Abstract

   Marginalized populations experience disproportionate rates of preterm
   birth and early term birth. Exposure to per- and polyfluoroalkyl
   substances (PFAS) has been reported to reduce length of gestation, but
   the underlying mechanisms are unknown. In the present study, we
   characterized the molecular signatures of prenatal PFAS exposure and
   gestational age at birth outcomes in the newborn dried blood spot
   metabolome among 267 African American dyads in Atlanta, Georgia between
   2016 and 2020. Pregnant people with higher serum perfluorooctanoic acid
   and perfluorohexane sulfonic acid concentrations had increased odds of
   an early birth. After false discovery rate correction, the effect of
   prenatal PFAS exposure on reduced length of gestation was associated
   with 8 metabolomic pathways and 52 metabolites in newborn dried blood
   spots, which suggested perturbed tissue neogenesis, neuroendocrine
   function, and redox homeostasis. These mechanisms explain how prenatal
   PFAS exposure gives rise to the leading cause of infant death in the
   United States.

   Subject terms: High-throughput screening, Environmental impact,
   Biomarkers, Epidemiology
     __________________________________________________________________

   Mechanisms of the impact of PFAS (also known as forever chemicals) on
   adverse birth outcomes remain largely unknown. Here, authors identified
   tissue neogenesis, neuroendocrine function, and redox homeostasis as
   imprints of prenatal PFAS exposures and reduced gestational age in the
   newborn metabolome.

Introduction

   In 2020, there were an estimated 364,487 infants born preterm (22– < 37
   completed gestational weeks) and 1,003,260 infants born early term
   (37–38 completed gestational weeks) in the United States (US)^[52]1.
   The annual rates of these adverse birth outcomes are consistently
   highest among Black Americans^[53]1. Preterm birth (PTB) and early term
   birth (ETB) are leading risk factors for morbidity and mortality during
   infancy, childhood, and early adulthood^[54]2–[55]5. A reduced length
   of gestation is also linked to cardiovascular disease, diabetes,
   neurodevelopmental disorders, cancer, and other prevalent chronic
   health conditions across the life course^[56]6–[57]11. The gestational
   age at birth is influenced by a complex interplay of psychosocial,
   behavioral, nutritional, and biological determinants, plus mounting
   evidence suggests environmental exposures potentiate the risk of PTB
   and ETB^[58]12–[59]16. Recent work has demonstrated per- and
   polyfluoroalkyl substances (PFAS) are commonly present in utero, which
   may explain poor fetal growth and development^[60]17–[61]20. However,
   most environmental epidemiologic studies have focused on white, highly
   educated populations and little is known about marginalized
   populations. Further, the underlying molecular mechanisms elicited by
   PFAS within the fetus’ metabolic, endocrine, and immune systems remain
   poorly understood.

   PFAS are anthropogenic surfactants used by industries throughout the
   world and have a long history of use by the US Department of
   Defense^[62]21. Legacy PFAS, including perfluorooctanoic acid (PFOA),
   perfluorononanoic acid (PFNA), perfluorooctane sulfonic acid (PFOS),
   and perfluorohexane sulfonic acid (PFHxS), share in common a lipophobic
   carbon-fluorine chain, hydrophilic functional group, and proteinophilic
   attraction towards albumin and various fatty acid binding
   proteins^[63]22–[64]24. These chemical properties result in long
   half-lives and foster persistence in the environment, which through a
   variety of exposure pathways lead to bioaccumulation in humans^[65]25.

   In the US population, 99% of pregnant people have detectable levels of
   PFOA and PFOS in their blood^[66]26. Similarly, PFOA, PFOS, PFNA, and
   PFHxS are detected in nearly all maternal serum samples collected in
   the late first trimester or early second trimester in the Atlanta
   African American Maternal-Child Cohort^[67]27. During pregnancy, a
   proportion of the maternal PFAS body burden is able to cross the
   placental barrier into the uterus where the fetus is exposed
   ^[68]28–[69]31. The direct effects of prenatal PFAS exposure on reduced
   length of gestation have been examined in several human
   populations^[70]32–[71]34. However, the inconsistent and limited data
   among marginalized groups warrant more research, plus an unfulfilled
   public health priority is to hone the causal pathways for these
   exposure-outcome relationships. Our group and others have proposed
   interference with homeostatic processes from such environmental
   exposures in utero leads to a cascade of bioenergetic perturbation,
   endocrine disruption, and oxidative stress production, which may
   synergistically promote adverse birth outcomes^[72]35–[73]38.

   Untargeted metabolomics by high-resolution mass spectrometry combined
   with a meet-in-the-middle (MITM) analysis may help to characterize the
   molecular signatures of PFAS in utero, biological processes integral to
   fetal programming, and adverse phenotypes in early life^[74]37.
   Specifically, MITM allows for environmental exposures and health
   outcomes to be linked to metabolomic profiles, the global set of
   metabolites and systemic responses to internal doses of exogenous and
   endogenous substances^[75]39,[76]40. Intermediate biomarkers and
   biological pathways for exposure-outcome relationships have been
   identified in several environmental epidemiologic studies using the
   MITM framework, including exposure to air pollution, tobacco smoke, and
   PFAS and fertility, PTB, and small-for-gestational age (SGA),
   respectively^[77]37,[78]41,[79]42. To our knowledge, no investigations
   have taken this approach to understand mechanistically how prenatal
   PFAS exposure influences the newborn metabolome and, in turn, how these
   responses are associated with gestational age at birth outcomes.

   In a prospective birth cohort, we sought to profile the neonatal
   metabolome for molecular signatures of maternal PFAS concentrations
   during early to middle pregnancy and gestational age at birth outcomes
   among African American mother-newborn dyads in Atlanta, Georgia. Based
   on prior work, we hypothesized that prenatal PFAS exposure interferes
   with gestational length and fetal growth^[80]37,[81]43. Additionally,
   we analyzed newborn dried blood spots (DBS), a minimally invasive
   biospecimen used for screening within 48 h of birth, with
   high-resolution metabolomics and the MITM framework to identify and
   measure the underlying metabolites and pathways.

   Here, we show that an increase in maternal serum PFAS concentrations
   was prospectively associated with ETB and medically indicated early
   birth prior to full-term. The newborn DBS metabolome revealed
   perturbations in biological pathways involving amino acids, bioactive
   lipids, and enzymes, coenzymes, and cofactors underly the PFAS and
   gestational age at birth outcome relationships. We further
   characterized the molecular network by identifying salient metabolites
   in the newborn circulatory system, including L-DOPA, linoleic acid, and
   β-NAD.

