Developments in reproductive biology and medicine Mitochondria as determinants of reproductive senescence and competence: implications for diagnosis of embryo competence in assisted reproduction 生殖生物学和医学的发展 线粒体作为生殖衰老和能力的决定因素：对辅助生殖中胚胎能力诊断的影响
Raziye Melike Yildirim (ID) and Emre Seli (ID)* Raziye Melike Yildirim （ID） 和 Emre Seli （ID）*Department of Obstetrics, Gynecology, and Reproductive Sciences, Yale School of Medicine, New Haven, CT, USA 耶鲁大学医学院妇产科和生殖科学系，美国康涅狄格州纽黑文市*Correspondence address. Department of Obstetrics, Gynecology, and Reproductive Sciences, Yale School of Medicine, 200 West Campus Drive, 2nd Floor/Room 211, Orange, CT 06477, USA. E-mail: emre.seli@yale.edu (D) https://orcid.org/0000-0001-7464-8203 *通讯地址。耶鲁大学医学院妇产科和生殖科学系，美国康涅狄格州奥兰治市西校区大道 200 号/211 室 06477。电子邮件： emre.seli@yale.edu （D） https://orcid.org/0000-0001-7464-8203
Abstract 抽象
Mitochondria are commonly recognized as the powerhouses of the cell, primarily responsible for energy production through oxidative phosphorylation. Alongside this vital function, they also play crucial roles in regulating calcium signaling, maintaining membrane potential, and modulating apoptosis. Their involvement in various cellular pathways becomes particularly evident during oogenesis and embryogenesis, where mitochondrial quantity, morphology, and distribution are tightly controlled. The efficiency of the mitochondrial network is maintained through multiple quality control mechanisms that are essential for reproductive success. These include mitochondrial unfolded protein response, mitochondrial dynamics, and mitophagy. Not surprisingly, mitochondrial dysfunction has been implicated in infertility and ovarian aging, prompting investigation into mitochondria as diagnostic and therapeutic targets in assisted reproduction. To date, mitochondrial DNA copy number in oocytes, cumulus cells, and trophectoderm biopsies, and fluorescent lifetime imaging microscopy-based assessment of NADH and flavin adenine dinucleotide content have been explored as potential predictors of embryo competence, yielding limited success. Despite challenges in the clinical application of mitochondrial diagnostic strategies, these enigmatic organelles have a significant impact on reproduction, and their potential role as diagnostic targets in assisted reproduction is likely to remain an active area of investigation in the foreseeable future. 线粒体通常被认为是细胞的动力源，主要负责通过氧化磷酸化产生能量。除了这一重要功能外，它们还在调节钙信号传导、维持膜电位和调节细胞凋亡方面发挥着关键作用。它们在卵子发生和胚胎发生过程中参与各种细胞途径变得尤为明显，其中线粒体数量、形态和分布受到严格控制。线粒体网络的效率是通过多种质量控制机制来维持的，这些机制对生殖成功至关重要。这些包括线粒体未折叠蛋白反应、线粒体动力学和线粒体自噬。毫不奇怪，线粒体功能障碍与不孕症和卵巢衰老有关，促使人们研究线粒体作为辅助生殖的诊断和治疗靶点。迄今为止，卵母细胞、卵丘细胞和滋养外胚层活检中的线粒体 DNA 拷贝数，以及基于荧光寿命成像显微镜的 NADH 和黄素腺嘌呤二核苷酸含量评估已被探索为胚胎能力的潜在预测因子，但成功率有限。尽管线粒体诊断策略的临床应用面临挑战，但这些神秘的细胞器对生殖有重大影响，并且在可预见的未来，它们作为辅助生殖诊断靶标的潜在作用可能仍将是一个活跃的研究领域。
Eukaryotes are organisms whose cells contain a membranebound nucleus. Most eukaryotic cells also harbor mitochondria in their cytoplasm, unique organelles characterized by a double membrane structure, their own DNA, and reproduction through binary fission, where one mitochondrion splits into two daughter mitochondria. 真核生物是其细胞包含膜结合核的生物。大多数真核细胞的细胞质中也含有线粒体，这种独特的细胞器以双膜结构、自身的 DNA 和通过二元裂变繁殖，其中一个线粒体分裂成两个子线粒体。
