Embryologie I OOGENESIS autumn 2024 Reproductive aging Zuzana Holubcová Department of Histology and Embryology zholub@med.muni.cz Reproductive aging - ↓ chance of natural conception - ↑ time to pregnancy - ↑ pregnancy loss/miscarriages - ↑ congenital defects - ↓number of oocytes retrieved after COS - ↓ fertility rate - ↓ developmental rate - ↑ implantation failure - ↑ incidence of genetically abnormal embryos Granulosa cells quality - mutilifactorial process - physiological factors - genetic factors - environmental and lifestyle factors - outcomes of donor cycles indicate that reproductive aging is related to ovarian not unterine factors - gradual age-related decline in the quatity and quality of oocytes in the ovaries Reproductive aging ❖Quantitative ovarian aging - age-related exhaution of ovarian reserve - continuous process - markers: - Age (chronological vs. biological) - Menstrual cycle characteristics - FSH - Estradiol (E2) - LH/FSH ratio - Inhibin B - Antimüllerian hormone (AMH) - Basal antral follicule count (AFC) - Basal ovarian volume - Basal stromal blood flow *amenorhea for >12 months * - age at menopause is highly heritable - extreme malnutrition related to earlier menopause Reproductive aging ❖Qualititative ovarian aging - diminishing of oocyte quality with advanced age ANEUPLOIDY Oocyte quality Granulosa cell deficiency Reduced vascularization and tissue remodelling Mitochondria dysfunction e.g. mtDNA mutation, lack of ATP production, ROS production Metabolic deficiency e.g. malnutrition, overglycation, impaired ubiquitinilation, lipoperoxidation, ER stress Oxidative stress and inflamation e.g. endometriosis, repeated ovulation Insuficient DNA damage repair Environmental and life style factors e.g. smoking, polutants, toxicants, medication Epigenetic changes and translational decline Aneuploidy = presence of abnormal number of chromosomes - chromome gain/loss resulting from unequal chromosome segregation Non-cancerous somatic cells <1% Sperm 1-4% Human MII oocyte ~20% Mouse MII oocyte <5% fetal losses 50% still births 4% live births 0.3% Maternal age effect = maternal age effect - incidence of oocyte aneuploidy increases with age Hassold and Hunt 2001 Egg aneuploidy ↑ trisomic pregnancies Prevalence of the common aneuploidies in newborns Aneuploidy Prevalence Trisomy 21 (Down syndrome) 1 : 700 Trisomy 18 (Edwards syndrome) 1 : 7.000 Trisomy 11 (Patau syndrome) 1 : 20.000 47, X (Turner syndrome) 1 : 2.5000 47, XXX ("super female") 1 : 1.200 females 47, XXY (Klinefelterův syndrome) 1 : 900 males 47, XYY ("supermale") 1 : 1000 males in women in their early 30s ~ 10-25% in women above 40 years ~ 50-90% Errors in maternal meiosis Segregation errors in female meiosis Nagaoka et al 2012 - recombination defects predisposes oocytes to chromosome segregation errors PREMATURE or NO separation of homologous chromosomes - recombination failure - inadequate number and position of crossovers random segregation of univalents Lane and Kauppi 2019 Webster and Schuh 2017 Embryonic aneuploidy Euploid embryo 50%100% 50% Segregation errors in female meiosis Meiosis I Most chromosome segregation errors occurr in meiosis I Some MI erros may be balanced in meiosis II Natural fertility courve Meiosis errors and aging Gruhn et al, Science 2019 Origin of human egg aneuploidy - Young age: nondisjunction of homologous chromosomes - Advanced age: premature separation of sister chromosomes Chromosome nondisjunction Reverse segregation Premature separation of sister chromatids Egg aneuploidy Natural fertility courveCohesion deterioration - age-dependent loss of cohesins from chromosome arms Zielinska et al 2015. Zielinska et al 2019. Melina Schuh - precocious splitting of sister kinetochores during meiosis I Natural fertility courve Separation of sister kinetochores in meiosis I promotes abnormal kinetochore-microtubule attachments Zielinska et al 2015.Thomas et al 2021. Meiosis I Cohesion deterioration Natural fertility courve Anomalous attachment of separated sister kinetochores may cause bivalent rotation, twisting and splitting in meiosis I Zielinska et al 2015.Thomas et al 2021. Meiosis I Cohesion deterioration Natural fertility courve Loss of cohesion induces kinetochore fragmentation in aged MII eggs. Zielinska et al 2019. Thomas et al 2021. Meiosis II Cohesion deterioration Natural fertility courveCohesion deterioration 40 years Mihalas et al. 2024 - reduced Sgo2 location at the pericentromeric bridge is associated with increased inter-sister kinetochore distance and incidence of single chromatids in MII oocytes METAPHASE II Chromosome lagging - distinct velocity types of chromosome laggards with different risk to result in aneuploidy Greg FitzHarris Mihajlovic et al 2021 - merotelic attachments promote chromosome lagging - controlled prolongation of meiosis I specifically lessens class-I lagging to prevent