Human ovarian aging is characterized by oxidative damage and mitochondrial dysfunction.

STUDY QUESTION: Are human ovarian aging and the age-related female fertility decline caused by oxidative stress and mitochondrial dysfunction in oocytes? SUMMARY ANSWER: We found oxidative damage in oocytes of advanced maternal age, even at the primordial follicle stage, and confirmed mitochondrial dysfunction in such oocytes, which likely resulted in the use of alternative energy sources. WHAT IS KNOWN ALREADY: Signs of reactive oxygen species-induced damage and mitochondrial dysfunction have been observed in maturing follicles, and even in early stages of embryogenesis. However, although recent evidence indicates that also primordial follicles have metabolically active mitochondria, it is still often assumed that these follicles avoid oxidative phosphorylation to prevent oxidative damage in dictyate arrested oocytes. Data on the influence of ovarian aging on oocyte metabolism and mitochondrial function are still limited. STUDY DESIGN, SIZE, DURATION: A set of 39 formalin-fixed and paraffin-embedded ovarian tissue biopsies were divided into different age groups and used for immunofluorescence analysis of oxidative phosphorylation activity and oxidative damage to proteins, lipids, and DNA. Additionally, 150 immature oocytes (90 germinal vesicle oocytes and 60 metaphase I oocytes) and 15 cumulus cell samples were divided into different age groups and used for targeted metabolomics and lipidomics analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ovarian tissues used for immunofluorescence microscopy were collected through PALGA, the nationwide network, and registry of histo- and cytopathology in The Netherlands. Comprehensive metabolomics and lipidomics were performed by liquid-liquid extraction and full-scan mass spectrometry, using oocytes and cumulus cells of women undergoing ICSI treatment based on male or tubal factor infertility, or fertility preservation for non-medical reasons. MAIN RESULTS AND THE ROLE OF CHANCE: Immunofluorescence imaging on human ovarian tissue indicated oxidative damage by protein and lipid (per)oxidation already at the primordial follicle stage. Metabolomics and lipidomics analysis of oocytes and cumulus cells in advanced maternal-age groups demonstrated a shift in the glutathione-to-oxiglutathione ratio and depletion of phospholipids. Age-related changes in polar metabolites suggested a decrease in mitochondrial function, as demonstrated by NAD+, purine, and pyrimidine depletion, while glycolysis substrates and glutamine accumulated, with age. Oocytes from women of advanced maternal age appeared to use alternative energy sources like glycolysis and the adenosine salvage pathway, and possibly ATP which showed increased production in cumulus cells. LIMITATIONS, REASONS FOR CAUTION: The immature oocytes used in this study were all subjected to ovarian stimulation with high doses of follicle-stimulating hormones, which might have concealed some age-related differences. WIDER IMPLICATIONS OF THE FINDINGS: Further studies on how to improve mitochondrial function, or lower oxidative damage, in oocytes from women of advanced maternal age, for instance by supplementation of NAD+ precursors to promote mitochondrial biogenesis, are warranted. In addition, supplementing the embryo medium of advanced maternal-age embryos with such compounds could be a treatment option worth exploring. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by the Amsterdam UMC. The authors declare to have no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Cytogenetic testing of pregnancy loss tissue: a meta-analysis.

Many clinics offer routine genetic testing of pregnancy loss tissue. This review presents a comprehensive literature search and meta-analysis on chromosomal abnormality rates of pregnancy loss tissue from women with a single or recurrent pregnancy loss. A total of 55 studies published since 2000 were included, analysed on the prevalence of test failure rates, abnormality detection rates and percentages of trisomy, monosomy X, structural abnormalities and other clinically (ir)relevant abnormalities detected by conventional karyotyping, array-comparative genomic hybridization (aCGH), single nucleotide polymorphism (SNP) array, fluorescence in-situ hybridization (FISH) and multiplex ligation-dependent probe amplification (MLPA). The detected prevalence of chromosomal abnormalities was 48% (95% confidence interval [CI] 39-57) using aCGH, 38% (95% CI 28-49) with FISH, 25% (95% CI 12-42) using MLPA, 60% (95% CI 58-63) using SNP array and 47% (95% CI 43-51) with conventional karyotyping. The percentage of detected abnormalities did not differ between women that suffered sporadic (46%; 95% CI 39-53) or recurrent (46%; 95% CI 39-52) pregnancy loss. In view of the high prevalence of chromosomal abnormalities in pregnancy loss tissue, and the low chance of recurrence of the same chromosomal aberration, it was concluded that detection of specific chromosomal abnormalities in pregnancy loss tissue has no clinical benefit. Therefore, routine testing of pregnancy loss tissue for chromosomal abnormalities is not recommended.

Development of the OPAL prediction model for prediction of live birth in couples with recurrent pregnancy loss: protocol for a prospective and retrospective cohort study in the Netherlands.

