Early Pregnancy Maternal Plasma Phospholipid Saturated Fatty Acids and Fetal Growth: Findings from a Multi-Racial/Ethnic Birth Cohort in US

Saturated fatty acids (SFAs) during pregnancy are associated with disrupted metabolic programming among offspring at birth and later growth. We examined plasma phospholipid SFAs in early pregnancy and fetal growth throughout pregnancy. We enrolled 321 pregnant women from the NICHD Fetal Growth Studies—Singleton Cohort at gestational weeks 8–13. Ultrasonogram schedules were randomly assigned to capture weekly fetal growth. We measured plasma phospholipid SFAs at early pregnancy using blood samples and modeled fetal growth trajectories across tertiles of SFAs with cubic splines using linear mixed models after full adjustment. We then compared pairwise weekly fetal growth biometrics referencing the lowest tertile in each SFA using the Wald test. We found that even-chain and very long even-chain SFAs were inversely associated, whereas odd-chain SFAs were positively associated with fetal weight and size. Compared with the lowest tertile, the highest tertile of pentadecanoic acid (15:0) had a greater fetal weight and size, starting from week 13 until late pregnancy (at week 39: 3429.89 vs. 3269.08 g for estimated fetal weight; 328.14 vs. 323.00 mm for head circumference). Our findings could inspire future interventions using an alternative high-fat diet rich in odd-chain SFAs for optimal fetal growth.


Introduction
A body of evidence stemming from human and animal research has shown that maternal nutrition during pregnancy is directly related to the adequate development of measurements and images, into the Clinical Trial Management System, which were then electronically transferred to the Data Coordinating Center.

Assessments of Plasma Phospholipid Saturated Fatty Acids (SFAs)
Upon enrollment at 8-13 weeks of gestation (visit 0), we collected blood from all participants and stored biospecimens at −80 • C until thawing prior to the assay. Phospholipid fatty acid profiles were extracted using a previously described method [21,22]. Briefly, lipids were extracted with chloroform/methanol, separated using thin-layer chromatography, and the phospholipid batch was derivatized to methyl esters. The final product was injected into a capillary Varian Cp7420 30-m column with a Hewlett Packard 5890 gas chromatograph with flame ionization detection, interphased with HP Chemstation software A.06. All SFA components were calculated in percentages (%) referencing the total weight of the plasma phospholipid FA fraction. The analytic assessments and inter-assay coefficients of variation (CVs) for all SFAs in the same cohort were published elsewhere [23]. We identified eight plasma phospholipid SFAs and 3 SFA-derived indices, including the sum of even-chain SFAs: myristic acid (14:0), palmitic acid (16:0) and stearic acid (18:0); the sum of odd-chain SFAs: pentadecanoic acid (15:0) and heptadecanoic acid (17:0); and the sum of very long even-chain SFAs: arachidic acid (20:0), behenic acid (22:0) and lignoceric acid (24:0). Table S1 shows the weight of the percentages of all SFAs among all plasma phospholipid FA fractions.

Fetal Growth Measurement throughout Pregnancy (10-40 Weeks of Gestational Age)
Women received an ultrasonographic examination at enrollment 8-13 weeks into gestation (visit 0) and another one at four following ultrasonography schedules via randomization, as follows: weeks 16 Table S2 and Figure S1. All study visits allowed ±1 week of the targeted GA to accommodate the subjects' availability. We captured weekly fetal growth data in a mixed longitudinal randomization scheme without exposing individual women to ultrasound every week [24].
At each ultrasonographic examination, trained sonographers performed standard operating procedures using identical equipment (Voluson E8; GE Healthcare, Boston, MA, USA) and assessed a series of fetal growth biometrics, including head circumference (HC, mm), biparietal diameter (BPD, mm), abdominal circumference (AC, mm), femur length (FL, mm) and HC/AC ratio. We then calculated the estimated fetal weight (EFW, g) using a Hadlock formula based on HC, AC and FL [25]. Furthermore, measurement errors in terms of fetal growth biometrics were minimized in our study due to high inter-and intra-grader reliability reported in our study, regardless of maternal obesity status [24].

Covariates
At study entry, trained research coordinators interviewed participants to collect information on maternal demographics, pregnancy history and lifestyle behaviors, as well as blood pressure measurements, anthropometric indices and infant sex from medical records. We calculated GA at delivery based on the ultrasound-verified last menstrual period (LMP) of mothers and the date of delivery. Among all covariates of interest, we identified maternal pre-pregnancy body mass index (BMI) as a key covariate [26]. Despite having matched maternal age and race/ethnicity between cases and controls, we continued to control for maternal age and race/ethnicity to obtain conservative estimates. We applied a final adjustment model for all statistical analyses, including maternal age, race/ethnicity, nulliparity, pre-pregnancy BMI and infant sex.

