Association between fetal sex and maternal plasma microRNA responses to prenatal alcohol exposure: evidence from a birth outcome-stratified cohort

Abstract

Most persons with fetal alcohol spectrum disorders (FASDs) remain undiagnosed or are diagnosed in later life. To address the need for earlier diagnosis, we previously assessed miRNAs in the blood plasma of pregnant women who were classified as unexposed to alcohol (UE), heavily exposed with affected infants (HEa), or heavily exposed with apparently unaffected infants (HEua). We reported that maternal miRNAs predicted FASD-related growth and psychomotor deficits in infants. Here, we assessed whether fetal sex influenced alterations in maternal circulating miRNAs following prenatal alcohol exposure (PAE). To overcome the loss of statistical power due to disaggregating maternal samples by fetal sex, we adapted a strategy of iterative bootstrap resampling with replacement to assess the stability of statistical parameter estimates. Bootstrap estimates of parametric and effect size tests identified male and female fetal sex-associated maternal miRNA responses to PAE that were not observed in the aggregated sample. Additionally, we observed, in HEa mothers of female, but not male fetuses, a network of co-secreted miRNAs whose expression was linked to miRNAs encoded on the X-chromosome. Interestingly, the number of significant miRNA correlations for the HEua group mothers with female fetuses was intermediate between HEa and UE mothers at mid-pregnancy, but more similar to UE mothers by the end of pregnancy. Collectively, these data show that fetal sex predicts maternal circulating miRNA adaptations, a critical consideration when adopting maternal miRNAs as diagnostic biomarkers. Moreover, a maternal co-secretion network, predominantly in pregnancies with female fetuses, emerged as an index of risk for adverse birth outcomes due to PAE.

Keywords: Bootstrap resampling; Extracellular miRNAs; Fetal alcohol spectrum disorders; Maternal miRNA co-secretion; Sex as a biological variable.

Fig. 1

Bootstrap resampling with ANOVA statistical testing identifies sex-specific miRNAs. a The schematic workflow for iterative bootstrap resampling with replacement, with the ANOVA as a sample parametric test. (1) Aggregated data in each group were resampled with replacement to generate a new sample with n number of observations, identical to the sample size of the original group. (2) A one-way ANOVA was conducted on the three resampled groups. (3) A p value from that iteration was recorded. (4) The resampling process was then repeated for 2000 iterations. (5) Steps 1–4 were then repeated for the male and female groups separately. (6) For each miRNA, we report the proportion of iterations in which the ANOVA p < 0.05. (7) Steps 1–6 are repeated for each of the miRNAs. b The proportion of bootstrap iterations in which a one-way ANOVA was significant (p < 0.05) for each miRNA during the second trimester and third trimester. Based on the analyses of the aggregated data (males and females combined, black dots), the miRNAs were ranked from those most likely to be significant in bootstrap reanalysis to those least likely to be significantly altered. Separate resampling was performed for pregnancies with male fetuses (blue dots) and female fetuses (red dots). The gray line indicates the chance probability of reaching statistical significance. c Number of “likely alcohol-sensitive, fetal sex-specific” miRNAs in each group

Fig. 2

Bootstrap resampling to assess the stability of Hedges’ g effect size estimates. Median (black dots) and 95% confidence intervals (gray shading) of the bootstrapped effect sizes for each miRNA for the total sample at the second and third trimester time points. Separate bootstrap analysis of the median effect size of miRNA expression from pregnancies with female (red dots) or male (blue dots) fetuses is overlaid. miRNAs are arranged on the x-axis in the order of decreasing median bootstrap effect size in the composite analyses. Dotted lines indicate moderate (0.5) and large (0.8) effect size cutoffs. The table shows the number of miRNAs with sex-segregated median effect size falling outside the 95% confidence interval for the composite sample

Fig. 3

Bootstrap resampling to determine the confidence interval and effect size estimates of the effects of fetal sex. Median bootstrap effect sizes (solid dot) and 95% confidence interval (error bars) for miRNAs which have a non-zero containing confidence interval during sex-segregated resampling. Second and third trimester HEa vs. UE estimates are shown. The miRNA identities of MIMAT numbers (unique accession numbers assigned by miRBase.org) represented in this figure are in Additional file 5. The dashed line indicates zero effect size. The 95% confidence intervals for the resampling distributions for each miRNA were estimated through computing the 2.5 and 97.5 quantiles of the bootstrapped values

Fig. 4

Correlated expression of circulating miRNAs. a Correlation plots show significant (p < 0.05) cross-correlated expression between miRNA pairs (hierarchically clustered) in each of the exposure groups in the second and third trimesters color-coded by Pearson’s correlation coefficient. b Histogram distributions of the number of significant miRNA cross-correlations (p < 0.05) in each of the 2000 bootstrap iterations at the second trimester and third trimester time points. The table shows the p values for the null hypothesis testing that the mean number of significant correlations in either the HEa or HEua group is not different from the UE group in the second and third trimesters.

Fig. 5

Sex-segregated bootstrap analysis of miRNA expression correlation. Histogram distributions of the number of significant cross-correlations (p < 0.05) in each of the 2000 bootstrap iterations at the second trimester and third trimester time points segregated by pregnancies with a male fetus and pregnancies with a female fetus. The table shows the p values for the null hypothesis testing that the mean number of significant correlations is not different between each group and the UE group and exposure group for each time point and fetal sex comparison

Fig. 6

Cross-chromosomal correlations in miRNA expression. Radar plots showing the percent change (HEa or HEua compared to UE) in the number of significant cross-correlations (p < 0.05) between miRNAs on each pair of chromosomes at the second- and third trimester time points