Prenatal maternal stress: Pregnant women especially vulnerable to chronic stress; potential challenging situations such as body image issues, lifestyle changes, fluctuating hormones, induce lasting changes to fetal stress response ("fetal programming")

Prenatal stress perturbs fetal iron homeostasis in a sex specific manner. Peter Zimmermann, Marta C. Antonelli, Ritika Sharma, Alexander Müller, Camilla Zelgert, Bibiana Fabre, Natasha Wenzel, Hau-Tieng Wu, Martin G. Frasch & Silvia M. Lobmaier. Scientific Reports volume 12, Article number: 9341, Jun 4 2022. https://www.nature.com/articles/s41598-022-13633-z

Abstract: The adverse effects of maternal prenatal stress (PS) on child’s neurodevelopment warrant the establishment of biomarkers that enable early interventional therapeutic strategies. We performed a prospective matched double cohort study screening 2000 pregnant women in third trimester with Cohen Perceived Stress Scale-10 (PSS-10) questionnaire; 164 participants were recruited and classified as stressed and control group (SG, CG). Fetal cord blood iron parameters of 107 patients were measured at birth. Transabdominal electrocardiograms-based Fetal Stress Index (FSI) was derived. We investigated sex contribution to group differences and conducted causal inference analyses to assess the total effect of PS exposure on iron homeostasis using a directed acyclic graph (DAG) approach. Differences are reported for p < 0.05 unless noted otherwise. Transferrin saturation was lower in male stressed neonates. The minimum adjustment set of the DAG to estimate the total effect of PS exposure on fetal ferritin iron biomarkers consisted of maternal age and socioeconomic status: SG revealed a 15% decrease in fetal ferritin compared with CG. Mean FSI was higher among SG than among CG. FSI-based timely detection of fetuses affected by PS can support early individualized iron supplementation and neurodevelopmental follow-up to prevent long-term sequelae due to PS-exacerbated impairment of the iron homeostasis.

Discussion

PS disrupts fetal iron homeostasis in a sex-specific manner

This study indicates a sex-dependent difference in fetal iron homeostasis and FSI due to PS in an otherwise healthy cohort, mainly driven by the male sex. Causal inference approach allowed us to independently verify fetal sex as an important effect modifier on the causal pathway between PS and cord blood ferritin. The findings strengthen previous published FSI results10.

The PS effect on the fetal iron biomarkers has been poorly understood. Rhesus monkey infants born to stressed mothers were more likely to develop iron deficiency7. Likewise, several studies in humans have shown a correlation between PS and cord blood zinc protoporphyrin/heme index as well as PS and ferritin levels4,5,6.

During pregnancy, maternal stress hormones such as cortisol influence the growing fetus and its neurodevelopment, presumably via epigenetic mechanisms9,19. Cortes and colleagues proposed an influence of chronic stress through a stress-induced altered expression of a variant of the enzyme acetylcholinesterase on the iron-regulating system in fetal sheep brain-derived primary microglia cultures15. They assumed the afferent cholinergic anti-inflammatory pathway signaling on microglial α7 nicotinic acetylcholine receptors to down-regulate metal ion transporter and ferroportin, which acts as a hepcidin receptor (Fig. 1).

Animal studies observed stress-dependent cognitive deficits mainly seen in males20,21. In humans, sex-specific PS effects are reflected by lower scores in conduct assessments and higher test scores for emotional disturbance in males compared to females13,22,23. Campbell et al. applied six specific PS questionnaires, each twice in the second and third trimester, to 428 ~ 28-years-old mothers and found newborns of pregnant women exposed to violence to be stronger associated with cord blood ferritin levels lower in boys than in girls4.

The relation of iron homeostasis biomarkers to PS

Our results show no relationship between the presence of maternal anemia, fetal iron deficiency and PS. These findings are in agreement with literature suggesting that the fetus is robust against moderate changes of the maternal iron homeostasis24,25.

