目的 探讨连续重复给予含左炔诺孕酮(levonorgestrel,LNG)紧急避孕药(emergency contraception pills,ECPs)对雌性大鼠生育力及其F1代仔鼠健康的影响.方法 成年SPF级雌性大鼠于每个动情周期连续给予3次LNG-ECPs,分别给予3个(P-3)、6个(P-6)和12个(P-12)动情周期.雌性大鼠每个给药时程下,采用Excel产生随机数按体质量分层随机分为2组,分别为LNG-ECPs组和溶媒对照组,分别灌胃给予0.12 mg/kg LNG-ECPs和等体积溶媒.各组于末次给药后4 h分别取1/2动物(12～18只)进行解剖(6～9只)和交配(6～9只);剩余1/2动物(12～18只)于停药恢复3个动情周期后再分别进行解剖(6～9只)和交配(6～9只),计算脏器系数.采用酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)检测雌性大鼠血清中卵泡刺激素(follicle-stimulating hormone,FSH)、黄体生成素(lutenizing hormone,LH)、雌激素、孕激素、睾酮、抗苗勒管激素(anti-Müllerian hormone,AMH)以及游离甲状腺激素3(free thyroid hormone,fT3)的水平.雌性大鼠卵巢组织切片苏木精-伊红(hematoxylin and eosin,HE)染色后进行卵泡计数.记录LNG-ECPs给药结束和停药恢复期雌性大鼠妊娠率及窝仔数,测定F1代仔鼠生长指数以及主动和被动运动能力等指标.取P-12大鼠卵巢组织进行转录组学测序,建立LNG-ECPs致卵巢差异表达基因谱,分析LNG-ECPs致卵巢损伤的差异基因,并进行基因本体(gene ontology,GO)和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路富集分析.结果 ①连续给予3个和6个动情周期后,LNG-ECPs组雌鼠的血清激素水平和生育力与溶媒对照组相比,差异均无统计学意义(均P＞0.05);F1代仔鼠生长指数和行为学结果与溶媒对照组仔鼠相比差异均无统计学意义(均P＞0.05).②连续给予12个动情周期后,与溶媒对照组相比,LNG-ECPs组大鼠血清中 FSH[(0.21±0.17)U/L]、LH[(0.27±0.08)U/L]和孕激素[(0.68±0.23)μg/L]水平显著低于溶媒对照组[(1.00±0.82)U/L,P=0.043;(1.00±0.50)U/L,P=0.006;(1.00±0.20)μg/L,P=0.027],雌二醇[(2.24±1.03)μg/L]和睾酮[(1.25±0.25)μg/L]水平明显高于溶媒对照组[(1.00±0.35)μg/L,P=0.019;(1.00±0.07)μg/L,P=0.044];卵巢中原始卵泡数量(4.88±2.36)显著少于溶媒对照组(16.13±9.36,P=0.005),闭锁卵泡数量(24.38±5.01)显著高于溶媒对照组(19.13±2.30,P=0.018);LNG-ECPs组中F1代仔鼠负重游泳时间[(157.13±32.29)s]明显低于溶媒对照组[(198.06±40.01)s,P=0.003].停药恢复3个动情周期后,LNG-ECPs 可造成大鼠血清中 FSH[(2.48±1.18)U/L]、LH[(1.60±0.41)U/L]、睾酮[(1.37±0.23)μg/L]及FSH/LH值(1.61±0.41)显著高于溶媒对照组[(1.00±0.67)U/L,P=0.024;(1.00±0.27)U/L,P=0.014;(1.00±0.18)μg/L,P=0.011;1.00±0.49,P=0.042],且雌激素水平[(0.49±0.15)μg/L]和 AMH 水平[(0.79±0.15)μg/L]显著低于溶媒对照组[(1.00±0.37)μg/L,P=0.011;(1.00±0.10)μg/L,P=0.016],同时,大鼠卵巢中原始卵泡数量(6.25±5.06)仍显著少于溶媒对照组(12.00±5.56,P=0.048);F1代仔鼠在旷场中的运动总距离[(89.85±36.98)m]以及负重游泳时长[(112.00±29.52)s]均显著低于溶媒对照组[(147.55±23.13)m,P＜0.001;(137.69±25.85)s,P=0.014].③转录组测序结果表明,与溶媒对照组相比,LNG-ECPs组雌性大鼠卵巢组织中存在Cd5、Cxcr1、Lexm、Fga、Mybphl和Gstm5等显著差异表达的基因,参与萜类骨架生物合成、卵巢类固醇生成和皮质醇的合成与分泌等类固醇激素合成相关过程,还涉及碳代谢、丁酸甲酯代谢和半胱氨酸等物质代谢过程,以及细胞因子-细胞因子受体相互作用、病毒蛋白与细胞因子及细胞因子受体的相互作用等免疫调节过程.结论 在雌性大鼠中,短期重复(＜12个周期)给予LNG-ECPs对雌鼠生育力及其F1代仔鼠生长发育和行为学未见明显影响;而长期重复(12个周期)给予LNG-ECPs会导致卵巢功能损伤,并会对F1代仔鼠的健康产生负面影响,停药恢复3个动情周期后仍未见明显改善.
