研究背景: 

多囊卵巢综合征（Polycystic Ovary Syndrome，PCOS）是育龄女性高发的生殖内分泌和代谢紊乱性疾病，全球发病率约11%~13%。动物实验证明，PCOS小鼠的生殖内分泌和代谢异常能跨代遗传至子二代（Second Filial Generation，F2）雌鼠，孕前干预是改善子代代谢健康的有效方法。临床实践证明，二甲双胍治疗肥胖PCOS患者可降低其高胰岛素血症、高雄激素血症，并恢复规律的月经周期，能够改善PCOS患者孕前代谢水平和激素水平。

研究目的：

探究二甲双胍孕前干预阻断PCOS小鼠通过卵母细胞跨代遗传给子代的生殖内分泌和代谢异常的作用及相关分子机制。

研究方法：

本研究采用脱氢表雄酮（Dehydroepiandrosterone，DHEA）诱导青春期小鼠的PCOS模型，探究二甲双胍治疗对PCOS小鼠的生殖与代谢表型的影响。生殖表型包括肛门生殖器距离（Anogenital Distance，AGD）、卵巢病理结构、血清睾酮水平、黄体生成素（Luteinizing Hormone，LH）水平、卵泡刺激素（Follicle Stimulating Hormone，FSH）水平、动情周期。代谢表型包括体重、空腹血糖、葡萄糖和胰岛素耐受能力、脂肪细胞大小、血脂水平、肝脏脂质含量、整体代谢水平。为了进一步探索二甲双胍治疗对PCOS小鼠卵母细胞基因表达谱的影响，我们收集生发泡（Germinal Vesicle，GV）期卵母细胞进行单细胞转录组测序，构建差异基因表达谱。利用透射电子显微镜观察卵巢颗粒细胞中内质网形态等亚细胞器特征性改变。

阴道涂片结果显示DHEA组小鼠持续处于动情期，二甲双胍治疗后PCOS小鼠的部分生殖和代谢异常得到纠正即可认为造模成功。为了排除亲代（Parental Generation，F0）高雄激素及宫内微环境对子代的影响，我们对F0小鼠实施超排卵、体外受精（In Vitro Fertilization，IVF），将胚胎移植入生理状态正常的受体母鼠完成妊娠，分娩得到子一代（First Filial Generation，F1）小鼠，并按照同样的方法得到F2雌鼠。

观察PCOS小鼠的F1和F2雌鼠的生殖表型和代谢表型的改变，以及F0小鼠孕前使用二甲双胍对其F1和F2雌鼠的生殖表型和代谢表型的影响。接着，我们收集F1和F2雌鼠的GV期卵母细胞进行单细胞转录组测序，以探索卵母细胞基因表达谱的变化以及二甲双胍治疗F0小鼠对其F1和F2雌鼠卵母细胞基因表达的影响。我们明确了PCOS组F0小鼠和F2雌鼠呈现同向调控的差异表达基因（Differentially Expressed Genes，DEGs），并对这些跨代改变的DEGs进行富集分析。接着，我们探索了PCOS小鼠卵母细胞跨代改变的DEGs在F0小鼠和F2雌鼠各个组GV期卵母细胞的基因表达情况，并明确二甲双胍治疗部分逆转mRNA表达变化趋势的跨代DEGs。

我们采集了PCOS患者和健康受试者的外周血单核细胞（Peripheral Blood Mononuclear Cells，PBMCs）进行转录组测序，并与前期获得的PCOS小鼠卵母细胞跨代改变的DEGs数据进行联合分析，鉴定在人类PBMCs和小鼠卵母细胞同向调控的关键DEGs。我们进一步分析了公共数据库中PCOS患者卵母细胞或PBMCs的DEGs与本研究中PCOS小鼠卵母细胞的跨代改变DEGs的重叠基因，并在F2雌鼠卵巢组织中验证其mRNA表达水平，从而筛选出在PCOS跨代遗传中具有保守性的关键调控分子，为揭示PCOS跨代遗传的分子机制提供证据。

我们明确了PCOS小鼠的F1雄鼠的生殖表型（如睾丸病理结构、血清睾酮水平、睾丸系数和附睾系数）和代谢表型（如体重、空腹血糖、葡萄糖耐受能力、胰岛素耐受能力、脂肪细胞大小、血脂水平和整体代谢水平）的改变，以及F0小鼠孕前使用二甲双胍对F1雄鼠的生殖表型和代谢表型的影响，并收集F1雄鼠的睾丸组织进行转录组测序。