Statistical Analysis
Because our study participants diagnosed with GDM (107 out of 321, 33.3%) were overrepresented compared with the general pregnant population in the US (107 out of 2802, 4%), we re-weighted all assessments following the idea of pseudolikelihood by Samuelsen (1997) [27] to represent the full cohort [24,28]. We used descriptive statistics to summarize crude and weighted characteristics of women and their neonates in the primary and nested case-control cohort (Table S3).
Tertiles of individual SFAs were treated as independent variables, and trajectories of all fetal biometrics were treated as dependent variables and modeled using a cubic spline model estimated using a restricted maximum likelihood approach [29]. Because of the skewed distribution of all fetal growth biometrics, we then log-transformed all assessments to stabilize variances across GA with an approximate normal distribution. The model initially included fixed effects of the linear, quadratic and cubic terms and cubic spline terms of GA (3 knots at the 25th, 50th and 75th percentiles), as well as a random intercept and random effects of the linear, quadratic, and cubic terms and cubic spline terms of weekly GA. The random effect covariance was unstructured, and the random effect of the cubic spline term of weekly GA was removed to facilitate model convergence.
We first applied global testing to investigate the overall difference in fetal growth trajectories across tertiles of SFAs in the full model. Then, we calculated the log-likelihood ratio by adding an interaction term between SFA tertiles and GA followed by Bonferroni correction [30]. Once a significant association was identified from all the steps mentioned above, we calculated the weekly means (back-transform fetal biometrics) and compared weekly differences in fetal growth biometrics across tertiles of each SFA (using the lowest tertile as a reference) in the full model using the Wald test. We further adjusted for family history of diabetes, maternal random glucose level at enrollment, maternal total cholesterol level at enrollment and sum of other SFA subgroups at visit 0 in the sensitivity analyses. We conducted all the analyses using SAS version 9.4 (SAS Institute, Cary, NC, USA) and R Software (version 4.2.1). We reported all estimates with a 95% confidence interval (CI) or p-value. We defined significance as a two-tailed p-value of 0.05.

Results
All SFAs and sums of SFA subgroups were significantly associated with fetal weight and size, with at least two or more fetal growth parameters, even after Bonferroni correction (Tables 1 and S4). Among all SFAs, myristic acid (14:0), pentadecanoic acid (15:0), stearic acid (18:0), lignoceric acid (24:0) and the sum of odd-chain SFAs were consistently and significantly associated with all fetal growth biometrics (p < 0.05).

Even-Chain SFAs and Fetal Growth
Overall, plasma phospholipid even-chain SFA levels were inversely associated with fetal growth throughout pregnancy. Among three even-chain SFAs, myristic acid (14:0) and stearic acid (18:0) were associated with all fetal growth parameters, whereas palmitic acid (16:0) was only associated with AC. Compared with the lowest tertile, the highest tertile of plasma phospholipid myristic acid (14:0) was significantly associated with a decrement in FL and HC, starting in early pregnancy (FL at week 11: 4.11 Figure S2). Interestingly, the second tertile of myristic acid (14:0) had a stronger and longer impact on reduced EFW and AC than that of the highest tertile compared with the lowest tertile, starting in early-mid pregnancy ( Figure S2).
Similarly, stearic acid (18:0) showed a comparable magnitude in the second tertile and highest tertiles in terms of EFW, AC, FL, HC and BPD. For example, compared with the lowest tertile, the highest tertile of stearic acid (18:0) was associated with a decrement in HC and BPD, starting from early pregnancy (HC at week 13 Figure S2). Compared with the lowest tertile, the second tertile of stearic acid (18:0) was also associated with a decrement yet with a longer duration in EFW and AC than that of the highest tertile, starting from early pregnancy (EFW at week 30 Figure S2).
Regarding the sum of even-chain SFAs, compared with the lowest tertile, the highest tertile was significantly associated with a reduced fetal weight and size, starting from early pregnancy (EFW at week 13 Figure S3).

