Study of Heart function in PRE-Eclampsia during and after PreGnancy (SHePREG): The Pilot Cohort

Abstract

Background

Pre-eclampsia with severe features (severe PreE) is associated with heart dysfunction, yet the impact beyond pregnancy, including its association with cardiomyopathic genetic polymorphisms, remains poorly understood.

Objective

We aimed to characterize the temporal impact of severe PreE on heart function through the 4^th trimester in women with and without deleterious cardiomyopathic genetic variants.

Methods

Pregnant women were enrolled to undergo transthoracic echocardiography (TTE) in late pregnancy and 3 months postpartum. In women with severe PreE a targeted approach to identify pathogenic cardiomyopathic genetic polymorphisms was undertaken, and heart function was compared in carriers and non-carriers.

Results

Pregnant women (32 ± 4 years old, severe PreE = 14, control = 8) were enrolled between 2019 – 2021. Women with severe PreE displayed attenuated myocardial relaxation (mitral e’ = 11.0 ± 2.2 vs. 13.2 ± 2.3 cm/sec, p < 0.05) in late pregnancy, and on in-silico analysis, deleterious cardiomyopathic variants were found in 58%. At 103 ± 33 days postpartum, control women showed stability in myocardial relaxation (Mitral e’ Entry: 13.2 ± 2.3 vs. Postpartum: 13.9 ± 1.7cm/sec, p = 0.464), and genetic negative severe PreE women (G−) demonstrated recovery of diastolic function to control level (Mitral e’ Entry: 11.0 ± 3.0 vs. Postpartum 13.7 ± 2.8cm/sec, p < 0.001), unlike their genetic positive (G+) counterparts (Mitral e’ Entry: 10.5 ± 1.7 vs. Postpartum 10.8 ± 2.4cm/sec, p = 0.853).

Conclusions

Postpartum recovery of heart function after severe PreE is attenuated in women with deleterious cardiomyopathic genetic polymorphisms.

Graphical Abstract

Created with BioRender

Introduction

Cardiovascular events are the most common cause of maternal mortality in the United States, resulting in 20 deaths per 100,000 live births.(1) Importantly, one of the major contributors to this risk are hypertensive disorders of pregnancy (HDP) such as pre-eclampsia (PreE), which have nearly doubled in the U.S. between the years 1993 and 2014.(2) For women who develop PreE, there is a significant risk for the future development of cardiovascular disease (CVD), including heart failure, coronary artery disease, and stroke.(3) Of all types of CVD, PreE has been shown to exert the greatest effect on postpartum risk for heart failure (HF), both within the first year after pregnancy (adjusted risk ratio 4.10), and up to 10 years after delivery (adjusted risk ratio 8.42).(3)

Human studies have characterized changes in heart function by transthoracic echocardiogram (TTE) in women with PreE at the time of diagnosis, typically at term or near-term in pregnancy.(4–9) However, only very recently have efforts turned toward meticulously evaluating and comparing pre-delivery vs. postpartum heart function indices in women with HDP,(10,11) with fewer detailing expected postpartum temporal changes in women with the most advanced form of PreE, termed PreE with severe features (severe PreE). Specific inquiry to both the expected and pathogenic changes in heart function beyond pregnancy complicated by severe PreE may provide clues about the link between peripartum heart disease and late CVD. One of the fundamental goals of this ongoing study is to better understand the relationship between PreE and future HF risk, more specifically, about the potential role that genetic changes implicated in cardiomyopathy may play in women with a recent PreE-associated pregnancy.

Mechanistically, contemporary data has suggested that PreE results from the effects of soluble anti-angiogenic factors released from the placenta that mediate maternal endothelial dysfunction and a systemic inflammatory response.(12) It is generally acknowledged that early onset and late onset PreE are likely different, with abnormal placentation largely mediating the former, and maternal genetic predisposition for CVD, the latter.(12) However, there is a major gap in knowledge about what genetic predisposition means, in particular, as it relates to heart function and failure in women with PreE. Previous studies have shown that large-scale next generation sequencing (NGS) to detect cardiac disease reveal likely pathogenic variations in ~ 30% of subjects, however report that the pathogenic variants were mainly observed in genes with definitive evidence of disease causation, supporting a targeted panel approach such as this over NGS.(13)There has been some controversy in targeted panel approach studies that have evaluated cardiomyopathic genetic testing in this group,(14,15) and therefore limited success in understanding the role that genetic risk for cardiomyopathy may play in modulating maternal heart function during and beyond a PreE-afflicted pregnancy. One of the major limitations of the prior work is that cardiomyopathic genetic results have not been evaluated in the context of real-time heart function by TTE.