研究结果：

1. 本研究成功构建DHEA诱导的PCOS小鼠模型，该模型呈现典型的代谢和生殖内分泌紊乱特征。代谢方面表现为体重增加；葡萄糖耐受能力下降和胰岛素抵抗；脂肪细胞面积增大；血清和肝脏的甘油三酯含量增加；夜间呼吸交换率升高，摄食量增加。生殖内分泌方面表现为AGD延长；血清睾酮水平及LH/FSH比值显著升高伴随FSH水平降低；持续动情期状态；以及多囊卵巢样病理特征。二甲双胍干预可显著改善PCOS小鼠代谢紊乱，表现为体重降低；空腹血糖下降，胰岛素敏感性增强；脂肪细胞面积缩小；血清甘油三酯、低密度脂蛋白胆固醇、及肝脏甘油三酯、游离脂肪酸含量下降；夜间呼吸交换率与摄食量同步下降。在生殖内分泌方面，该治疗显著缩短AGD，降低LH/FSH比值并提升血清FSH水平，但对PCOS小鼠的血清睾酮水平、动情周期、卵巢病理结构未见显著的改善。

2. F0小鼠GV期卵母细胞的单细胞测序结果的富集分析表明二甲双胍可能调控自噬和内质网应激相关通路。同时，卵巢颗粒细胞透射电镜观察显示，二甲双胍干预可显著缓解DHEA诱导的细胞器病理改变，包括自噬体异常积累和内质网结构紊乱。这些结果共同提示，二甲双胍可能通过调节卵母细胞和颗粒细胞的自噬及内质网应激反应，从而发挥对PCOS生殖和代谢异常的改善作用。

3. PCOS小鼠的F1雌鼠仅表现为AGD增加、血清睾酮水平降低、动情前期比例下降，而未出现其他代谢紊乱。然而，F2雌鼠出现空腹血糖升高、血清甘油三酯水平升高等代谢异常和血清睾酮水平升高、LH水平和LH/FSH比值升高、FSH水平降低，AGD延长等生殖内分泌紊乱。二甲双胍治疗PCOS组F0小鼠可显著改善其子代的表型，F1雌鼠的6周龄体重降低、AGD缩短、脂肪细胞面积减少，血清睾酮水平升高。此外，F2雌鼠的6周龄体重降低、空腹血糖降低、血清甘油三酯和低密度脂蛋白胆固醇水平减少、脂肪细胞面积缩小、AGD缩短、血清LH水平和LH/FSH比值降低，FSH水平升高。

4. 单细胞转录组分析表明，DHEA组和对照组F2雌鼠GV卵母细胞的DEGs主要富集于自噬调控和内质网应激相关通路；DHEA+二甲双胍组和DHEA组F2雌鼠GV卵母细胞的DEGs富集于线粒体呼吸、内质网应激和自噬调控等生物学过程。

5. 在F0和F2雌鼠中，DHEA组和对照组的GV期卵母细胞的基因表达差异存在跨代改变：F0和F2雌鼠中共鉴定出11个共同上调的DEGs和28个共同下调的DEGs。这些跨代改变的DEGs主要富集于内质网应激相关通路，提示内质网应激可能在PCOS通过卵母细胞跨代遗传的过程中发挥重要作用。

6. 转录组分析显示，PCOS患者和健康受试者的PBMCs的DEGs主要富集于对活性氧和氧化应激的反应、线粒体释放细胞色素C、脂质代谢、类固醇激素生物合成等生物学过程。PCOS患者PBMCs的DEGs和PCOS小鼠GV期卵母细胞跨代改变的DEGs有一个共同下调的基因DUSP5。通过比较PCOS小鼠GV期卵母细胞的跨代DEGs与公共数据库中PCOS患者卵母细胞或PBMCs的DEGs，我们鉴定出四个同向改变的保守基因(Fam110a、Clcn3、PIAS4和TBCEL)。

