Female fertility is inversely correlated with maternal age due to a depletion of the oocyte pool and a reduction in oocyte developmental competence. Few studies have addressed the effect of maternal age on the human mature oocyte (MII) transcriptome, which is established during oocyte growth and maturation, however, the pathways involved remain unclear. Here, we characterize and compare the transcriptomes of a large cohort of fully grown germinal vesicle stage (GV) and in vitro matured (IVM‐MII) oocytes from women of varying reproductive age. First, we identified two clusters of cells reflecting the oocyte maturation stage (GV and IVM‐MII) with 4445 and 324 putative marker genes, respectively. Furthermore, we identified genes for which transcript representation either progressively increased or decreased with age. Our results indicate that the transcriptome is more affected by age in IVM‐MII oocytes (1219 genes) than in GV oocytes (596 genes). In particular, we found that transcripts of genes involved in chromosome segregation and RNA splicing significantly increased representation with age, while genes related to mitochondrial activity showed a lower representation. Gene regulatory network analysis facilitated the identification of potential upstream master regulators of the genes involved in those biological functions. Our analysis suggests that advanced maternal age does not globally affect the oocyte transcriptome at GV or IVM‐MII stages. Nonetheless, hundreds of genes displayed altered transcript representation, particularly in IVM‐MII oocytes, which might contribute to the age‐related quality decline in human oocytes.

Mitochondria are fundamental organelles in eukaryotic cells that provide ATP through oxidative phosphorylation. During this process, reactive oxygen species (ROS) are produced, and an imbalance in their concentrations can induce oxidative stress (OS), causing cellular damage. However, mitochondria and ROS play also an important role in cellular homeostasis through a variety of other signaling pathways not related to metabolic rates, highlighting the physiological relevance of mitochondria–ROS interactions. In reproduction, mitochondria follow a peculiar pattern of activation, especially in gametes, where they are relatively inactive during the initial phases of development, and become more active towards the final maturation stages. The reasons for the lower metabolic rates are attributed to the evolutionary advantage of keeping ROS levels low, thus avoiding cellular damage and apoptosis. In this review, we provide an overview on the interplay between mitochondrial metabolism and ROS during gametogenesis and embryogenesis, and how OS can influence these physiological processes. We also present the possible effects of assisted reproduction procedures on the levels of OS, and the latest techniques developed to select gametes and embryos based on their redox state. Finally, we evaluate the treatments developed to manage OS in assisted reproduction to improve the chances of pregnancy.

The finding that certain somatic cells can be directly converted into cells of other lineages by the delivery of specific sets of transcription factors paves the way to novel therapeutic applications. Here we show that human cord blood (CB) CD133 ⁺ cells lose their hematopoietic signature and are converted into CB-induced neuronal-like cells (CB-iNCs) by the ectopic expression of the transcription factor Sox2, a process that is further augmented by the combination of Sox2 and c-Myc. Gene-expression analysis, immunophenotyping, and electrophysiological analysis show that CB-iNCs acquire a distinct neuronal phenotype characterized by the expression of multiple neuronal markers. CB-iNCs show the ability to fire action potentials after in vitro maturation as well as after in vivo transplantation into the mouse hippocampus. This system highlights the potential of CB cells and offers an alternative means to the study of cellular plasticity, possibly in the context of drug screening research and of future cell-replacement therapies.

Advanced maternal age is associated with a decline in oocyte quality, which often leads to reproductive failure in humans. However, the mechanisms behind this age-related decline remain unclear. To gain insights into this phenomenon, we applied plexDIA, a multiplexed data-independent acquisition, single-cell mass spectrometry method, to analyze the proteome of oocytes from both young women and women of advanced maternal age. Our findings primarily revealed distinct proteomic profiles between immature fully grown germinal vesicle and mature metaphase II oocytes. Importantly, we further show that a woman’s age is associated with changes in her oocyte proteome. Specifically, when compared to oocytes obtained from young women, advanced maternal age oocytes exhibited lower levels of the proteasome and TRiC complex, as well as other key regulators of proteostasis and meiosis. This suggests that aging adversely affects the proteostasis and meiosis networks in human oocytes. The proteins identified in this study hold potential as targets for improving oocyte quality and may guide future studies into the molecular processes underlying oocyte aging.

Microtubules are intracellular filaments that define in space and in time a large number of essential cellular functions such as cell division, morphology and motility, intracellular transport and flagella and cilia assembly. They are therefore essential for spermatozoon and oocyte maturation and function, and for embryo development. The dynamic and functional properties of the microtubules are in large part defined by various classes of interacting proteins including MAPs (Microtubule Associated Proteins), microtubule dependent motors, and severing and modifying enzymes. Multiple mechanisms regulate these interactions. One of them is defined by the high diversity of the microtubules themselves generated by the combination of different tubulin isotypes and by several tubulin post-translational modifications (PTMs). This generates a so-called tubulin code that finely regulates the specific set of proteins that associates with a given microtubule thereby defining the properties and functions of the network. Here we provide an in depth review of the current knowledge on the tubulin isotypes and PTMs in spermatozoa, oocytes, and preimplantation embryos in various model systems and in the human species. We focus on functional implications of the tubulin code for cytoskeletal function, particularly in the field of human reproduction and development, with special emphasis on gamete quality and infertility. Finally, we discuss some of the knowledge gaps and propose future research directions.