Within the DAG framework, we estimated that PS reduced the cord blood serum ferritin levels by approximately 15%. These findings are exceeding the adaption factor for inflammatory processes in infants the WHO uses in a current guideline26. We assume that during pregnancy even relatively small additional shifts in fetal iron homeostasis, especially in ferritin levels, may induce sex-specific neurodevelopmental effects27. Our observations regarding the link between PS, fetal iron homeostasis and the postnatal neurodevelopmental trajectories warrant further investigations, because this condition may be corrected therapeutically via targeted prenatal and/or postnatal iron supplementation2.

The PS effect transmitted by maternal cortisol on the fetal neurodevelopment may depend on the time course of exposure28,29. Hypothetically, taking our explanation further antepartum, i.e., to ~ 3.5 weeks earlier at the time of taECG recording, we speculate that PS-induced differences in hepcidin at that time may lead to the reported changes in iron parameters that could still be detected in the cord blood1. Our exploratory findings of higher FSI within certain ranges of at-birth iron biomarkers support this notion. The absence of group differences of cord blood iron parameters including the whole cohort may reflect adaptions (more pronounced in females) that occur as pregnancy progresses20.

The role of the immune system

Our data in leukocytes showed no evidence of increased inflammatory processes in SG neonates (Table S1). Nevertheless, acute inflammatory processes, a common phenomenon during delivery, may have had an effect on our cord blood findings transmitted by other cellular messengers such as the cytokine IL-6 (Fig. 1). In general, inflammation upregulates the acute phase protein ferritin influencing its role as a biomarker of the iron storage30. Inflammation also upregulates hepcidin levels leading to an intestinal sequestration of iron14. Cord blood interleukin levels were increased in chronically stressed mother’s infants31. Taken together, the effects of PS can be mediated by inflammatory processes and this link should be investigated further in future studies including a broader characterization of the maternal and neonatal inflammatory profiles32.

FSI as a potential biomarker of PS in late gestation

The present findings confirm that FSI is increased in PS during the third trimester of pregnancy10. Because the FSI showed poor association with the measured iron biomarkers, we assume that PS influences fHR and mHR coupling by different pathways. Moreover, in our DAG framework it is conceivable that FSI may serve as an indicator of subsequent altered neurodevelopmental trajectories, even in the absence of biochemical PS correlates such as alterations in iron homeostasis33,34.

ML-based predictions of PS

With our ML approach, we mimicked a real-life scenario to identify mother–fetus dyads affected by PS. Our results are consistent with findings in other clinical settings where electronic medical record mining identified patients at risk even without additional biophysical assessments, such as ECG35. Notably, adding biophysical characteristics improves ML model performance, thus emphasizing the potential of antepartum mother–child monitoring using taECG to improve the early detection of health abnormalities such as PS.

Strengths and limitations

Strengths of the FELICITy study are the prospective design preventing recall bias and the definition of criteria for a matching system to exclude possible confounders. Additionally, eventual confounding factors such as the intake of iron supplement and ethnic group showed no group differences (Fig. S1). This is the first prospective longitudinal study starting in utero aiming to assess PS and fetal biomarkers. Additionally, it is the first study to use causal inference and machine learning approaches to investigate sex-dependent influence of PS on the fetal iron homeostasis. There are certain limitations. Our inclusion criteria prevented us from enrolling non-German-speaking patients. This may have biased how the PS effects are represented in the multicultural Munich population. Also, we used a matching system that could not include every screened CG patient. Due to the uncertainties of a human study, several subject numbers for different sub-analyses were lower. Furthermore, we focused on measuring PS in the third trimester which necessarily neglected earlier stages of pregnancy and a possible temporal dynamic of PS over the entire course of pregnancy.

We chose not to include other potential effect modifiers on the causal pathway of the DAG such as inflammatory processes as they are difficult to define quantitatively and were not the focus of this study. However, future studies could further refine estimates of PS → Iron Biomarker average exposure effect by adjusting for these covariates.

This study did not differentiate between arterial or venous origin of the analyzed cord blood samples. To our knowledge this issue has not been addressed in literature so far. In general, the placental iron transfer and the assessment of the fetal iron status using cord blood parameters are poorly understood36,37. As of the date of the manuscript’s submission no commonly used normal ranges of cord blood iron parameters exist. The established ranges start with the child's birth26 but are not applicable to cord blood ranges since in cord blood usually, iron parameters are higher38. These issues warrant further research to identify potential biasing effects on cord blood analysis.