7. PCOS小鼠的F1雄鼠也出现PCOS样的生殖内分泌和代谢紊乱，具体表现为体重增加、空腹血糖升高、葡萄糖耐受量下降、脂肪细胞面积增加、夜间呼吸交换率和夜间摄食量增加、血清甘油三酯水平和睾酮水平升高。二甲双胍治疗PCOS小鼠后，F1雄鼠的体重、空腹血糖、脂肪细胞面积、血清甘油三酯和低密度脂蛋白胆固醇水平、脂肪细胞面积、夜间呼吸交换率和夜间摄食量、血清睾酮水平、葡萄糖耐受量均得到显著改善，表明二甲双胍治疗PCOS小鼠对其F1雄鼠的生殖内分泌和代谢异常具有显著的改善作用。

研究结论：

DHEA诱导的PCOS小鼠可模拟PCOS患者的代谢紊乱和生殖内分泌异常，而二甲双胍可以有效改善PCOS小鼠的部分生殖内分泌和代谢紊乱。PCOS小鼠的生殖内分泌和代谢异常可以独立通过卵母细胞部分遗传给F1~F2雌鼠和F1雄鼠。二甲双胍孕前干预不仅可以改善PCOS小鼠的生殖内分泌和代谢异常，还可以缓解F2雌鼠和F1雄鼠的异常表型。分子机制研究表明，在F0和F2雌鼠中，DHEA组和对照组的GV期卵母细胞的基因表达差异存在跨代改变，共鉴定出11个共同上调和28个共同下调的DEGs，这些基因富集于内质网应激通路，提示内质网应激可能在PCOS通过卵母细胞跨代遗传的过程中发挥重要作用。PCOS小鼠卵母细胞的跨代改变的部分DEGs在PCOS患者PBMCs或卵母细胞转录组数据中显示出一致的mRNA表达变化趋势。这些基因可能在PCOS的跨代遗传中起重要作用，可以作为PCOS患者将生殖和代谢异常表型跨代遗传给子代的潜在风险标志物。未来可以针对这些关键基因开发靶向干预策略，通过孕前干预阻断PCOS跨代遗传，为临床防治提供新思路。

Background

Polycystic ovary syndrome (PCOS) is a highly prevalent reproductive endocrine and metabolic disorder among women of reproductive age, with a global prevalence rate of approximately 11%-13%. Animal experiments have demonstrated that the reproductive endocrine and metabolic abnormalities observed in PCOS mice can be inherited transgenerationally, affecting the second filial generation (F2) of female mice. Preconception intervention is an effective method for improving the metabolic health of offspring. Clinical evidence shows that metformin treatment in obese PCOS patients can reduce hyperinsulinemia and hyperandrogenemia, restore regular menstrual cycles, and improve pre-pregnancy metabolic and hormonal levels in PCOS patients.

Objective

This study aims to investigate the role of metformin preconception intervention in blocking the oocyte-mediated transgenerational inheritance of reproductive endocrine and metabolic abnormalities from PCOS mice to offspring, and to elucidate the underlying molecular mechanisms.

Methods

This study employed a dehydroepiandrosterone (DHEA)-induced PCOS mouse model during the pubertal phase to investigate the therapeutic effects of metformin intervention on both reproductive and metabolic abnormalities of PCOS. Reproductive outcomes included anogenital distance (AGD), ovarian pathological structure, serum testosterone levels, luteinizing hormone (LH) levels, follicle stimulating hormone (FSH) levels, and estrous cycle. Concurrently, metabolic parameters involved body weight, fasting blood glucose, glucose tolerance, insulin tolerance, adipocyte size, serum lipid levels, liver lipid content, and overall metabolic status. Additionally, germinal vesicle (GV) stage oocytes were collected for single-cell RNA sequencing to further explore the impact of metformin treatment on the gene expression profile of oocytes in PCOS mice. Transmission electron microscopy (TEM) was employed to observe characteristic alterations in subcellular organelles, including endoplasmic reticulum morphology, in ovarian granulosa cells.

Vaginal cytology analysis revealed persistent estrus phase maintenance in DHEA-induced PCOS mice. The successful establishment of the disease model was confirmed by the partial restoration of reproductive parameters and metabolic indices following metformin intervention. To eliminate the influence of parental generation (F0) hyperandrogenism and the intrauterine microenvironment on the offspring, we performed superovulation and in vitro fertilization (IVF) on F0 mice. The resulting embryos were transferred into recipient female mice with normal physiological status to achieve pregnancy, yielding first filial generation (F1) mice. The F2 female mice were obtained by following the same method.