It has been demonstrated that women with cardiomyopathy are more likely to develop HDP,(16) and conversely, that those with a history of PreE have increased risk for the development of future HF.(3) However, for women with presumably normal pre-pregnancy heart function, there is no reliable way to identify those with PreE who are at highest risk for the development of future HF and necessitate close follow-up and future screening. Recognizing this gap, and the limitations in prior work, we sought to determine if women with severe PreE harbor pathogenic cardiomyopathic genetic polymorphisms, and if so, whether variant presence affects temporal heart function up to 3 months postpartum after severe PreE.

Methods

Data Collection and Sample Processing

Patients 18 years of age or older pregnant with viable pregnancy were prospectively recruited to participate in this study. Subjects were evaluated for the presence of PreE with severe features according to the most recent American College of Obstetricians and Gynecologists (ACOG) practice bulletin. (17) Additional inclusion/exclusion criteria, enrollment details, and sample processing are available in the methods section of the supplemental data file.

Transthoracic Echocardiogram

Transthoracic echocardiographic studies were reviewed in a blinded fashion independently by three advanced echocardiography trained investigators. Digital grayscale 2-dimensional cine loops were collected from the standard apical 4-chamber, 2-chamber, and long-axis views. Image acquisition occurred during breath hold and gain settings were adjusted for routine 2-dimensional images to optimize endocardial border definition. Left ventricular function was evaluated by biplane Simpson’s rule using manual tracings. Long-axis views were used for assessment of end diastolic dimension. Ventricular diastology was assessed according to the most recent American Society of Echocardiography guidelines,(18) and strain as outlined by the 2D speckle tracking task force document.(19)

Genetic Analyses

Informed by the data that supports a targeted genetic testing,(13) and recognizing that the main objective was to evaluate for heart dysfunction on TTE, we chose a targeted genetic testing approach. For severe PreE samples only, genomic DNA was extracted locally (Qiagen FlexiGene DNA Kit) and sent to Invitae© for comprehensive analysis of genes associated with inherited cardiomyopathy (Invitae Corporation, cardiomyopathy comprehensive panel, test 02251). Additional genetic analyses methods available in the supplemental data file.

Statistical Analysis and Funding Support

Any advanced statistical analyses utilized are reported where appropriate in the methods sections. For general analyses, basic demographic information was reviewed, and descriptive statistics were generated (means and standard deviations for quantitative variables, and frequencies and relative frequencies for categorical variables). Primary and secondary outcomes data were analyzed and visually inspected for result distrubution and outliers. This analysis was performed in all patients, and then individually for both the control and severe PreE group at baseline and 3 months follow-up. The between-group difference was evaluated using two-sample T-test, nonparametric Wilcoxon Rank-sum test, or Fisher’s exact test, depending on the type of variable being summarized and its distribution. The outcome variables (i.e., TTE findings) was summarized by group at each one of the two time points (study entry, and 3-mo postpartum) using descriptive statistics. Two types of comparisons were performed: 1) within-group comparisons showing the change of cardiac function over time within each group; completed using paired t-test, or non-parametric Wilcoxon Signed-rank test for the differences between pairs, and 2) between-group comparisons showing the group difference at each of the of two time points (entry and 3 months postpartum); this was accomplished using two-sample t-test or nonparametric Wilcoxon Rank-sum test, where appropriate. Data is expressed as mean ± SD or frequency (%) where appropriate, with significance occurring at an α level of ≤ 0.05. The data were analyzed using JMP^® Pro, Version 16.0 SAS Institute Inc, Cary, NC, 2021. This study was supported in part, by an investigator-initiated grant from F. Hoffman-La Roche Ltd and by the National Institute of Health (HL148701). The funding sources had no rule in study design, data collection, data analysis, data interpretation, writing of the manuscript, or the decision to submit the manuscript for publication. The authors are solely responsible for the design and conduct of this study, all study analyses, and drafting and editing of the paper and its final contents.

Results

Subject Characteristics and Serum Analysis

Between November 1, 2019, and June 30, 2021 we prospectively recruited 22 pregnant women (average age 32 ± 4 years old, 21 (88%) self-reported as non-Hispanic White, gestational age at delivery 36 ± 3 weeks) into either a control (n = 8) or severe PreE (n = 14) cohort. Women were similar in both groups except for gestational age (34 ± 2 vs. 38 ± 2 weeks, p < 0.05) and body mass index, which was slightly higher in the severe PreE women (30 ± 5 vs. 25 ± 4kg/m², p < 0.05). Full baseline characteristics of the study participants are described in Table 1 and Supplementary Table 1.

Table 1.