Results

Study population characteristics

   The characteristics of 267 African American pregnant people and
   newborns included in our study are summarized in Table [82]1. In early
   to middle pregnancy, the majority of mothers had a BMI considered
   overweight (n = 58; 22%) or with obesity (n = 109; 41%), were parous
   (n = 155; 58%), and did not use tobacco (n = 239; 90%) or marijuana
   (n = 177; 66%). At enrollment, the average participant age was 25.6
   years (SD = 5.2) and 163 (61%) of the mothers were in the first
   trimester. Participants predominantly had a high school education or
   less (n = 153; 57%), public health insurance with Medicaid (n = 218;
   82%), and an income level 132% or lower times that of the Federal
   Poverty Level (n = 153; 57%).

Table 1.

   Characteristics of 267 pregnant African American people and newborns in
   the Atlanta African American Maternal-Child cohort, 2016–2020
                 Characteristic               Participants, No. (%)^a
   Delivery year
   2016                                       44 (16)
   2017                                       95 (36)
   2018                                       64 (24)
   2019                                       33 (12)
   2020                                       31 (12)
   Maternal age, mean (SD), y                 25.6 ± 5.2
   Education
   Less than high school                      38 (14)
   High school                                115 (43)
   Some college                               68 (25)
   College graduate or above                  46 (17)
   Income-poverty ratio^b
   <100%                                      113 (42)
   100 – 132%                                 40 (15)
   133 – 149%                                 22 (8)
   150 – 199%                                 49 (18)
   200 – 299%                                 13 (5)
   300 – 399%                                 11 (4)
   ≥400%                                      19 (7)
   Married or cohabitating
   Yes                                        114 (43)
   No                                         153 (57)
   Health insurance
   Private                                    49 (18)
   Public                                     218 (82)
   Hospital
   Emory University Hospital (private)        103 (39)
   Grady Hospital (public)                    164 (61)
   Parity                                     1.1 ± 1.2
   Nulliparous                                112 (42)
   Primiparous                                72 (27)
   Multiparous                                83 (31)
   Prenatal BMI, kg/m^2 c                     29.0 ± 7.6
   Underweight                                8 (3)
   Normal weight                              92 (34)
   Overweight                                 58 (22)
   Obesity                                    109 (41)
   Marijuana use one month before pregnancy
   Yes                                        90 (34)
   No                                         177 (66)
   Tobacco use one month before pregnancy
   Yes                                        28 (10)
   No                                         239 (90)
   Trimester serum sample collected^d         11.3 ± 2.2
   1st trimester                              163 (61)
   2nd trimester                              104 (39)
   Delivery mode
   Vaginal                                    120 (45)
   Cesarean section                           31 (12)
   Missing                                    116 (43)
   Neonatal sex
   Female                                     139 (52)
   Male                                       128 (48)
   Gestational age at birth^e
   Preterm                                    31 (12)
   Early term                                 82 (31)
   Full-term                                  154 (57)
   Gestational age at birth, mean (SD), weeks 38.7 (2.0)
   Labor and delivery course for early births^f
   Spontaneous                                82 (69)
   Medically indicated                        31 (26)
   [83]Open in a new tab

   y year, BMI body mass index (calculated as weight in kilograms divided
   by height in meters squared), w week.

   ^aReported percentages are composition ratios of each horizontal
   characteristic.

   ^bIncome-poverty ratio calculated as total family income divided by the
   Federal poverty threshold.

   ^cBMI categorized as follows: underweight, <18.5 kg/m^2; normal weight,
   18.5 – 24.9 kg/m^2; overweight, 25.0 – 29.9 kg/m^2; and obesity,
   ≥30 kg/m^2.

   ^dTrimesters categorized as follows: 1^st trimester, 6 – 12 gestational
   weeks; 2^nd trimester, 13 – 17 gestational weeks.

   ^eGestational age at birth categorized as follows: preterm, 22 – <37
   gestational weeks; early term, 37 – 38 gestational weeks; full-term,
   ≥39 gestational weeks.

   ^fReported percentages for labor and delivery course are composition
   ratios of preterm birth and early term birth versus 118 healthy
   full-term births.

   There were 139 (52%) newborns assigned female sex at birth. The average
   gestational age at delivery was 38.7 weeks (SD = 2.0), with a total of
   118 (51%) healthy and full-term, 31 (12%) preterm, and 82 (31%) early
   term. Among the early births (PTB or ETB) prior to full-term, 82 (69%)
   followed spontaneous labor and 31 (26%) followed medically indicated
   induction or C-section (Fig. [84]S2).

   All four PFAS were detected in 98–100% of maternal serum samples
   collected during early to middle pregnancy (Table [85]S1). The GM (GSD)
   concentrations of PFOS were highest 1.43 ng/mL (2.72), followed by
   PFHxS 1.09 (2.30), then PFOA 0.57 (2.31), and lastly PFNA 0.25 (2.26).
   The log[2]-transformed PFAS concentrations (ng/mL) were weakly to
   moderately correlated with each other (Pearson
   [MATH: <mi>ρ</mi> :MATH]
   range = 0.23–0.64) (Table [86]S2).

Prenatal PFAS in maternal serum and neonatal birth outcomes

   The effects of maternal PFAS concentration on gestational age at birth
   (continuous and categorical gestational weeks) and labor and delivery
   (spontaneous or medically indicated) outcomes relative to healthy,
   full-term birth are presented in Fig. [87]1 and Table [88]S3. For every
   log[2]-unit increase in PFOA concentrations, the odds of ETB were 1.59
   (95% CI: 1.15, 2.21) compared to healthy full-term birth. The odds
   ratio (OR) of ETB was also significantly increased among those in the
   2nd quartile (OR = 2.85; 95% CI 1.16, 7.02) and 4th quartile
   (OR = 4.59; 95% CI 1.78, 11.89) of PFOA concentration versus the
   referent. Concentrations of PFOA categorized as the 3rd quartile
   increased the odds of ETB, but did not reach statistical significance
   (p > 0.05). Quartiles of PFHxS concentrations demonstrated a similar
   dose-response relationship with PTB and medically indicated early
   birth. Log[2]-transformed and quartile PFOA concentrations were
   associated with moderate increases in the odds of spontaneous labor.
   Finally, gestational age at delivery was inconsistently associated with
   PFAS concentrations.