Effects of consecutively repeated application of emergency contraceptive pills containing levonorgestrel on female fertility and the health of F1 offspring
Objective To explore the effects of consecutively repeated application of emergency contraception pills(ECPs)containing levonorgestrel(LNG)on the female fertility and the health outcomes of F1 generation rats.Methods Female SPF rats were intragastric administered with LNG-ECPs consecutively for 3(P-3),6(P-6)and 12(P-12)estrous cycles(three times in each estrous cycle),respectively.Under each administration schedule,rats were randomly divided into 2 groups according to body weight stratification using random numbers generated in Excel,i.e.LNG-ECPs group and solvent control group,administered with 0.12 mg/kg LNG-ECPs and corresponding volumes of 0.5％CMC-Na,respectively.Four hours after the last dosing,half of the animals(12-18)in each group were allotted randomly for dissection(6-9)and mating(6-9),respectively.The remaining half(12-18)were recovered for 3 estrous cycles,and then were randomly allocated for dissection(6-9)and mating(6-9).Organ coefficients were calculated.Serum levels of follicle-stimulating hormone(FSH),luteinizing hormone(LH),estradiol,progesterone,testosterone,anti-Müllerian hormone(AMH)and free thyroid hormone 3(fT3)were examined by enzyme linked immunosorbent assay(ELISA).Ovarian tissues were sectioned and stained with hematoxylin and eosin(HE)for follicle counting.In addition,the pregnancy rate and litter size of the female rats were recorded,and the growth indexes and behavioral parameters of the cubs were measured.Moreover,RNA sequencing(RNA-seq)of the ovarian tissues was performed to establish the differential expression gene profile of ovarian injury induced by LNG-ECPs.Then gene ontology(GO)function and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment were analyzed.Results 1)After consecutive administration for 3 and 6 estrous cycles,LNG-ECPs showed no significant impact on the serum hormone levels and female fertility(all P＞0.05),and the growth indexes and behavioral parameters of the F1 generation(all P＞0.05).2)After consecutive administration for 12 estrous cycles,the serum levels of FSH[(0.21±0.17)U/L],LH[(0.27±0.08)U/L]and progesterone[(0.68±0.23)μg/L]in LNG-ECPs group decreased significantly compared with those in solvent control group[(1.00±0.82)U/L,P=0.043;(1.00±0.50)U/L,P=0.006;(1.00±0.20)μg/L,P=0.027],while the level of estradiol[(2.24±1.03)μg/L]and testosterone[(1.25±0.25)μg/L]increased noticeably compared with those in solvent control group[(1.00±0.35)μg/L,P=0.019;(1.00±0.07)μg/L,P=0.044].The number of primordial follicles(4.88±2.36)lost distinctly,while the number of atretic follicles(24.38±5.01)increased markedly in LNG-ECPs group compared with those in solvent control group(16.13±9.36,P=0.005;19.13±2.30,P=0.018).In addition,the weight-loaded swimming(WLS)time of the F1 generation rats from the LNG-ECPs group[(157.13±32.29)s]reduced obviously compared with those from the solvent control group[(198.06±40.01)s,P=0.003].Moreover,after recovering for 3 estrous cycles,LNG-ECPs significantly increased the levels of FSH[(2.48±1.18)U/L],LH[(1.60±0.41)U/L],testosterone[(1.37±0.23)μg/L]and the ratio of FSH/LH(1.61±0.41)compared with those in solvent control group[(1.00±0.67)U/L,P=0.024;(1.00±0.27)U/L,P=0.014;(1.00±0.18)μg/L,P=0.011;1.00±0.49,P=0.042],respectively.Additionally,the serum levels of estradiol[(0.49±0.15)μg/L]and AMH[(0.79±0.15)μg/L]were significantly lower than those in solvent control group[(1.00±0.37)μg/L,P=0.011;(1.00±0.10)μg/L,P=0.016].In addition,the number of primordial follicles in rats of LNG-ECPs group(6.25±5.06)were obviously less than that in solvent control group(12.00±5.56,P=0.048).Furthermore,the total distance in open field[(89.85±36.98)m]and the swimming time in WLS[(112.00±29.52)s]in rats treated with LNG-ECPs both decreased distinctly compared with those in solvent control group[(147.55±23.13)m,P＜0.001;(137.69±25.85)s,P=0.014].3)According to transcriptomic analysis,Cd5,Cxcr1,Lexm,Fga,Mybphl and Gstm5 were the significant differential expressed genes(DEGs)in the ovarian tissues of rats.These DEGs were involved in pathways related to steroid hormone biosynthesis,including terpenoid backbone biosynthesis,ovarian steroidogenesis,cortisol synthesis and secretion.Additionally,these genes were involved in metabolic processes,such as carbon metabolism,butanoate metabolism,cysteine and methionine metabolism.And the genes were also involved in immunoregulatory processes including cytokine-cytokine receptor interaction,viral protein interaction with cytokine and cytokine receptors.Conclusion Consecutively repeated administering LNG-ECPs to the female rats in a short-term period(＜12 cycles)did not demonstrate significant adverse effects on female fertility and the growth and development and the behaviors of their F1 generation cubs.However,long-term repeated treatment with LNG-ECPs(12 cycles)caused ovarian injury on female rats and showed negative impacts on the health of the F1 generation cubs,and no significant improvement was observed after recovering for 3 estrous cycles.
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