衰老影响人类卵母细胞DNA修复反应、代谢活性和精子DNA损伤：阐明配子质量与生殖结果的相互联系

IF 6 1区医学 Q1 OBSTETRICS & GYNECOLOGY

Human reproduction Pub Date : 2025-06-28 DOI:10.1093/humrep/deaf097.608

F Horta, H Newman, S Mercer, M Di Battista, J Mullen, B Vollenhoven, S Catt, R Gilchrist

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引用次数: 0

摘要

配子生物标志物如卵母细胞DNA修复反应、卵母细胞代谢活性和精子DNA损伤与生殖衰老和抗逆转录病毒治疗结果有关吗？随着年龄的增长，人类卵母细胞的DNA损伤反应和代谢活动发生了变化，而精子DNA损伤的增加与年龄的增长有关。已经知道的是，母亲年龄的增加会显著影响生育能力、生殖成功率、后代健康，并与卵母细胞质量的下降密切相关。尽管决定因素仍然难以捉摸，但DNA损伤修复能力和代谢活动正在成为支持与年龄相关的卵母细胞质量恶化的关键因素。必需代谢辅助因子烟酰胺腺嘌呤二核苷酸磷酸，[NAD(P)H]和黄素腺嘌呤二核苷酸（FAD）与体细胞组织和未成熟小鼠卵母细胞的衰老呈负相关。代谢与DNA修复蛋白的表达、激活或抑制有关，DNA修复蛋白是修复卵母细胞和精子DNA损伤的关键。研究设计、规模、持续时间进行了为期1年的前瞻性队列研究。对ART周期（n = 32）中GV（生发囊）和MI（中期I）期的卵母细胞（n = 63）进行卵母细胞DNA修复/反应生物标志物评估，如磷酸化ATM （pATM）、YH2AX和非侵入性代谢活性辅助因子NAD(P)H和FAD。对每个伴侣都进行了精子DNA损伤测试。考虑到年龄是主要的共同影响因素，对女性和男性的生物标志物进行了分析，包括ART生殖结果。参与者/材料、环境、方法参与者是在墨尔本诺丁山城市生育中心出现原发性或继发性不孕症的夫妇。使用免疫细胞化学方法分析不同年龄患者的卵母细胞的pATM和H2AX，包括使用共聚焦显微镜（Olympus FV1200）在GV期或MI期测量的NAD(P)H和FAD水平。图像分析使用斐济软件（v2.0.0-rc-69/1.52n），使用任意单位。采用线性混合模型来确定DNA修复和代谢生物标志物与年龄的关系。主要结果和作用是GV卵母细胞表现出对H2AX和pATM的核表达，而MI卵母细胞表现出对H2AX的核表达，而pATM在gvbd（生发囊泡破裂）后表现出细胞质定位。代谢辅助因子的研究表明，NAD(P)H和FAD在细胞质中表达，在线粒体中表达量较高，尤其是FAD。未成熟人卵母细胞中年龄和分子标记物之间的关联检测显示，年龄和α - H2AX之间没有相关性（n = 49）。而心肌梗死卵母细胞中，年龄与pATM呈显著正相关（p = 0.010）。在代谢辅助因子的背景下，光氧化还原比[ORR: NAD(P)H/ NAD(P)H + FAD]在MI卵母细胞中呈显著负相关（P = 0.020），而在GV卵母细胞中无显著负相关(P = 0.484；N = 50)。采用Pearson检验分析DNA损伤反应标志物、H2AX、DNA修复蛋白、pATM、代谢辅助因子NAD(P)H、FAD和ORR的相关性。pATM、NAD(P)H、ORR与年龄的关系均有统计学意义（n = 32、0.496、P = 0.002、0.470、P = 0.003、-0.455、P = 0.004）。使用TUNEL法评估精子DNA损伤与年龄之间的显著正相关(n = 31， Pearson相关性0.302,p <；0.05)。本研究为进一步研究无创检测卵母细胞代谢活性提供了有价值的基础。该研究使用ART周期中剩余的人类卵母细胞进行，这些卵母细胞不一定代表成熟卵母细胞。此外，体外培养条件也会影响未成熟卵母细胞的结果。老年妇女代谢特征改变与较差生殖结果之间的联系应进一步调查。随着年龄的增长，DNA损伤反应的基础水平没有变化，这表明尽管衰老的卵母细胞可能会积累DNA损伤，但它们不能有效地激活DNA损伤反应标志物。因此，卵母细胞DNA修复能力可能随着年龄的增长而受到影响，从而影响卵母细胞的代谢活性。试验注册号