Baseline Demographics, All Subjects

Characteristic
Mean ± SD or frequency (%)	Control (n = 8)	Severe PreE (n = 14)	P Value
Age (years)	32 ± 4	31 ± 5	0.868
Gestational age, weeks	38 ± 2	34 ± 2	0.002
Biologic Race (non-Caucasian)	1 (13%)	1 (7%)	0.230
Pre-pregnancy Weight (kg)	71 ± 10	83 ± 18	0.047
Height (m)	1.68 ± 0.05	1.68 ± 0.08	1.000
Pre-pregnancy BMI (kg/m2)	25 ± 4	30 ± 5	0.029
Diabetes, Pregestational	0	0	1.000
Diabetes, Gestational	2 (25%)	1 (7%)	0.250
Hypertension, pregestational	0	0	1.000
Smoking	0	0	1.000

BMI: body mass index, NS = not significant, PreE: pre-eclampsia, SD = standard deviation. The average gravida status of the entire cohort was G2, and there were a total of 9 nulliparous women included.

With subjects enrolling at term, or at the time of induction for delivery in the case of women with severe PreE, we also collected obstetric and fetal characteristics of the cohort. Not surprisingly, women with severe PreE were more likely to require induction and/or cesarean delivery. The same women were more likely to present with fetal growth restriction (5 (36%) vs. 0 (0%), p < 0.05), and had a longer length of stay after delivery. The offspring of women with severe PreE weighed less (2,009 ± 629 vs. 3,110 ± 546 grams, p = 0.001), demonstrated reduced APGAR 1 and APGAR 2 scores (6.4 ± 2.4 vs. 8.4 ± 0.5, p = 0.01 and 8.1 ± 0.9 vs. 9.1 ± 0.4, p < 0.01), had higher rates of respiratory support (CPAP use), and demonstrated significantly longer fetal length of stay (14.4 ± 12.7 vs. 2.9 ± 4.5 days, p < 0.01) (Supplementary Table 2).

There were few clinically meaningful significant differences in serum chemistry analyses at baseline, including the sFlt1:PlGF ratio, known to correlate with the presence of PreE,(20–22) and was as expected, elevated in women with severe PreE at study entry (201 ± 187 vs. 12 ± 14, p < 0.05). Interestingly, the purine metabolic product uric acid was elevated at baseline in women with severe PreE (5.6 ± 0.5 vs. 3.9 ± 0.6 mg/dL, p < 0.05), which may be important, given the association with hypertension, metabolic syndrome, and CVD,(23,24) particularly in women.(25) Cardiac biomarker analysis was also performed and demonstrated modest elevation in Pro-BNP in women with severe PreE (164 ± 120 vs. 57 ± 51 pg/mL, p < 0.05), with no significant changes in non-fasting serum cholesterol levels, troponin, or creatinine kinase myoglobin fraction (CKMB) (Supplementary Table 3). To further investigate potential markers that may correlate with cardiac function in the setting of severe PreE, we performed metabolomic analyses of the cohort (Supplementary Figures 1,2, Supplementary Table 4), however did not demonstrate significant changes in myocardial metabolomic fatty acid oxidation pathway substrates (long chain fatty acids).

Finally, given the shared hormonal influence between PreE and peripartum cardiomyopathy/PPCM,(26) (27,28)we evaluated the circulating miRNA profile in the women with severe PreE to determine if the 16 kDa PRL-miRNA-ERBB4 axis may be responsible for changes in cardiac function in women with severe PreE, similar to what has been reported in PPCM.(27) Quantification of relative miRNA expression was performed for six of the most highly implicated miRNAs in PPCM (severe PreE delivery n = 13, severe PreE postpartum n = 10, control delivery n = 8, control postpartum n = 7). Results of miRNA profiling are shown in Supplemental Figure 3. For all six miRNAs tested (miR-146a-5p, miR-199a-5p, miR-221–3p, miR-23a-3p, miR-130a-3p, and miR-135a-5p), no statistically significant difference in expression was found between the severe PreE and control groups, either at the time of delivery or postpartum (Supplementary Figure 4), suggesting that miRNA triggered inhibition of the cardioprotective tyrosine kinase receptor ERBB4 is an unlikely contributor to heart function/dysfunction in severe PreE.

Cardiomyopathic Genetics and In-Silico Analysis Results

High quality DNA samples from 12 of the 14 women with severe PreE were available for genetic analysis. Opting for specific methods to prioritize analysis of putative functional impact over general computational methods (see methods section), we performed comprehensive cardiomyopathic gene panel testing, which identified 14 potentially pathogenic variants. We then performed in-silico analyses to assess variant pathogenicity. All variants had available Polyphen2 scores; using this tool we found 8 deleterious variants in 7 (58% of those tested) women with severe PreE, which are shown in Figure 1. Remaining variants, additional in-silico methods such as REVEL score, and data including those initially classified as ‘likely benign’ based on Polyphen2 scores < 0.5 are shown in Supplementary Table 5.

Figure 1.

