Supplementary Material for: Protective effect of glutathione administration on ovarian function in female rats with cyclophosphamide-induced ovarian damage

Objectives:
We investigated the potential of glutathione to protect ovarian function in rats exposed to cyclophosphamide by measuring serum anti-Mullerian hormone levels, follicle counts, and related parameters.
Design:
Forty-two adult female Sprague-Dawley rats were randomly divided into six groups and treated with various combinations of cyclophosphamide, glutathione, and sodium chloride. On day 21, the rats were anaesthetized, and their ovaries were removed for examination.
Participants/Materials, Setting, Methods:
Histopathological examination, serum anti-Mullerian hormone (AMH) concentrations, follicle counts, anti-Mullerian hormone-positive staining of follicle percentages were analyzed. Statistical analysis was performed using a one-way analysis of variance and Tukey’s test, with significance set at p<0.05.
Primary evaluations included serum anti-Mullerian hormone concentrations and ovarian follicle counts. Secondary measures encompassed histopathological examination and percentages of anti-Mullerian hormone-positive staining of follicles.
Results
Significant differences were observed in follicle counts, anti-Mullerian hormone-positive follicle parameters, and serum anti-Mullerian hormone concentrations among the six groups. Group 2 (treated with cyclophosphamide) had the lowest primordial, primary, secondary, and antral follicle counts and the highest atretic count. Group 6, treated with cyclophosphamide and 200 mg/kg glutathione, showed improved follicle counts compared to those in group 2. Reducing the glutathione dose to 100 mg/kg was ineffective.
Limitations
This was an experimental animal investigation with a comparatively modest sample size. Experimental studies should be conducted to determine the optimal dosage and duration of glutathione therapy. Information gathered from an experimental animal model may not yield precisely similar outcomes in humans; therefore, additional investigations are necessary to examine the impact of glutathione on women experiencing POI.
Conclusions
The anti-oxidative protective effect of directly administered glutathione was demonstrated for the first time. Low-dose glutathione was ineffective, whereas a high dose yielded significant ovarian protection against cyclophosphamide. Our findings provide valuable insights for supplementing clinical trials on the protective effects of glutathione against ovarian damage.

研究目的：本研究旨在通过检测血清抗缪勒管激素（anti-Mullerian hormone, AMH）水平、卵泡计数及相关参数，探究谷胱甘肽（glutathione）对环磷酰胺（cyclophosphamide）暴露大鼠卵巢功能的保护潜力。
实验设计：将42只成年雌性Sprague-Dawley（SD）大鼠随机分为6组，分别给予环磷酰胺、谷胱甘肽与氯化钠的不同组合干预。于第21天时对大鼠实施麻醉并摘除卵巢用于后续检测。
研究对象、材料、实验环境与方法：本研究对卵巢组织进行组织病理学检测，同时分析血清抗缪勒管激素（AMH）浓度、卵泡计数以及卵泡抗缪勒管激素阳性染色百分比。统计学分析采用单因素方差分析（one-way analysis of variance, ANOVA）与Tukey多重比较检验，以p<0.05作为差异具有统计学意义的判定标准。
主要评估指标包括血清AMH浓度与卵巢卵泡计数；次要评估指标涵盖组织病理学检测以及卵泡AMH阳性染色百分比。
实验结果：六组大鼠的卵泡计数、抗缪勒管激素阳性卵泡相关参数及血清AMH浓度均存在显著组间差异。第2组（仅给予环磷酰胺干预）的原始卵泡、初级卵泡、次级卵泡与窦状卵泡计数均为各组最低，闭锁卵泡计数则为各组最高。第6组（给予环磷酰胺联合200 mg/kg谷胱甘肽干预）的各类型卵泡计数均较第2组显著改善；但将谷胱甘肽剂量降至100 mg/kg时则未产生保护效果。
研究局限性：本研究为动物实验，样本量相对较小。未来需开展进一步实验以确定谷胱甘肽治疗的最优剂量与干预时长。由于动物实验模型的研究结果未必完全适用于人类，因此需开展更多研究以探究谷胱甘肽对早发性卵巢功能不全（Primary Ovarian Insufficiency, POI）女性患者的影响。
研究结论：本研究首次证实直接给药的谷胱甘肽具有抗氧化保护作用。低剂量谷胱甘肽未产生卵巢保护效果，而高剂量谷胱甘肽可显著对抗环磷酰胺诱导的卵巢损伤。本研究结果为后续开展谷胱甘肽对卵巢损伤保护作用的临床试验提供了重要参考依据。