Odd-Chain SFAs and Fetal Growth
In contrast to even-chain SFAs, higher levels of individual and subgroup odd-chain SFAs were associated with a larger fetal weight and size. Compared with the lowest tertile, the highest tertile of pentadecanoic acid (15:0) was associated with a larger EFW, AC, FL, HC and BPD throughout pregnancy, starting from early pregnancy (EFW at week 13 Figure 1).      Even though there were similar effect sizes, heptadecanoic acid (17:0) exerted a relatively smaller impact on fetal growth than that of pentadecanoic acid (15:0). For instance, compared with the lowest tertile, the highest tertile of heptadecanoic acid (17:0) was associated with EFW, AC, FL, HC and BPD since early-mid pregnancy, yet it attenuated in mid-late pregnancy (EFW at week 23 Figure 2).
By combing pentadecanoic acid (15:0) and heptadecanoic acid (17:0), the sum of oddchain SFAs was found to be significant with a larger EFW, FL, HC and BPD, but not with AC. Compared with the lowest tertile, the highest tertile of the sum of SFAs was associated with a greater fetal weight and size, only being significant starting from mid-pregnancy  (Table S9).

Very Long Even-Chain SFAs and Fetal Growth
Overall, higher levels of arachidic acid (20:0), behenic acid (22:0) and lignoceric acid (24:0) were associated with a reduced fetal weight and/or size. Compared with the lowest tertile, the highest tertile of lignoceric acid (24:0) was significantly associated with a reduced AC and BPD since early-mid pregnancy (AC at week 29 Figure S4A). Interestingly, the impacts of arachidic acid (20:0) and behenic acid (22:0) on reduced fetal weight and size were much weaker and shorter in duration (Tables S11 and S12).
In terms of the sum of very long even-chain SFAs, the impact on reduced fetal weight and size was more significant and longer in duration in the second tertile than that in the highest tertile, compared with the lowest tertile. For example, women in the second tertile of the sum of very long even-chain SFAs exerted an early-pregnancy decrement in fetal growth parameters (EFW at week 13 Figure S4B).
The sensitivity analysis of additional adjustments on family history of diabetes, maternal plasma random glucose levels, total cholesterol levels and the sum of other subgroups of SFAs at visit 0 in the global test did not attenuate any significant associations in the relationships identified above (Tables S14-S17).

Figure 2.
Back-transformed geometric means of femur length, head circumference and biparietal diameter by gestational weeks to tertiles of heptadecanoic acid (17:0) within the NICHD Fetal Growth Studies-Singletons cohort, 10-40 weeks gestational age. The 1st (lowest) tertile curve is in blue, the 2nd (middle) tertile curve is in red, and the 3rd (highest) tertile is in green. The gray shaded area and the blown-up graph on the right indicate a significant increment in estimated femur length from 19 to 23 week of gestation (A), a significant increment in head circumference from 20 to 29 weeks of gestation (B), and a significant increment in biparietal diameter from 20 to 32 weeks of gestation (C) in both the 2nd and 3rd tertiles, compared with the 1st tertile.

