Education Program in Reproduction & Development, Department of Obstetrics and Gynecology, Monash University, Melbourne, VIC 3168, Australia.
Peter MacCallum Cancer Centre, Monash Health, Melbourne, VIC 3164, Australia.
Hum Reprod. 2020 Mar 27;35(3):529-544. doi: 10.1093/humrep/dez308.
Does female ageing have a negative effect on the DNA repair capacity of oocytes fertilised by spermatozoa with controlled levels of DNA damage?
Compared to oocytes from younger females, oocytes from older females have a reduced capacity to repair damaged DNA introduced by spermatozoa.
The reproductive lifespan in women declines with age predominantly due to poor oocyte quality. This leads to decreased reproductive outcomes for older women undergoing assisted reproductive technology (ART) treatments, compared to young women. Ageing and oocyte quality have been clearly associated with aneuploidy, but the range of factors that influence this change in oocyte quality with age remains unclear. The DNA repair activity prior to embryonic genomic activation is considered to be of maternal origin, with maternal transcripts and proteins controlling DNA integrity. With increasing maternal age, the number of mRNAs stored in oocytes decreases. This could result in diminished efficiency of DNA repair and/or negative effects on embryo development, especially in the presence of DNA damage.
STUDY DESIGN, SIZE, DURATION: Oocytes from two age groups of 30 super-ovulated female mice (young: 5-8 weeks old, n = 15; old: 42-45 weeks old, n = 15) were inseminated with sperm from five males with three different controlled DNA damage levels; control: ≤10%, 1 Gray (Gy): 11-30%, and 30 Gy: >30%. Inseminated oocytes (young: 125, old: 78) were assessed for the formation of zygotes (per oocyte) and blastocysts (per zygote). Five replicates of five germinal vesicles (GVs) and five MII oocytes from each age group were analysed for gene expression. The DNA damage response (DDR) was assessed in a minimum of three IVF replicates in control and 1 Gy zygotes and two-cell embryos using γH2AX labelling.
PARTICIPANTS/MATERIALS, SETTING, METHODS: Swim-up sperm samples from the cauda epididymidis of C57BL6 mice were divided into control (no irradiation) and 1- and 30-Gy groups. Treated spermatozoa were irradiated at 1 and 30 Gy, respectively, using a linear accelerator Varian 21iX. Following irradiation, samples were used for DNA damage assessment (Halomax) and for insemination. Presumed zygotes were cultured in a time-lapse incubator (MIRI, ESCO). Gene expression of 91 DNA repair genes was assessed using the Fluidigm Biomark HD system. The DNA damage response in zygotes (6-8 h post-fertilisation) and two-cell embryos (22-24 h post-fertilisation) was assessed by immunocytochemical analysis of γH2AX using confocal microscopy (Olympus FV1200) and 3D volumetric analysis using IMARIS software.
The average sperm DNA damage for the three groups was statistically different (control: 6.1%, 1 Gy: 16.1%, 30 Gy: 53.1%, P < 0.0001), but there were no significant differences in fertilisation rates after IVF within or between the two age groups [(young; control: 86.79%, 1 Gy: 82.75%, 30 Gy: 76.74%) (old; control: 93.1%, 1 Gy: 70.37%, 30 Gy: 68.18%) Fisher's exact]. However, blastocyst rates were significantly different (P < 0.0001) among the groups [(young; control: 86.95%, 1 Gy: 33.33%, 30 Gy: 0.0%) (old; control: 70.37%, 1 Gy: 0.0%, 30 Gy: 0.0%)]. Between the age groups, 1-Gy samples showed a significant decrease in the blastocyst rate in old females compared to young females (P = 0.0166). Gene expression analysis revealed a decrease in relative expression of 21 DNA repair genes in old GV oocytes compared to young GV oocytes (P < 0.05), and similarly, old MII oocytes showed 23 genes with reduced expression compared to young MII oocytes (P < 0.05). The number of genes with decreased expression in older GV and MII oocytes significantly affected pathways such as double strand break (GV: 5; MII: 6), nucleotide excision repair (GV: 8; MII: 5) and DNA damage response (GV: 4; MII: 8). There was a decreased DDR in zygotes and in two-cell embryos from old females compared to young regardless of sperm treatment (P < 0.05). The decrease in DNA repair gene expression of oocytes and decreased DDR in embryos derived from older females suggests that ageing results in a diminished DNA repair capacity.
LARGE-SCALE DATA: N/A.
LIMITATIONS, REASONS FOR CAUTION: Ionising radiation was used only for experimental purposes, aiming at controlled levels of sperm DNA damage; however, it can also damage spermatozoa proteins. The female age groups selected in mice were intended to model effects in young and old women, but clinical studies are required to demonstrate a similar effect.
Fertilisation can occur with sperm populations with medium and high DNA damage, but subsequent embryo growth is affected to a greater extent with aging females, supporting the theory that oocyte DNA repair capacity decreases with age. Assessment of the oocyte DNA repair capacity may be a useful diagnostic tool for infertile couples.
