Sepsis, an extreme immune response to an infection of the bloodstream, is a leading cause of neonatal morbidity and mortality. In the last decade, there were an estimated 3,930 cases of neonatal sepsis per 100,000 live births worldwide and nearly 20% of cases were fatal (1). While proximal risk factors such as preterm labor, prolonged rupture of membranes, and intra-amniotic infections have been recognized (2), preventing neonatal sepsis remains a key challenge (3). Identifying more distal risk factors for neonatal sepsis that are modifiable may be a promising strategy, as it is often too late to prevent fetal and neonatal infections by the time medical attention is sought (3). One such plausible risk factor is prenatal exposure to environmental metallic and metalloid trace elements, which are ubiquitous in the air, soil, water, and food supply (4). Several non-essential trace elements, including arsenic (a metalloid) (5), cadmium (a metal) (6), lead (a metal) (7), and mercury (a metal) (8), are recognized as immunotoxicants. In contrast, other nutritionally-essential trace elements such as copper (a metal) (9), manganese (a metal) (10), selenium (a metalloid) (11), and zinc (a metal) (10) may be necessary for optimal immune function, with experimental studies in animals indicating adverse effects from deficiencies and excesses (12–15).

Non-essential metallic and metalloid trace elements have distinct mechanisms of immunotoxicity, with many implicated as immunosuppressants, particularly when exposures occur early in life. For example, arsenic exposures in childhood have been linked to reductions in the proportion of CD4 (“helper”) T cells in peripheral blood (16), which capture and kill bacteria from infected dendritic cells. Prenatal exposures to cadmium have also been tied to alterations in lymphocyte composition in infancy (17), and prenatal methylmercury exposures to alterations that persist into childhood (18). At the same time, non-essential metals and metalloids could promote inflammation, which is an appropriate response to the presence of infection but can be dangerous when excessive (19). Research on lead has identified links between in utero exposures with lower concentrations of the anti-inflammatory cytokines interleukin (IL)-4 and IL-8, as well as higher concentrations of the pro-inflammatory cytokine tumor necrosis factor alpha (TNF-α) in early childhood (20), suggesting exposures may have implications for infection elimination and/or tissue damage (19). In the United States, over 85% of women of reproductive age have measurable levels of arsenic, cadmium, lead, and mercury in their bodies (21), making immunotoxic effects of prenatal exposures a pertinent concern.

It has been posited that non-essential metals and metalloids gain entry into human cells by “mimicking” and potentially competing with nutritionally-essential trace elements (22). For this reason, epidemiologic studies of exposure mixtures encompassing both non- and nutritionally-essential trace elements have become popular under the premise that nutritional interventions could counteract environmental metal/metalloid toxicity (23). Although few epidemiologic studies to date have considered the role of complex trace element mixtures in the development of immune-mediated and infectious diseases (23), nutritionally-essential elements are recognized for their important roles in the immune system. Zinc, for example, modulates the inflammatory response by binding to and inhibiting IKK-β, a protein important for cytokine-activated intracellular signaling. In a mouse model of polymicrobial sepsis, a zinc-deficient diet promoted insufficient control of IKK-β, leading to excessive inflammation (24). Therefore, we hypothesized that greater prenatal exposures to nutritionally-essential trace elements, namely copper, manganese, selenium, and zinc would be associated with a reduced risk of neonatal sepsis, whereas the opposite would be true for the non-essential trace elements arsenic, cadmium, mercury, and lead (i.e., greater prenatal exposures to non-essential trace elements would be associated with an increased risk of neonatal sepsis). To test these hypotheses, we analyzed each element individually and complemented this approach with quantile g-computation to assess how combinations of exposures in utero influences the risk of sepsis within the first few weeks of life. We conducted all analyses within the Extremely Low Gestational Age Newborns (ELGAN) cohort, which is comprised of infants born extremely preterm who are at a markedly increased risk of developing neonatal bacterial sepsis due to having immature immune systems and requiring invasive medical procedures and devices (25).

Neonatal bacterial sepsis is a leading cause of infant morbidity and mortality, resulting in a significant healthcare burden (1, 41). Survivors often face neurological sequelae such as cerebral palsy and impaired motor function that have a persistent impact on quality of life (42). Although research suggests that the contribution of environmental factors to sepsis is just as strong as genetics (43), there have been no epidemiologic studies to date on the role of environmental chemical exposures. In this prospective cohort of infants born extremely preterm, we found suggestive evidence that higher levels of prenatal exposure to cadmium were associated with an increased risk of developing bacterial sepsis during the first 28 days of life. However, the results from mixture analyses indicated that joint prenatal exposures to arsenic, copper, and selenium may have a mitigating effect.

