Polycystic ovary syndrome is transmitted via a transgenerational epigenetic process

Abstract

Polycystic ovary syndrome (PCOS) is the most common reproductive and metabolic disorder affecting women of reproductive age. PCOS has a strong heritable component, but its pathogenesis has been unclear. Here, we performed RNA sequencing and genome-wide DNA methylation profiling of ovarian tissue from control and third-generation PCOS-like mice. We found that DNA hypomethylation regulates key genes associated with PCOS and that several of the differentially methylated genes are also altered in blood samples from women with PCOS compared with healthy controls. Based on this insight, we treated the PCOS mouse model with the methyl group donor S-adenosylmethionine and found that it corrected their transcriptomic, neuroendocrine, and metabolic defects. These findings show that the transmission of PCOS traits to future generations occurs via an altered landscape of DNA methylation and propose methylome markers as a possible diagnostic landmark for the condition, while also identifying potential candidates for epigenetic-based therapy.

Keywords: AMH; PCOS; developmental programming; epigenetics; fertility; inheritance; metabolic disorder; methylation; neuroendocrine; transgenerational.

Graphical abstract

Figure 1

Prenatal AMH exposure induces transgenerational transmission of PCOS neuroendocrine traits to multiple generations (A) Schematic illustration of experimental design employed to generate F1, F2, and F3 offspring. (B) Anogenital distance (AGD) measurement over postnatal days (P) 30, 40, 50, and 60 in adult control females (n = 14), PAMH F1 (n = 13–16), PAMH F2 (n = 14), and PAMH F3 (n = 14). (C) Plasma testosterone concentration in adult females (P60–P90) in diestrus (CNTR F1, n = 12; PAMH F1, n =12; PAMH F2, n = 14; PAMH F3, n = 15). (D) Plasma LH levels in adult (P60–P90) diestrus females (CNTR F1, n = 14; PAMH F1, n = 11; PAMH F2, n = 17; PAMH F3, n = 17). (E) Quantification of the number of corpora lutea (CL) in the ovaries of adult diestrus female mice (CNTR F1, n = 8; PAMH F1, n = 3; PAMH F3, n = 3). (F) Representative estrous cyclicity of 8 mice/treatment group during 16 consecutive days. M/D: metestrus/diestrus phase, P, proestrus; E, estrus. (G) Quantitative analysis of estrous cyclicity in adult (P60–P90) mice from control and PAMH lineages. Scatterplot representing the percentage (%) of time spent in each estrous cycle in CNTR F1 (n = 19), PAMH F1 females (n = 19), PAMH F2 females (n = 14), and PAMH F3 females (n = 12), respectively. The horizontal line in each scatter plot corresponds to the median value. The vertical line represents the 25^th–75^th percentile range. (H) Number of pups per litter. (I) Time to first litter (number of days to first litter after pairing). (J) Fertility index: number of litters per females over 3 months, quantified per generation and pairing. Data in (B)–(E) and (H)–(J) are represented as mean ± SEM. For statistical analysis, p values were calculated by Kruskal-Wallis followed by Dunn's multiple comparisons post hoc test (B and G) or by one-way ANOVA followed Tukey’s multiple comparison post hoc test. ^∗p < 0.05; ^∗∗p < 0.005; ^∗∗∗p < 0.0005; ^∗∗∗∗p < 0.0001.

Figure 2

Prenatal AMH exposure causes a transgenerational transmission of metabolic derangements in 6-month-old female offspring (A) Body composition of CNTR (n = 16; 6 months old), PAMH F1 (n = 16; 6 months old), PAMH F2 (n = 11–12; 6 months old), and PAMH F3 (n = 16; 6 months old), presented as body weight (g), percent fat mass normalized to body weight (g), and percent of lean mass. (B and C) Oral glucose tolerance test (GTT) upon 14 h of fasting (B) and insulin tolerance test (ITT) upon 4-h fasting (C) in CNTR (n = 7; 6 months old) and PAMH F1 adult female offspring (n = 7; 6 months old). (D) Glucose levels upon 12 h of fasting in CNTR (n = 10; 6 months old), PAMH F1 (n = 10; 6 months old), and PAMH F3 (n = 7; 6 months old) female offspring. (E) LSFM images of solvent-cleared pancreata dissected from 6-month-old CNTR and PAMH F1 female mice. Left: 3D projection of immunostaining for insulin (red) and glucagon (white); scale bars, 150 μm. Middle: 3D analysis of rendered pancreatic islets expressing insulin; scale bars, 200 μm. Right: single plane optical reslice of the pancreata; scale bars, 200 μm. (F) Plasma insulin levels upon 12 h of fasting in CNTR (n = 10; 6 months old) and PAMH F1 (n = 10; 6 months old) female offspring. Values are represented as the mean ± SEM. For statistical analysis, p values were calculated by one-way ANOVA followed by Tukey’s multiple comparison post hoc test (A, body mass), by Kruskal-Wallis followed by Dunn's multiple comparisons post hoc test (A, % fat mass, % lean mass; D) or by an unpaired two-tailed Student’s t test (B and E). Statistical significance for all analyses were ^∗p < 0.05; ^∗∗p < 0.005; ^∗∗∗p < 0.0005; ^∗∗∗∗p < 0.0001.