One of the most well-known and crucial functions of mitochondria is to generate energy required for various biochemical and physiological processes within the cell. This energy production primarily occurs through oxidative phosphorylation (OXPHOS), facilitated by the electron transport chain (ETC) (Scott et al., 2018). However, a notable consequence of this energy generation process is the production of reactive oxygen species (ROS) as a by-product. The accumulation of ROS, often observed in the presence of mitochondrial dysfunction, induces cellular stress. If left unaddressed, this stress may ultimately culminate in cellular senescence and death (Bratic and Larsson, 2013). 线粒体最著名和最关键的功能之一是产生细胞内各种生化和生理过程所需的能量。这种能量产生主要通过氧化磷酸化 （OXPHOS） 发生，由电子传递链 （ETC） 促进（Scott 等人，2018 年）。然而，这种能源产生过程的一个显着后果是产生活性氧 （ROS） 作为副产品。ROS 的积累（通常在线粒体功能障碍存在的情况下观察到）会诱导细胞应激。如果不加以解决，这种压力最终可能导致细胞衰老和死亡（Bratic 和 Larsson，2013 年）。
In addition to their role in ATP production, mitochondria are also important in the regulation of various cellular processes, including calcium signaling, membrane potential maintenance, and apoptosis, all of which are essential for cellular function. Emerging studies identify mitochondria as key determinants of subfertility and accelerated ovarian aging due to their pivotal roles in cellular homeostasis, including the generation of energy essential for the normal assembly of spindles and the segregation of chromosomes, and regulation of programmed cell death during follicular atresia (Babayev et al., 2016; May-Panloup et al., 2016; Pasquariello et al., 2018). As mitochondrial processes are relatively well characterized, they also have the potential to 除了在 ATP 产生中的作用外，线粒体在各种细胞过程的调节中也很重要，包括钙信号传导、膜电位维持和细胞凋亡，所有这些都对细胞功能至关重要。新兴研究确定线粒体是不育力和加速卵巢衰老的关键决定因素，因为它们在细胞稳态中起着关键作用，包括产生纺锤体正常组装和染色体分离所必需的能量，以及调节滤泡闭锁期间的程序性细胞死亡（Babayev et al.， 2016;May-Panloup 等人，2016 年;Pasquariello et al.， 2018）。由于线粒体过程的特征相对较好，因此它们也有可能
serve as biomarkers for enhancing the prediction of gamete and embryo viability and competence. 作为增强配子和胚胎活力和能力预测的生物标志物。
In this review, our first objective is to provide a summary of the current knowledge regarding mitochondrial features and mechanisms contributing to female reproductive competence. To this end, we will examine the basic functions of mitochondria during oocyte and embryo development and how the integrity of mitochondrial quality control mechanisms may affect female fertility throughout the reproductive lifespan. Subsequently, in assessing evidence for age-related changes in morphology and topography of mitochondria and the association between mitochondrial dysfunction and infertility, we will discuss the potential role of mitochondria as prognostic biomarkers of embryo competence within the realm of assisted reproduction. 在这篇综述中，我们的第一个目标是总结有关线粒体特征和促进女性生殖能力的机制的当前知识。为此，我们将研究线粒体在卵母细胞和胚胎发育过程中的基本功能，以及线粒体质量控制机制的完整性如何影响女性在整个生殖生命周期中的生育能力。随后，在评估线粒体形态和地形与年龄相关的变化以及线粒体功能障碍与不孕症之间的关联的证据时，我们将讨论线粒体作为辅助生殖领域内胚胎能力的预后生物标志物的潜在作用。
Mitochondrial determinants of reproductive competence Mitochondrial energy production 生殖能力的线粒体决定因素线粒体能量产生
