Department of Obstetrics and Gynaecology, Faculty of Medical and Health Sciences, University of Auckland, Auckland 1023, New Zealand Fertility Associates, Remuera, Auckland 1051, New Zealand
Department of Obstetrics and Gynaecology, Faculty of Medical and Health Sciences, University of Auckland, Auckland 1023, New Zealand.
Hum Reprod. 2015 Jun;30(6):1410-20. doi: 10.1093/humrep/dev066. Epub 2015 Mar 27.
Does maternal ageing and ovarian stimulation alter mitochondrial DNA (mtDNA) copy number and gene expression of oocytes and cumulus cells from a novel bovine model for human IVF?
Oocytes collected from females with identical nuclear genetics show decreased mtDNA copy number and increased expression of an endoplasmic reticulum (ER) stress gene with repect to ovarian stimulation, whilst differences in the expression of genes involved in mitochondrial function, antioxidant protection and apoptosis were evident in relation to maternal ageing and the degree of ovarian stimulation in cumulus cells.
Oocyte quality declines with advancing maternal age; however, the underlying mechanism, as well as the effects of ovarian stimulation are poorly understood. Human studies investigating these effects are often limited by differences in age and ovarian stimulation regimens within a patient cohort, as well as genetic and environmental variability.
STUDY DESIGN, SIZE, DURATION: A novel bovine cross-sectional maternal age model for human IVF was undertaken. Follicles were aspirated from young (3 years of age; n = 7 females) and old (10 years of age; n = 5 females) Holstein Freisian clones following multiple unstimulated, mild and standard ovarian stimulation cycles. These bovine cloned females were generated by the process of somatic cell nuclear transfer (SCNT) from the same founder and represent a homogeneous population with reduced genetic and environmental variability. Maternal age and ovarian stimulation effects were investigated in relation to mtDNA copy number, and the expression of 19 genes involved in mitochondrial function, antioxidant protection, oocyte-cumulus cell signalling and follicle development in both oocytes and cumulus cells.
MATERIALS, SETTING, METHODS: Young (3 years of age; n = 7 females) and old (10 years of age; n = 5 females) Holstein Freisian bovine clones were maintained as one herd. Stimulation cycles were based on the long GnRH agonist down-regulation regimen used in human fertility clinics. Follicle growth rates, numbers and diameters were monitored by ultrasonography and aspirated when the lead follicles were >14 mm in diameter. Follicle characteristics were analysed using a mixed model procedure. Quantitative PCR (qPCR) was used to determine mtDNA copy number and reverse transcriptase-qPCR (RT-qPCR) was used to measure gene expression in oocytes and cumulus cells.
Method of ovarian stimulation (P = 0.04), but not maternal age (P > 0.1), was associated with a lower mtDNA copy number in oocytes. Neither factor affected mtDNA copy number in cumulus cells. In oocytes, maternal age had no effect on gene expression; however, ovarian stimulation in older females increased the expression of GRP78 (P = 0.02), a gene involved in ER stress. In cumulus cells, increasing maternal age was associated with the higher expression of genes involved in mitochondrial maintenance (TXN2 P = 0.008 and TFAM P = 0.03), whereas ovarian stimulation decreased the expression of genes involved in mitochondrial oxidative stress and apoptosis (TXN2 P = 0.002, PRDX3 P = 0.03 and BAX P = 0.03).
LIMITATIONS, REASON FOR CAUTION: The low number of oocyte and cumulus cell samples collected from the unstimulated cycles limited the analysis. Fertilization and developmental potential of the oocytes was not assessed because these were used for mtDNA and gene expression quantification.
Delineation of the independent effects of maternal age and ovarian stimulation regimen on mtDNA copy number gene expression in oocytes and cumulus cells was enabled by the removal of genetic and environmental variability in this bovine model for human IVF. Therefore, these extend upon previous knowledge and findings provide relevant insights that are applicable for improving human ovarian stimulation regimens.
