Genetic insights into biological mechanisms governing human ovarian ageing

Abstract

Reproductive longevity is essential for fertility and influences healthy ageing in women^1,2, but insights into its underlying biological mechanisms and treatments to preserve it are limited. Here we identify 290 genetic determinants of ovarian ageing, assessed using normal variation in age at natural menopause (ANM) in about 200,000 women of European ancestry. These common alleles were associated with clinical extremes of ANM; women in the top 1% of genetic susceptibility have an equivalent risk of premature ovarian insufficiency to those carrying monogenic FMR1 premutations³. The identified loci implicate a broad range of DNA damage response (DDR) processes and include loss-of-function variants in key DDR-associated genes. Integration with experimental models demonstrates that these DDR processes act across the life-course to shape the ovarian reserve and its rate of depletion. Furthermore, we demonstrate that experimental manipulation of DDR pathways highlighted by human genetics increases fertility and extends reproductive life in mice. Causal inference analyses using the identified genetic variants indicate that extending reproductive life in women improves bone health and reduces risk of type 2 diabetes, but increases the risk of hormone-sensitive cancers. These findings provide insight into the mechanisms that govern ovarian ageing, when they act, and how they might be targeted by therapeutic approaches to extend fertility and prevent disease.

Extended Data Figure 1. Overview of ovarian reserve and follicular activity across reproductive life.

a, Key processes involved in follicular activity from fetal development to menopause showing the numbers of oocytes at each stage; b, Summary of key biological pathways involved in follicular activity and their relationship to stage of reproductive life. Follicles, consisting of oocytes and surrounding granulosa cells are formed in utero and maintained as resting primordial follicles in the cortex constituting the ovarian reserve. Follicles are sequentially recruited from the ovarian reserve at a rate of several hundred per month in childhood, peaking at around 900 per month at ~15 years of age. Following recruitment, follicles grow by mitotic division of granulosa cells and expansion of oocyte volume for almost 6 months until meiosis is reinitiated at ovulation and the mature oocyte is released into the oviduct. Waves of atresia (follicle death) accompany developmental transitions and growing follicles are continuously induced to undergo cell death such that, typically, only a single follicle matures to ovulate each month. As ovarian reserve declines the rate of follicle recruitment decreases, but the preovulatory follicles continue to produce substantial amounts of oestrogen, while other important hormones such as anti-Müllerian hormone and inhibin-B decline, leading to upregulation of the hypothalamus-pituitary gonadal axis.

Extended Data Figure 2. Overview of performed analyses.

Extended Data Figure 3. Consistency of effect estimates across analyses methods and strata.

Comparison of effect estimates from: a, Cox proportional hazards regression in UK Biobank with linear regression effect estimates from the overall meta-analysis (“Effect full metaanalysis”); b, Cox proportional hazards regression in UK Biobank with linear regression effect estimates from the meta-analysis excluding UK Biobank (“Effect 1KG+BCAC”); c, linear regression in UK Biobank with linear regression effect estimates from the meta-analysis excluding UK Biobank (“Effect 1KG+BCAC”). Comparison of linear regression effect estimates from: d, UK Biobank GWAS vs. the meta-analysis of 1000 Genomes imputed studies; e, UK Biobank GWAS vs. meta-analysis of samples from the Breast Cancer Association Consortium (BCAC); f, meta-analysis of BCAC samples vs. the meta-analysis of 1000 Genomes imputed studies; g, 23andMe replication analysis (rescaled) vs. overall meta-analysis. HR, hazard ratio from Cox proportional hazards model; r, Pearson correlation coefficient; blue line is y=x for reference. Note: P values < 1×10^-300 are shown as 1×10^-300.