Deleterious Cardiomyopathic Genetic Polymorphisms in Women with Severe Pre-Eclampsia

In-silico analyses (Polyphen2 data shown) of variant pathogenicity in severe pre-eclamptic patients is shown ranging from benign to damaging, with actual score represented by the black X (A). Sequence conservation across species is shown for each of the likely pathogenic variants (B).

Heart Function, All Subjects

Heart function was assessed at study entry, and women with severe PreE displayed evidence of impaired myocardial relaxation compared to controls (mitral e’ = 11.0 ± 2.2 vs. 13.2 ± 2.3 cm/sec, p < 0.05), and demonstrated a trend supporting left ventricular (LV) remodeling (LV posterior wall 1.01 ± 0.03 vs. 0.93 ± 0.09cm, p = 0.102) and augmentation of right heart function (RV S’ 15.7 ± 3.1 vs. 13.8 ± 1.3cm, p = 0.57), similar to prior studies (Table 2, Figure 2).

Table 2.

Heart Function at Study Entry, All Subjects

Characteristic
Mean ± SD or frequency (%)	Control (n = 8)	Severe PreE (n = 14)	P Value
LVEF (%)	60 ± 5	59 ± 5	0.541
LV Fractional shortening (%)	36 ± 4	34 ± 6	0.517
LV Septum (cm)	0.92 ± 0.17	1.00 ± 0.13	0.240
LV Posterior Wall (cm)	0.93 ± 0.09	1.01 ± 0.03	0.102
LV, GLS (%)	−21 ± 3	−20 ± 3	0.241
LV, GLS >−18%	0 (0%)	5 (36%)	0.028
LV, Tei Index	0.43 ± 0.07	0.53 ± 0.13	0.066
Mitral E Velocity (cm/sec)	89 ± 17	82 ± 13	0.368
Mitral A Velocity (cm/sec)	59 ± 8	73 ± 6	0.172
Mitral e’ Velocity (cm/sec)	13.2 ± 2.3	11.0 ± 2.2	0.022
Mitral E/e’	6.8 ± 1.4	8.0 ± 1.9	0.120
Mitral E/A	1.6 ± 0.5	1.2 ± 0.3	0.054
Mitral Deceleration Time (msec)	212 ± 31	171 ± 41	0.018
RV Length (cm)	7.2 ± 0.3	7.5 ± 0.2	0.581
RV Base (cm)	4.0 ± 0.5	3.8 ± 0.6	0.446
RV, FAC (%)	45 ± 9	46 ± 8	0.783
TAPSE (cm)	2.5 ± 0.4	2.5 ± 0.4	0.986
RV S’ (cm)	13.8 ± 1.3	15.7 ± 3.1	0.057
RV, GLS (%)	−25 ± 5	−23 ± 3	0.365
LA Volume (mL)	42 ± 15	51 ± 10	0.173
LA Volume, indexed (mL/m2)	22 ± 8	26 ± 5	0.268
RA Volume (mL)	38 ± 9	37 ± 3	0.858
RA Volume, indexed (mL/m2)	20 ± 4	18 ± 5	0.428

FAC: Fractional area change, G+ = cardiomyopathy gene positive, G− = cardiomyopathy gene negative, GLS: global longitudinal strain, LA: left atrium, LV: left ventricle, LVEF: left ventricular ejection fraction, PreE: pre-eclampsia, RA: right atrium, RV: right ventricle, SD: standard deviation, TAPSE: tricuspid annular plane systolic excursion

Figure 2.

Heart Function in Severe Pre-Eclampsia

Select measures of cardiovascular function in women (control = 8 (blue), Severe PreE/+DCGP = 7 (dark pink), Severe PreE/-DCGP = 5 (light pink)) at the time of delivery and 3 months postpartum are shown. All women with severe PreE demonstrated attenuated mitral annular velocity (e’) during pregnancy, with persistence of attenuated e’ 3-months postpartum in those that were G+, unlike their G− counterparts, who recovered function. * p< 0.05, † < 0.01. DCGP = deleterious cardiomyopathic genetic polymorphism, PreE: pre-eclampsia.

At 103 ± 33 days after delivery, we assessed the control and severe PreE groups both together, and more importantly independent of each other, understanding that there are expected heart function changes that occur with pregnancy, and should return to normal postpartum. To assess for divergence from the expected ‘return to normal’, we performed within group comparisons of study entry (peak pregnancy) vs. postpartum heart function independently in each group. In the control group we found that there was good stability in diastolic function between peak pregnancy (study entry) and postpartum (Mitral e’ Entry: 13.2 ± 2.3 vs. Postpartum: 13.9 ± 1.7cm/sec, p = 0.464) with further reduction of E/e’ postpartum, consistent with resolution of the volume loaded state of pregnancy. Similarly, mild compensatory hypertrophy (LV posterior wall) and systolic function (LV, GLS) were more robust postpartum, after the volume load of peak pregnancy resolved, supporting that there are expected compensatory changes in pregnancy that return to normal within 3 months of delivery (Tables 3,4).

