妊娠前应激暴露对小鼠后续妊娠的影响

doi:10.3877/cma.j.issn.1674-0785.2023.04.012

目的

探讨给予糖皮质激素作为应激因素对小鼠后续妊娠结局的影响及可能机制。

方法

将40只BALB/C雌鼠随机分为2组：正常对照组和实验组，实验组小鼠腹腔注射20 mg/kg/d皮质酮（CORT），连续处理24天后，与对照组比较应激相关指标变化。应激小鼠造模成功后，小鼠休息2周后合笼。观察比较2组小鼠妊娠结局相关指标并统计分析。收集妊娠E13.5~15.5天的胎盘组织，免疫组化检测皮质酮代谢基因糖皮质激素受体（GR）和血清/糖皮质激素诱导激酶1（SGK1）表达情况。

结果

CORT实验处理组小鼠与对照组比较，强迫游泳不动时间明显延长（P=0.004），血清CORT和促肾上腺皮质激素（ACTH）水平明显升高（P=0.048，P=0.040）；实验组平均产仔量、仔鼠平均出生体重均显著降低（P=0.025，P=0.003），妊娠中期吸收胎比率显著增高（P＜0.001）。免疫组化结果显示实验组蜕膜区和迷路区GR表达显著多于对照组（P=0.037，P=0.001）；而蜕膜区SGK1阳性染色显著低于对照组（P=0.048）。

结论

通过腹腔注射CORT成功诱导小鼠应激模型，后续妊娠的应激小鼠吸收胎比率显著升高，产仔数量，仔鼠出生体重显著降低。妊娠前应激暴露引起后续不良妊娠结局，与后续妊娠中皮质酮代谢基因GR表达增多，以及胎盘蜕膜组织SGK1表达减少密切相关。本研究为妊娠前高糖皮质激素暴露对后续妊娠的影响提供了研究基础。

关键词: 应激, 皮质酮, 糖皮质激素受体, 血清/糖皮质激素诱导激酶1, 不良妊娠结局

Objective

To investigate the effect of pre-pregnancy glucocorticoid administration as a stress factor on subsequent pregnancy outcomes in mice and the potential mechanism involved.

Methods

Forty female BALB/c mice were randomly divided into a corticosterone (CORT) administration group and a control group. Mice in the CORT administration group were peritoneally injected with CORT at a dose of 20 mg/kg/d for 24 days, while the control group was given equal volume of the vehicle. Serum level of CORT, adrenocorticotropic hormone (ACTH), and 5-HT (5-hydroxytryptamine) were compared between the two groups. CORT treated female mice were bred with male BALB/c mice 2 weeks after the final injection and checked for vaginal plugs daily. The indexes of pregnancy outcomes were observed and compared between the two groups. E13.5-15.5 placentas were collected to analyze the expression of glucocorticoid receptor (GR) and serum-and glucocorticoid-inducible kinase-1 (SGK1) by immunohistochemistry.

Results

Compared with the control mice, the immobility time in the forced swimming test were significantly longer in CORT treated mice (P=0.004), and serum levels of CORT and ACTH were significantly increased (P=0.048, P=0.040). The litter size (P=0.025) and litter weight (P=0.003) were significantly decreased with a significantly increased rate of resorption (P＜0.001) in CORT treated mice. Immunohistochemical analysis showed that the expression of GR in the decidua basalis and labyrinth (P=0.037 and P=0.001, respectively) in the CORT administration group significantly increased, while the expression of SGK1 in the decidua (P=0.048) was significantly decreased.

Conclusion

A mouse stress model has been successfully induced by CORT, and there is a significantly increased rate of implantation resorption, and reduced litter size and litter weight during subsequent pregnancy in model mice. The pregnancy loss is related with increased GR in the decidua basalis and labyrinth but decreased SGK1 in the decidua. High level of CORT exposure before pregnancy has a significant effect on subsequent pregnancy outcomes.