Fig. 1. Dot-and-whisker plots showing the associations between prenatal serum
PFAS levels and gestational age at birth outcomes among African American
mother-newborn dyads in Atlanta, 2016–2020.

   [89]Fig. 1
   [90]Open in a new tab

   Statistical tests were performed with two-sided multivariable linear or
   logistic regression with a significance level of p-value < 0.05. The
   sample size of independent dyads was as follows: N = 267 for
   gestational age at birth, N = 200 for early term birth and spontaneous
   early birth, N = 149 for preterm birth and medically indicated early
   birth. Data are presented as coefficient estimates (β)
   [MATH: <mo>±</mo> :MATH]
   95% confidence intervals (CI) or odds ratios (ORs)
   [MATH: <mo>±</mo> :MATH]
   95% confidence intervals (CI). The coefficient estimates (β) for
   gestational age and ORs for preterm birth, early term birth, medically
   indicated early birth (preterm birth or early term birth), and
   spontaneous early birth (preterm birth or early term birth) are on the
   X-axis. For the binary birth outcomes, the reference group was healthy
   full-term births. The vertical gray dashed line is the null value.
   Exposure to PFAS for every log[2]-unit increase and categorized by
   quartiles are on the Y-axis. Dots represent quartile exposures and
   triangles represent continuous exposures. Dots or triangles and
   whiskers color coded as purple are statistically significant at
   p-value < 0.05. The quartile cutoffs for PFOA (ng/mL) were as follows:
   Q1: <LOD – 0.42, Q2: 0.42 – 0.63, Q3: 0.63 – 0.96, Q4: 0.96 – 3.42. The
   quartile cutoffs for PFNA (ng/mL) were as follows: Q1: <LOD – 0.16, Q2:
   0.16 – 0.28, Q3: 0.28 – 0.46, Q4: 0.46 – 1.51. The quartile cutoffs for
   PFOS (ng/mL) were as follows: Q1: <LOD – 1.04, Q2: 1.04 – 1.64, Q3:
   1.64 – 2.46, Q4: 2.46 – 9.59. The quartile cutoffs for PFHxS (ng/mL)
   were as follows: Q1: <LOD – 0.66, Q2: 0.66 – 1.07, Q3: 1.07 – 1.93, Q4:
   1.93 – 6.17. Note: Exact p-values are provided in Supplemental Table 3.
   PFAS, perfluoroalkyl substances; PFHxS, perfluorohexane sulfonic acid;
   PFOS, perfluorooctane sulfonic acid; PFOA, perfluorooctanoic acid;
   PFNA, perfluorononanoic acid; Q, quartile. Source data are provided as
   a Source Data file.

Neonatal metabolites, prenatal PFAS in maternal serum, and birth outcomes

   We successfully obtained 6981 signals in the untargeted metabolomics
   dataset after data preprocessing, quality control procedures, and data
   filtering (Fig. [91]S3). To increase the rigor of our study, we
   selected the MWAS with ≥100 signals enriched at the most conservative
   significance threshold for the remaining analyses, including pathway
   enrichment and molecular phenotyping using the MITM framework. In the
   PFAS MWAS, there were 435, 559, 154, and 1262 signals significantly
   associated with prenatal serum concentrations of PFOA (raw
   p-value < 0.05), PFOS (raw p-value < 0.05), PFNA (Bonferroni
   q-value < 0.01), and PFHxS (Bonferroni q-value < 0.01), respectively
   (Table [92]S4). In the gestational age at birth outcomes MWAS, there
   were 318, 199, 244, 669, and 142 signals significantly associated with
   medically indicated early birth (FDR q-value < 0.20), ETB (FDR
   q-value < 0.20), spontaneous early birth (FDR q-value < 0.05), PTB (FDR
   q-value < 0.05), and gestational weeks (Bonferroni q-value < 0.01),
   respectively (Table [93]S5). The number of significantly enriched
   signals associated with PFAS concentrations did not materially change
   when gestational week at sample collection was removed from the models
   (Table [94]S6).

   In total, we found 664 overlapping signals in one or more of the PFAS
   MWAS and one or more of the gestational age at birth outcomes MWAS when
   the significance threshold was set to p-value < 0.05 for PFOA and PFOS
   and FDR < 0.05 for all others (Table [95]S7). Upon signal
   identification and annotation, a total of 56 overlapping metabolites
   were matched with high confidence ontology levels, including nine
   labeled as OL1, 12 labeled as OL2a, 10 labeled as OL2b, and 25 labeled
   as PDa (Table [96]S8 and Table [97]S9). The metabolite profile
   consisted of carnitines, bile acids, amino acids and proteins, chemical
   messengers, redox reactions, fatty acids and lipids, and xenobiotics
   (Figs. [98]2 and  [99]3). Amino acids and proteins were the largest of
   these classes with 11 metabolites, which were predominantly associated
   with maternal serum PFNA and PFHxS levels plus gestational age and PTB.
   Carnitines and bile acids overlapped with the fewest number of PFAS
   exposures and gestational age at birth outcomes whereas chemical
   messengers overlapped across all nine MWAS. The polyunsaturated ω-6
   fatty acids, arachidonic acid and linoleic acid, as well as
   tetradecenoyl-L-carnitine, which shuttles saturated long-chain fatty
   acids to mitochondria, were top metabolites and shared in common an
   association with PFHxS and gestational weeks (Fig. [100]2C, D, E, H).
   Another group of metabolites with a high frequency of overlap included
   indole-3-methyl acetate, 4-hydroxy-3-methyoxyphenylglycol, and
   β-nicotinamide adenine dinucleotide (β-NAD) (Fig. [101]2A–D, F, H).

Fig. 2. Volcano plots of identified metabolites associated with prenatal
serum PFAS levels and gestational age at birth outcomes in Atlanta, 2016 –
2020.