Glycolysis is the metabolic pathway where a single glucose molecule transforms into two pyruvate molecules in the cytoplasm, yielding two ATP and two NADH molecules, for each glucose molecule (Fig. 1). Pyruvate can then be transported into the mitochondrial matrix, where it is metabolized into acetyl coenzyme A (acetyl CoA) by pyruvate dehydrogenase, generating another NADH. Within the mitochondrial matrix, acetyl CoA enters the tricarboxylic acid (TCA) cycle, also known as the citric acid or Krebs cycle; three NADH, one flavin adenine dinucleotide (FADH2), and one GTP molecules are generated for each acetyl CoA. OXPHOS, essential for mitochondrial energy production, proceeds through the ETC embedded in the inner mitochondrial 糖酵解是一种代谢途径，其中单个葡萄糖分子在细胞质中转化为两个丙酮酸分子，每个葡萄糖分子产生两个 ATP 分子和两个 NADH 分子（图 1）。然后丙酮酸可以被运输到线粒体基质中，在那里它被丙酮酸脱氢酶代谢成乙酰辅酶 A（乙酰辅酶 A），产生另一种 NADH。在线粒体基质中，乙酰辅酶 A 进入三羧酸 （TCA） 循环，也称为柠檬酸或克雷布斯循环;为每个乙酰辅酶 A 生成三个 NADH、一个黄素腺嘌呤二核苷酸 （FADH2） 和一个 GTP 分子。OXPHOS 对线粒体能量产生至关重要，通过嵌入线粒体内的 ETC 进行
Figure 1. Glycolysis and oxidative phosphorylation: the metabolic cooperation between the oocyte and surrounding cumulus cells. 图 1.糖酵解和氧化磷酸化：卵母细胞与周围卵丘细胞之间的代谢合作。
Oocytes exhibit limited glycolytic activity, prompting glucose metabolism to occur predominantly within cumulus cells. Pyruvate generated in cumulus cells by glycolysis is transported to the oocyte via gap junctions. In the oocyte, pyruvate is transported into the mitochondrial matrix, where it undergoes oxidation via the tricarboxylic acid cycle (TCA), resulting in the production of reduced cofactors NADH and flavin adenine dinucleotide (FADH2). These cofactors play a crucial role in driving the electron transport chain (ETC), which facilitates ATP synthesis by establishing a proton gradient through electron transfer. Cytochrome C (Cyt C), complex I, II, III, and IV (I, II, III, IV). Created with BioRender.com. 卵母细胞表现出有限的糖酵解活性，促使葡萄糖代谢主要发生在卵丘细胞内。糖酵解在卵丘细胞中产生的丙酮酸通过间隙连接转运到卵母细胞。在卵母细胞中，丙酮酸被转运到线粒体基质中，在那里它通过三羧酸循环 （TCA） 发生氧化，导致产生还原的辅因子 NADH 和黄素腺嘌呤二核苷酸 （FADH2）。这些辅因子在驱动电子传递链 （ETC） 中起着至关重要的作用，ETC 通过电子转移建立质子梯度来促进 ATP 合成。细胞色素 C （Cyt C），复合物 I、II、III 和 IV （I、II、III、IV）。使用 BioRender.com 创建。
membrane, involving five protein complexes. NADH and FADH _(2){ }_{2} molecules produced in earlier steps enter the ETC and undergo oxidation to contribute electrons to the system. ATP synthesis occurs via ATP synthase, utilizing the proton gradient generated by the repetitive transfer of electrons (Scott et al., 2018). Oxidation of each NADH molecule results in the generation of three ATP molecules, whereas FADH_(2)\mathrm{FADH}_{2} oxidation generates two ATPs. In total, 36 ATP molecules can be generated per glucose molecule through OXPHOS, in addition to the two ATPs produced during glycolysis (Scott et al., 2018). 膜，涉及 5 种蛋白质复合物。在早期步骤中产生的 NADH 和 FADH _(2){ }_{2} 分子进入 ETC 并发生氧化，为系统提供电子。ATP 合成通过 ATP 合酶发生，利用电子重复转移产生的质子梯度（Scott 等人，2018 年）。每个 NADH 分子的氧化导致产生三个 ATP 分子，而 FADH_(2)\mathrm{FADH}_{2} 氧化产生两个 ATP。除了糖酵解过程中产生的两种 ATP 外，每个葡萄糖分子总共可以通过 OXPHOS 产生 36 个 ATP 分子（Scott 等人，2018 年）。
Half a century ago, Biggers et al. (1967) demonstrated somatic cells’ involvement in providing nutritional support for oocytes. They found that oocyte maturation was inhibited in a glucosecontaining medium unless either cumulus cells (CCs) or pyruvate were also present. This was corroborated by subsequent discoveries indicating that CCs can synthesize pyruvate from glucose or lactate in vitro, and that oocytes depend on pyruvate as an energy source for growth and resumption of meiosis (Donahue and Stern, 1968; Leese and Barton, 1985). The exchange of nutrients and endocrine signals between the oocyte and CCs within the cumulus-oophorus complex is facilitated by gap junctions and plays a pivotal role in the survival and maturation of the oocyte (Sutton-McDowall et al., 2010). Confirming this, the targeted deletion of connexin-37, which leads to the absence of the oocyte-specific gap junctional subunit, causes impaired follicle development and ovulation (Ackert et al., 2001). 