In human in vitro fertilization, the main uncertainty factor impacting on success is oocyte quality, which largely depends on the follicular status at the time of collection. Decades of debate ensued to find the perfect stimulation protocol demonstrated the complexity of the ovarian response to exogenous gonadotropins and the dynamic nature of late folliculogenesis. Although several follicular markers, proteins, RNA from granulosa cells or microRNA, and follicular fluid metabolites have been associated with outcome, the possibility to influence them during stimulation remains elusive. The heterogeneity of the follicle's maturity following control ovarian stimulation is also an important factor to explain average poor oocyte quality still observed today. In this review, the analogy between the apple ripening on the tree and follicular development is presented to focus the attention on a biphasic process: growth and differentiation. The molecular analysis of the progressive follicular differentiation indicates two competing phenomena: growth and differentiation, where a delicate balance must operate from one to the other to ensure proper maturity at ovulation. As long as follicle-stimulating hormone (FSH) stimulates growth, follicles remain green, and it is only when FSH is replaced by luteinizing hormone that the ripening process begins, and “apples” become red. Both fruits, follicles and apples, depend on a perfect timing of events to generate offspring.

Summary Sentence

The importance of the final differentiation process during follicular dominance in relation to the resulting oocyte quality.

Graphical Abstract

In vitro oocyte maturation is an assisted reproductive technology in which a meiotically immature oocyte (prophase I or germinal vesicle stage) is recovered from an antral follicle and matured in vitro prior to fertilization. This technology, although in widespread use in domestic livestock, is not typically implemented during human in vitro fertilization cycles. This review examines how in vitro oocyte maturation is currently used in the clinical setting, including the various ways in vitro oocyte maturation is defined in practice. The role of in vitro oocyte maturation in patient care and the major challenges for implementation are described. Efficiency and safety are critically explored. The role of in vitro oocyte maturation in oncofertility will also be discussed. Finally, the outlook for the future of clinical in vitro oocyte maturation is considered.

Summary Sentence

This review summarizes the current state of the art of in vitro oocyte maturation as used in the treatment of human infertility and fertility preservation, its advantages, and shortcomings, and examines what is next for this technology.

Oocytes are maintained in a state of meiotic arrest following the first meiotic division until ovulation is triggered. Within the antral follicle, meiotic arrest is actively suppressed in a process facilitated by the cyclic nucleotides cGMP and cAMP. If removed from this inhibitory follicular environment and cultured in vitro, mammalian oocytes undergo spontaneous meiotic resumption in the absence of the usual stimulatory follicular stimuli, leading to asynchronicity with oocyte cytoplasmic maturation and lower developmental competence. For more than 50 years, pharmacological agents have been used to attenuate oocyte germinal vesicle (GV) breakdown in vitro. Agents that increase intra-oocyte cAMP or prevent its degradation have been predominantly used; however, agents such as kinase and protein synthesis inhibitors have also been trialed. Twenty years of research demonstrates that maintaining GV arrest for a period before in vitro maturation (IVM) improves oocyte developmental competence, and is likely attributed to maintenance of bidirectional communication with cumulus cells leading to improved oocyte metabolic function. However, outcomes are influenced by various factors including the mode of action of the modulators, dose, treatment duration, species, and the degree of hormonal priming of the oocyte donor. Cyclic GMP and/or cAMP modulation in a prematuration step (called pre-IVM) prior to IVM has shown the greatest consistency in improving oocyte developmental competence, whereas kinase and protein synthesis inhibitors have proven less effective at improving IVM outcomes. Such pre-IVM approaches have shown potential to alter current use of artificial reproductive technologies in medical and veterinary practice.

The purpose of meiosis is to generate developmentally competent, haploid gametes with the correct number of chromosomes. For reasons not completely understood, female meiosis is more prone to chromosome segregation errors than meiosis in males, leading to an abnormal number of chromosomes, or aneuploidy, in gametes. Meiotic spindles are the cellular machinery essential for the proper segregation of chromosomes. One unique feature of spindle structures in female meiosis is spindles poles that lack centrioles. The process of building a meiotic spindle without centrioles is complex and requires precise coordination of different structural components, assembly factors, motor proteins, and signaling molecules at specific times and locations to regulate each step. In this review, we discuss the basics of spindle formation during oocyte meiotic maturation focusing on mouse and human studies. Finally, we review different factors that could alter the process of spindle formation and its stability. We conclude with a discussion of how different assisted reproductive technologies could affect spindles and the consequences these perturbations may have for subsequent embryo development.

Summary Sentence This review consolidates information about how spindles form in human and mouse oocytes and how this process can be altered.