Table 3.

Heart Function After the 4^th Trimester, All Subjects

Characteristic
Mean ± SD or frequency (%)	Control (n = 8)	Severe PreE (n = 14)	P Value
LVEF (%)	60 ± 3	58 ± 3	0.442
LV Fractional shortening (%)	28 ± 16	32 ± 5	0.651
LV Septum (cm)	0.87 ± 0.11	0.91 ± 0.10	0.544
LV Posterior Wall (cm)	0.83 ± 0.09	0.88 ± 0.11	0.388
LV, GLS (%)	−24 ± 3	−21 ± 2	0.109
LV, Tei Index	0.46 ± 0.07	0.45 ± 0.11	0.863
Mitral E	72 ± 13	78 ± 13	0.480
Velocity (cm/sec) Mitral A	39 ± 12	50 ± 12	0.155
Velocity (cm/sec)
Mitral e’ Velocity (cm/sec)	13.9 ± 1.7	11.8 ± 2.9	0.097
Mitral E/e’	5.3 ± 1.1	6.8 ± 1.7	0.049
Mitral E/A	2.0 ± 0.5	1.6 ± 0.5	0.294
Mitral Deceleration Time (msec)	244 ± 35	196 ± 27	0.033
RV Length (cm)	7.0 ± 0.8	7.1 ± 0.7	0.869
RV Base (cm)	3.6 ± 0.4	3.5 ± 0.5	0.695
RV, FAC (%)	41 ± 6	43 ± 7	0.562
TAPSE (cm)	2.1 ± 0.4	2.3 ± 0.3	0.299
RV S’ (cm)	13.3 ± 0.5	11.2 ± 1.3	0.001
RV, GLS (%)	−26 ± 4	−21 ± 6	0.090
LA Volume (mL)	38 ± 12	44 ± 6	0.389
LA Volume, indexed (mL/m2)	21 ± 6	22 ± 3	0.533
RA Volume (mL)	33 ± 10	31 ± 8	0.749
RA Volume, indexed (mL/m2)	18 ± 5	16 ± 3	0.482

FAC: Fractional area change, G+ = cardiomyopathy gene positive, G− = cardiomyopathy gene negative, GLS: global longitudinal strain, LA: left atrium, LV: left ventricle, LVEF: left ventricular ejection fraction, PreE: pre-eclampsia, RA: right atrium, RV: right ventricle, SD: standard deviation, TAPSE: tricuspid annular plane systolic excursion

Table 4.

Heart Function at Delivery Compared to 4th Trimester, Control

Control	Control (n = 8) at Delivery	Control (n = 8) 4th Trimester	P Value
LV Septum (cm)	0.92 ± 0.17	0.87 ± 0.11	0.212
LV Posterior Wall (cm)	0.93 ± 0.09	0.83 ± 0.09	0.054
LV, GLS (%)	−21 ± 3	−24 ± 3	0.046
LV, Tei Index	0.43 ± 007	0.46 ± 0.07	0.078
Mitral E Velocity (cm/sec)	89 ± 17	72 ± 13	0.063
Mitral A Velocity (cm/sec)	59 ± 8	39 ± 12	0.057
Mitral e’ Velocity (cm/sec)	13.2 ± 2.3	13.9 ± 1.7	0.464
Mitral E/e’	6.8 ± 1.4	5.3 ± 1.1	0.004
Mitral E/A	1.6 ± 0.5	2.0 ± 0.5	0.053
Mitral Deceleration Time (msec)	212 ± 31	244 ± 35	0.015
TAPSE (cm)	2.5 ± 0.4	2.1 ± 0.4	0.194
RV S’ (cm)	13.8 ± 1.3	13.3 ± 0.5	0.436
RV, GLS (%)	−25 ± 5	−26 ± 4	0.835
LA Volume, indexed (mL/m2)	22 ± 8	21 ± 6	0.940

FAC: Fractional area change, G+ = cardiomyopathy gene positive, G− = cardiomyopathy gene negative, GLS: global longitudinal strain, LA: left atrium, LV: left ventricle, LVEF: left ventricular ejection fraction, PreE: pre-eclampsia, RA: right atrium, RV: right ventricle, SD: standard deviation, TAPSE: tricuspid annular plane systolic excursion

In women with severe PreE, we found insignificant regression of LV wall thickness, perhaps supporting improvement in remodeling postpartum (LV posterior wall 1.01 ± 0.03 vs. 0.88 ± 0.11, p = 0.059). While there appeared to be return to normal of the E/e’ ratio and left atrial volume postpartum, reflecting resolution of increased whole-body volume at term, mitral tissue e’ velocity remained attenuated and unchanged postpartum (Entry: 11.0 ± 2.2 vs. Postpartum: 11.8 ± 2.9cm/sec, p = 0.356), supporting persistence of dampened e’ levels, different than their control counterparts. While women in the control group displayed stability in RV function both at term and postpartum, those with severe PreE demonstrated hyperdynamic RV function at term, that dropped below expected levels postpartum when compared to control subjects (RV S’ Pre: 15.7 ± 3.1 vs. 11.2 ± 1.3, p < 0.05) (Tables 3,5).

