Zavos P M, Correa J R, Zarmakoupis-Zavos P N
Andrology Institute of America, Lexington, KY 40523, USA.
Tohoku J Exp Med. 1998 Apr;184(4):277-84. doi: 10.1620/tjem.184.277.
The objective of this study was to develop new techniques for the cryopreservation of washed spermatozoa. Two media (Ham's F-10 and nonthermoprecipitated TEST-yolk buffer [NT-TYB]) containing 7% (v/v) glycerol were compared to semen cryopreservation by adding glycerol directly to the semen. Twenty four men collected a semen specimen each after 4 days of sexual abstinence via the use of a semen collection device at intercourse. Specimens were assessed for volume (ml), count (x 10(6)), percentage and grade of motility, morphology (% normal) and acrosomal status (% intact acrosomes). Each ejaculate was split into 3 aliquots (Aliquots 1 to 3) and processed for freezing. Aliquot 1 was prepared for cryopreservation by adding glycerol (7% [v/v] final concentration) directly via a dropwise mode. Aliquot 2 and 3 were diluted 1:1 (v/v) with Ham's F-10 and NT-TYB, respectively. Aliquots 2 and 3 were then centrifuged (400 x g for 10 minutes) and resuspended into the corresponding media containing 7% (v/v) glycerol to complete the sperm wash procedure. All aliquots were frozen in 0.5 ml french straws. Sperm specimens were frozen in liquid nitrogen (LN2) vapor from +23 degrees C to -68 degrees C at a slow rate (2.3 degrees C/minute), after which the specimens were plunged directly into LN2 and stored for 30 days. The quality of the spermatozoa were monitored throughout each step of the overall procedure by measuring the motility characteristics of the spermatozoa. Straws corresponding to each aliquot were thawed in a water bath at 37 degrees C for 2 minutes, followed by assessment of sperm motility and acrosomal status. The percentage of motility after thawing was 31.6 +/- 5.6%, 32.8 +/- 1.8% and 37.3 +/- 1.9% in Aliquots 1 to 3, respectively. Similarly, the grade of motility was 2.4 +/- 0.2, 2.6 +/- 0.1 and 3.0 +/- 0.1 in Aliquots 1 to 3, respectively. The acrosomal status (% intact acrosomes) in Aliquots 1 to 3 was 41.2 +/- 2.6, 43.1 +/- 3.6 and 51.6 +/- 4.5, respectively. The results suggest that the characteristics of spermatozoa washed and frozen in NT-TYB (Aliquot 3) were improved over those spermatozoa prepared via direct addition of glycerol to the semen (Aliquot 1) or by using Ham's F-10 (Aliquot 2). The most significant reduction noted during freezing was in the loss of acrosomal integrity. The results obtained in this study point out that washed spermatozoa can be cryopreserved with some success and that the recovered spermatozoa could be used for intrauterine insemination in an artificial insemination program using husband's or donor sperm, or for the various assisted reproductive technology procedures. It is the opinion of the authors that the information generated in this study is of importance for those scientists and clinicians involved in the handling and manipulation of cryopreserved spermatozoa.
本研究的目的是开发用于洗涤精子冷冻保存的新技术。将含有7%（v/v）甘油的两种培养基（哈姆氏F-10和非热沉淀TEST-蛋黄缓冲液[NT-TYB]）与直接向精液中添加甘油进行精液冷冻保存的方法进行比较。24名男性在禁欲4天后，通过性交时使用精液采集装置每人采集一份精液标本。对标本进行体积（ml）、计数（x 10⁶）、活力百分比和等级、形态（%正常）以及顶体状态（%完整顶体）的评估。每份射精标本分成3份等分试样（等分试样1至3）并进行冷冻处理。等分试样1通过逐滴方式直接添加甘油（最终浓度7%[v/v]）制备用于冷冻保存。等分试样2和3分别用哈姆氏F-10和NT-TYB按1:1（v/v）稀释。然后将等分试样2和3离心（400 x g，10分钟），并重悬于含有7%（v/v）甘油的相应培养基中以完成精子洗涤程序。所有等分试样均在0.5 ml法式细管中冷冻。精子标本在液氮（LN₂）蒸气中从+23℃以缓慢速率（2.3℃/分钟）冷冻至-68℃，之后标本直接投入LN₂中并储存3天。通过测量精子的活力特征在整个程序的每个步骤中监测精子的质量。对应于每个等分试样的细管在37℃水浴中解冻2分钟，随后评估精子活力和顶体状态。解冻后的活力百分比在等分试样1至3中分别为31.6±5.6%、32.8±1.8%和37.3±1.9%。同样，活力等级在等分试样1至3中分别为2.4±0.2、2.6±0.1和3.0±0.1。等分试样1至3中的顶体状态（%完整顶体）分别为41.2±2.6、43.1±3.6和51.6±4.5。结果表明，在NT-TYB中洗涤和冷冻的精子（等分试样3）的特征优于通过直接向精液中添加甘油制备的精子（等分试样1）或使用哈姆氏F-10制备的精子（等分试样2）。冷冻过程中最显著的减少是顶体完整性的丧失。本研究获得的结果指出，洗涤后的精子可以成功冷冻保存，并且回收的精子可用于使用丈夫或供体精子的人工授精程序中的宫内授精，或用于各种辅助生殖技术程序。作者认为，本研究产生的信息对于那些参与冷冻保存精子处理和操作的科学家和临床医生具有重要意义。