半个世纪前，Biggers 等人（1967 年）证明了体细胞参与为卵母细胞提供营养支持。他们发现，除非还存在卵丘细胞 （CC） 或丙酮酸，否则卵母细胞成熟在含葡萄糖的培养基中受到抑制。随后的发现证实了这一点，表明 CCs 可以在体外从葡萄糖或乳酸合成丙酮酸，并且卵母细胞依赖丙酮酸作为减数分裂生长和恢复的能量来源（Donahue 和 Stern，1968 年;Leese 和 Barton，1985 年）。卵丘-卵母细胞复合体内卵母细胞和 CCs 之间的营养物质和内分泌信号交换由间隙连接促进，并在卵母细胞的存活和成熟中起着关键作用（Sutton-McDowall et al.， 2010）。证实了这一点，连接蛋白 37 的靶向缺失导致卵母细胞特异性间隙连接亚基的缺失，导致卵泡发育和排卵受损（Ackert et al.， 2001）。
Consistent with these observations, oocytes primarily rely on pyruvate via OXPHOS to fuel their growth and maturation (illustrated in Fig. 1). Notably, pyruvate consumption is significantly higher during the maturation of MI (metaphase I) oocytes compared to immature (prophase I) or mature (MII, metaphase II) oocytes (Downs et al., 2002). Oocyte-specific deletion of Pdha1 (pyruvate dehydrogenase E1 alpha 1), the gene encoding the enzymatic subunit of the pyruvate dehydrogenase complex, results in impairments in oocyte maturation (Johnson et al., 2007). Similarly, while key enzymes involved in the glycolytic pathway 与这些观察结果一致，卵母细胞主要依靠丙酮酸通过 OXPHOS 来促进其生长和成熟（如图 1 所示）。值得注意的是，与未成熟（前期 I）或成熟（MII，中期 II）卵母细胞相比，丙酮酸消耗量在 MI（中期 I）卵母细胞成熟期间显着更高（Downs 等人，2002 年）。Pdha1（丙酮酸脱氢酶 E1 α 1）的卵母细胞特异性缺失是编码丙酮酸脱氢酶复合物酶促亚基的基因，导致卵母细胞成熟受损（Johnson et al.， 2007）。同样，虽然参与糖酵解途径的关键酶
are abundantly expressed in CCs, their expression in oocytes is minimal in mice (Sugiura et al., 2005). Pyruvate consumption predominates during the cleavage stage of embryo development as well. However, as the embryo advances to the blastocyst stage, glycolysis becomes active, coinciding with an increase in metabolic activity, in preparation for implantation (Riley and Moley, 2006; Leese, 2012). 在 CCs 中大量表达，它们在卵母细胞中的表达在小鼠中最小（Sugiura et al.， 2005）。丙酮酸消耗在胚胎发育的卵裂阶段也占主导地位。然而，随着胚胎进入囊胚阶段，糖酵解变得活跃，同时代谢活动增加，为植入做准备（Riley 和 Moley，2006 年;Leese，2012 年）。
Regulation of spindle formation, chromosome separation, translation, intracellular signaling, and calcium homeostasis hinges upon the availability of high-energy substrates, such as ATP, and contribute to the oocyte quality. Research has shown that oocytes experience a notable reduction in ATP content as they age, reflecting a decrease in their energy efficiency over time (Simsek-Duran et al., 2013). In mice, aging oocytes demonstrate an impaired ability to adjust intracellular ATP levels during fertilization, leading to compromised developmental potential (Igarashi et al., 2005; Eichenlaub-Ritter et al., 2011). In humans, higher ATP levels observed in oocytes have been linked with superior embryo development and higher rates of successful implantation (Van Blerkom et al., 1995; Zhao and Li, 2012). Thus, the vitality of mitochondrial energy production is critical for achieving reproductive success (Eichenlaub-Ritter et al., 2011). 纺锤体形成、染色体分离、翻译、细胞内信号传导和钙稳态的调节取决于高能底物（如 ATP）的可用性，并有助于卵母细胞质量。研究表明，随着年龄的增长，卵母细胞的 ATP 含量会显着降低，这反映了它们的能量效率随着时间的推移而降低（Simsek-Duran et al.， 2013）。在小鼠中，衰老的卵母细胞在受精过程中调节细胞内 ATP 水平的能力受损，导致发育潜力受损（Igarashi 等人，2005 年;Eichenlaub-Ritter et al.， 2011）。在人类中，在卵母细胞中观察到的较高 ATP 水平与良好的胚胎发育和较高的成功植入率有关（Van Blerkom 等人，1995 年;Zhao 和 Li，2012 年）。因此，线粒体能量产生的活力对于实现生殖成功至关重要（Eichenlaub-Ritter et al.， 2011）。