This study observed alterations in reproductive and metabolic phenotypes in F1 and F2 female offspring of PCOS mice, as well as the impact of preconceptional metformin administration in F0 mice on these phenotypes in their F1 and F2 female offspring. Subsequently, we collected GV-stage oocytes from F1 and F2 female offspring for single-cell RNA sequencing to investigate the alterations in oocyte gene expression profiles. Additionally, we examined the effects of metformin treatment in F0 mice on the gene expression of oocytes in their F1 and F2 offspring. We identified co-directionally regulated differentially expressed genes (DEGs) in F0 mice and F2 female offspring of the PCOS group, followed by enrichment analysis on these transgenerational DEGs. Next, we explored the gene expression of transgenerational DEGs in all groups of F0 and F2 female mice, and identified the transgenerational DEGs whose mRNA expression changes could be partially reversed by metformin treatment.

We performed transcriptome sequencing of peripheral blood mononuclear cells (PBMCs) from PCOS patients and healthy controls. These data were integrated with previously obtained transgenerational DEGs data from PCOS mouse oocytes through joint analysis, identifying key co-directionally regulated DEGs in both human PBMCs and mouse oocytes. We further analyzed the overlapping genes between DEGs from either oocytes or PBMCs of PCOS patients in public databases and the transgenerational DEGs in PCOS mouse oocytes from our study. After identifying conserved DEGs, we quantitatively validated their mRNA expression patterns in F2 female ovarian tissues. This approach enabled screening of key regulatory molecules exhibiting conserved characteristics in PCOS transgenerational inheritance, thereby providing mechanistic evidence for PCOS transmission across generations.

We identified alterations in reproductive phenotypes (testicular histopathology, serum testosterone levels, testicular coefficient, and epididymal coefficient) and metabolic phenotypes (body weight, fasting blood glucose, glucose tolerance, insulin tolerance, adipocyte size, blood lipid profiles, and overall metabolic status) in F1 male offspring of PCOS mice, and investigated the effects of preconception metformin administration in parental PCOS mice on these reproductive and metabolic phenotypes in F1 male offspring. We also collected the testicular tissues of F1 male offspring for transcriptome sequencing.

Results

1. This study successfully established a DHEA-induced PCOS mouse model that exhibited both metabolic and reproductive endocrine abnormalities. Metabolic manifestations included increased body weight, impaired glucose tolerance and insulin sensitivity, enlarged adipocyte size, elevated serum and hepatic triglyceride levels, along with increased nocturnal respiratory exchange ratio (RER) and heightened nighttime food intake. Reproductive endocrine abnormalities manifested as prolonged AGD, significantly elevated serum testosterone levels and LH/FSH ratio accompanied by decreased FSH levels, persistent estrus phase, and polycystic ovary-like pathological features. Metformin intervention significantly ameliorated metabolic disturbances in PCOS mice, manifested as reduced body weight; decreased fasting blood glucose and improved insulin sensitivity; diminished adipocyte size; lowered serum triglycerides and low-density lipoprotein cholesterol (LDL-C) levels, along with reduced hepatic triglyceride and nonestesterified fatty acid content; and concomitant decreases in nocturnal RER and food intake. Regarding reproductive endocrine outcomes, the treatment notably shortened AGD, lowered LH/FSH ratio, and elevated serum FSH levels. However, it failed to significantly improve serum testosterone levels, estrous cycle regularity, or ovarian histopathological features in PCOS mice.

2. The enrichment analysis of single-cell RNA sequencing data from F0 mice GV stage oocytes demonstrated that metformin may regulate pathways associated with autophagy and endoplasmic reticulum stress. TEM observation of ovarian granulosa cells revealed that metformin intervention significantly ameliorated DHEA-induced pathological alterations in organelles, including abnormal autophagosome accumulation and endoplasmic reticulum structural disorganization. These results collectively suggested that metformin may ameliorate reproductive and metabolic dysfunction in PCOS by modulating autophagy and endoplasmic reticulum stress responses in both oocytes and granulosa cells.