Table 5.

Heart Function at Delivery Compared to 4^th Trimester, Severe Pre-Eclampsia by Genetic Status

Severe Pre-Eclampsia	Severe PreE (n = 14) at Delivery	Severe PreE (n = 14) 4th Trimester	P Value
	1.00 ± 0.13	0.91 ± 0.10	0.058
	G+ 1.02 ± 0.18	G+ 0.98 ± 0.05	0.648
LV Septum (cm)	G− 0.97 ± 0.08	G− 0.83 ± 0.17	0.208
	1.01 ± 0.03	0.88 ± 0.11	0.059
	G+ 1.00 ± 0.12	G+ 0.92 ± 0.07	0.124
LV Posterior Wall (cm)	G− 0.98 ± 0.12	G− 0.84 ± 0.17	0.429
	−20 ± 3	−21 ± 2	0.814
	G+ −19 ± 4	G+ −21 ± 2	0.797
LV, GLS (%)	G− −21 ± 3	G− −21 ± 3	0.898
	0.53 ± 0.13	0.45 ± 0.11	0.205
	G+ 0.51 ± 0.14	G+ 0.42 ± 0.12	0.241
LV, Tei Index	G− 0.58 ± 0.08	G− 0.53 ± 0.06	0.830
	82 ± 13	78 ± 13	0.238
	G+ 85 ± 12	G+ 78 ± 15	0.527
Mitral E Velocity (cm/sec)	G− 85 ± 14	G−77+15	0.289
	73 ± 6	50 ± 12	0.029
	G+ 75 ± 30	G+ 49 ± 5	0.208
Mitral A Velocity (cm/sec)	G− 75 ± 17	G−46+17	0.061
	11.0 ± 2.2	11.8 ± 2.9	0.346
	G+ 10.5 ± 1.7	G+ 10.8 ± 2.4	0.853
Mitral e’ Velocity (cm/sec)	G− 11.0 ± 3.0	G− 13.7 ± 2.8	<0.001
	8.0 ± 1.9	6.8 ± 1.7	0.146
	G+ 8.2 ± 1.7	G+ 7.3 ± 1.4	0.383
Mitral E/e’	G− 8.1 ± 2.4	G− 5.7 ± 1.6	0.076
	1.2 ± 0.3	1.6 ± 0.5	0.067
	G+ 1.3 ± 0.5	G+ 1.6 ± 0.3	0.443
Mitral E/A	G− 1.1 ± 0.2	G− 1.8 ± 0.6	0.086
	171 ± 41	196 ± 27	0.389
	G+ 173 ± 44	G+ 203 ± 36	0.487
Mitral Deceleration Time (msec)	G− 167 ± 50	G−196+6	0.493
	2.5 ± 0.4	2.3 ± 0.3	0.025
	G+ 2.7 ± 0.4	G+ 2.2 ± 0.3	0.098
TAPSE (cm)	G− 2.5 ± 0.3	G− 2.5 ± 0.1	0.124
	15.7 ± 3.1	11.2 ± 1.3	0.002
	G+ 15.5 ± 3.4	G+ 10.4 ± 1.3	0.018
RV S’ (cm)	G− 15.7 ± 3.4	G− 11.9 ± 0.9	0.151
	−23 ± 3	−21 ± 6	0.272
	G+ −26 ± 2	G+ −22 ± 7	0.228
RV, GLS (%)	G− −22 ± 4	G− −23 ± 5	0.834
	26 ± 5	22 ± 3	0.058
	G+ 26 ± 4	G+ 24 ± 1	0.297
LA Volume, indexed (mL/m2)	G− 25 ± 7	G− 21 ± 4	0.038

FAC: Fractional area change, G+ = cardiomyopathy gene positive, G− = cardiomyopathy gene negative, GLS: global longitudinal strain, LA: left atrium, LV: left ventricle, LVEF: left ventricular ejection fraction, PreE: pre-eclampsia, RA: right atrium, RV: right ventricle, SD: standard deviation, TAPSE: tricuspid annular plane systolic excursion