aneuploidy - smaller chromosomes actively moved to the center of metaphase plate - in the inner region, the chromosomes are pulled by the stronger bipolar MT forces, which facilitates premature chromosome separation Chromosome lagging Tomoya Kitajima Takenouchi et. 2024 - flourescent probes for invidual chromosomes - 3D tracking of meiosis I in live mouse oocytes → ↑risk of chromosome missegregation in aged oocytes with weakend cohesins Maternal age effectOrigin of human egg aneuploidy Human egg aneuploidy Chromosome nondisjunction Premature separation of sister chromatids Spindle instability Sub-proficient Spindle Assembly Checkpoint Holubcova et al. 2015 So et al. 2022. Zielinska et al. 2015Grugn et al 2019 Mihalas et al 2023 (preprint) Zielinska et al. 2019 Zielinska et al. 2015 Sakakibara et al., 2015 Patel et al. 2016 Ottollini et al. 2015 DNA damage response DSB meiotic recombination induced DNA damage DNA repair tolerance H2AX loss of ovarian reserve developmental arrest misscarriage birth defects genetic mutations reduced reproductive lifespan ROS UV Chemical toxicants Low tolerance for DNA damage Stringent quality control Sensitiveness to DNA damage TAp63 - isoform of p63 - homologue of p53 - proapoptotic factor Role of p53 and p73? - stage-dependent vulnerability to DNA damage DNA repair DNA damage survey DNA damage response DNA damage response Sharma et al 2024 DNA repair proteins are organized into distinct repair compartments in GV oocyte nucleus DNA damage response - detection of DSBs by prophase I oocyte - increased DSBs during meiotic maturation result in segregation errors due to chromosome fragmentation - no cell cycle arrest or significant delay in anaphase onset Mayer et al 2016 Neocarzinostatin(NCS) DNA damage response during aging Neocarzinostatin - age-related deterioration of DNA damage sensing and repairing machinery DNA damage response during aging Aged oocytes accumulate DNA damage and take longer than young oocytes to repair DNA damage DNA repair proteins are organized into distinct nuclear DNA repair compartments Aged oocytes show changes in DNA repair machinery, favoring error-prone NHEJ repair pathway Age-related cohesin loss results in reduced DNA damage repair efficiency Reduced capacity for DNA repair in aged oocytes Epigenetic regulation - regulation of gene expression without DNA sequence modifiation - affect DNA accessibility for transcription factors - gene trascription switched „ON“ and „OFF“ depending on cell needs Ubiquitylation Sumoylation - explains complexity of multicellular organism from a single genetic blueprint - enables cellular plasticity/genomic integrity during development and in response to environmental factors CpG 5mC/5hmC Global erasure of epigenetic marks fertilization PGCs Epigenetic reprograming during development (In)complete erasure? Transgenerational epigenetic inheritance?? Oocyte epigenomic profile DNA methylation Histone acetylation Histone methylation - Epigenetic marks established during oogenesis - Specific heterochromatin profile - Different histone variants and epigenic modifiers Global changes Translational decline - reduced translation in late-stage oocytes during ovarian aging might contribute to meiotic defects Danielson et al 2024 - mRNA polyadenylation, translation and protein levels are decreased in old mouse - oocytes premature CDK1 activation, and accelerated reentry into meiosis - dysfunction in the oocyte translation program associated with the decline in oocyte quality during aging in mouse o Radiation o Air pollution o PFAS o Endocrine disruptors o Plastics o Heavy metals etc.... 100.000 human-made chemicals released to environment 70.000 unknown effects Environmental factors Environmental factors ❖Plastics - Microplastics - particles 5 mm - 1 m - Nanoplastics - particles < 1 m ← degradation of plastics - found in human blood, milk, urine, placenta, meconium, follicular fluid, altered microbiome,... - difficult identification, classification and quantification - unknown effect on environment and human health altered epigenetics ❖PFAS = per- and polyfluoroalkyl substances - synthetic chemicals containg carbone-fluorine bonds - low biodegradability („forever chemicals“) Environmental factors Plasticizers Plasticizers ❖Endocrine disruptors = natural or man-made chemicals that may mimic or interfere with synthesis, secretion, transport, bidning, action or elimination of hormones in the human or animal body Herbicides Pesticides Oocyte quality hierarchy - limited oocyte pool hypothesis (Warburton 1989) Scaramuzzi et al 2011 - poor quality oocyte would not become dominant in young female ovary because of abundance of better quality oocytes - in older women with small ovarian reserve a defective oocyte would be more likely ovulated Human egg misery? Evolutionary advantage of female subfertility? Preference of quality over quantity?