3. F1 female offspring derived from PCOS mice had no other PCOS-like metabolic disorders except increased AGD, significantly reduced serum testosterone level, and decreased proestrus phase proportion. However, F2 female mice of PCOS mice manifested metabolic disturbances including elevated fasting blood glucose and heightened serum triglycerides levels. F2 female mice of PCOS mice also showed increased serum testosterone levels, elevated LH concentrations and LH/FSH ratio, reduced FSH levels, increased AGD and other reproductive endocrine disorders. Preconception metformin administration in PCOS mice significantly improved offspring phenotypes: F1 female mice exhibited reduced 6-week body weight, shortened AGD, decreased adipocyte area, and elevated serum testosterone levels. Additionally, F2 females demonstrated decreased 6-week body weight, reduced fasting blood glucose, lowered serum triglycerides and LDL-C levels, diminished adipocyte area, shortened AGD, along with reduced serum LH levels and LH/FSH ratio, but increased FSH levels.

4. Single-cell RNA sequencing analysis revealed that in F2 female mice oocytes, DEGs from the DHEA group versus control group were enriched in the autophagy regulation and endoplasmic reticulum stress-related pathways, whereas DEGs from DHEA+metformin group and DHEA groups were enriched in biological processes including mitochondrial respiration, endoplasmic reticulum stress, and autophagy regulation.

5. In F0 and F2 female mice, there were transgenerational changes in gene expression in GV stage oocytes between the DHEA group and the control group. Specifically, there were 11 co-upregulated DEGs and 28 co-downregulated DEGs in F0 and F2 female mice. These transgenerational DEGs were enriched in pathways related to the endoplasmic reticulum stress response, suggesting that endoplasmic reticulum stress may play a crucial role in the transgenerational inheritance of PCOS via oocytes.

6. Transcriptome analysis revealed that DEGs in PBMCs of PCOS patients and healthy controls were enriched in biological processes including response to reactive oxygen species and oxidative stress, mitochondrial cytochrome c release, lipid metabolism, and steroid hormone biosynthesis. The DEGs in PBMCs of PCOS patients and the transgenerational DEGs in GV-stage oocytes of PCOS mice share one commonly downregulated gene DUSP5. Comparative analysis between transgenerational DEGs in PCOS mice GV-stage oocytes and DEGs from either oocytes or PBMCs of PCOS patients in public datasets identified four evolutionarily conserved genes (FAM110A, CLCN3, PIAS4, and TBCEL) exhibiting concordant expression alterations.

7. F1 male mice of PCOS mice also showed PCOS-like reproductive endocrine and metabolic disorders, which were manifested as increased body weight, elevated fasting blood glucose, impaired glucose tolerance, adipocyte hypertrophy, heightened nocturnal RER and food intake, along with elevated serum triglycerides and testosterone levels. After preconception metformin administration in PCOS mice, these F1 male mice demonstrated significant reductions in body weight, fasting glucose, adipocyte area, serum triglyceride and LDL-C levels, nocturnal RER and food intake, and serum testosterone levels, accompanied by restored glucose tolerance. These findings indicated that metformin preconception intervention in PCOS mice can significantly improve the metabolic abnormalities and serum testosterone levels of their F1 male offspring.

Conclusions

DHEA-induced PCOS mouse can simulate the metabolic disorders and reproductive endocrine abnormalities in PCOS patients, and metformin can significantly improve some reproductive endocrine and metabolic disorders in PCOS mice. Some of the reproductive endocrine and metabolic abnormalities observed in PCOS mice can be independently transmitted to both F1-F2 female offspring and F1 male offspring through oocytes. Preconception intervention with metformin not only improves some of these reproductive endocrine and metabolic abnormalities in PCOS mice but also ameliorates similar abnormalities in F2 female mice and F1 male mice. Mechanistic studies revealed that in F0 and F2 female mice, the gene expression differences in GV stage oocytes between the DHEA group and control group exhibited transgenerational changes. Specifically, there were 11 co-upregulated and 28 co-downregulated DEGs between F0 and F2 female mice. These transgenerational DEGs were enriched in pathways related to endoplasmic reticulum stress, suggesting that endoplasmic reticulum stress may play a crucial role in the transgenerational inheritance of PCOS through oocytes. A subset of transgenerational DEGs in PCOS mouse oocytes demonstrated consistent mRNA expression trends in transcriptomic data from either PBMCs or oocytes of PCOS patients. These genes may play an important role in the transgenerational inheritance of PCOS and could serve as potential risk markers for PCOS patients to transmit their abnormal reproductive and metabolic phenotypes to their offspring across generations. In the future, targeted intervention strategies against these key genes could be developed for preconception intervention in PCOS patients, thereby blocking the transgenerational inheritance of PCOS-like phenotypes, providing novel clinical avenues for PCOS prevention and management.