Heart Function, Severe PreE Genetic Cohorts

To better understand the relationship between cardiomyopathic genetic risk and heart function in severe PreE, we divided those with and without deleterious cardiomyopathic genetic polymorphisms and re-analyzed heart function. Severe PreE women who harbored a deleterious cardiomyopathic genetic variant (G+, n = 7) were compared to those with benign genetics or no evidence of a deleterious cardiomyopathic variant (G-, n = 5). We found that severe PreE women without pathogenic variants (G-) demonstrated recovery of myocardial relaxation to control levels (Mitral e’ in G- Entry: 11.0 ± 3.0 vs. Postpartum 13.7 ± 2.8cm/sec, p < 0.001) unlike their G+ counterparts (Mitral e’ in G+ Entry: 10.5 ± 1.7 vs. 10.8 ± 2.4cm/sec, p = 0.853). Similarly, an insignificant improvement in E/e’ was found in the G- women (Entry: 8.1 ± 2.4 vs. 5.7 ± 1.6, p = 0.076) (Figure 2, Table 5). While all women with severe PreE demonstrated augmentation of RV function (RV, S’) at the time of delivery, with depression of RV function postpartum, the postpartum impact of severe PreE appeared to be greater in the G+ women (Entry RV, S’: 15.5 ± 3.4 vs. Postpartum 10.4 ± 1.3cm, p < 0.05) when compared to their G- counterparts (Entry RV, S’: 15.7 ± 3.4 vs. Postpartum: 11.9 ± 0.9cm, p = 0.151) (Table 5).

Discussion

The data presented here demonstrate that over half of women studied with severe PreE harbored at least one deleterious cardiomyopathic genetic polymorphism. In severe PreE women with pathogenic variants, there was lack of postpartum recovery of myocardial relaxation to control levels, a new finding suggesting that heart function in women with severe PreE may derive influence at least in part, from genetic-based cardiomyopathic risk factors.

In the current study we begin to identify clues about the chronicity of PreE’s effect on heart function, extending from delivery through the 4^th trimester. The association between PreE and late cardiovascular risk in women has been well established,(3) and while this association has been demonstrated, the direct impact of severe PreE on heart function has only recently been studied. Vaught and colleagues demonstrated in their prospective study that women with severe PreE have elevated right heart pressure, more diastolic dysfunction, left-sided heart remodeling and higher rates of peripartum pulmonary edema. (4) Subsequent work has demonstrated that echocardiographic parameters used in conjunction with clinical findings in a prediction model can identify women with PreE who will most likely experience postpartum hypertension.(10) Attempts to study the temporal impact of PreE-associated heart dysfunction at 1- and 2-years postpartum have been undertaken, but have included subjects with chronic hypertension and lacked reporting of discreet TTE parameters, limiting the degree to which conclusions about postpartum heart function can be made. (29) More recently, postpartum TTE data has been investigated in those with HDP, but lack stratification of the types of HDP and also lack a control group for comparison.(11) The data presented here is unique, in that we provide a 4-way analysis to compare between group (control and severe PreE) changes in late pregnancy (1) and 3-months postpartum (2), as well as perform within group analysis between late pregnancy and 3-months postpartum in controls (3) and severe PreE women (4). This analysis allows for examination of expected heart function changes that occur with pregnancy, and assessment for divergence of hemodynamics from the expected ‘return to normal’ that should occur after resolution of the volume load associated with pregnancy.

Utilizing this analysis schema, in late pregnancy we demonstrated changes in myocardial relaxation and showed evidence of augmentation in right heart function in women with severe PreE. In other forms of cardiomyopathy, RV function at diagnosis has been associated with outcomes from left sided heart failure,(30–33) including in PPCM, (34) highlighting the importance of biventricular interactions in systolic heart failure. While in this prior work the focus has been on LV systolic dysfunction, we postulate that impaired myocardial relaxation, such as in this PreE cohort, may trigger compensatory hyperdynamic RV function to augment cardiac output in early stages before systolic dysfunction develops. However, we recognize this is an area that requires future study.

Following the cohort through the 4^th trimester, we demonstrated that control women retain normal myocardial relaxation at all timepoints, but that those with severe PreE continue to demonstrate attenuated mitral e’ wave tissue velocity, despite adequate resolution of volume load (E/e’ normalization). Importantly, over half of women with severe PreE studied demonstrated the presence of a deleterious cardiomyopathic genetic polymorphism (G+); The G+ women from the severe PreE group did not recover myocardial relaxation to control levels postpartum, unlike their G- counterparts, suggesting that cardiomyopathic genetic risk may impact the development of persistent, and/or late HF, in women with recent severe PreE. It may not seem surprising that this is the case, since genetic risk for cardiomyopathy has been implicated in the development of the related condition, PPCM,(28,35) yet, has not been fully explored in the context of PreE-associated heart dysfunction until now. Previous studies have used targeted and non-targeted whole exome sequencing to evaluate whether cardiomyopathic genetic polymorphisms may be associated with PreE, as a potential explanation for heart dysfunction.(14,36) However, the results of these studies appear to be contradictory, particularly in describing the frequency of variants in the PreE population when compared to reference populations. Gammill and colleagues reported that prevalence for cardiomyopathic variants was elevated in PreE women above control populations,(14) however this was called to question when considering large consortia level data,(37) and further supported by a similar small study evaluating 30 genes implicated in the development of cardiomyopathy in women with PreE.(36) Despite the diametric results from these groups, we must acknowledge that the major limitation is that genetic results were not studied in the context of real-time heart function data, as we did in this study.

