Comparison of Rapid Freezing and Vitrification for Human Sperm Cryopreservation Using Trehalose as a Cryoprotective Agent

Rapid freezing and vitrification are becoming popular for human sperm cryopreservation;however, it remains unclear which method is better. The aims of the present study were to determine the optimal trehalose concentration and to compare the cryoprotective effects of rapid freezing and vitrification. The results showed that: 1) The optimal trehalose concentration was 0.25 mol/L;2) The post-thaw recovery rates of total and progressive sperm motilities after rapid freezing (38.6% ± 3.0% and 41.1% ± 5.0%) were significantly higher (P 0.05) than that after vitrification (26.1% ± 3.1% and 27.2% ± 1.3%) when 0.5 mL straws were used;3) However, the recovery rates of total and progressive motilities after rapid freezing in 0.5 mL straw (26.7% ± 9.6% and 26.8% ± 8.7%) were significantly lower (P 0.05) than that after vitrification in a novel straw-in-straw system (43.1% ± 4.2% and 41.8% ± 15.5%);and 4) The post-thaw sperm nuclear DNA damage level after rapid freezing in 0.5 mL straw (8.7% ± 2.8%) was not significantly different from that of sperm after vitrification in the straw-in-straw system (9.2% ± 2.5%). It was concluded that rapid freezing is superior to vitrification when using 0.5 mL straws;however, vitrification is superior to rapid freezing when using the straw-in-straw systems.

Human Sperm Freezing: Mini Update

Sperm freezing is widely used in ART clinics around the globe. Very little has actually changed with respect to cryopreservation protocols and methodology of freezing over the last 50 years. The aim of this paper is to briefly review the basic principles that underlie freezing and ice crystal formation and also provide a brief overview of newer sperm freezing techniques like sperm vitrification and freeze drying of sperm.

褪黑素对无渗透性冷冻保护剂的精子玻璃化冷冻影响

目的探讨褪黑素在无渗透性冷冻保护剂微量精子玻璃化冷冻技术中的研究。方法选取2022年4-9月在荆门市人民医院生殖医学中心就诊的30例男性患者的精液样本。每份标本分为常规冷冻组、微滴冷冻组、无保护剂微滴冷冻组、褪黑素+无保护剂微滴冷冻组。通过计算机辅助精液分析法(CASA)观察冷冻前及冷冻后精子前向运动(PR),伊红-苯胺黑染色检测精子存活率,改良巴氏染色检测正常形态率以及顶体完整率,低渗肿胀试验检测质膜完整率,SCD法检测DNA完整性检测,试剂盒检测MDA、SOD、GSH-Px水平等。结果微滴冷冻组、无保护剂微滴冷冻组复苏后PR、复苏率及复苏后存活率高于常规冷冻组(P<0.05);褪黑素+无保护剂微滴冷冻组复苏后PR和复苏率高于无保护剂微滴冷冻组(P<0.05)。褪黑素+无保护剂微滴冷冻组复苏后正常形态率和顶体完整性明显高于其他3组(P<0.05)。微滴冷冻组、无保护剂微滴冷冻组复苏后质膜完整率高于常规冷冻组(P<0.05);褪黑素+无保护剂微滴冷冻组复苏后质膜完整率高于微滴冷冻组(P<0.05),而DFI明显低于微滴冷冻组(P<0.05)。与常规冷冻组相比,无保护剂微滴冷冻组、褪黑素+无保护剂微滴冷冻组MDA明显降低(P<0.05),SOD以及GSH-Px明显升高(P<0.01);与微滴冷冻组相比,褪黑素+无保护剂微滴冷冻组MDA明显降低(P<0.05)。褪黑素+无保护剂微滴冷冻组SOD、GSH-Px较其他3组明显升高(P<0.05)。结论无渗透性冷冻保护剂添加褪黑素能够提高复苏后精子的PR、活力、正常形态、DFI、质膜和顶体完整性,主要与降低MDA,增加SOD以及GSH-Px表达有关。

Improving native human sperm freezing protection by using a modified vitrification method