INTRODUCTION: Recurrent pregnancy loss (RPL) is defined as the loss of two or more conceptions before 24 weeks gestation. Despite extensive diagnostic workup, in only 25%-40% an underlying cause is identified. Several factors may increase the risk for miscarriage, but the chance of a successful pregnancy is still high. Prognostic counselling plays a significant role in supportive care. The main limitation in current prediction models is the lack of a sufficiently large cohort, adjustment for relevant risk factors, and separation between cumulative live birth rate and the success chance in the next conception. In this project, we aim to make an individualised prognosis for the future chance of pregnancy success, which could lead to improved well-being and the ability managing reproductive choices. METHODS AND ANALYSIS: In this multicentre study, we will include both a prospective and a retrospective cohort of at least 931 and 1000 couples with RPL, respectively. Couples who have visited one of the three participating university hospitals in the Netherlands for intake are eligible for the study participation, with a follow-up duration of 5 years. General medical and obstetric history and reports of pregnancies after the initial consultation will be collected. Multiple imputation will be performed to cope for missing data. A Cox proportional hazards model for time to pregnancy will be developed to estimate the cumulative chance of a live birth within 3 years after intake. To dynamically estimate the chance of an ongoing pregnancy, given the outcome of earlier pregnancies after intake, a logistic regression model will be developed. ETHICS AND DISSEMINATION: The Medical Ethical Research Committee of the Leiden University Medical Center approved this study protocol (N22.025). There are no risks or burden associated with this study. Participant written informed consent is required for both cohorts. Findings will be published in peer-reviewed journals and presentations at international conferences. TRIAL REGISTRATION NUMBER: NCT05167812.

Longevity pathways are associated with human ovarian ageing.

STUDY QUESTION: Are genes known to be involved in somatic cell ageing, particularly related to longevity pathways, associated with the accelerated ageing process of the ovary? SUMMARY ANSWER: Growth, metabolism, and cell-cycle progression-related pathways that are involved in somatic cell ageing are also associated with ovarian ageing. WHAT IS KNOWN ALREADY: Ovarian ageing is characterized by a gradual decline in ovarian follicle quantity, a decline in oocyte quality, and lower chances of pregnancy. Genetic pathways modulating the rate of somatic cell ageing have been researched intensively. Ovarian ageing does not follow the same timeline as somatic cell ageing, as signs of ovarian ageing occur at a younger female age, while the somatic cells are still relatively young. It is not known whether the generally recognized somatic cell longevity genes also play a role during ovarian ageing. Looking at somatic cell longevity genes can lead to new hypotheses and possible treatment options for subfertility caused by ovarian ageing. STUDY DESIGN SIZE DURATION: In this observational study, we analysed a dataset of individual gene expression profiles of 38 germinal vesicle (GV) oocytes from 38 women aged between 25 and 43 years. We correlated female age (calendar age in years) and biological age (factors known to be associated with ovarian ageing such as dosage of FSH needed for ovarian hyperstimulation, and antral follicle count (AFC)) with gene expression signatures of longevity pathways. PARTICIPANTS/MATERIALS SETTING METHODS: Transcripts of 38 GV oocytes were used for individual gene expression analysis. R version 3.5.1 was used to process and analyse data. The GeneAge database (build 19) was used to obtain mouse ageing-related genes. Human to mouse orthologues were obtained using the R package biomaRt. Correlations and significance between gene expression data and age were tested for using Pearson's product moment correlation coefficient using ranked expression data. Distributions were compared with an ANOVA, and the Tukey Honest Significant Difference method was used to control for the Type I error rate across multiple comparisons. MAIN RESULTS AND THE ROLE OF CHANCE: Of the 136 genes in the GeneAge database, the expression of 15 anti-longevity genes identified in oocytes showed a positive correlation with female calendar age and FSH dosage administered during ICSI treatment, and a negative correlation with AFC. Expression of 32 pro-longevity genes was negatively correlated with calendar age and FSH dosage, and positively correlated with AFC. In general, anti- and pro-longevity genes changed in opposing directions with advancing maternal age in oocytes. Notably, the anti-longevity genes include many 'growth'-related genes involved in the mechanistic target of rapamycin (mTOR) Complex 1 pathway, such as EIF5A2, EIF3H, EIF4E, and mTOR. The pro-longevity genes include many cell-cycle progression-related genes involved in DNA damage repair (e.g. XRCC6, ERCC2, and MSH2) or cell-cycle checkpoint regulation genes (e.g. ATM, BRCA1, TP53, TP63, TP73, and BUB1B). LIMITATIONS REASONS FOR CAUTION: Using mature oocytes instead of GV-stage oocytes discarded from ICSI treatments may provide different results. No correction for multiple testing was carried out on individual genes because a small set of longevity-related genes was selected a priori for the analysis. The global trend was corrected for multiple testing and remained significant. This work was an observational study and, as no additional experimental work was performed, the associations described do not directly demonstrate the involvement of such genes in oocyte ageing. WIDER IMPLICATIONS OF THE FINDINGS: Growth, metabolism, and cell-cycle progression-related pathways that are known to be involved in somatic cell ageing were associated with ovarian ageing. If experimental data are obtained to support these associations, we suggest that interventions known to modulate these processes could benefit women suffering from ovarian ageing. STUDY FUNDING/COMPETING INTERESTS: G.E.J. is supported by a VENI grant from ZonMw (https://www.zonmw.nl). Work in the Houtkooper group is financially supported by an ERC Starting grant (No. 638290), a VIDI grant from ZonMw (No. 91715305), and the Velux Stiftung (No. 1063). M.G. declares several research and educational grants from Guerbet, Merck and Ferring (all location VUmc), outside the scope of the submitted work. The other authors report no competing interest. TRIAL REGISTRATION NUMBER: N/A.