Extended Data Figure 4. Deviation from additive effects and distribution of estimated heritability across chromosomes.

a-d, Genome-wide significant signals showing departure from an additive model. We tested the identified signals for departure from an additive allelic model. a, rs11668344 shows no deviation from an additive allelic model; b, rs11670032 and c, rs28416520 show deviation from the additive allelic model and a recessive effect; and d, rs75770066 shows a heterozygote effect. The mean and 95% confidence interval around the mean estimate are shown for each genotype. The expected mean ANM for the heterozygotes is the average of the mean ANM in the homozygote groups. The dashed orange line shows the effect estimate by genotype from linear regression based on an additive allelic model. Estimated ANM for each genotype was calculated as constant from regression model + number alleles × effect estimate from regression model. The dashed grey line indicates the additive effect estimate by genotype from a model adjusting for the dominance deviation effect of the heterozygote group (solid grey line). All regression models were adjusted for centre, genotyping chip and genetic principal components. ANM, age at natural menopause; dom dev, dominance deviation. e, The percentage of the total heritability explained that was attributable to each chromosome (observed heritability) is compared with the expected proportion calculated on the basis of chromosome size. The heritability of ANM was not uniformly distributed across chromosomes in proportion to their size. The X-chromosome did not explain more heritability than expected given its size, however chromosome 19 explained 2.36% [1.98-2.75] of the trait variance – greater than the individual contributions of nearly all larger chromosomes (weighted average for chromosomes 1-18: 1.7%, s.e 0.2%) and ~2.5x more than expected given its size. This was partially attributable to a single locus at 19q13 which explained ~0.75% trait variance and where we mapped 6 independent signals (Supplementary Table 2). The dashed line shows the mean ratio of expected to observed heritability across all chromosomes. Chromosome size was estimated based on the number of genetic variants.

Extended Data Figure 5. Gene co-regulation networks for age at menopause genes with those co-regulated with MCM8 highlighted.

a, Gene co-regulation network for genes relating to age at menopause. Nodes indicate genes that either in a cis region from the GWAS or have been prioritized by Downstreamer, edges indicate a co-regulation relationship with a Z-score >4. Co-regulation is defined as the Pearson correlation between genes in a scaled eigenvector matrix derived from a multi-tissue gene network. Cis genes are defined as genes that are within +/-300kb of a GWAS top hit for age at menopause. Trans genes are defined as having been prioritized by Downstreamer’s co-regulation analysis and are not within +/-300kb of a GWAS top hit. Downstreamer prioritizes genes by associating the gene p-value profile of the GWAS (calculated using PASCAL) to the co-regulation profile of each protein coding gene. Only genes where this association passes Bonferroni significance are shown as trans genes. Colours of nodes indicate the following: Teal indicates Cis genes, Dark Teal indicates Trans genes and Yellow indicates genes with a 1st degree relation to MCM8. b, Gene co-regulation network showing the genes that have a first degree relationship with MCM8 with a Z-score >4. Width of the edge indicates the Z-score of the co-regulation relationship. Colours indicate the same as in a, with the exception of Yellow, as all genes indicated have a 1st degree relation to MCM8.

Extended Data Figure 6. DNA damage response and repair pathways implicated in reproductive ageing in humans.

a, Consequences of replication stress annotated with genes involved that were within 300kb of the age at natural menopause (ANM) signals; b, Genes involved in downstream DNA damage response and repair pathways with those within 300kb of an ANM signal shown in blue. A full list of genes involved in DNA damage response and apoptosis annotated with genome-wide signals for ANM is provided in Supplementary Table 19. MRN, MRN-MRE11-RAD50-NBS1 complex; RPA, Replication Protein A including a subunit encoded by RPA1; RFC, Replication Factor C including a subunit encoded by RFC1; 9-1-1, RAD9-HUS1-RAD1 complex.

Extended Data Figure 7. Cluster plot of expression of consensus genes identified from the genome-wide analyses in germ cells across different developmental stages.

Genes were selected from the GWAS signals, based on in silico prioritisation (Supplementary Table 5). Of the 283 consensus genes highlighted by the GWAS, 258 passed QC and were available in the expression dataset. Gene expression was measured in human fetal primordial germ cells^,, and oocytes and granulosa cells in adult follicles (dataset generated in this study). Plot shows Z-scores, calculated by subtracting the mean transcripts per million (TPM) in all samples for a gene and dividing by the standard deviation. GC, granulosa cell; MII, meiosis II; PGC, primordial germ cell; Wks, weeks.

Extended Data Figure 8. Relationship between decreased ovarian reserve and gene expression.

