药物非临床生殖毒性试验中动物妊娠的主要难点及应对方法

doi:10.12300/j.issn.1674-5817.2024.177

摘要/Abstract

摘要：

药物非临床生殖毒性试验多选用大鼠、家兔和食蟹猴等哺乳动物，而动物妊娠是目前影响非临床生殖毒性试验的关键环节，本文主要针对动物妊娠环节中存在的难点及应对方法进行讨论。大鼠可应用于生育力评估与早期胚胎发育毒性试验（Ⅰ段）、胚胎-胎仔发育毒性试验（Ⅱ段）和围产期毒性试验（Ⅲ段），可以通过阴道涂片法确定雌性大鼠的动情周期。在雌、雄大鼠按1∶1比例合笼过夜后，次日通过阴道栓检查来确认大鼠是否发生交配行为。家兔一般应用于胚胎-胎仔发育毒性试验（Ⅱ段），可通过观察雄性家兔和处于发情期的雌性家兔合笼后是否发生交配行为来确定受孕时间。家兔在生殖毒性动物试验中常出现雌兔发情不典型、不同批次间家兔发情情况差异显著、交配失败等问题，可通过延长光照时间、增加蛋白质摄入、选择合适的交配方式、改变饲养环境（雌兔和雄兔邻近饲养）等方式改善。非人灵长类动物一般应用于围产期毒性试验（Ⅲ段），准确判断雄性非人灵长类动物是否性成熟是其药物非临床生殖毒性试验的难点之一，可通过综合评估动物年龄、体重和睾丸体积等指标来判断它们是否性成熟。一般雄性猕猴年龄在4.5岁以上，体重大于4.5 kg，单侧睾丸体积超过10 mL，双侧睾丸总体积大于20 mL则表示性成熟。关于雌性非人灵长类动物是否妊娠，可通过超声检查是否能观察到明显孕囊进行判定，但这需要经验丰富的临床兽医制定完善的B超检查方法。综上所述，在非临床生殖毒性动物研究中，应根据不同种属动物的生殖系统解剖学特性，选择适合的检测/观察方法和结果判定标准，以确保动物成功交配，使非临床生殖毒性试验顺利开展。

关键词: 非临床生殖毒性试验, 动物妊娠, 动物发情及交配

Abstract:

Non-clinical reproductive toxicity studies typically employ mammals like rats, rabbits, and cynomolgus monkeys, with animal pregnancy being a key challenge in such testing. This article focuses on the difficulties encountered in the animal pregnancy process and potential countermeasures. Rats can be used for fertility and early embryonic development toxicity studies (Segment Ⅰ), embryo-fetal development toxicity studies (Segment Ⅱ), and perinatal toxicity studies (Segment Ⅲ). The estrous cycle of female rats can be determined by vaginal smear, and mating behavior is confirmed through copulatory plug checks the following day after pairing one female with one male in the same cage. Rabbits are commonly used in embryo-fetal development toxicity studies (Segment Ⅱ). Mating behavior between male rabbits and estrous females is observed to determine the time of conception. However, challenges such as atypical estrus of female rabbits, large variations in estrus between batches, and mating failure often occur in reproductive toxicity testing, which may be addressed through prolonged light exposure, increased protein supplementation, optimized mating strategies, and environmental modifications like female and male rabbits are raised adjacent to each other. Non-human primates (NHPs) are typically employed in perinatal toxicity studies (Segment Ⅲ), where one of the key challenges lies in accurately determining sexual maturity in males - a critical factor for reproductive toxicity testing, which can be assessed through comprehensive evaluation of age, body weight, and testicular volume. Generally, male macaques are considered sexually mature when they meet the following criteria: age >4.5 years, body weight >4.5 kg, single testis volume >10 mL, and combined testicular volume >20 mL. For pregnancy confirmation, ultrasound examination demonstrating visible gestational sacs is required, though this necessitates experienced veterinary clinicians to establish standardized ultrasound examination protocols. In conclusion, reproductive toxicity studies should employ species-appropriate detection methods and evaluation criteria based on anatomical characteristics of the reproductive system to ensure successful mating and proper study execution.

Key words: Non-clinical reproductive toxicity testing, Animal pregnancy, Estrus and mating in animals

中图分类号:

Q95-33

刘鹍,兰青,易兵,等. 药物非临床生殖毒性试验中动物妊娠的主要难点及应对方法[J]. 实验动物与比较医学, 2025, 45(4): 449-456. DOI: 10.12300/j.issn.1674-5817.2024.177.

LIU Kun,LAN Qing,YI Bing,et al. Key Challenges and Mitigation Strategies for Animal Pregnancy in Non-clinical Reproductive Toxicity Testing of Drugs[J]. Laboratory Animal and Comparative Medicine, 2025, 45(4): 449-456. DOI: 10.12300/j.issn.1674-5817.2024.177.

图/表 2

图1 成年雌性大鼠动情周期各阶段的典型阴道组织涂片注：A，动情前期；B，动情期；C，动情后期；D，间情期。黑色箭头所指的是有核上皮细胞；红色箭头所指的是无核上皮细胞；绿色箭头所指的是白细胞。涂片的染色方法为HE染色。图像的放大倍数为40，标尺为100 μm。

Figure 1 Typical vaginal smear characteristics from adult female rats at each stage of estrous cycleNote：A， proestrus； B， estrus； C， metestrus； D， diestrus. The black arrow indicates nucleated epithelial cells； the red arrow indicates anucleated epithelial cells； the green arrows indicate leukocytes. The staining method of the smear was hematoxylin and eosin （HE） staining. The magnification of the images is 40 and the scale bars are 100 μm.

图2 妊娠初期雌猴孕囊B超切面注：图中箭头所指区域可见成型的孕囊结构。

Figure 2 Ultrasound cross-sectional view of gestational sac in a female monkey during early pregnancyNote：The area indicated by the arrow in the figure shows a well-formed gestational sac structure.

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