   [102]Fig. 2
   [103]Open in a new tab

   Blue dots represent metabolites with negative associations, red dots
   represent metabolites with positive associations, and gray dots
   represent metabolites with non-significant associations among those
   confirmed and annotated. The coefficient estimate (β) from the MWAS is
   on the X-axis. The significance threshold is on the Y-axis. The
   vertical gray dashed line represents the null value. Select metabolites
   are annotated on the plots. A Volcano plot of metabolites associated
   with PFOA. Statistical tests were performed with two-sided
   multivariable linear regression. There were N = 267 independent dyads
   in the analytic sample. No adjustments were made for multiple
   comparisons. The horizontal gray dashed line represents the log[10]
   p-value = 0.05. B Volcano plot of metabolites associated with PFOS.
   Statistical tests were performed with two-sided multivariable linear
   regression. There were N = 267 independent dyads in the analytic
   sample. No adjustments were made for multiple comparisons. The
   horizontal gray dashed line represents the log[10] p-value = 0.05. C
   Volcano plot of metabolites associated with PFNA. Statistical tests
   were performed with two-sided multivariable linear regression. There
   were N = 267 independent dyads in the analytic sample. FDR from
   multiple comparisons was corrected with the Benjamini–Hochberg
   procedure. The horizontal gray dashed line represents the log[10] FDR
   q-value = 0.05. D Volcano plot of metabolites associated with PFHxS.
   Statistical tests were performed with two-sided multivariable linear
   regression. There were N = 267 independent dyads in the analytic
   sample. FDR from multiple comparisons was corrected with the
   Benjamini–Hochberg procedure. The horizontal gray dashed line
   represents the log[10] FDR q-value = 0.05. E Volcano plot of
   metabolites associated with gestational weeks at birth. Statistical
   tests were performed with two-sided multivariable linear regression.
   The reference group was healthy full-term births. There were N = 267
   independent dyads in the analytic sample. FDR from multiple comparisons
   was corrected with the Benjamini-Hochberg procedure. The horizontal
   gray dashed line represents the log[10] FDR q-value = 0.05. F Volcano
   plot of metabolites associated with preterm birth. Statistical tests
   were performed with two-sided multivariable logistic regression. The
   reference group was healthy full-term births. There were N = 149
   independent dyads in the analytic sample. FDR from multiple comparisons
   was corrected with the Benjamini–Hochberg procedure. The horizontal
   gray dashed line represents the log[10] FDR q-value = 0.05. G Volcano
   plot of metabolites associated with early term birth. Statistical tests
   were performed with two-sided multivariable logistic regression. The
   reference group was healthy full-term births. There were N = 200
   independent dyads in the analytic sample. FDR from multiple comparisons
   was corrected with the Benjamini-Hochberg procedure. The horizontal
   gray dashed line represents the log[10] FDR q-value = 0.05. H Volcano
   plot of metabolites associated with spontaneous early birth.
   Statistical tests were performed with two-sided multivariable logistic
   regression. The reference group was healthy full-term births. There
   were N = 200 independent dyads in the analytic sample. FDR from
   multiple comparisons was corrected with the Benjamini-Hochberg
   procedure. The horizontal gray dashed line represents the log[10] FDR
   q-value = 0.05. I Volcano plot of metabolites associated with medically
   indicated early birth. Statistical tests were performed with two-sided
   multivariable logistic regression. The reference group was healthy
   full-term births. There were N = 149 independent dyads in the analytic
   sample. FDR from multiple comparisons was corrected with the
   Benjamini-Hochberg procedure. The horizontal gray dashed line
   represents the log[10] FDR q-value = 0.05. MWAS, metabolome-wide
   association study; PFAS, per- and polyfluoroalkyl substances; PFHxS,
   perfluorohexane sulfonic acid; PFOS, perfluorooctane sulfonic acid;
   PFOA, perfluorooctanoic acid; PFNA, perfluorononanoic acid; FDR, false
   discovery rate. Source data are provided as a Source Data file.

Fig. 3. Alluvial plots of identified metabolites that relate the association
of prenatal serum PFAS levels with gestational age at birth outcomes in
Atlanta, 2016–2020.

   [104]Fig. 3
   [105]Open in a new tab

   The sample size of independent dyads was as follows: N = 267 for
   gestational age at birth, N = 200 for early term birth and spontaneous
   early birth, N = 149 for preterm birth and medically indicated early
   birth. The gray stacked bars indicate the frequency and relative
   proportion of PFAS exposure or gestational age at birth outcomes
   associated with the metabolite. The colored stacked bars indicate the
   frequency and relative proportion of metabolites associated with PFAS
   exposure and gestational age at birth outcomes. The legend shows which
   color corresponds to one of the 56 metabolites. Abbreviations: PFAS,
   perfluoroalkyl substances; PFHxS, perfluorohexane sulfonic acid; PFOS,
   perfluorooctane sulfonic acid; PFOA, perfluorooctanoic acid; PFNA,
   perfluorononanoic acid; PTB, preterm birth; ETB, early term birth;
   MIEB, medically indicated early birth; SEB, spontaneous early birth;
   GA, gestational weeks at birth. Source data are provided as a Source
   Data file.

Neonatal pathways, prenatal PFAS in maternal serum, and birth outcomes

   We identified 14 unique, overlapping pathways associated with PFAS
   exposure and gestational age at birth outcomes, which were broadly
   related to micronutrients, bioenergetics, bioactive lipids, and enzymes
   and cofactors (Fig. [106]4 and Table [107]S10). Gestational weeks at
   birth had no pathways that were present in the PFAS pathway enrichment
   analyses after removing those with <10% overlap. PFOS and PFHxS,
   containing a sulfonic acid moiety, shared in common the pathway for
   drug metabolism with ETB. PFOA and PFNA, containing a carboxylic acid
   moiety, shared in common lysine metabolism with PTB and leukotriene
   metabolism with medically indicated early birth. Glycerophospholipid
   metabolism overlapped with PFOA and PFOS and medically indicated early
   birth. The metabolic pathway for amino acids in the urea cycle was
   significantly enriched in all pathway enrichment analyses performed,
   except for PFOS. Tryptophan metabolism was significantly enriched in
   seven out of the nine pathway enrichment analyses, excluding PFNA and
   PTB. Lastly, across the nine pathway enrichment analyses performed, the
   percent of the proportion of significant putative metabolic signals
   versus the overall pathway size ranged from 10% to 64%.

Fig. 4. Enriched pathways that relate the association of prenatal serum PFAS
levels with gestational age at birth outcomes in Atlanta, 2016–2020.