STUDY QUESTION: Is there a relationship between the mitochondrial activity and the meiotic progression of oocytes from germinal vesicle (GV) to metaphase II (MII) stages in young and advanced maternal age (AMA) women? SUMMARY ANSWER: Poor mitochondrial metabolism impairs the meiotic progression of human GV oocytes, contributing to a lower oocyte maturation capacity of AMA oocytes. WHAT IS KNOWN ALREADY: AMA oocytes are characterized by diminished quality, mostly due to the higher rates of chromosomal segregation errors occurring during meiosis I. Another hallmark of AMA oocytes is impaired mitochondrial metabolism. Studies in mice have suggested a link between metabolic dysfunction and meiotic failure, but this relationship has not been fully elucidated in humans. Metabolic dynamics can be visualized by indirect measurements through mitochondrial staining and quantified more directly using fluorescence lifetime imaging microscopy (FLIM). This live-imaging approach can generate metabolic timelapse profiles of oocytes throughout meiosis. In the present study, we explored mitochondrial distribution and functionality in human oocytes at the GV and MII stages, obtained from young and AMA women, to establish the role of mitochondrial metabolism in meiosis progression. STUDY DESIGN, SIZE, DURATION: A total of 340 GV oocytes from young (≤34 years) and AMA (>37 years) women were included in the study. Denuded GVs were matured in vitro in G2-plus medium for 30 h. Maturation was determined by the presence of the extruded first polar body (PB1). The collected oocytes were processed for mitochondrial protein imaging (n = 80), or for live imaging (n = 171). Moreover, 89 oocytes were used for loss-of-function analysis by treating young GVs with 1 μM trifluoromethoxy-carbonylcyanide-phenylhydrazone (FCCP) for 30 min before in vitro maturation. PARTICIPANTS/MATERIALS, SETTING, METHODS: The proteins dihydrolipoamide-S-acetyltransferase (D-LAT) and translocase-of-outer mitochondrial-membrane (TOMM20) were analyzed in young and AMA oocytes by immunofluorescence to assess mitochondrial activity and localization, respectively. Fluorescence mean intensities (arbitrary-unit, AU) were quantified with ImageJ and compared by t-test; maturation rates were compared by chi-squared test. FLIM comprehensive metabolism (NAD(P)H; FAD+) was taken at GV stage. Different FLIM parameters (fluorescence intensity, fraction bound, short/long lifetime) and the Redox ratio (NAD(P)H intensity/FAD+ intensity) were evaluated. MAIN RESULTS AND THE ROLE OF CHANCE: The findings revealed that active mitochondria are specifically localized in the subcortical area, while mitochondria in general are distributed across the whole oocyte. This pattern was substantially maintained in AMA oocytes, which were in turn characterized by a lower mitochondrial activity (D-LAT intensity of 78 614 ± 58 534 AU in young, 12 517 ± 10 187 AU in AMA, P = 0.003), while a lower number of mitochondria was observed In AMA patients but the difference did not reach statistical significance (TOMM20 intensity of 61 674 ± 24 322 AU in young, 32 186 ± 33 414 AU in AMA, P = 0.195). Using non-invasive FLIM, we assessed the metabolic dynamics of maturing oocytes (Redox ratio in young 2e + 00 ± 0.15, in AMA 1e + 00 ± 0.16, P = 2.969e-05), confirming a similar pattern observed by immunofluorescence. Specifically, FLIM microscopy revealed that GV oocytes from young women slightly increased their metabolism, by 4% on average, after the GV breakdown, and the increase was very consistent across different oocytes. On the contrary, in AMA maturing oocytes, little to no increase in metabolism was observed; they were characterized instead by higher variability, and more AMA oocytes failed to successfully reach the MII stage [AMA oocytes (62.3%; 38/61) compared with young oocytes (86.3%; 63/73; P = 0.002). These differential trends observed in AMA oocytes compared to the young oocytes suggest that impaired metabolic activity significantly compromises maturation capacity, revealing a functional link between adequate metabolic levels and successful meiosis progression. LIMITATIONS, REASONS FOR CAUTION: Maturation rates were assessed by the presence of an extruded PB1 and variations in spindle assembly timings may have been overlooked. The quantification of mitochondrial activity in loss-of-function studies was assessed only by immunofluorescence staining. Additionally, the oocytes included in the present study were collected from women who underwent ovarian stimulation and may not faithfully recapitulate physiological maturation. WIDER IMPLICATIONS OF THE FINDINGS: Our findings demonstrate the presence of a functional link between oocyte mitochondrial metabolism and meiosis progression, which may contribute to the decline of oocyte quality with aging. Overall, we provided evidence to understand the biological mechanisms in mitochondrial metabolism that might contribute to driving the decay in oocyte quality in AMA women. STUDY FUNDING/COMPETING INTEREST(S): This project received intramural funding from the Eugin Group and funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No 860960. T.S. is a former owner and former stock owner of Optiva Fertility Inc (company closed) and filed two patents for Optiva Fertility Inc (both abandoned). D.S.: Presenter EMD Senoro and Dep. Editor of Human Reproduction. All of the other authors (S.P., M.M., M.B., E.I., M.P., R.V., and F.Z.) have no conflicts of interest to declare. All of the authors contributed substantially to the manuscript and approve its submission. TRIAL REGISTRATION NUMBER: N/A.