Open bar/dot groups – control maternal diet, normal ovarian reserve. Grey bar/dot groups: obesogenic maternal diet, reduced ovarian reserve. a, Ovarian follicular reserve in young adulthood in wild-type mice. Total follicles/mm ovarian tissue at 12 weeks. Dots: individual observations. Bar heights and error bars: mean± SEM. n= 8 biologically independent animals from different litters in each group. P=0.0091 derived from 2-way ANOVA after correction for multiple hypothesis testing. b, Brsk1 expression in the same animals, measured using qrtPCR and expressed as average copy number. Dots: individual observations. Bar heights and error bars: mean± SEM. n= 8 biologically independent animals from different litters in each group. P=0.0001 derived from 2-way ANOVA after correction for multiple hypothesis testing. c, Wee1 expression in the same animals, measured using qrtPCR and expressed as average copy number. Dots: individual observations. Bar heights and error bars: mean± SEM. n= 8 biologically independent animals from different litters in each group. P=0.0256 derived from 2-way ANOVA after correction for multiple hypothesis testing. d, Dmc1 expression in the same animals, measured using qrtPCR and expressed as average copy number. Dots: individual observations. Bar heights and error bars: mean± SEM. n= 8 biologically independent animals from different litters in each group. P=0.00001 derived from 2-way ANOVA after correction for multiple hypothesis testing. e, Mapt expression in the same animals, measured using qrtPCR and expressed as average copy number. Dots: individual observations. Bar heights and error bars: mean± SEM. n= 8 biologically independent animals from different litters in each group. P=0.0378 derived from 2-way ANOVA after correction for multiple hypothesis testing. qrtPCR, quantitative reverse transcription polymerase chain reaction; SEM, standard error of mean. *, P<0.05; **, P<0.01; ***, P<0.001.

Extended Data Figure 9. Chek2 deletion increases reproductive lifespan in mouse.

a, Representative images of ovarian sections of 1.5-and 13.5-month-old wild type (WT) and Chek2^-/- mice stained with PAS-Hematoxylin. Primordial follicles (inset (i)), primary follicles (inset (ii)), secondary follicle (white arrow) and antral follicle (black arrow) are shown. Scale bar represents 200 μm. b-e, Quantification of the number of follicles (by class and total) present in WT and Chek2^-/- mice ovaries: b, c, 1.5-month-old; d, e, 13.5-month-old. The numbers in parentheses correspond to the total number of ovaries analysed. f, Serum AMH (ng/ml) in 16-17 months old Chek2^-/- mice. The numbers in parentheses correspond to the number of mice assessed. g-i, Diagram illustrates the gonadotrophin stimulation of 13.5-month old females. Numbers in parentheses show: g, the number of MII oocytes retrieved per female; h, the number of MII oocytes fertilized; and i, the number of fertilized oocytes assessed for blastocyst formation. j, Litter size of WT and Chek2^-/- females throughout the reproductive life span. Litter sizes from 9 WT and 5 Chek2^-/- females are shown. Breeding cages contained one male and one female. Generalized linear model analysis showed maternal age effect, but no effect on genotype on litter sizes. k, Image of healthy pups born to 13 month-old Chek2^-/- females. b-i, Two sample t and Fisher’s exact tests were used to compare WT and Chek2^-/- for statistical significance: *, P<0.05; **, P<0.025; ***, P<0.001. All P-values are two sided. Error bars indicate standard error of mean. Box-and-whisker plots show interquartile range and median (b-g). an, antral follicle; hCG, human chorionic gonadotrophin; pMSG, pregnant mare serum gonadotrophin; pri, primary follicle; P0, primordial follicle; sec, secondary follicle; WT=wildtype. Mouse strain: maintained on a mixed background, C57BL/6 129Sv, accession number BRC03481 at the RIKEN Bioresource Centre.