Discussion
Our prospective longitudinal data suggest opposite associations of subgroups of SFAs with fetal growth throughout pregnancy. Specifically, higher maternal plasma phospholipid odd-chain SFAs in early pregnancy were positively associated with fetal growth. In contrast, higher maternal plasma phospholipid even-chain and very long even-chain SFAs were inversely associated with fetal growth in early pregnancy. The relevance of timing in fetal growth increments or decrements with different subgroups of SFAs significantly started in early-mid pregnancy (10-15 weeks of gestation) and attenuated in mid-late pregnancy (23-40 weeks of gestation).
Traditional evidence indicates that SFAs are strongly associated with impaired insulin sensitivity, glucose intolerance and lipotoxicity, which could be biased by failing to separate the metabolic beneficial components, such as odd-chain SFAs, from conventional cardiometabolic risk components, such as even-chain SFAs [17]. However, instead of avoiding all high-fat diets, emerging research investigated the fat composition and differentiated the good high-fat pattern from the bad [31]. For example, maternal n-3 polyunsaturated fatty acids (PUFAs) levels were proven to be beneficial not only for cardiometabolic health in the general population [32] but also for fetal growth throughout pregnancy [33]. However, current evidence only focuses on growth biometrics after delivery, and the assessment of maternal SFAs is diverse in techniques and gestational age. Therefore, studies regarding plasma phospholipid maternal SFA compositions and fetal growth are sparse in the research scope.
Even-chain SFAs are FAs that are totally hydrogenated, with a linear chain without double bonds between carbon atoms. They are in a stable state at room temperature and include lauric acid (12:0), myristic acid (14:0), palmitic acid (16:0) and stearic acid (18:0) [1]. All even-chain SFAs can be derived from both exogenous intake (e.g., Western diets rich in butter, palm oil and red meat) and endogeneous synthesis (e.g., the de novo lipogenesis [DNL] pathway) [34,35], from which palmitic acid (16:0) and stearic acid (18:0) were mainly synthesized. Such even-chain FAs seemed to mediate multiple biological mechanisms, including increasing oxidative stress [17], inducing insulin resistance via the proteasomal degradation of key insulin-signaling molecules [36] and activating proinflammatory signaling via Toll-like receptor 4 [37]. Our study found that the total sum of maternal even-chain SFAs during early pregnancy was associated with reduced fetal growth from early to mid-late pregnancy. Among them, myristic acid (14:0) and stearic acid (18:0) imposed a much stronger impact on fetal weight and size than that of palmitic acid (16:0). We speculated two possible underlying pathophysiological mechanisms to our observation. First, increased levels of different even-chain SFAs might be attributable to elevated levels of oxidative stress and inflammation in vivo [17,38], and such changes could viciously disrupt the placental circulation and supply of nutrients and oxygen that are vital for fetal growth. Second, it is suggested that placental tissue has lipoprotein receptors and expresses enzymes with lipase and phospholipase activities [39][40][41]. Such activities were involved in the mechanism of maternal FA transfer across the placenta to sustain the fetal lipid requirements. In other words, the elevation of even-chain SFAs might compete with other beneficial yet much larger molecules of FAs (e.g., n-3 PUFAs) through the placenta, indirectly contributing to inadequate fetal growth [13,14].
The origin of odd-chain SFAs has long been attributed to the diet, especially dairy product intake [42]. However, emerging evidence suggests that circulating pentadecanoic acid (15:0) and heptadecanoic acid (17:0) are independently derived. For instance, pentadecanoic acid (15:0) correlated directly with dietary intake, and heptadecanoic acid (17:0) is a product of biosynthesis regulated by dietary intake [43,44]. Several epidemiological studies have shown that both pentadecanoic acid (15:0) and heptadecanoic acid (17:0) have protective effects on glucose homeostasis via the inhibition of hepatic oxidation [45]. Therefore, odd-chain SFAs are inversely associated with metabolic diseases [46][47][48], including reducing the risk of type 2 diabetes (T2D) [17]. Our findings indicate a beneficial impact of elevated maternal plasma phospholipid odd-chain SFAs during early pregnancy and fetal growth throughout pregnancy, indirectly aligned with the optimal effect of odd-chain SFAs suggested a priori. For example, Santaren et al. suggested that circulating pentadecanoic acid (15:0) is inversely associated with plasminogen activator inhibitor-1 (PAI-1), tumor necrosis factor-α (TNF-α) and interleukin-18 (IL-18) [49]. In another study by Zheng et al., research findings also indicated inverse associations between higher levels of odd-chain SFAs and lower levels of major lipids (i.e., total cholesterol, triglycerides, apolipoprotein A-1 and apolipoprotein B) and hepatic markers [50]. We postulate that odd-chain SFAs reduced the inflammation levels both in the maternal environment and placental perfusion, contributing to sufficient fetal nutrients and blood supply.
Regarding very long even-chain SFAs, they have not been extensively studied except for their known roles in hereditary peroxisomal disorders and special food formulations [51]. Common knowledge on very long even-chain SFAs suggests that they are derived from a limited food source, such as peanuts, macadamia nuts and canola oil [52], and from endogenous metabolism from stearic acid (18:0) [53]. Studies have shown that very long even-chain SFAs are the major components of insulin resistance and reduced β-cell mass and function [52,54,55]. Our cohort shows that the sum of very long even-chain SFAs, especially lignoceric acid (24:0), is associated with a reduced fetal weight and size, which might be related to inflammation and disrupted glycemic metabolism. However, emerging evidence suggests that circulating very long even-chain SFAs are protective against cardiovascular outcomes, such as incident heart failure, atrial fibrillation, coronary heart disease, sudden cardiac arrest and even better aging [51]. Further research on such SFA clusters regarding their biological functions and impacts on fetal growth and child health is warranted.