   [108]Fig. 4
   [109]Open in a new tab

   The sample size of independent dyads was as follows: N = 200 for early
   term birth and spontaneous early birth, N = 149 for preterm birth and
   medically indicated early birth. A Heat map of the γ-adjusted p-values.
   A γ p-value is calculated based on permutations that randomly resamples
   the list of total features for a number of features equal to the
   significant set many times to create a γ null distribution. Cell color
   corresponds to the p-value for a metabolic pathway that overlaps in at
   least one PFAS metabolome-wide association study (MWAS) and gestational
   age at birth outcome MWAS. The reference group was healthy full-term
   births for the following MWAS: preterm birth, early term birth,
   medically indicated early birth (PTB or ETB), and spontaneous early
   birth (PTB or ETB). The significant signals are the average numbers of
   significant putative metabolites enriched in the overlapping pathways
   and associated an exposure or outcome. The pathway size is the average
   number of significant putative metabolites in the overlapping metabolic
   pathways. Overlap % is the relative proportion of significant signals
   to the pathway size. The metabolic pathways are grouped by amino acids,
   enzymes, coenzymes, and cofactors, and bioactive lipids. B Sunburst
   charts showing the frequency of overlap for the serum PFAS levels
   (green), overlapping pathways (gold), and gestational age at birth
   outcomes (blue). The largest sections indicate the greatest amount of
   overlap and the smallest sections indicate the least amount of overlap.
   Abbreviations: PFAS, per- and polyfluoroalkyl substances; PFHxS,
   perfluorohexane sulfonic acid; PFOS, perfluorooctane sulfonic acid;
   PFOA, perfluorooctanoic acid; PFNA, perfluorononanoic acid; PTB,
   preterm birth; ETB, early term birth; MIEB, medically indicated early
   birth; SEB, spontaneous early birth; GA, gestational weeks at birth.
   Source data are provided as a Source Data file.

   The pathways associated with PFAS exposure and gestational age at birth
   outcomes were generally consistent across the enrichment analyses
   performed at serially reduced significance thresholds (Table [110]S11
   and Table [111]S12). Additionally, the overlapping pathways
   significantly enriched in the PFAS sensitivity analyses did not greatly
   differ from the results obtained in the main analyses (Table [112]S13).

Discussion

   To our knowledge, this is the first study that has used newborn DBS
   metabolomics to explore the molecular mechanisms between prenatal PFAS
   exposure and gestational age at birth outcomes. We found maternal serum
   PFAS levels during early to middle pregnancy, which were surrogate
   measurements for fetal exposure to PFAS, were associated with early
   birth (PTB or ETB) prior to full-term among African American pregnant
   people and their newborns. Furthermore, the neonatal metabolome had an
   intermediate role for this relationship and offered insights into the
   underlying molecular network, as presented in Fig. [113]5. The
   biological pathways and biomarkers indicated perturbations in tissue
   neogenesis, neuroendocrine function, and redox homeostasis, which may
   have long-term health consequences beyond time of birth.

Fig. 5. Proposed molecular network of mechanisms and biomarkers in the
newborn dried blood spot metabolome underlying the association of prenatal
serum PFAS levels with gestational age at birth outcomes in Atlanta,
2016–2020.

   [114]Fig. 5
   [115]Open in a new tab

   Abbreviations: PFAS, per- and polyfluoroalkyl substances; AHR, aryl
   hydrocarbon receptor; PPAR, peroxisome proliferator-activated receptor;
   AR, androgen receptor; ER, estrogen receptor; GR, glucocorticoid
   receptor; MR, mineralocorticoid receptor; PR, progestin receptor; FXR,
   farnesoid X receptor; TCA cycle, tricarboxylic acid; L-DOPA, levodopa;
   β-NAD, β-nicotinamide adenine dinucleotide; 15-cyclohexyl pentanor
   PGF2α, 15-cyclohexyl pentanor prostaglandin F2α. Source data are
   provided as a Source Data file.

   In comparing pregnant people in the Atlanta African American
   Maternal-Child Cohort versus participants matched on sex, age, and race
   in NHANES, the detection rate and geometric average of PFOA, PFOS, and
   PFNA are similar while PFHxS is significantly higher in our study
   population^[116]27. Relative to other prospective cohorts in the US,
   maternal serum PFAS concentrations collected during early pregnancy are
   comparable to those observed in Chemicals In Our Bodies and Illinois
   Kids Infant Development Study, and lower than those observed in Project
   Viva, LIFECODES, and Healthy Start Study, which have a majority white
   and higher SES study population^[117]44–[118]47.

   While a growing body of evidence suggests exposure to PFAS increase the
   risk of PTB, as supported by our findings, other studies linking
   spontaneous or medically indicated early birth in relation to serum
   PFAS levels have proven
   inconclusive^[119]34,[120]48–[121]51,[122]32,[123]52. Similarly, the
   relationship between various PFAS measured in pregnant people and
   gestational age of newborns has been difficult to replicate across
   environmental epidemiologic studies^[124]20,[125]53,[126]54. A
   potential explanation for these observed differences is the proportion
   of PTB and ETB were higher in the present study relative to prior work,
   which mirrors the current trends of maternal and child health outcomes
   among African American families. Our findings provide credence to a
   relationship between prenatal PFAS exposure and adverse birth outcomes
   in populations routinely excluded from environmental epidemiologic
   studies.

   When compared to adults, the developing fetus is exposed to a higher
   dose of environmental chemicals due to immature size and has
   insufficient enzymatic capacity to protect against xenobiotics^[127]55.
   Our analyses suggest maternal PFAS levels may affect fetal P450 and
   other drug-metabolizing enzymes, as made evident in the newborn DBS
   metabolome, and are associated with PTB and ETB, respectively. Such
   exposures may interfere with developmental transitions in CYP
   expression, which occur throughout gestation and postnatally, thereby
   influencing the disposition of PFAS^[128]56. Since mitochondrial P450
   catabolize cholesterol, these enzymatic mechanisms may also point to
   perturbed steroidogenesis, bile acid synthesis, and vitamin D3
   activation, which our group and others have shown to be affected by
   PFAS^[129]57,[130]58,[131]37,[132]59–[133]67.