Extended Data Figure 10. Conditional knockout Chek1 females are infertile due to requirement for Chek1 during preimplantation embryo development.

a, Schematic of the conditional-knockout mouse model of Chek1 (Chek1 cKO) in the female germline using the Ddx4-Cre. A similar approach was used for Zp3-Cre. b, In the ovarian sections stained with haematoxylin and eosin, we found follicles, corpora lutea (CL) and oocytes which contain nuclear structures (indicated with arrowheads in the magnified right hand panel). These findings suggest that estrus cycles and ovulation followed by corpus luteum formation are independent from Chek1 disruption in oocytes in vivo. c, Litter size of Chek1 cKO females. Three females older than 5 weeks age were mated with C57BL/6J males. Five independent littermate females (F/+, Tg-/Tg-; F/F, Tg-/Tg-; or F/+, Tg+/Tg-) were used as Chek1 controls (ctrl). While Chek1 ctrl females delivered normally, Chek1 cKO females delivered no litters (**, Mann Whitney test P=0.0179). Thus, these results indicate that CHEK1 is essential in the female germline. d, Litter size of Chek1-cKO and controls using the Zp3-Cre during follicular growth. 3 months old control (Chek1 F/F; Chek1 ctrl, n=4) and conditional knockout (Chek1 F/F; Chek1 cKO with Zp3-Cre, n=4) were three-times consecutively mated with wild-type (Chek1^+/+) males, and the number of live (left) and dead (right) pups was monitored. While Chek1 ctrl delivered a normal amount of live pups, Chek1 cKO had only a reduced amount of perinatally dead pups (Mann-Whitney U Test: ***, P<0.001; **, P<0.01). Numbers in parentheses show the number of litters. e, The mean number of all ovulated eggs (the sum of MII oocytes and fertilized MII oocytes) per mouse with SEM (Mann Whitney U Test, P=0.126). Each data point presents the no. of eggs per mouse. 3-5 months old Chek1 ctrl (n=3) and Chek1 cKO (n=5) females were mated with wild-type (Chek1^+/+) males after pMSG + hCG stimulation. The number of ovulated eggs isolated 18 h post hCG stimulation and additional 10 h cultured in vitro was scored. The number of mice is shown in brackets. f, The proportion of fertilized MII oocytes to all ovulated eggs with a binomial confidence interval (*, Fisher’s Exact Test, P=0.012; 95% CI 1.9–6.0; OR: 2.62). Numbers in parentheses show the total number of analysed eggs. g, The proportion of embryos that developed to blastocysts with binomial confidence interval (***, Fisher’s Exact Test, P<0.0001). Fertilized MII oocytes (zygotes) were isolated from pMSG + hCG stimulated females 18h post hCG administration and cultured in vitro for 96 hours (~ E3.5) when development to blastocyst was scored. Data are pooled from four independent experiments. The number of embryos is shown in brackets. h, Fertilized eggs from Chek1 ctrl (n=18) and Chek1 cKO (n=13) females were fixed and stained for DNA (DAPI). All fertilized eggs from both genotypes showed normal pronuclei formation. The data were pooled from two independent experiments. Asterisks mark polar bodies. i, The majority of Chek1 ctrl embryos formed blastocyst (g), but Chek1 cKO embryos were arrested mainly in 3-8 cell stages. Representative bright-field images are shown. j, Proportion of developmental stages 2 cell, 3-4 cell and 5-8 cell (**, Cochran-Armitage Trend Test, P=0.0073). Chek1 ctrl and Chek1 cKO zygotes were isolated from 13 Chek1 ctrl and 6 Chek1 cKO pMSG + hCG stimulated females 18h post hCG administration and cultured in vitro for 49 hours. Embryos were fixed and stained for ƔH2AX by immunofluorescence. DNA was visualized by DAPI (l). k, Proportion of embryos with genome fragmentation with binomial confidence interval (***, Fisher’s Exact Test, P<0.0001). Data are pooled from two independent experiments. The number of embryos is shown in brackets. l, Chek1 ctrl and Chek1 cKO zygotes (j,k) were fixed and stained for ƔH2AX (magenta) by immunofluorescence. DNA (gray) was visualized by DAPI. Arrows indicate genome fragments. Asterisks indicate polar bodies. These findings suggest that maternally expressed Chek1 is critical for genome integrity protection during first divisions of preimplantation embryos in mice. All P-values are two sided. Box-and-whisker plots show interquartile range and median. Strains: C57BL/6-FVB mixed background for a-c (Chek1 cKO, Ddx4-Cre); C57BL6-CD1 mixed background (Chek1 cKO, Zp3-Cre) for panels d-l.