Cadmium is found in tobacco smoke and is a contaminant of certain foods (e.g., cereals, bread, and leafy vegetables) (44). Although smoking during pregnancy was associated with notably higher levels of cadmium in umbilical cord tissue, the low prevalence in ELGAN (9.3%) makes it likely that diet was the predominant source of cadmium exposure for this population (45). During pregnancy, cadmium is partially sequestered in the placenta but does cross the placental barrier to reach the developing fetus (46). We found that nearly all umbilical cord tissue samples (98.1%) had detectable levels of cadmium and the median concentration was 1.33 ng/g, a value in line with other studies examining trace elements in cord tissue (47, 48). Prenatal exposure to cadmium has been tied to immunosuppressive endpoints in both animal and epidemiologic studies, including lower levels of T helper memory and natural killer cells (17, 49), potentially increasing susceptibility to infections. There is also evidence to suggest the immunosuppressive effects of prenatal cadmium exposure differ by fetal sex. In mice exposed to environmentally-relevant levels of cadmium in utero and later immunized with the streptococcal vaccine, females exhibited a greater reduction in vaccine-induced antibody production and in the number of antibody secreting cells as compared to males (49). In our study, stratified models revealed stronger associations between prenatal cadmium exposure and incident sepsis in female infants, highlighting the potential for sex-based differences in the immunotoxic effects related to prenatal cadmium exposure.

While our results suggested greater prenatal exposure to cadmium increases the risk of neonatal bacterial sepsis, copper, selenium, and arsenic seemed to do the opposite. Although we anticipated that copper and selenium, as essential nutrients, would confer protection, the results for arsenic were contrary to our hypothesis. Arsenic is a non-essential metalloid and prenatal exposures have previously been linked to immunosuppression and inflammation (50, 51). However, it is possible that arsenic exposure could reduce the risk of sepsis, as it was historically used as a treatment for syphilis due to its antimicrobial properties (52). In 1909, Paul Ehrlich and Sahachiro Hata discovered that the organic arsenic compound arsphenamine (later sold under the brand name Salvarsan) was remarkably effective at killing the parasitic bacterium Treponema pallidum without overtly harming the host (53). One explanation for our findings is that the infants with the highest cord tissue arsenic concentrations had predominantly been exposed to organic arsenic compounds (e.g., arsenobetaine, arsenosugars, arsenolipids), which are frequently found in seafood and are believed to be far less toxic than inorganic forms (54). Since only total arsenic content was measured for this study, we were unable to consider exposure to different arsenic species. It is also plausible that the inverse association observed for arsenic is an artifact of uncontrolled negative confounding by seafood intake (55). In addition to organic arsenicals, seafood, including fish, shellfish, and seaweed, contains nutrients like omega-3 fatty acids that have been shown in an in vitro study to induce anti-inflammatory effects relevant for the prevention of neonatal sepsis (56). The ELGAN cohort did not assess prenatal dietary intakes so we were unable to examine seafood consumption during pregnancy as a source of arsenic and/or mercury exposure. However, the strongest element-by-element correlation we observed was between arsenic and mercury. Methylmercury, in particular, is a common seafood contaminant and, like arsenic, requires more complex sample preparation and speciation analysis techniques to determine specific biomarker concentrations (57). Given the very small sample masses of umbilical cord tissue available, speciation analysis was not feasible for this study. Future cohort studies examining environmental trace element exposures should consider collecting prospective dietary data and sufficient biological samples for performing speciation analyses to better disentangle beneficial and harmful effects.

The apparent protective associations observed for copper and selenium in this study are noteworthy and raise the possibility of dietary interventions as preventive or therapeutic strategies. Similar to the issue of seafood consumption during pregnancy, the findings for copper and selenium may be an artifact of uncontrolled confounding by prenatal supplement use, for which detailed information was not collected in ELGAN. Still, experimental data and the biological role of nutritionally essential trace elements provide a rationale for further investigation in the context of neonatal bacterial sepsis prevention (58–60). In a randomized trial of very low birth weight and preterm infants (<1500 g and <32 weeks', n = 90), supplementation of 10 μg selenium per day for 28 days significantly reduced the risk of culture-proven sepsis by 13.3% (60). Both copper and selenium are cofactors for antioxidant enzymes, which suppress the formation of free radicals that, if left unchecked, can lead to cellular and tissue damage (61). Specifically, glutathione peroxidase depends on selenium (62), whereas superoxide dismutase, in forms SOD1 and SOD3, requires copper (in addition to zinc) (63). In the United States, it is estimated that <10% of pregnant women have inadequate copper and selenium intakes, but virtually no one exceeds the respective tolerable upper intake levels (64), indicating there may be an opportunity to increase amounts present in prenatal dietary supplements. Another potential avenue could be including selenium in parenteral nutrition formulations used by preterm infants. In spite of a recommendation by the American Society for Parenteral and Enteral Nutrition, selenium is not a component in the only multi-trace element product currently marketed in North America for neonates (Multitrace-4 Neonatal by American Regent) (65). Investigating supplementation, either during pregnancy or after delivery, for the prevention of neonatal sepsis is a key area for future research (66), and could be expanded to examine efficacy in sub-groups highly exposed to environmental contaminants.