   In early life and beyond, bioenergetic processes rely on vitamin B3 to
   sustain the reduced nicotinamide adenine dinucleotide phosphate (NADPH)
   pool, which is involved in mitochondrial oxidative phosphorylation,
   redox homeostasis, and protein synthesis and function^[134]68,[135]69.
   We found maternal serum PFOA and PFHxS levels affected neonatal vitamin
   B3 metabolism, which was associated with medically indicated early
   birth prior to full-term. Additionally, the vitamin B3 metabolite β-NAD
   was attenuated by PFOS and PFHxS. Taken altogether, a reduced length of
   gestation may arise in part from prenatal PFAS exposure targeting vital
   pathways for energy metabolism.

   Biopterins are cofactors for the biosynthesis of neurotransmitters,
   hormone precursors, and vasodilators plus indicate oxidative stress
   since reactive oxygen species (ROS) limit their
   bioavailability^[136]70–[137]74. PFAS generate an excessive amount of
   ROS in vitro and deplete antioxidant reserves in vitro and in
   vivo^[138]75–[139]78. We found several antioxidative metabolites,
   including benzoic acid and S-lactyolglutathione, were associated with
   maternal serum PFHxS concentrations and gestational age, which may be
   in response to exposure. In addition, oxidative stress biomarkers have
   been linked to an increased risk of preterm birth^[140]79. Our
   metabolomic findings need to be validated in other fetal and neonatal
   populations, but illustrate the potential for oxidative stress to serve
   as a shared link between prenatal PFAS exposure and reduced length of
   gestation.

   We observed most of the intermediate metabolomic signatures between
   PFAS in pregnant people and gestational age at birth outcomes were
   related to amino acids, consistent with our prior work using maternal
   serum metabolomics^[141]37. Complex systems of amino acid transporters
   in the placenta tightly regulate the flux of amino acids between mother
   and fetus to ensure the demand for tissue neogenesis is met and so
   byproducts of protein metabolism are removed^[142]80,[143]81. It has
   been shown that PFAS exposure during gestation reduces amino acid
   transporter expression and system activity, which subsequently limits
   fetal growth and development. For example, there is reduced expression
   of SNAT4, an isoform of the system A amino acid transporter, in the
   placentas of pregnant mice exposed to PFOS^[144]82. In the future,
   these early life mechanisms of amino acid metabolism may serve as
   therapeutic targets for reduced length of gestation caused by PFAS in
   utero.

   Prenatal exposure to PFAS may potentiate risk of PTB and ETB by
   disrupting the metabolism of tryptophan and tyrosine. Tryptophan
   results in serotonin, kynurenine, tryptamine, and indole production,
   which take part in neurological, hormonal, and immunological processes
   during pregnancy and afterwards^[145]83–[146]85. Perturbations to this
   amino acid during pregnancy have been implicated in chronic
   inflammation disease states among mothers and adverse
   neurodevelopmental outcomes among offspring^[147]86. Similarly,
   tyrosine has a prominent role in the dopaminergic system of the
   placenta-brain-axis. Tyrosine hydroxylase synthesizes the dopamine
   precursor, levodopa (L-DOPA), from a combination of tyrosine, oxygen,
   and biopterin to support fetal neurodevelopment and
   survival^[148]72,[149]87. Our results suggest the indirect effects of
   PFAS on reduced length of gestation may occur through neuroendocrine
   pathways, and follow-up research should prioritize the investigation of
   related chemical messengers among premature and early newborns.

   The newborn DBS metabolome was enriched with metabolic pathways for
   glycerophospholipids and leukotrienes along with arachidonic acid,
   linoleic acid, and 15-cyclohexyl pentanor prostaglandin F2α
   metabolites. In other populations with occupational, community, and
   background exposure, dyslipidemia is commonly present regardless of
   age, possibly suggesting PFAS affect a lipid pathway conserved across
   the life course^[150]60,[151]88–[152]92. Specific to fetal growth and
   development, perturbed bioactive lipids may contribute to the
   mechanisms underlying the relationship between PFAS toxicity in utero
   and reduced length of gestation.

   The relationship between serum PFAS levels and lipids has been
   primarily attributed to suppressed mitochondrial fatty acid
   β-oxidation, attenuated farsenoid x receptor (FXR) via bile acids, and
   activated peroxisome proliferator-activated receptors
   (PPAR)^[153]61,[154]93,[155]94. These three processes have the
   potential to drive bioenergetic perturbation and oxidative stress
   production during gestation, which may be antecedent molecular events
   to adverse birth outcomes. One proposed mechanism is upregulated PPAR
   signaling from PFAS exposure alters arachidonic acid metabolism and its
   downstream metabolites, which leads to oxidative stress and
   inflammation, based on experimental work in rodents using metabolomics,
   proteomics, and transcriptomics^[156]95–[157]98. Pregnancy may
   represent a sensitive window for PFAS to interact with eicosanoids and
   their precursors because of their role in the onset of labor, which has
   long been recognized by clinicians^[158]99. The application of targeted
   methods is needed to validate and expand upon the results presented
   here, particularly the roles of bioactive lipids and inflammatory
   processes that precede or concomitantly occur with a reduced length of
   gestation.

   The exact role of prenatal PFAS exposure, as well as their overall
   significance, in driving maternal and child health disparities among
   African Americans remains unclear. Perhaps, exposure to PFAS compounds
   the physiological effects of gendered racial stress and discrimination
   during pregnancy, ultimately leading to an early birth. In
   consideration of the mechanisms identified here, more research is
   needed to understand the potential joint effects of environmental
   chemicals and non-chemical stressors on perinatal health outcomes.

   A combination of systems biology methods and tools, sampling
   techniques, and biostatistical approaches strengthened our study.
   First, we leveraged an untargeted high-resolution metabolomics platform
   with the MITM framework, which together positioned us to identify and
   measure the links among exposures, molecular signatures, and outcomes.
   Our group initially examined maternal metabolomic perturbations
   associated with prenatal PFAS exposure and fetal growth outcomes in the
   Atlanta African American Maternal-Child Cohort^[159]37. As a step to
   validate and expand upon such findings, we used newborn DBS samples to
   make inferences about how the fetal biological system is affected by
   PFAS exposure in utero, a major advancement over studies that have
   relied on cord blood. Our study also serves as a proof-of-concept for
   integrating high-throughput -omics, newborn screening protocols, and
   environmental molecular epidemiology, which will be increasingly
   important as public health and translational science advance toward a
   precision medicine model. Third, our phenotypic classification of
   gestational age at birth outcomes was based on medical record
   abstraction by medical personnel, minimizing the chance of
   misclassification that might affect studies based on self-report. Our
   research protocol also considered the phenotypic classification of
   births as early (PTB or ETB) or full-term based on early pregnancy
   dating with further phenotypic classification of early births following
   spontaneous labor or medically indicated labor, allowing us to
   distinguish these different pathways to early birth in our analyses.
   There is a recognized need for further research into factors increasing
   the risk of ETB as this is an understudied birth outcome and infants
   born in this category disproportionately experience excess morbidity
   relative to full-term infants^[160]100. Lastly, our study population
   included exclusively African Americans, a population that is sorely
   underrepresented in the extant literature.

