Effects of consecutively repeated application of emergency contraceptive pills containing levonorgestrel on female fertility and the health of F1 offspring
10.3760/cma.j.cn101441-20230711-00272
- VernacularTitle:连续重复给予含左炔诺孕酮紧急避孕药对雌性大鼠生育力及其子代健康的影响
- Author:
Yingyi HU
1
;
Qicheng NI
;
Ruihua ZHONG
;
Wenjie YANG
;
Guoting LI
;
Jieyun ZHOU
;
Xiangjie GUO
;
Shuwu XIE
;
Jialing ZHOU
;
Yan ZHU
Author Information
1. 复旦大学药学院，上海 200032
- Publication Type:Journal Article
- Keywords:
Levonorgestrel;
Fertility;
Ethology;
Ovarian injury;
Transcriptomics
- From:
Chinese Journal of Reproduction and Contraception
2024;44(6):562-573
- CountryChina
- Language:Chinese
-
Abstract:
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.
