Nakata Kumiko, Yoshida Kaoru, Yamashita Naoki
Reproductive Medicine Research Center, Yamashita Shonan Yume Clinic, Kanagawa 251-0025, Japan.
Faculty of Life and Environmental Sciences, University of Yamanashi, Yamanashi 400-8510, Japan.
J Reprod Dev. 2019 Aug 9;65(4):297-304. doi: 10.1262/jrd.2017-086. Epub 2019 Apr 11.
The aim of this study was to develop a new container for cryopreservation of a limited number of spermatozoa. To evaluate the efficacy and safety of this new container, we performed preclinical evaluations using human sperm or mouse oocytes and sperm. First, using human sperm that was frozen and then thawed, we demonstrated that the sperm recovery rate using the new container was 96.7% (58/60), which was significantly higher (P < 0.05) than the recovery rate of 21.2% (11/52) when using the Cryotop. Sperm motility rates were 19.2% (10/52) using the Cryotop and 35.0% (21/60) using the new container. Second, murine epididymal spermatozoa were divided into three groups: fresh spermatozoa, spermatozoa frozen using a straw, and spermatozoa frozen using the new container. Sperm motility, sperm membrane and DNA integrity, in vitro development of fertilized eggs, and offspring development after embryo transfer were assessed. The motility of freeze-thawed sperm was lower in spermatozoa that were frozen using the new container than in fresh spermatozoa or those that were frozen using a straw. After intracytoplasmic sperm injection, the survival rate was 96.7% (145/150), the 2-cell development rate was 90.3% (131/145), and the blastocyst development rate was 77.2% (112/145), when using the new container. There were no differences in the sperm membrane, DNA integrity, or in the embryo development rates to the blastocyst stage among the different frozen groups. Six offspring were derived from spermatozoa freeze-thawed in the new container, and they developed normally. Thus, the new container allows easy handling of a small number of sperms and minimizes sperm loss during cryopreservation.
本研究的目的是开发一种用于冷冻保存少量精子的新型容器。为了评估这种新型容器的有效性和安全性，我们使用人类精子或小鼠卵母细胞及精子进行了临床前评估。首先，使用冷冻后再解冻的人类精子，我们证明使用新型容器时精子回收率为96.7%（58/60），显著高于使用Cryotop时21.2%（11/52）的回收率（P<0.05）。使用Cryotop时精子活力率为19.2%（10/52），使用新型容器时为35.0%（21/60）。其次，将小鼠附睾精子分为三组：新鲜精子、用细管冷冻的精子和用新型容器冷冻的精子。评估了精子活力、精子膜和DNA完整性、受精卵的体外发育以及胚胎移植后的后代发育。使用新型容器冷冻的精子中，冻融后精子的活力低于新鲜精子或用细管冷冻的精子。使用新型容器进行胞浆内精子注射后，存活率为96.7%（145/150），2细胞发育率为90.3%（131/145），囊胚发育率为77.2%（112/145）。不同冷冻组之间在精子膜、DNA完整性或囊胚阶段的胚胎发育率方面没有差异。有6只后代来自于在新型容器中冻融的精子，且发育正常。因此，这种新型容器便于处理少量精子，并在冷冻保存过程中使精子损失最小化。