Key words: Stress, Corticosterone, Serum- and glucocorticoid-inducible kinase-1, Glucocorticoid receptor, Adverse pregnancy outcomes

图1 小鼠应激模型的验证。图a为应激暴露期间2组小鼠体重随时间变化的比较；图b为2组小鼠强迫游泳实验的不动时间比较；图c为2组小鼠血清应激标志物血清水平比较注：^*P＜0.05；^**P＜0.01；^***P＜0.001

表1 不同时间点各组小鼠体重的比较（mean±sem/［M（P₂₅，P₇₅）］）

分组	体重（g）
分组	基础	第3天	第6天	第9天	第12天	第15天	第18天	第21天	第24天
对照组	18.13±0.13	18.24±0.18	18.66±0.15	18.82±0.20	18.59（18.45，19.12）	19.32±0.16	19.63±0.20	20.25±0.31	21.19±0.32
皮质酮处理组	18.19±0.13	18.38±0.16	18.43±0.15	18.77±0.15	18.49±0.16	18.69±0.13	18.30±0.14	18.89±0.20	19.04±0.14
t/Z值	-0.305	-0.593	1.064	0.222	-1.041	3.026	5.542	3.837	6.126
P值	0.763	0.557	0.295	0.826	0.302	0.005	P＜0.001	0.001	P＜0.001

表1 不同时间点各组小鼠体重的比较（mean±sem/［M（P₂₅，P₇₅）］）

分组	体重（g）
分组	基础	第3天	第6天	第9天	第12天	第15天	第18天	第21天	第24天
对照组	18.13±0.13	18.24±0.18	18.66±0.15	18.82±0.20	18.59（18.45，19.12）	19.32±0.16	19.63±0.20	20.25±0.31	21.19±0.32
皮质酮处理组	18.19±0.13	18.38±0.16	18.43±0.15	18.77±0.15	18.49±0.16	18.69±0.13	18.30±0.14	18.89±0.20	19.04±0.14
t/Z值	-0.305	-0.593	1.064	0.222	-1.041	3.026	5.542	3.837	6.126
P值	0.763	0.557	0.295	0.826	0.302	0.005	P＜0.001	0.001	P＜0.001

表2 各组血清应激标志物血清水平（

x ¯

±s）

	CORT	ACTH	5-HT
对照组	0.15±0.03	0.45±0.16	443.93±35.97
皮质酮处理组	0.63±0.13	1.26±0.27	426.59±39.43
t值	-4.012	-2.747	0.302
P值	0.048	0.040	0.769

表2 各组血清应激标志物血清水平（

x ¯

±s）

	CORT	ACTH	5-HT
对照组	0.15±0.03	0.45±0.16	443.93±35.97
皮质酮处理组	0.63±0.13	1.26±0.27	426.59±39.43
t值	-4.012	-2.747	0.302
P值	0.048	0.040	0.769

图2 2组小鼠妊娠结局比较。图a和b分别为小组小鼠平均产仔量、仔鼠体重及E14天胎盘及胎儿重量比较；图c为2组妊娠小鼠子宫；图d为2组小鼠吸收胎占比比较注：^*P＜0.05；^**P＜0.01；^***P＜0.001

图3 2组小鼠胎盘组织GR和SGK1表达情况（×100）。图a和b分别为对照组和皮质酮处理组胎盘组织SGK1免疫组化染色；图c为2组小鼠胎盘各区域SGK1平均光密度比较；图d和e分别为对照组和皮质酮处理组GR免疫组化染色；图f为2组小鼠胎盘各区域GR平均光密度比较注：^*P＜0.05；^**P＜0.01；GR为糖皮质激素受体；SGK1为血清/糖皮质激素诱导激酶1

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Effect of stress exposure before pregnancy on subsequent pregnancy outcomes in mice

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Effect of stress exposure before pregnancy on subsequent pregnancy outcomes in mice