STUDY FUNDING/COMPETING INTEREST(S): Funded by the Education Program in Reproduction and Development, Department of Obstetrics and Gynaecology, Monash University. None of the authors has any conflict of interest to report.
女性衰老是否会对经精子处理后具有受控水平 DNA 损伤的卵母细胞的 DNA 修复能力产生负面影响？
与年轻女性的卵母细胞相比，老年女性的卵母细胞修复由精子引入的受损 DNA 的能力降低。
女性的生殖寿命随着年龄的增长而下降，主要是由于卵母细胞质量差。这导致接受辅助生殖技术 (ART) 治疗的老年女性的生殖结果比年轻女性差。衰老和卵母细胞质量与非整倍体明显相关，但影响卵母细胞随年龄变化的这种质量变化的因素范围尚不清楚。胚胎基因组激活前的 DNA 修复活性被认为来自母体，母体转录物和蛋白质控制 DNA 完整性。随着母体年龄的增长，卵母细胞中储存的 mRNA 数量减少。这可能导致 DNA 修复效率降低，或对胚胎发育产生负面影响，尤其是在存在 DNA 损伤的情况下。
研究设计、规模、持续时间：从五只雄性的精子中分别选取精子样本，对 30 只超排卵雌性老鼠的两个年龄组（年轻：5-8 周龄，n=15；年老：42-45 周龄，n=15）的卵母细胞进行受精。精子样本分别接受三种不同的 DNA 损伤处理，即对照（≤10%）、1 戈瑞（Gy）（11-30%）和 30 Gy（>30%）。评估受精卵母细胞（年轻：125，年老：78）的形成情况受精卵（每个卵母细胞）和囊胚（每个受精卵）。分析每个年龄组的五个生殖泡（GV）和五个 MII 卵母细胞的五个重复实验中的基因表达。采用γH2AX 标记法，在至少三个 IVF 对照和 1 Gy 受精卵和二细胞胚胎中评估 DNA 损伤反应 (DDR)。
参与者/材料、设置、方法：从 C57BL6 雄性的附睾尾获得游泳精子样本，将其分为对照组（无辐照）和 1-和 30-Gy 组。分别用线性加速器 Varian 21iX 将处理后的精子照射到 1 和 30 Gy。照射后，使用 Halomax 评估样品的 DNA 损伤情况，并用于受精。假定的受精卵在时间延迟培养箱（MIRI，ESCO）中培养。使用 Fluidigm Biomark HD 系统评估 91 个 DNA 修复基因的表达。使用共聚焦显微镜（Olympus FV1200）和 IMARIS 软件的 3D 体积分析评估受精卵（受精后 6-8 小时）和二细胞胚胎（受精后 22-24 小时）中的 DNA 损伤反应。
三组的平均精子 DNA 损伤存在统计学差异（对照组：6.1%，1 Gy：16.1%，30 Gy：53.1%，P<0.0001），但两组内或两组间 IVF 后的受精率无显著差异[（年轻；对照组：86.79%，1 Gy：82.75%，30 Gy：76.74%）（年老；对照组：93.1%，1 Gy：70.37%，30 Gy：68.18%）Fisher 精确检验]。然而，囊胚率存在显著差异（P<0.0001），组间差异明显[（年轻；对照组：86.95%，1 Gy：33.33%，30 Gy：0.0%）（年老；对照组：70.37%，1 Gy：0.0%，30 Gy：0.0%）]。在年龄组之间，与年轻女性相比，1 Gy 组的老年女性囊胚率显著降低（P=0.0166）。基因表达分析显示，与年轻 GV 卵母细胞相比，老年 GV 卵母细胞中 21 个 DNA 修复基因的相对表达减少（P<0.05），同样，与年轻 MII 卵母细胞相比，老年 MII 卵母细胞中 23 个基因的表达减少（P<0.05）。在老年 GV 和 MII 卵母细胞中表达减少的基因数量显著影响了双链断裂（GV：5；MII：6）、核苷酸切除修复（GV：8；MII：5）和 DNA 损伤反应（GV：4；MII：8）等途径。与年轻女性相比，无论精子处理如何，来自老年女性的受精卵和二细胞胚胎的 DDR 均减少（P<0.05）。来自老年女性的卵母细胞 DNA 修复基因表达减少和胚胎中的 DDR 减少表明，衰老导致 DNA 修复能力下降。
无。
局限性、谨慎的原因：仅使用实验性的电离辐射来控制精子 DNA 损伤水平；然而，它也可能损害精子蛋白。在小鼠中选择的雌性年龄组旨在模拟年轻和老年女性的影响，但需要临床研究来证明类似的效果。
可以用中高 DNA 损伤的精子群体进行受精，但随着雌性年龄的增长，随后的胚胎生长受到更大的影响，这支持了卵母细胞 DNA 修复能力随年龄增长而下降的理论。评估卵母细胞的 DNA 修复能力可能是诊断不孕夫妇的有用工具。
研究基金/利益冲突：由 Monash 大学产科和妇科教育计划资助。作者均无利益冲突。