Figure 3

RNA-seq analysis of ovarian tissue in control and F3 PCOS animals points to altered gene expression linked to ovarian and metabolic functions and to inflammatory response (A) Schematic illustration of the experimental design. (B) MA plot of gene expression changes in the PAMH F3 ovaries (n = 4) versus control (prenatally PBS-treated; CNTR, n = 3) for all experimental conditions. Ovaries were dissected from CNTR or PAMH F3 adult females (P60) at diestrus. The MA plot represents the estimated log₂ fold change as a function of the mean of normalized counts. Significant genes were selected when adjusted p value lower than 0.05. For significant genes, a selection of first gene names according to the adjusted p value is displayed in red for the upregulated genes and blue for the downregulated genes. (C) Pie chart refers to the number of genes upregulated and downregulated when comparing PAMH F3 with CNTR ovaries (p_adj ≤ 0.05). (D) STRING protein network analysis of the downregulated genes revealed strong interaction between genes involved in regulating IGF transport and uptake by IGFBPs. (E–H) Functional annotation charts using DAVID performed on the differentially regulated genes either decreased in PAMH F3 versus CNTR (blue, E and F) or increased in PAMH F3 versus CNTR (red, G and H). Significance is indicated as −log10 p value. (I–L) Histograms significantly show enrichment in the PAMH F3 ovaries versus CNTR of genes involved in the negative regulation of insulin secretion (I), follistatin (Fst; J), lipid metabolism (K), and inflammatory response (L). ^∗p_adj < 0.05; ^∗∗p_adj < 0.005; ^∗∗∗p_adj < 0.0005.

Figure 4

Preponderance of hypomethylations in the ovarian tissue of F3 PCOS animals compared with controls and biological process of hypomethylated and hypermethylated genes (A) Schematic illustration of the experimental design. Methylated DNA immunoprecipitation and deep sequencing (MeDIP-seq) was performed in control ovaries (CNTR n = 3, independent samples at diestrus, P60) and PAMH F3 ovaries (n = 3, independent samples at diestrus, P60). (B) MA plot of MeDIP-seq reads. The MA plot represents the log₂ fold change as a function of the mean of normalized counts. Log₂ fold change corresponds to shrunk fold change as calculated with the method proposed in Love et al. (2014). Red dots correspond to significantly different hypermethylated and hypomethylated regions in the ovaries of PAMH F3 animals as compared with controls, adjusted p ≤ 0.05. (C) Total number of methylated regions detected as hyper- or hypomethylated after comparing MeDIP-seq data of the two groups (PAMH3 F3 versus CNTR). (D) Functional annotation plot showing the proportion of methylated regions falling into several genomic features. Upstream refers to upstream regulatory regions. These are regions located −20/−1 kb away from the TSS. Promoter-TSS refers to regions located −1 kb/+100 bp around the TSS and TTS refers to regions located −100 bp/+1 kb around the TTS. Plots are shown for hypomethylated and hypermethylated regions. (E) Venn diagram shows the overlap between genes associated with hypermethylated and hypomethylated regions in ovarian tissues of PAMH F3 mice (MeDIP-seq data) with the 102 DEGs obtained from the RNA-seq analysis. (F) The table shows the list of the 4 common genes found between the MeDIP-seq and the RNA-seq. It details the methylation status, the gene expression changes, and gene ontology related either to ovarian function or PCOS and references when it applies. (G and H) Functional enrichment analysis performed by DAVID on the genes associated with hypomethylated regions when comparing PAMH F3 versus CNTR. GO biological processes (top 20 most significant processes) and KEGG pathway are shown. Significance is indicated as −log10 p value. (I) STRING protein network prediction interaction of proteins associated with glucose metabolism, insulin signaling, insulin response, and insulin receptor binding. (J and K) Functional enrichment analysis performed by DAVID on the genes associated with hypermethylated regions when comparing PAMH F3 versus CNTR. GO biological processes and KEGG pathway are shown. Significance is indicated as −log10 p value.