   Our study also had several limitations. Only the individual effects of
   PFAS were included in this study, but mixture effects are plausible and
   alternative PFAS with lower detection rates and/or shorter half-lives
   may contribute to the observed associations. There is a potential for
   interaction of the studied PFAS with other environmental chemicals from
   co-exposure, which may be antagonistic, synergistic, or additive, and
   mask the true health effects of PFAS. Future population-based studies
   are needed to investigate environmental chemical mixtures and emerging
   PFAS, for which there are thousands, in both the intrauterine and
   extrauterine environments. Our limited sample size also necessitates a
   larger-scale study, which newborn DBS sampling could facilitate. There
   was a relatively small number of pregnant people who experienced PTB,
   which may explain why we did not observe a significant dose-response
   relationship when participants were categorized by exposure quartiles.
   Furthermore, the pathogenesis of adverse birth outcomes is likely
   multifactorial and influenced by genetics, socioeconomic status and
   behaviors, diet, environment, and notably for minority racial and
   ethnic groups, structural factors such as social inequality and
   systemic racism. As a result, we cannot rule out the possibility of
   residual confounding. By the same token, there is an ongoing discussion
   in environmental epidemiology about how maternal BMI is associated with
   serum PFAS concentrations during pregnancy. Based on the body of
   literature available today, we treated maternal BMI as a confounder,
   although more research is needed to clarify the direction of effect
   between adiposity and PFAS in pregnant populations^[161]101,[162]102.
   The newborn DBS samples and panel of gestational age at birth outcomes
   were measured during the same time period, which hinders causal
   inference for the metabolomic signal-outcome associations. Specific to
   the newborn DBS samples collected via heel-stick, critics have raised
   concerns over potential uneven cell distribution, cell lysis,
   hematocrit variation, and pre-analytical clotting when analyzing this
   biomatrix using untargeted metabolomics^[163]103–[164]105. Although the
   untargeted DBS metabolomics analysis was normalized by the size and
   weight of the DBS, we were unable to conduct additional, potentially
   more optimal normalization methods, including adjustment for
   hemoglobin, specific gravity, protein, or potassium due to limited
   volume of available sample^[165]106. Finally, there are fewer
   assumptions required for using the MITM framework compared to formal
   mediation analysis and the results should be interpreted with caution.

   Taken altogether, our study provides novel evidence and supports the
   current mechanistic understanding of how PFAS in utero influence PTB
   and ETB risk. Additional research is needed to validate our findings in
   other populations and to investigate the potential of these metabolic
   mechanisms and biomarkers for detection, treatment, and prevention
   strategies in public health and clinical settings.

Methods

Study design and population

   Participants from the Atlanta African American Maternal-Child Cohort
   (ATL AA hereafter) were included in the present analysis. This ongoing,
   prospective birth cohort enrolls pregnant African Americans between 6
   and 17 weeks gestation at Emory Midtown Hospital and Grady Hospital,
   which serve socioeconomically diverse populations in Atlanta, Georgia,
   and extends dyad follow-up through age six. Additional information
   regarding the cohort profile and data collection is described in detail
   elsewhere^[166]107,[167]108. Participants were eligible for inclusion
   if they self-identified as African American or Black, and were born in
   the US, between 18 and 40 years old, pregnant with a singleton, fluent
   in English, and had no chronic medical conditions. All participants
   provided written, informed consent to participate in the study, which
   was approved by the Institutional Review Board at Emory University
   (approval reference number 68441). Participant data are confidential
   and proprietary information to the ATL AA cohort.

   A total of 279 mothers and newborns enrolled in the ATL AA cohort had
   serum and DBS samples, respectively. At the time of our analysis, there
   were 273 participants with complete data available for PFAS
   measurements and DBS metabolomics (Fig. [168]S1). The live births
   occurred between June 2016 and June 2020. We excluded newborns with
   congenital anomalies (n = 6) from our analysis. The remaining 267 dyads
   were included in the PFAS metabolome-wide association studies (MWAS),
   gestational age MWAS, and prenatal PFAS-gestational age analysis.

Covariates

   A thorough review of the literature was completed a priori to identify
   additional covariates that potentially confound the relationships
   between our exposures and outcomes. In previous analyses conducted in
   the ATL AA Cohort, we found maternal body mass index (BMI), use of
   tobacco and marijuana, education, parity, and age are significant
   predictors of prenatal PFAS exposure^[169]27. Tobacco smoke is also
   associated with perturbations in the maternal serum metabolome and
   adverse birth outcomes^[170]42. Altogether, this information guided our
   construction of a directed acyclic graph (DAG; Fig. [171]S2). Parity
   (0–8) and use of tobacco and marijuana one month prior to pregnancy
   (yes/no) were abstracted from prenatal medical records. Other social,
   economic, and demographic factors were obtained by questionnaire. The
   mothers self-reported age (years), highest attained educational level
   (less than high school, high school, some college, college and above),
   number of members in household and household income for determining
   income-to-poverty ratio (<100%, 100–150%, 150–300%, 300–399%, ≥400%),
   health insurance type (private/public), and partner status (married or
   cohabitating, unmarried or not cohabitating) at time of enrollment were
   collected. The mother’s BMI (kg/m^2) was calculated from the first
   clinical visit’s weight and height measurements during early pregnancy
   and categorized as follows: underweight <18.5 kg/m^2, normal weight
   18.5– < 25 kg/m^2, overweight 25– < 30 kg/m^2, and with obesity
   ≥30 kg/m^2. Since PFAS undergo trans-placental transfer throughout the
   course of pregnancy, the gestational week when the serum samples were
   collected from the mothers was included as a continuous covariate in
   the models which included PFAS as the exposures. Finally, neonatal sex
   was believed to affect the exposures and outcomes, and thus adjusted in
   the models.

