 60.6% (40/66), respectively (P = 0.052). The mean embryo cleavage rates in the fresh and frozen-thawed groups were 88% (22/25) and 85% (34/40), respectively (P = 0.990). This cryopreservation method for individual or small numbers of human spermatozoa was efficient and simple. These findings make this method a promising technique for the clinical application of ejaculated sperm from oligozoospermic patients.
authors.affiliation: Department of Assisted Reproduction, Shanghai 9th People's Hospital, School of Medicine, Shanghai Jiao Tong University, 639 ZhiZaoJu Road, 200011, Shanghai, China. pengqiuping@hotmail.com
authors.forename: Qiu-Ping
authors.initials: QP
authors.lastname: Peng
authors.forename: Shao-Feng
authors.initials: SF
authors.lastname: Cao
authors.forename: Qi-Feng
authors.initials: QF
authors.lastname: Lyu
authors.forename: Song-Guo
authors.initials: SG
authors.lastname: Xue
authors.forename: Wei
authors.initials: W
authors.lastname: Jin
authors.forename: Xiao-Yin
authors.initials: XY
authors.lastname: Liu
authors.forename: Wen-Jie
authors.initials: WJ
authors.lastname: Zhang
authors.forename: H Ingolf
authors.initials: HI
authors.lastname: Nielsen
authors.forename: Yan-Ping
authors.initials: YP
authors.lastname: Kuang
mesh_terms: D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000737:chemistry; D004867:Equipment Design; D005260:Female; D005306:Fertilization / Q000502:physiology*; D006801:Humans; D008297:Male; D009865:Oocytes / Q000166:cytology; D012662:Semen Preservation / Q000379:methods*; D020554:Sperm Injections, Intracytoplasmic / Q000379:methods; D013081:Sperm Motility / Q000502:physiology*; D013094:Spermatozoa / Q000166:cytology
chemical_list: D003451:Cryoprotective Agents