Mitochondrial biogenesis and dynamics 线粒体生物发生和动力学
In contrast to mature mitochondria, which form highly organized networks in fully differentiated somatic cells (Kirillova et al., 2021), mitochondrial morphology and distribution display developmental stage-specific changes in fetal and adult female germ cells (Motta et al., 2000). In fetal oogonia, mitochondria exhibit an oval to elongated shape characterized by a dense matrix and tubular cristae. Upon formation of the primordial follicles, oocyte mitochondria transition to a rounded morphology with a less dense matrix, and the inner mitochondrial membrane takes on the form of a few irregular cristae. In subsequent stages of growing and maturing oocytes, as well as in very early embryos, mitochondria predominantly assume a spherical to oval shape, with a diameter ranging from 0.3 to 0.5 mum0.5 \mu \mathrm{~m}. Their appearance is inert, featuring a dense matrix and a few arch-like cristae located peripherally or transversely (Sathananthan and Trounson, 2000). 与在完全分化的体细胞中形成高度组织化网络的成熟线粒体相反（Kirillova 等人，2021 年），线粒体形态和分布显示胎儿和成年女性生殖细胞的发育阶段特异性变化（Motta 等人，2000 年）。在胎儿卵子中，线粒体呈椭圆形至细长状，其特征是致密的基质和管状嵴。在原始卵泡形成后，卵母细胞线粒体转变为基质密度较低的圆形形态，线粒体内膜呈现出一些不规则嵴的形式。在卵母细胞生长和成熟的后续阶段，以及在非常早期的胚胎中，线粒体主要呈球形至椭圆形，直径从 0.3 到 0.5 mum0.5 \mu \mathrm{~m} 不等。它们的外观是惰性的，具有致密的基质和一些位于外围或横向的拱状嵴（Sathananthan 和 Trounson，2000）。
Mitochondrial microtopography also undergoes significant changes during oocyte maturation. Specifically, before germinal vesicle breakdown, individual mitochondria and small clusters are concentrated in the perinuclear area and are absent from the outer cytoplasmic regions. In MI oocytes, numerous mitochondria populate the cortical region, showing a tendency to approach randomly distributed endoplasmic reticulum (ER) structures. Upon maturation, most mitochondria become physically associated with ER membranes and the ooplasm of mature (MII) oocytes exhibits characteristic ‘necklace-like’ complexes, featuring a spherical sac (ER) adorned with circularly arranged mitochondria (Trebichalská et al., 2021). These strictly regulated characteristics of mitochondrial morphology, distribution, and biogenesis make any deviations or changes associated with subfertility or aging a suitable target for the investigation of their impact on reproductive competence. 线粒体微地形图在卵母细胞成熟过程中也会发生显着变化。具体来说，在生发囊泡分解之前，单个线粒体和小簇集中在核周区域，而在细胞质外部区域不存在。在 MI 卵母细胞中，许多线粒体填充在皮质区域，显示出接近随机分布的内质网 （ER） 结构的趋势。成熟后，大多数线粒体与 ER 膜物理相关，成熟 （MII） 卵母细胞的卵质表现出特征性的“项链状”复合物，其特征是球形囊 （ER） 装饰有圆形排列的线粒体（Trebichalská等人，2021 年）。线粒体形态、分布和生物发生的这些严格调节的特征使与不育或衰老相关的任何偏差或变化成为研究其对生殖能力影响的合适目标。
The process of oocyte maturation is intricate, demanding significant energy levels to facilitate the requisite transformations within both the nucleus and the cytoplasm. Nuclear maturation primarily focuses on chromosome segregation, while cytoplasmic maturation involves the reorganization of organelles and the storage of mRNAs and proteins essential for oocyte maturation, fertilization, and early embryogenesis (Kang et al., 2023). Upon initiation of oocyte maturation, meiotic reactivation ensues in the prophase I-arrested oocyte and is accompanied by 卵母细胞成熟的过程错综复杂，需要大量的能量水平来促进细胞核和细胞质内必要的转化。核成熟主要集中在染色体分离，而细胞质成熟涉及细胞器的重组以及卵母细胞成熟、受精和早期胚胎发生所必需的 mRNA 和蛋白质的储存（Kang 等人，2023 年）。卵母细胞成熟开始时，前期 I 期阻滞的卵母细胞发生减数分裂再激活，并伴有
