Fetally-encoded GDF15 and maternal GDF15 sensitivity are major determinants of nausea and vomiting in human pregnancy

Abstract

Human pregnancy is frequently accompanied by nausea and vomiting that may become severe and life-threatening, as in hyperemesis gravidarum (HG), the cause of which is unknown. Growth Differentiation Factor-15 (GDF15), a hormone known to act on the hindbrain to cause emesis, is highly expressed in the placenta and its levels in maternal blood rise rapidly in pregnancy. Variants in the maternal GDF15 gene are associated with HG. Here we report that fetal production of GDF15, and maternal sensitivity to it, both contribute substantially to the risk of HG. We found that the great majority of GDF15 in maternal circulation is derived from the feto-placental unit and that higher GDF15 levels in maternal blood are associated with vomiting and are further elevated in patients with HG. Conversely, we found that lower levels of GDF15 in the non-pregnant state predispose women to HG. A rare C211G variant in GDF15 which strongly predisposes mothers to HG, particularly when the fetus is wild-type, was found to markedly impair cellular secretion of GDF15 and associate with low circulating levels of GDF15 in the non-pregnant state. Consistent with this, two common GDF15 haplotypes which predispose to HG were associated with lower circulating levels outside pregnancy. The administration of a long-acting form of GDF15 to wild-type mice markedly reduced subsequent responses to an acute dose, establishing that desensitisation is a feature of this system. GDF15 levels are known to be highly and chronically elevated in patients with beta thalassemia. In women with this disorder, reports of symptoms of nausea or vomiting in pregnancy were strikingly diminished. Our findings support a causal role for fetal derived GDF15 in the nausea and vomiting of human pregnancy, with maternal sensitivity, at least partly determined by pre-pregnancy exposure to GDF15, being a major influence on its severity. They also suggest mechanism-based approaches to the treatment and prevention of HG.

Figure 1. Circulating GDF15 is elevated in women experiencing nausea and vomiting in pregnancy and hyperemesis gravidarum.

A: Dot and box plots illustrating the distribution of circulating GDF15 levels in women of ~15 weeks’ gestation with a history of vomiting in pregnancy vs those reporting no nausea and vomiting in pregnancy. P-value is from an unadjusted linear regression model using natural-log transformed GDF15 concentrations. B: Dot and box plots illustrating the distribution of GDF15 levels (Mean gestational age ~ 10 weeks) in women presenting with hyperemesis gravidarum (HG) and those with low levels of nausea and vomiting in pregnancy. P-value is from an unadjusted linear regression model using natural-log transformed GDF15 concentrations. C: Scatter plot illustrating the relationship between gestational age and GDF15 in the first trimester. The trend lines show predicted values of GDF15 levels (+/− 95% CI) in women with and without HG from a linear regression model of natural-log transformed GDF15 with gestational age and HG status included as predictor variables. The P-values are derived from the same regression model for the effect of HG (HG vs Con) and gestational age (Gest. Age). 5 participants (HG = 1, Control = 4) included in the analysis in panel (B) are not plotted or included in this model as they were recruited after the first trimester.

Figure 2. Circulating GDF15 in human pregnancy is predominantly of fetal origin.

A: Schema of experimental design. The GDF15 dimer for maternal and fetal GDF15 is extracted and then digested with the endopeptidase GluC, cutting the N-terminal region into two distinct peptides with glutamic acid C-termini. The stoichiometry of the H and D peptides can then be monitored using LC-MS/MS to determine the relative levels of maternal or fetal derived GDF15 in the maternal circulation. B: Representative LC-MS retention time of H and D peptides from maternal plasma where the mother is heterozygous at H202D and the fetus is homozygous for the H or D allele as indicated. C-E: Scatter plots of the relative quantitation of H peptide vs the D peptide in plasma from pregnancies with the indicated genotypes. The dashed coloured lines in (C-E) indicate the expected relationships between the H and D peptides for the given circulatory origins of GDF15. C: N=20 samples from 5 pregnancies, D: N=8 samples from 2 pregnancies, E: N = 47 samples from 12 pregnancies.

Figure 3. Rare and common hyperemesis gravidarum risk variants lower circulating GDF15 in the non-pregnant state.

A: We studied the secretion of a rare hyperemesis gravidarum risk variant GDF15 C211G. C211G impairs the secretion of GDF15 as determined by western blotting of cell culture medium of cells expressing Flag-tagged wild-type GDF15 (WT-Flag) or GDF15 C211G (C211G-Flag). B: GDF15 C211G impairs the secretion of wild-type GDF15 in a dominant negative manner as co-expression of the mutant inhibited secretion of wild-type GDF15 from 293T cells co-transfected with different amounts (shown in nanograms) of wild-type WT-Flag and Myc-tagged GDF15 C211G (C211G-Myc), as indicated. For A and B representative images from 3 independent experiments are presented. EV represents transfection with the empty plasmid backbone. C: Dot and box plots showing GDF15 levels measured using the Ansh Total GDF15 assay in carriers of GDF15 C211G variant (N=10) identified in an exome-sequencing study of a Croatian population and age and sex matched controls (N=60) derived from the same study. The P-value is from a linear regression model of natural log transformed GDF15 ~ C211G status. D: Forest plot illustrating the effect (standardised betas) of previously described HG risk SNPS on circulating GDF15 measured in 18,184 participants in the Generation Scotland Study. The effect estimates for the rs1054221 variant presented are from an analysis conditioned on the the lead HG variant rs45543339. The beta estimates for GDF15 represent the effect of the HG risk allele on circulating GDF15 in standard deviations. The beta estimates for HG (Hyperemesis Gravidarum) represent the effect of the SNP on risk of HG in log-odds. E: Scatterplot of HG GWAS effect estimates (ie log-odds) vs Roche-based GDF15 pQTL effect estimates derived from cis-Mendelian randomization at the GDF15 locus. MR was performed using m=259 SNPs with genome-wide evidence of pQTL effects on GDF15 levels within 1Mb GDF15 locus and adjusted using LD estimates from UK Biobank WGS individuals (n=138335; see Methods). Causal effect estimates obtained using LD-aware MR and reflected as regression lines.

Figure 4. Treatment with long acting GDF15 influences the response to the anorectic actions of acute GDF15 treatment in mice.

A: Schema of the experimental paradigm. Single housed, adult male and female C57Bl/6J mice were injected with 0.01mg/kg of Fc-GDF15–15 fusion protein (Fc_GDF15) or vehicle control (PBS). Food intake was measured overnight (from 5pm to 9am) before (black bar) and after treatment (red bar) with standard, short acting human recombinant GDF-15 (hrGDF15 0.1mg/kg). Body weight was measured at the same timepoints. B: Food intake recorded overnight (5pm-9am) the day before (black dots) and after an acute bolus of hrGDF15 (red dots) in mice with and without pre-treatment with Fc_GDF15. C: Body weight at 9am the day before (black dots) and 9 am the day after (red dots) an acute bolus of hrGDF15 in mice with and without pre-treatment with Fc_GDF15. N=17 (12 male, 5 female) in Control and 19 in FC_GDF15 group (13 male, 6 female). Data were analysed using mixed-effect analysis, post-hoc testing comparing before and after acute GDF-15 treatment was undertaken with the Sidak test to correct for multiple testing. *P<0.05, ***P<0.001, ****P<0.0001, ns = non-significant, P>0.05.