Quantification of perfluoroalkyl substances

   Whole blood samples were collected from pregnant participants between 6
   and 17 gestational weeks via venipuncture in an antecubital vein. These
   samples were transported to the National Institute of Environmental
   Health Sciences (NIEHS) Children’s Health Exposure Analysis Resource
   (CHEAR) laboratories, including Emory University’s Laboratory of
   Exposure Assessment and Development for Environmental Research
   (LEADER), New York University’s Laboratory Hub (NYU), and the Wadsworth
   Center Laboratory in New York. The samples were centrifuged (2000g) to
   obtain the serum fraction, which was used to quantify PFOA, PFNA, PFOS,
   and PFHxS, and subsequently stored at −80 °C. The analytical procedures
   and methods to quantify PFAS have been described in detail
   elsewhere^[172]27,[173]109. Briefly, after randomization, the sera were
   spiked with isotopic internal standards followed by solid-phase
   extraction and analysis by high-performance liquid
   chromatography-tandem mass spectrometry (LC-MS/MS). Individual PFAS
   species were quantified by isotope dilution calibration.

   The Human Health Exposure Analysis Resource (HHEAR) replaced CHEAR in
   2019 and requires adherence to strict quality control procedures so
   services at any of the laboratories are harmonized and comparable to
   globally recognized, external laboratories^[174]110–[175]112.
   Additionally, all of the laboratories participate in the German
   External Quality Assessment Scheme ([176]https://app.g-equas.de/web/)
   to demonstrate data quality assurance in serum PFAS analysis.

Birth outcomes

   Gestational age at birth (weeks) was calculated from the estimated date
   of conception, based on the mother’s self-reported last menstrual
   period and/or early pregnancy ultrasound, to the date of
   delivery^[177]113,[178]114. In accordance with the American College of
   Obstetricians and Gynecologists (ACOG) and the Society for Maternal
   Fetal Medicine (SMFM), gestational age at birth was categorized as
   follows: gestational age 22– < 37 weeks was considered PTB; gestational
   age 37–38 weeks was considered ETB; full-term births had a gestational
   age ≥39 weeks^[179]100. Information about the presence or absence of
   pregnancy complications and the labor and delivery course was
   abstracted from the medical record and used to further categorize the
   early births (those that were PTB or ETB) as spontaneous (i.e.,
   following spontaneous labor or premature rupture of membranes) or
   medically indicated (i.e., following C-section or induction for a
   maternal or fetal indication); in addition, full-term births were
   further categorized as healthy full-term births if the pregnancy was
   without complications (such as gestational hypertension, gestational
   diabetes, or preeclampsia) and these healthy full-term births were used
   as the referent category in analyses.

Untargeted High-Resolution Metabolomics

   Newborn DBS samples are routinely collected at the time of birth for
   medical screening and public health surveillance then archived for
   future biomonitoring purposes. Metabolomics analysis of newborn DBS
   samples has recently emerged as a powerful method to phenotype
   exposures and health outcomes, such as those in the present study, at
   the population level^[180]115–[181]118. In comparison to whole blood
   samples containing serum and plasma fractions, newborn DBS samples are
   also reliable, reproducible, and representative biospecimens of the
   circulating metabolome in humans, plus carry the advantage of novel
   insights into the intrauterine environment, which are largely
   uncharacterized^[182]118.

   DBS samples were collected within 24–48 h after birth from a heel stick
   performed by trained hospital nursery personnel using a standardized
   protocol that involves cleaning the skin of the heel with 75%
   isopropanol (wiping off any excess alcohol and allowing the skin to
   air-dry), using a sterile 2.5 mm lancet to obtain the sample, and
   collecting the sample onto a standard Guthrie card by saturating each
   circle with ~75 uL of blood^[183]119. On the day of collection, card
   specimens were transported to the Georgia Department of Public Health
   Laboratory for storage in a walk-in refrigerator (2–8 °C) without a
   desiccant for up to three months and then transported to an Emory
   University Laboratory for storage in freezer (−80 °C) within
   gas-impermeable bags with dessicant until assay^[184]119. For this
   study, we obtained single 15-mm punches (equivalent to ~50 uL whole
   blood) that were collected from 279 children between 2016 and
   2020^[185]116. An additional set of 15-mm punches was obtained from
   adjacent portions of filter paper from the same Guthrie cards to use as
   blanks.

   Untargeted high-resolution metabolomics profiling was conducted in a
   single run by the North Carolina HHEAR Hub (NC HHEAR Hub) in UNC
   Nutrition Research Institute, North Carolina Research Campus
   (Kannapolis, NC) using established methods. Blood spot samples were
   extracted with 1 mL ice-cold methanol containing 500 ng/mL
   L-tryptophan-d5, vortexed at 5,000 rpm for 10 min, and then sonicated
   for another 20 min. After centrifugation at 16,000 rcf for 10 min at
   4 °C, 600 µL of the extracted supernatant was transferred into a 2.0 mL
   low-bind microfuge tube. An additional 70 µL of the extracted
   supernatant from each of the study samples was pooled together and then
   redistributed into new set of tubes with 600 µL per tube to generate
   total study pool samples. The empty blood spot card (without blood)
   matched with each of the study samples in terms of size and material
   was used as a blank and processed using the same procedures as the
   study sample. Supernatant (70 µL) from each of the blanks were mixed
   together and then distributed into multiple low-bind 2.0 mL tubes with
   a volume of 600 µL per tube. All supernatant aliquots (600 µL)
   including study samples, study pools, and blanks were dried by vacuum
   concentrator overnight and reconstituted with 100 µL water-methanol
   (95:5, v/v) for the ultra-high performance (UHP) LC-HR-MS analysis.
   Aliquots of 50 µL of NIST reference plasma (SRM 1950) were also
   prepared as external quality control samples. Briefly, 400 µL of
   methanol containing 500 ng/mL L-tryptophan-d5 was vortexed at 5,000 rpm
   for 2 min. Afterwards, extracts were centrifuged at 16,000 rcf for
   10 min at 4 °C, and 350 µL of the supernatant was dried by vacuum
   concentrator overnight and reconstituted with 100 µL water-methanol
   (95:5, v/v) prior to analysis. Study samples were randomized before
   sample preparation and data acquisition. Quality control study pools,
   NIST plasma references, and blanks were interspersed at a rate of 10%