STUDY FUNDING/COMPETING INTERESTS: Funding was provided by Fertility Associates and the University of Auckland. J.C.P. is a shareholder of Fertility Associates and M.P.G. received a fellowship from Fertility Associates. The other authors of this manuscript declare no conflict of interest that could be perceived as prejudicing the impartiality of the reported research.
母体年龄增长和卵巢刺激是否会改变来自用于人类体外受精的新型牛模型的卵母细胞和卵丘细胞中的线粒体 DNA（mtDNA）拷贝数和基因表达？
从具有相同核遗传学的女性中采集的卵母细胞显示出 mtDNA 拷贝数减少和内质网（ER）应激基因表达增加，而在卵丘细胞中与母体年龄增长和卵巢刺激程度相关的线粒体功能、抗氧化保护和细胞凋亡相关基因的表达差异明显。
随着母体年龄的增长，卵母细胞质量下降；然而，其潜在机制以及卵巢刺激的影响了解甚少。人类研究通常受到患者队列中年龄和卵巢刺激方案的差异、遗传和环境变异性以及个体差异的限制。
研究设计、大小、持续时间：采用了用于人类体外受精的新型牛母体年龄模型。从年轻（3 岁；n = 7 名女性）和老年（10 岁；n = 5 名女性）荷斯坦弗里森克隆牛中抽吸卵泡，在多次未经刺激、轻度和标准卵巢刺激周期后。这些牛克隆雌性是通过体细胞核转移（SCNT）从同一供体产生的，代表具有减少遗传和环境变异性的同质群体。研究了 mtDNA 拷贝数以及线粒体功能、抗氧化保护、卵母细胞-卵丘细胞信号转导和卵泡发育相关的 19 个基因的表达与卵母细胞和卵丘细胞中的母体年龄和卵巢刺激效应的关系。
材料、设置、方法：将年轻（3 岁；n = 7 名女性）和老年（10 岁；n = 5 名女性）荷斯坦弗里森克隆牛维持在一个牛群中。刺激周期基于人类生育诊所中使用的长 GnRH 激动剂下调方案。通过超声监测卵泡生长速度、数量和直径，并在主导卵泡直径>14mm 时抽吸卵泡。使用混合模型程序分析卵泡特征。使用定量 PCR（qPCR）确定 mtDNA 拷贝数，并使用逆转录定量 PCR（RT-qPCR）测量卵母细胞和卵丘细胞中的基因表达。
卵巢刺激方法（P = 0.04）而不是母体年龄（P>0.1）与卵母细胞中 mtDNA 拷贝数降低有关。两种因素都不影响卵丘细胞中的 mtDNA 拷贝数。在卵母细胞中，母体年龄对基因表达没有影响；然而，老年女性的卵巢刺激增加了 GRP78 的表达（P = 0.02），这是一种与内质网应激相关的基因。在卵丘细胞中，与线粒体维持相关的基因（TXN2，P = 0.008 和 TFAM，P = 0.03）的表达随着母体年龄的增加而增加，而卵巢刺激降低了与线粒体氧化应激和细胞凋亡相关的基因（TXN2，P = 0.002、PRDX3，P = 0.03 和 BAX，P = 0.03）的表达。
局限性、谨慎的原因：从未刺激的周期中收集的卵母细胞和卵丘细胞样本数量较少，限制了分析。由于这些样本用于 mtDNA 和基因表达定量，因此未评估卵母细胞的受精和发育潜力。
通过从用于人类体外受精的牛模型中消除遗传和环境变异性，能够确定母体年龄和卵巢刺激方案对卵母细胞和卵丘细胞中的 mtDNA 拷贝数和基因表达的独立影响。因此，这些研究扩展了以前的知识，并提供了相关的见解，这些见解适用于改善人类卵巢刺激方案。
研究资助/利益冲突：这项研究得到了生育协会和奥克兰大学的资助。J.C.P.是生育协会的股东，M.P.G.从生育协会获得了奖学金。这份手稿的其他作者没有声明可能影响报告研究公正性的利益冲突。