Finally, we acknowledge that serum chemistries did not uncover any apparently meaningful biomarkers. Given the shared hormonal influence between PreE and PPCM,(26) we felt it was important to evaluate the circulating miRNA profile in the women with severe PreE to determine if the 16 kDa PRL-miRNA-ERBB4 axis may be responsible for observed cardiovascular dysfunction in PreE, as has been demonstrated in PPCM.(27) However, examining the 6 most highly implicated miRNAs for heart dysfunction in PPCM, we found no difference between control women and those with PreE, suggesting that miRNA triggered inhibition of cardiomyocyte ERBB4 is likely not the mechanism responsible for cardiac dysfunction in PreE, dissimilar from PPCM.

Strengths and Limitations

To our knowledge, this is the first study that aims to link cardiomyopathic genetic data directly to heart function in the setting of severe PreE. The most notable limitation is that this is a small, relatively racially homogenous, pilot cohort. While the rate of PreE is significantly higher in non-Hispanic Black women and other races,(38) in this pilot study, we were not able to examine the impact of PreE among unique racial or ethnic groups. Enrolling a racially diverse group for similar evaluation is the goal of our ongoing work on this topic. We postulate that heart dysfunction may be impacted to a greater degree in racial subgroups at higher risk for PreE, and so this should be a critical area for future study. While some studies have shown that genetic changes may modify risk for PreE among racially diverse groups,(39,40) these studies do not consider the impact of environmental, social, system-, or other levels of influence upon disease expression. That said, there is evidence that differential disease expression, while clearly impacted by inequities in healthcare access and delivery, may also be influenced by the types of genetic variation and differential pathogenicity found on genetic testing, as has been the case in hypertrophic cardiomyopathy,(41) and therefore will also need to be considered in future scientific inquiry. Genetic testing, and the chosen methodology itself, are often subject to critique, and that is no different for this study. Given that most pathogenic variants identified by whole genome sequencing are observed in genes with already evident causation, (13) we chose a targeted approach, recognizing that significant findings would also then have the ability for wider clinical applicability. Budgeting for the pilot cohort required prioritization of TTE data in all subjects and biomarker analysis, and therefore genetic testing was not completed on the control cohort. This is a recognized limitation of the study and will be included in the larger cohort we continue to enroll and report on in future work. Finally, the impact of comorbid diabetes and elevated BMI both in concert with and independent of genetic status, could not reliably be assessed in this small cohort. However, given the relationship of BMI and clinical cardiomyopathy in young women,(42) this will need to be examined in future work.

Conclusions

Pre-eclampsia remains a critical contributor to maternal morbidity and mortality, with heart failure recognized as the most common short- and long-term cardiovascular effect. Contemporary data has shown the acute effects of severe PreE on heart function, and this study identifies the early temporal pattern of persistence in attenuation of myocardial relaxation in severe PreE women with deleterious cardiomyopathic genetic polyrmophisms. This data suggests that cardiomyopathic genetics may influence heart function in women with a PreE-associated pregnancy, and investigative efforts will be required to better characterize and understand this finding.

Tweetable Statement: Cardiomyopathic genetic polymorphisms may impact the recovery of heart function in women with severe pre-eclampsia (* We recommend using the central illustration in the tweet) #CardioObstetrics, #ACCCardioOB, #PregnancyHeartTeam

Clinical Implications:

Deleterious cardiomyopathic genetic polymorphisms were found in over half of a cohort of women with severe pre-eclampsia. All women with pre-eclampsia demonstrated impaired myocardial relaxation during pregnancy, however, those with pathogenic variants failed to recover heart function 3-months postpartum, unlike their genetic negative pre-eclamptic counterparts.

Translational Outlook:

Our study demonstrates that cardiomyopathic genetics may impact heart function during and beyond pregnancy in women who develop pre-eclampsia. Further investigation is critical to better understand the role that cardiomyopathic genetic risk may play in modulating peri- and postpartum heart function in women afflicted with a pre-eclampsiaassociated pregnancy, and will be required if we aim to reduce maternal cardiovascular morbidity and mortality.

Abbreviations

CVD

Cardiovascular Disease

HDP

Hypertensive Disorders of Pregnancy

HF

Heart failure

PreE

Pre-eclampsia

TTE

Transthoracic echocardiogram