Figure 5

Chromosomal distribution of DNA methylation reads and methylation signatures in the ovary and hypothalamus of PAMH F3 mice (A) Manhattan plot showing the association of methylated positions along the chromosomal positions. x axis represents methylated regions along the chromosomes. y axis is the −log₁₀ (adjusted p value), which is the significance of differentially methylated regions when comparing PAMH F3 versus CNTR. Sign of −log₁₀ (adjusted p value) corresponds to the direction of methylation change (hyper- or hypomethylated). Red dots show the peaks related to genes associated to hypomethylated regions and whose functional annotations are associated with insulin stimulus, glycolysis/gluconeogenesis and T2D, as depicted in the STRING analysis in Figure 4I. Numbers depicted below the Manhattan plot refer to the total number of significant deregulated peaks with p_adj ≤ 0.05 per chromosome. (B) Representative UCSC Genome Browser views of Tet1 and Uhrf1 locuses with DNA methylation peaks in ovarian tissues of CNTR versus PAMH F3 mice. Differential methylation analyses revealed that the 5-mC is decreased at the highlighted regions in PAMH F3 mice compared with the CNTR. Tet1, p_adj = 0.018; Uhrf1, p_adj = 0.01 (peak 1)/0.02 (peak 2). (C) Genomic DNA was isolated from hypothalami dissected from CNTR (n = 6–7) and PAMH F3 offspring (n = 4–5) and MeDIP-PCR experiments performed in the two groups of animals. Unpaired two-tailed Mann-Whitney U test, ^∗p < 0.05, n.s. not significant. (D) qRT-PCR analyses using primers against the genes listed were performed in hypothalamic tissues of CNTR (n = 6–8) and PAMH F3 offspring (n = 4–5). Unpaired one-tailed Mann-Whitney U test, ^∗p < 0.05, ^∗∗p < 0.005; n.s. not significant. Data in (C) and (D) are presented as mean ± SEM.

Figure 6

Epigenetic therapy restores PCOS neuroendocrine, reproductive, and metabolic traits in PAMH F3 adult females (A) Schematic of experimental design whereby adult (6 months old) PAMH F3 females have been treated or not with i.p. injections of SAM. SAM functions as the primary methyl donor for transmethylation reactions and acts by adding 5′ methylcytosine groups to the otherwise hypomethylated DNA. (B) Representative estrous cyclicity and experimental design. Prenatally PBS-treated, group 1 (n = 5, 6 months old); PAMH F3 animals, group 2 (n = 5, 6 months old); and SAM-treated, group 3 (n = 5, 6 months old). The y axis refers to the different stages of the estrous cycle: metestrus/diestrus (M/D), estrus (E), and proestrus (P). The x axis represents the time course of the experiments (days). Tail-blood samples were collected for LH and T measurements at day 10 (diestrus), before the beginning of the treatment, and trunk blood was collected at day 25 (diestrus) at the moment of the sacrifice, corresponding to the end of the treatment period. (C) Scatterplot representing the percentage (%) of time spent in each estrous cycle in the three groups of animals, respectively. The horizontal line in each scatter plot corresponds to the median value. The vertical line represents the 25th–75th percentile range. (D) Mean LH levels were measured in diestrus CNTR mice (n = 10) and in group 2 (n = 5) and group 3 (n = 5) before the treatment (day 10) and after the treatment (day 25). (E) Mean T levels measured in diestrus in CNTR mice (n = 10) and in group 2 (n = 5) and group 3 (n = 5) before the treatment (day 10) and after the treatment (day 25). (F) Body composition in the three experimental groups (n = 5 for each group, 6 months old) presented as body weight (grams), percent fat mass normalized to body weight (grams), and percent lean mass normalized to body weight (grams). (G) Mean total glucose levels (mg/dL) in diestrus CNTR mice (n = 11), in PAMH F3 mice (group 2; n = 10), and in PAMH F3 mice after SAM treatment (group 3; n = 5). (H) Representative photomicrographs showing insulin-expressing β cells (red) and glucagon-expressing α cells (white) in the pancreata of CNTR, PAMH F3, and PAMH F3 + SAM mice (females, 6 months old). Scale bar, 50 μm. (I) Quantitative analysis of the mean area of the islets in CNTR (n = 4), PAMH F3 (n = 5), and PAMH F3 after SAM treatment (n = 5). Values in (D)–(G) and (I) are represented as the mean ± SEM. For statistical analysis, p values were calculated by Kruskal-Wallis test followed by Dunn’s multiple comparison post hoc test (C), by one-way ANOVA followed by Tukey’s multiple comparison post hoc test (D, E, G, and I), or by Kruskal-Wallis followed by Dunn's multiple comparisons post hoc test (F). Statistical significance for all analyses was ^∗p < 0.05; ^∗∗p < 0.005; ^∗∗∗p < 0.0005; ^∗∗∗∗p < 0.0001; n.s. not significant.

Figure 7

Common epigenetic signatures in human blood samples from women with PCOS (A) Schematic illustration of the experimental design. Genomic DNA was isolated from blood samples of a case-control study comprising two cohorts of women. Group 1: women with and without PCOS (CNTR). Group 2: post-pubertal control daughters born to mothers without PCOS (CNTR-D) and daughters with PCOS born to mothers with PCOS (PCOS-D). Methylated DNA immunoprecipitation using antibody against anti-5mC, followed by PCR (MeDIP-PCR) using specific primers against the genes listed in (B) and (C) was performed in the two groups. (B) MeDIP-PCR analyses in CNTR women (n = 15) and women with PCOS (n = 32). (C) MeDIP-PCR analyses in daughters from the control group (CNTR-D, n = 3) and daughters with PCOS of women with PCOS (PCOS-D, n = 5). Data are presented as mean ± SEM. Unpaired two-tailed Mann-Whitney U test. ^∗p < 0.05; ^∗∗p < 0.005; n.s. not significant.