Graphical Abstract

Oocyte activation occurs at the time of fertilization and is a series of cellular events initiated by intracellular Ca²⁺ increases. Consequently, oocytes are alleviated from their arrested state in meiotic metaphase II (MII), allowing for the completion of meiosis. Oocyte activation is also an essential step for somatic cell nuclear transfer and an important tool to overcome clinical infertility. Traditional artificial activation methods aim to mimic the intracellular Ca²⁺ changes which occur during fertilization. Recent studies emphasize the importance of cytoplasmic Zn²⁺ on oocyte maturation and the completion of meiosis, thus suggesting artificial oocyte activation approaches that are centered around the concentration of available Zn²⁺ in oocytes. Depletion of intracellular Zn²⁺ in oocytes with heavy metal chelators leads to successful oocyte activation in the absence of cellular Ca²⁺ changes, indicating that successful oocyte activation does not always depends on intracellular Ca²⁺ increases. Current findings lead to new approaches to artificially activate mammalian oocytes by reducing available Zn²⁺ contents, and the approaches improve the outcome of oocyte activation when combined with existing Ca²⁺-based oocyte activation methods. Here, we review the important role of Ca²+ and Zn²⁺ in mammalian oocyte activation and development of novel oocyte activation approaches based on Zn²⁺ availability.

Summary Sentence Artificial oocyte activation is essential for the application of Assisted Reproductive Technology and the ability to control the level of cytoplasmic zinc can facilitate oocyte activation process.

Oocyte quality is perhaps the most important limiting factor in female fertility; however, the current methods of determining oocyte competence are only marginally capable of predicting a successful pregnancy. We aim to review the predictive value of non-invasive techniques for the assessment of human oocytes and their related cells and biofluids that pertain to their developmental competence. Investigation of the proteome, transcriptome, and hormonal makeup of follicular fluid, as well as cumulus-oocyte complexes are currently underway; however, prospective randomized non-selection-controlled trials of the future are needed before determining their prognostic value. The biological significance of polar body morphology and genetics are still unknown and the subject of debate. The predictive utility of zygotic viscoelasticity for embryo development has been demonstrated, but similar studies performed on oocytes have yet to be conducted. Metabolic profiling of culture media using human oocytes are also limited and may require integration of automated, high-throughput targeted metabolomic assessments in real time with microfluidic platforms. Light exposure to oocytes can be detrimental to subsequent development and utilization of time-lapse imaging and morphometrics of oocytes is wanting. Polarized light, Raman microspectroscopy, and coherent anti-Stokes Raman scattering are a few novel imaging tools that may play a more important role in future oocyte assessment. Ultimately, the integration of chemistry, genomics, microfluidics, microscopy, physics, and other biomedical engineering technologies into the basic studies of oocyte biology, and in testing and perfecting practical solutions of oocyte evaluation, are the future for non-invasive assessment of oocytes.

Summary Sentence

Direct and/or indirect non-invasive assessments of oocyte quality are reviewed and discussed in relation to clinical needs in assisted reproductive technologies and as informative underpinnings of basic reproductive biology spanning follicle to embryo development.

In vitro follicle development (IVFD) is an adequate model to obtain basic knowledge of folliculogenesis and provides a tool for ovarian toxicity screening. IVFD yielding competent oocytes may also offer an option for fertility and species preservation. To promote follicle growth and oocyte maturation in vitro, various culture systems are utilized for IVFD in rodents, domestic animals, wild animals, nonhuman primates, and humans. Follicle culture conditions have been improved by optimizing gonadotropin levels, regulatory factors, nutrient supplements, oxygen concentration, and culture matrices. This review summarizes quality assessment of oocytes generated from in vitro-developed antral follicles from the preantral stage, including oocyte epigenetic and genetic profile, cytoplasmic and nuclear maturation, preimplantation embryonic development following in vitro fertilization, as well as pregnancy and live offspring after embryo transfer. The limitations of oocyte quality evaluation following IVFD and the gaps in our knowledge of IVFD to support proper oocyte development are also discussed. The information may advance our understanding of the requirements for IVFD, with a goal of producing competent oocytes with genetic integrity to sustain embryonic development resulting in healthy offspring.

Summary Sentence

Although advances in current IVFD systems have greatly enhanced oocyte growth and maturation, further optimization is required to improve oocyte competence with genetic integrity for proper embryonic development.

Vitrification, is an ultra-rapid, manual cooling process that produces glass-like (ice crystal-free) solidification. Water is prevented from forming intercellular and intracellular ice crystals during cooling as a result of oocyte dehydration and the use of highly concentrated cryoprotectant. Though oocytes can be cryopreserved without ice crystal formation through vitrification, it is still not clear whether the process of vitrification causes any negative impact (temperature change/chilling effect, osmotic stress, cryoprotectant toxicity, and/or phase transitions) on oocyte quality, which translates to diminished embryo developmental potential or subsequent clinical outcomes. In this review, we attempt to assess the technique's potential effects and the consequence of these effects on outcomes.

Summary Sentence: The osmolarity changes and water movement during oocyte vitrification and warming procedures are illustrated.