Slow freezing is the most commonly used technique for the cryopreservation of spermatozoa in clinical practice.However,it has been shown to have a negative impact on sperm function and structure.Vitrification as a successful alternative method has been proved to have better protective effects on human embryos,but vitrification of spermatozoa is still subject to low recovery rates.In this study,a modified vitrification method for native spermatozoa was developed.A total of 28 semen samples were included;each sample was divided into three equal parts and assigned to fresh,slow freezing,and vitrification groups.Sperm vitality,motility,morphology,DNA integrity,and acrosome reaction were assessed for each of the groups.The results showed that vitrification achieves better results for several sperm protection parameters than slow freezing;vitrification achieves a higher recovery rate(P<0.05),motility(P<0.05),morphology(P<0.05),and curve line velocity(P<0.05)than slow freezing.Furthermore,DNA fragmentation was decreased(P<0.05)and better acrosome protection(P<0.05)was exhibited in the spermatozoa after vitrification.Principal component analysis of all sperm parameters revealed that the vitrification cluster was closer to the fresh cluster,indicating that spermatozoa are better preserved through vitrification.In conclusion,while both slow freezing and vitrification have negative effects on sperm function and structure,the vitrification protocol described here had a relatively better recovery rate(65.8%)and showed improved preservation of several sperm quality parameters compared with slow freezing.

Update on techniques for cryopreservation of human spermatozoa

In the 1960s,sperm cryopreservation was developed as a method to preserve fertility.Currently,techniques for the cryopreservation of human spermatozoa have been widely used in assisted reproduction.However,although sperm cryobiology has made notable achievements,the optimal method for the recovery of viable spermatozoa after cryopreservation remains elusive.Postthawing sperm quality can be affected by cryoprotectants,ice formation,storage conditions,and osmotic stress during the freezing process.This review discusses recent advances in different cryopreservation techniques,cryoprotectants,and freezing and thawing methods during cryopreservation and new indications for the use of cryopreserved spermatozoa.

不同精子来源质量冷冻方式与妊娠结局的关联性分析

目的探讨冷冻微量精子相关参数与体外受精周期实验室及临床结局的关系。方法回顾性分析2016年1月至2020年12月在生殖医学科行稀少精子冷冻复苏后卵胞浆内单精子显微注射(ICSI)的118个周期相关资料。根据冷冻精子的冷冻方法分为微量冷冻组、传统常规冷冻组;按精子来源分为丈夫射精组、附睾穿刺组、睾丸穿刺组;根据精子质量分为Ⅰ型、Ⅱ型、Ⅲ型严重少弱组。分别比较各组之间一般资料、实验室及临床结局等;采用多因素二元Logistic回归分析冷冻精子相关参数对临床结局的影响。结果①一般资料:按冷冻精子的冷冻方法、精子来源和精子质量分组,女方的年龄、体质量指数、不孕年限、基础促卵泡素(FSH)、抗苗勒氏管激素(AMH)、Gn天数、Gn总量、人绒毛膜促性腺激素(HCG)日雌二醇(E_(2))水平、获卵数、MⅡ卵数各组之间差异均无统计学意义(P>0.05);②实验室指标及临床结局:微量冷冻组与传统常规冷冻组两组组间胚胎利用率、临床妊娠率之间差异均无统计学意义(P>0.05),2PN率(P=0.002)、2PN卵裂率(P=0.036)、优胚率(P=0.002)差异均有统计学意义。丈夫射精组、附睾穿刺组和睾丸穿刺组组间比较,2PN率、临床妊娠率差异均无统计学意义(P>0.05);2PN卵裂率(P<0.001)、胚胎利用率(P=0.048)、优胚率(P=0.002)差异均有统计学意义。Ⅰ型、Ⅱ型和Ⅲ型严重少弱组3组间比较,2PN率、胚胎利用率、优胚率之间差异均无统计学意义(P>0.05);2PN卵裂率(P<0.001)、种植率(P=0.005)、临床妊娠率(P=0.048)差异均有统计学意义;③多因素二元Logistic回归分析结果显示,Ⅲ型严重少弱精组相对Ⅰ型严重少弱精组是低临床妊娠率危险因素[OR:0.259,95%CI、0.075~0.887]。结论微量冷冻和常规冷冻可获得相似临床妊娠率,精子来源不影响临床妊娠率,极严重少弱精可能降低临床妊娠率,但还需要扩大样本量进一步证实。