Although there is some evidence for adverse immunological effects of lead (20), mercury (18), manganese (67), and zinc at certain levels (24), associations for these trace elements appeared null in this analysis. This could be due to the particular exposure ranges represented in ELGAN. For instance, compared to umbilical cord tissue concentrations reported in the literature (47, 48), we found lower concentrations of lead and higher concentrations of zinc, suggesting low levels of exposure to lead pollution and adequate zinc nutriture within the ELGAN cohort. Another important consideration is the relatively small sample size, which may have hampered our ability to detect modest associations and left us underpowered to conduct some proposed analyses. Notably, we were unable to investigate early-onset cases of sepsis independently. In models restricted to late-onset cases, the association for the non-essential trace elements mixture was near null. While this finding may indirectly support in utero exposures to non-essential trace elements as a more salient risk factor for early as opposed to late-onset sepsis, validation in a larger cohort is needed. Alternatively, we acknowledge that although many non-essential trace elements show evidence for immunotoxicity, it may be that neonatal bacterial sepsis is not a particularly sensitive outcome to exposures in the context of extremely preterm infants for whom other factors (e.g., in utero infection status, immune system immaturity, and the need for invasive medical procedures) likely play an outsized role in determining risk.

Despite limitations of this study, notably its small sample size, lack of important dietary information, and inability to perform trace element speciation, it is the first to investigate environmental chemical exposures as a risk factor for neonatal bacterial sepsis. Beyond novelty, there are notable analytic strengths, including the prospective study design, classification of sepsis status through blood culture, and assessment of multiple trace element exposures through biomarkers. Sepsis status was defined by the presence of bacteria in the (usually sterile) bloodstream, indicating bacteremia. At first glance, this definition may seem incomplete because it does not include measures of systemic inflammation. However, markers like C-reactive protein (CRP) and white blood cell counts are generally uninformative in neonates (68). By contrast, blood culture is considered the “gold standard” for diagnosing bacterial sepsis in neonates and was performed serially in ELGAN, improving the internal validity of this study (68). In addition, the use of umbilical cord tissue as a biospecimen to measure prenatal exposure to metallic and metalloid trace elements is a unique advantage. Although most studies rely on maternal samples such as blood or urine collected during pregnancy, maternal biomarkers may not reflect fetal exposures due to toxicokinetics. At present, the literature on trace element content within umbilical cord tissue is sparse and limited data suggest that, compared to umbilical cord blood, tissue is acceptable for assessing in utero exposures to mercury and selenium, whereas it may be the inferior matrix for cadmium, lead, copper, and zinc, and there are no data on arsenic or manganese either way (48). In addition, there is the possibility that the location of the tissue sampled affects the amount of trace element present; given that the tissue assessed in this study was obtained from the portion of umbilical cord closest to the abdominal wall, we believe our study appropriately characterized prenatal trace element exposures of mercury and selenium but acknowledge that instead measuring concentrations of the other trace elements in cord blood would have likely enhanced study validity. An additional strength of this study was the availability of rich covariate information, including sociodemographic factors and pregnancy complications that are known risk factors for neonatal sepsis. We leveraged these data to control for potential confounders in primary analyses and hypothesized intermediates in supplemental analyses. The results from both were nearly identical, indicating that, under certain assumptions (69), prenatal metal and metalloid exposures likely influence the risk of neonatal bacterial sepsis directly, rather than through pregnancy complications or infant characteristics.

In summary, we identified a positive association for prenatal cadmium exposure with incident bacterial sepsis in a cohort of extremely preterm infants, which was consistent when cadmium was examined in isolation and within the context of joint exposures to other metals and metalloids. This association was more pronounced among female infants, suggesting cadmium-associated immunotoxicity may differ by sex. It also did not appear to be mediated through pregnancy complications or infant characteristics, indicating cadmium may directly affect the developing immune system. Interestingly, higher levels of in utero exposures to arsenic, copper, and selenium seemed to mitigate the risk associated with high cadmium exposures. Future studies are needed to understand whether exposure to specific arsenic species are contributing to this unexpected finding, whether the results could be explained by negative confounding by seafood consumption during pregnancy, and whether copper and selenium supplementation are viable strategies for reducing rates of bacterial sepsis in high-risk infants.

The data that support the findings of this study are available upon reasonable request from the corresponding author, RCF. The data are not available publicly due to their containing information that could compromise the privacy of the ELGAN study participants.

The ELGAN study was approved by the Institutional Review Boards at each of the 14 participating study sites and informed consent was provided by all enrolled mothers for themselves and their infants.

CB conceptualized the study, conducted all statistical analyses, created all tables and figures, and wrote the original draft of the manuscript. LS and AG prepared umbilical cord biospecimens for metals assessment. AG and PP developed the ICP-MS/MS methodology, measured metal concentrations, and provided critical review of the manuscript. KR curated the data. LE provided critical review of the manuscript. TO'S and RF acquired the funding, provided project administration, and provided critical review of the manuscript. All authors contributed to the article and approved the submitted version.

This study was funded in part by funding from the National Institutes of Health (UH3OD023348, R01HD092374, and 1U2CES026542).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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