Graphical Abstract

The prodigious rise of cancer survival rates enables many cancer survivors to live long lives. Therefore, the side effects of cancer treatments as well as the long-term quality of life after cancer have become more relevant. Ovarian toxicity is a major off-target effect of anticancer agents for childhood and young adult female cancer patients. Both chemotherapy and irradiation have been demonstrated to damage the ovary and increase the risks of premature ovarian failure (POF), early menopause, ovarian endocrine disorders, and sub- or infertility. Oncofertility is an emerging and multidisciplinary research and medical field that focuses on providing cancer patients with fertility preservation options. Oocyte quality and quantity are one of the most important factors to determine women's fertility success; therefore, preserving oocytes is paramount for maintaining the ability of young female cancer patients' reproduction after their recovery. This review summarizes peer-reviewed literature on current oocyte preservation options in oncofertility. We describe in-depth oocyte and embryo cryopreservation, ovarian suppression, ovarian tissue cryopreservation, in vitro maturation, ovarian transposition, and adjuvant therapy. Further, we discuss current guidelines and practices of female fertility preservation that cover preserving oocytes.

Summary sentence Preserving oocytes is fundamental for maintaining the ability of young female cancer patients' reproduction.

Graphical Abstract

The prevalence of obesity in adults worldwide, and specifically in women of reproductive age, is concerning given the risks to fertility posed by the increased risk of type 2 diabetes, metabolic syndrome, and other noncommunicable diseases. Obesity has a multi-systemic impact in female physiology that is characterized by the presence of oxidative stress, lipotoxicity, and the activation of pro-inflammatory pathways, inducing tissue-specific insulin resistance and ultimately conducive to abnormal ovarian function. A higher body mass is linked to Polycystic Ovary Syndrome, dysregulated menstrual cycles, anovulation, and longer time to pregnancy, even in ovulatory women. In the context of assisted reproductive technology (ART), compared to women of normal body mass index, obese women have worse outcomes in every step of their journey, resulting in reduced success measured as live birth rate. Even after pregnancy is achieved, obese women have a higher chance of miscarriage, gestational diabetes, pregnancy complications, birth defects, and most worryingly, a higher risk of stillbirth and neonatal death. The potential for compounding effects of ART on pregnancy complications and infant morbidities in obese women has not been studied. There is still much debate in the field on whether these poorer outcomes are mainly driven by defects in oocyte quality, abnormal embryo development, or an unaccommodating uterine environment, however the clinical evidence to date suggests a combination of all three are responsible. Animal models of maternal obesity shed light on the mechanisms underlying the effects of obesity on the peri-conception environment, with recent findings pointing to lipotoxicity in the ovarian environment as a key driver of defects in oocytes that have not only reduced developmental competence but long-lasting effects in offspring health.

The ovary is the first organ to age in humans with functional decline evident already in women in their early 30s. Reproductive aging is characterized by a decrease in oocyte quantity and quality, which is associated with an increase in infertility, spontaneous abortions, and birth defects. Reproductive aging also has implications for overall health due to decreased endocrinological output. Understanding the mechanisms underlying reproductive aging has significant societal implications as women globally are delaying childbearing and medical interventions have greatly increased the interval between menopause and total lifespan. Age-related changes inherent to the female gamete are well-characterized and include defects in chromosome and mitochondria structure, function, and regulation. More recently, it has been appreciated that the extra-follicular ovarian environment may have important direct or indirect impacts on the developing gamete, and age-dependent changes include increased fibrosis, inflammation, stiffness, and oxidative damage. The cumulus cells and follicular fluid that directly surround the oocyte during its final growth phase within the antral follicle represent additional critical local microenvironments. Here we systematically review the literature and evaluate the studies that investigated the age-related changes in cumulus cells and follicular fluid. Our findings demonstrate unique genetic, epigenetic, transcriptomic, and proteomic changes with associated metabolomic alterations, redox status imbalance, and increased apoptosis in the local oocyte microenvironment. We propose a model of how these changes interact, which may explain the rapid decline in gamete quality with age. We also review the limitations of published studies and highlight future research frontiers.

Summary Sentence Aging is associated with genetic, epigenetic, transcriptomic, proteomic, metabolomic, and redox status changes in the local oocyte microenvironment: cumulus cells and follicular fluid.

The development of oocytes and early embryos is dependent on mitochondrial ATP production. This reliance on mitochondrial activity, together with the exclusively maternal inheritance of mitochondria in development, places mitochondria as central regulators of both fertility and transgenerational inheritance mechanisms. Mitochondrial mass and mtDNA content massively increase during oocyte growth. They are highly dynamic organelles and oocyte maturation is accompanied by mitochondrial trafficking around subcellular compartments. Due to their key roles in generation of ATP and reactive oxygen species (ROS), oocyte mitochondrial defects have largely been linked with energy deficiency and oxidative stress. Pharmacological treatments and mitochondrial supplementation have been proposed to improve oocyte quality and fertility by enhancing ATP generation and reducing ROS levels. More recently, the role of mitochondria-derived metabolites in controlling epigenetic modifiers has provided a mechanistic basis for mitochondria–nuclear crosstalk, allowing adaptation of gene expression to specific metabolic states. Here, we discuss the multi-faceted mechanisms by which mitochondrial function influence oocyte quality, as well as longer-term developmental events within and across generations.

Summary Sentence Oocyte mitochondria as potential therapeutic targets.