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P-300 Aging impacts human oocyte DNA repair response, metabolic activity and sperm DNA damage: elucidating gamete quality interlink on reproductive outcomes

Study question Are gamete biomarkers such as oocyte DNA repair response, oocyte metabolic activity and sperm DNA damage linked to reproductive aging and ART outcomes? Summary answer Human oocytes showed an altered DNA damage response and metabolic activity with aging, while increased sperm DNA damage was associated with aging. What is known already Advancing maternal age significantly impacts fertility, reproductive success, offspring health, and is closely associated with a decline in oocyte quality. Although the determinants remain elusive, DNA damage repair capacity and metabolic activity are emerging as crucial factors underpinning the age-associated deterioration in oocyte quality. The essential metabolic cofactors nicotinamide adenine dinucleotide phosphate, [NAD(P)H] and flavin adenine dinucleotide (FAD) have been negatively associated with aging in somatic tissue and immature mouse oocytes. Metabolism has been linked to expression, activation or inhibition of DNA repair proteins, which is key to repair DNA damage from both oocytes and spermatozoa. Study design, size, duration A 1-year prospective cohort study was conducted. Oocytes (n = 63) at the GV (germinal vesicle) and MI (metaphase I) stage from ART cycles (n = 32) underwent assessment for oocyte DNA repair/response biomarkers such as phosphorylated ATM (pATM), YH2AX and non-invasive metabolic activity cofactors NAD(P)H and FAD. Testing for sperm DNA damage was conducted on every partner. Analysis female and male biomarkers, including ART reproductive outcomes was conducted considering age as main cofounder factor. Participants/materials, setting, methods Participants were couples presenting with either primary or secondary infertility at City Fertility, Notting Hill, Melbourne. Oocytes from patients of different age were analysed using immunocytochemistry for pATM and ɣH2AX, including NAD(P)H and FAD levels measured at the GV or MI phase using a confocal microcopy (Olympus FV1200). Images were analysed using FIJI software (v2.0.0-rc-69/1.52n), using arbitrary units. Linear mixed models were employed to determine the relationship between DNA repair and metabolic biomarkers with age. Main results and the role of chance GV oocytes exhibited nuclear expression for ɣH2AX and pATM, whereas MI oocytes displayed nuclear expression for ɣH2AX, with pATM showing a cytosolic localization post-GVBD (Germinal Vesicle Breakdown). Investigation of metabolic cofactors revealed that NAD(P)H and FAD were expressed in the cytosol, with higher expression in mitochondria, particularly for FAD. Examination of the association between age and molecular markers in immature human oocytes showed no correlation between age and ɣH2AX (n = 49). However, a significant positive relationship between age and pATM was observed in MI oocytes (p = 0.010). In the context of metabolic cofactors, the optical redox ratio [ORR: NAD(P)H/ NAD(P)H + FAD] had a significant negative relationship in MI oocytes (p = 0.020), but no in GV oocytes (p = 0.484; n = 50). DNA damage response marker, ɣH2AX, DNA repair protein, pATM, metabolic cofactors NAD(P)H and FAD and ORR were also analysed for correlation using Pearson tests. pATM, NAD(P)H and ORR had statistically significant relationships with age (n = 32, 0.496, p = 0.002, 0.470, p = 0.003 and -0.455, p = 0.004 respectively). A significant positive correlation between sperm DNA damage and age was observed when assessed using the TUNEL assay (n = 31, Pearson correlation 0.302, p < 0.05). This study offers a valuable foundation for further investigation into non-invasive tests to assess oocyte metabolic activity. Limitations, reasons for caution The study was conducted using left-over human oocytes from ART cycles, which do not necessarily represent mature oocytes. Additionally, in vitro culture conditions could also influence results of immature oocytes. The link between altered metabolic signatures and poorer reproductive outcomes in older women should be further investigated. Wider implications of the findings There were no changes in basal levels of the DNA damage response with age, indicating that while aged oocytes may accumulate DNA damage, they do not effectively activate the DNA damage response markers. Thus, oocyte DNA repair capacity maybe impacted with age, affecting oocyte metabolic activity. Trial registration number No

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来源期刊

Human reproduction 医学-妇产科学

CiteScore

10.90

自引率

6.60%

发文量

1369

审稿时长

1 months

期刊介绍： Human Reproduction features full-length, peer-reviewed papers reporting original research, concise clinical case reports, as well as opinions and debates on topical issues. Papers published cover the clinical science and medical aspects of reproductive physiology, pathology and endocrinology; including andrology, gonad function, gametogenesis, fertilization, embryo development, implantation, early pregnancy, genetics, genetic diagnosis, oncology, infectious disease, surgery, contraception, infertility treatment, psychology, ethics and social issues.