Extended Data Figure 11. Extended reproductive lifespan in females carrying an extra copy of Chek1 (sChek1).

a, mRNA expression levels of Chek1 in oocytes, numbers in parentheses show the number of mice stimulated for retrieving the oocytes. b, Representative images of ovarian sections of 1.5 and 13.5-month-old wild type (WT) and sChek1 mice stained with PAS-hematoxylin. Primordial follicles (inset (i)), primary follicles (inset (ii)), secondary follicle (white arrow) and antral follicle (black arrow) are shown. Scale bar: 200 μm. c-f, Quantification of the number of follicles (by class and total) present in WT and sChek1 littermates: c, d, 1.5-month-old; e, f, 13.5-month-old. The numbers in parentheses correspond to the total number of ovaries analysed. g-j, MII oocytes retrieved in response to pMSG and hCG, proportion of euploid oocytes, proportion fertilized and proportion developed to blastocysts at different ages of WT and sChek1 mice. Numbers in parentheses show: g, the number of MII oocytes retrieved per female; h, the number of oocytes assessed for aneuploidy; i, the number of MII oocytes fertilized; and j, the number of fertilized oocytes assessed for blastocyst development. k, Proportion of live births relative to transferred embryos from in vitro fertilized oocytes from aged mice (16 months), the numbers in parenthesis show the embryos transferred. l, Photo of healthy pups born to 16-month old sChek1 females after IVF. m, Litter sizes from F2 females or males from aged sChek1 females after IVF treatment in k, compared to females of equivalent ages that were naturally breeding. Note that for natural breeding there were two females and one male per breeding cage, whereas F2 cages contained a single male and one female. Therefore, litter sizes are an underestimate for the IVF-conceived pups. n, Litter sizes of WT and sChek1 females throughout their reproductive life span. Data are from six breeding cages, three for each genotype. Each breeding cage contained one WT male and two females that were either WT or sChek1. Generalized linear model analysis showed maternal age effect, but no effect on genotype on litter sizes. a-k, Two sample t and Fisher’s exact tests were used to compare WT and sChek1 for statistical significance: *, P<0.05; **, P<0.025; ***, P<0.001. All P-values are two sided. Error bars indicate standard error of mean. Box-and-whisker plots show interquartile range and median (c-g, m). an=antral follicle; hCG= human chorionic gonadotrophin; IVF=in vitro fertilization; NB=natural breeding; F2-f= F2 female; F2-m= F2 male; pMSG=pregnant mare serum gonadotrophin; pri=primary follicle; P0=primordial follicle; sec=secondary follicle; WT=wild type. Mouse strain: inbred from mixed background C57BL/6 129Sv.

Figure 1. Manhattan plot representing GWAS discovery analysis.

Previously identified loci in purple, novel loci in blue. Plotted variants have P<0.01 with P<1x10^-300 truncated. Insert: Effect sizes and minor allele frequencies of the loci. LOF, loss of function

Figure 2. Polygenic prediction of age at menopause.

a, Mean polygenic score (PGS; scaled to have mean=0, SD=1) for a given age at natural menopause (ANM). Higher PGS indicates later ANM. b, c, Association of each centile of PGS vs the 50th with, b, early menopause and, c, premature ovarian insufficiency. Higher PGS indicates earlier ANM.

Figure 3. Genetic manipulation of Chek1 or Chek2 extends reproductive lifespan in mouse models.

Numbers of follicles in young and aged, a, Chek2^-/- or, b, sChek1 females. Numbers of ovaries analysed in parentheses. c, Response to gonadotrophin stimulation of 13.5-month-old Chek2^-/- and sChek1 females assessed by the number of MII oocytes retrieved. Numbers of stimulated females in parentheses. Box-and-whisker plots show interquartile range and median. Two-sample t and Fisher’s exact tests used for comparisons: *, P<0.05; **, P<0.025; ***, P<0.001.