Prenatal androgen exposure causes a sexually dimorphic transgenerational increase in offspring susceptibility to anxiety disorders

Abstract

If and how obesity and elevated androgens in women with polycystic ovary syndrome (PCOS) affect their offspring's psychiatric health is unclear. Using data from Swedish population health registers, we showed that daughters of mothers with PCOS have a 78% increased risk of being diagnosed with anxiety disorders. We next generated a PCOS-like mouse (F₀) model induced by androgen exposure during late gestation, with or without diet-induced maternal obesity, and showed that the first generation (F₁) female offspring develop anxiety-like behavior, which is transgenerationally transmitted through the female germline into the third generation of female offspring (F₃) in the androgenized lineage. In contrast, following the male germline, F₃ male offspring (mF₃) displayed anxiety-like behavior in the androgenized and the obese lineages. Using a targeted approach to search for molecular targets within the amygdala, we identified five differentially expressed genes involved in anxiety-like behavior in F₃ females in the androgenized lineage and eight genes in the obese lineage. In mF₃ male offspring, three genes were dysregulated in the obese lineage but none in the androgenized lineage. Finally, we performed in vitro fertilization (IVF) using a PCOS mouse model of continuous androgen exposure. We showed that the IVF generated F₁ and F₂ offspring in the female germline did not develop anxiety-like behavior, while the F₂ male offspring (mF₂) in the male germline did. Our findings provide evidence that elevated maternal androgens in PCOS and maternal obesity may underlie the risk of a transgenerational transmission of anxiety disorders in children of women with PCOS.

Fig. 1. Risk of having an anxiety in daughters of women with PCOS.

A, B Crude and adjusted hazard ratios (HRs) and 95% confidence intervals (CIs) for the association between maternal PCOS and daughter’s diagnosis of anxiety in the Swedish nation-wide register-based study. Adjusted model is adjusted for maternal age, maternal country of birth, maternal education, maternal and paternal psychiatric history, and year of birth of child.

Fig. 2. Prenatal androgen exposure causes transgenerational anxiety-like behavior in female offspring.

A Schematic illustration of experimental design. Prior to mating with male mice fed control diet, F₀ mothers were fed control diet or high fat-high sucrose (HFHS) diet for 6 weeks. During embryonic day (E) 16.5−E18.5 pregnant mice were injected subcutaneously with 250 µg of dihydrotestosterone (DHT), a non-aromatizable androgen, dissolved in 50 µL of sesame oil or 50 µL of sesame oil alone (vehicle) resulting in four lineages; CD+Vehicle (control); CD+DHT (androgenized); HFHS+Vehicle (obese); and HFHS+DHT (obese- androgenized). F₁ female and male offspring were mated with unrelated males and females fed CD, respectively, to generate F₂ and thereafter to generate F₃ offspring. Anxiety-like behavior was tested in female and male offspring according to the graph. Male germline refers to F₂ and F₃ male offspring from F₁ males. F₂ and F₃ male siblings are brothers to F₂ and F₃ female offspring. B–D Time spent in the open arms in the elevated plus maze (EPM); time spent in the closed arm of the EPM; time spent in the center of the open filed (OF); time spent in the periphery of the OF. F1: two-way ANOVA, Tukey’s post hoc analysis; F₂ and F₃: one-way ANOVA, Dunnett’s post hoc analysis. All data are presented as mean ± s.e.m. DHT dihydrotestosterone, CD control diet, HFHS high-fat high-sugar, Mat maternal, GMat grand-maternal, GGMat great-grand maternal. Numbers of mice are stated in the bars of each group.

Fig. 3. Prenatal androgen exposure with and without maternal obesity causes transgenerational anxiety-like behavior in male offspring following the male germline.

A–C Time spent in the open arms in the elevated plus maze; time spent in the closed arms of the elevated plus maze; time spent in the center of the open field; time spent in the periphery of the open field. F₁– mF₃: two-way ANOVA, Tukey’s post hoc analysis. All data are presented as mean ± s.e.m. DHT dihydrotestosterone; CD control diet, HFHS high-fat high-sugar, Mat maternal, GMat grand-maternal, GGMat great-grand maternal. The numbers of mice are stated in the bars of each group.

Fig. 4. Prenatal androgen exposure with and without maternal obesity causes anxiety-related gene expression changes in the amygdala of F₃ female and mF₃ male offspring.

A Schematic illustration showing low-density TaqMan array of the amygdala. B Violin plots showing the expression level of selected DEGs in MII oocytes from each lineage. Violin plots showing the oocyte-specific log-normalized gene expression in the y-axis of MII oocytes from each lineage (four animals per lineage; n = 42 oocytes in control diet + vehicle, n = 48 oocytes in control diet + DHT and n = 47 oocytes in HFHS + vehicle). C–M Violin plots showing on the y-axis gene expression by 2^−ΔΔCT of C Camk2n1, D Cacna2d1, E Slc17a7, F Btg2, G Dbh, H Foxp2, I Tial1, J Adora2a, K Btg2, L Comt, and M Otx1 in the amygdala of C–J F₃ female offspring (control lineage n = 5, androgenized lineage n = 4, and obese lineage n = 5) and K–M mF₃ male offspring (control lineage n = 5, androgenized lineage n = 5, obese lineage n = 5 and obese and androgenized lineage n = 5). The data are present in violin plot (C–J) showing the frequency distribution curves. The median and quartiles values are shown in dotted and dashed lines, respectively. Two-way ANOVA was followed by Bonferroni post hoc analysis and one-way ANOVA by Bonferroni post hoc analysis. All data are presented as mean ± s.e.m. DHT dihydrotestosterone, CD control diet, HFHS high-fat high-sugar, GGMat great-grand maternal.

Fig. 5. Germ cells versus in utero environment in the transmission of anxiety like phenotype in offspring.

A Schematic illustration showing prepubertal mouse model and generation of F₁ and F₂ offspring by using IVF. B–D Time spent in the open arms in the elevated plus maze (EPM); time spent in the closed arms of the EPM; time spent in the center of the open filed (OF); time spent in the periphery of the OF. Data were analyzed with Student’s t-test. All data are presented as mean ± s.e.m. CNT control, DHT dihydrotestosterone.