Clinical Implications
Our study investigated the relationship between maternal plasma phospholipid SFAs in early pregnancy and fetal growth trajectories throughout pregnancy and further identified the relevance of timing for significant associations, in a relatively healthier pregnant population in the US with a lower prevalence of GDM (~4%). Such results remain significant even after adjustment for family history of diabetes, maternal early pregnancy glucose, total cholesterol levels and other subgroups of SFAs. Therefore, findings from the present study are robust even for a generally healthy population with fewer pregnancy complications. Further, this study is able to differentiate the physiological roles of even-chain, odd-chain and very long even-chain SFAs underlying fetal growth throughout pregnancy. Most of our observations on fetal growth were significant from early to mid-pregnancy, a stage of which provided useful information for the development of pre-eclampsia and pre-term birth [56] and even for the assessment of weight at birth [57]. Key findings regarding the beneficial impact of pentadecanoic acid (15:0) and heptadecanoic acid (17:0) in fetal growth can inspire future directions in oral supplementation. Thus, our data might offer evidence to support the targeting of plasma phospholipid odd-chain SFAs in early pregnancy to benefit fetal growth in the general population with pregnancy.

Strengths and Limitations
Our study has a few notable strengths. The prospective and longitudinal data collection included plasma phospholipid SFAs, abundant forms of circulating SFAs representing both exogenous and endogenous sources of SFAs, and longitudinal fetal growth ultrasound measures. We reported the temporal relationship between plasma phospholipid SFAs in early pregnancy and fetal growth throughout gestation and also identified the relevance of timing for significant associations. In addition, such a comprehensive panel of plasma phospholipid SFAs may further help elucidate the different roles of SFAs underlying the physiology of fetal development.
However, our study was not without limitations. Even though this is one of the largest prospective pregnancy cohorts investigating fetal growth throughout pregnancy, the relatively small sample size of 321 subjects may limit the statistical power of identifying the relevance of timing using pairwise comparisons at each gestation week. Second, we cannot eliminate residual confounding due to the study's observational nature, even though we controlled for known major confounders in our modeling. Third, dietary patterns or other lifestyle factors in mid-to-late pregnancy might modify the associations found in our study. However, such factors collected via dietary questionnaires are prone to measurement errors due to the subjectivity of self-reporting, and adjusting such factors could incur collider bias because they could be the potential mediators for subsequent fetal growth. Fourth, our observations were found to be significant mostly during early to mid-pregnancy instead of late pregnancy. It could be affected by the reduced power of fetal growth biometrics comparisons between tertiles because subjects gave birth at term. Last, there could be interactive effects among SFAs or between SFAs and other types of FAs, even though we accounted for the confounding effects among subgroups of SFAs for individual SFAs investigated in this study. Further studies with a larger sample size should investigate the underlying interaction among all FAs and are also warranted to verify our findings.

Conclusions
In summary, our study differentiates the inverse associations of even-chain and very long even-chain SFAs, in contrast to the positive associations of odd-chain SFAs in early pregnancy with fetal growth throughout pregnancy. Considering the modifiable nature of plasma phospholipid odd-chain SFAs due to their exogenous origins, such as pentadecanoic acid (15:0), our data might provide a potential target toward odd-chain SFAs (e.g., via oral supplements) to benefit fetal growth during pregnancy.  Table S13A. Back-transformed and pairwise comparison of weekly fetal growth biometrics across sum of very-long-even-chain saturated fatty acids tertiles in the NICHD Fetal Growth Studies-Singletons cohort; Supplementary Table S13B. Back-transformed and pairwise comparison of weekly fetal growth biometrics across sum of very long even-chain saturated fatty acid tertiles in the NICHD Fetal Growth Studies-Singletons cohort; Supplementary Table S14. Association between individual SFAs and fetal growth throughout 10-40 weeks of gestation using cubic spline model with gestational weeks after adjusting for maternal family history of diabetes; Supplementary Table S15. Association between individual SFAs and fetal growth throughout 10-40 weeks of gestation using cubic spline model with gestational weeks after adjusting for maternal random glucose level at visit 0; Supplementary Table S16. Association between individual SFAs and fetal growth throughout 10-40 weeks of gestation using cubic spline model with gestational weeks after adjusting for maternal total cholesterol level at visit 0; Table S17: Association between individual SFA and fetal growth throughout between 10-40 weeks gestation using cubic spline model with gestational weeks after adjusting for subgroup SFAs at visit 0. Supplementary Figure S1. Study flow chart; Supplementary Figure S2. Back-transformed geometric means of estimated fetal growth, abdominal circumference, head circumference and biparietal diameter by gestational weeks for tertiles of myristic acid (14:0) and stearic acid (18:0), respectively, within the NICHD Fetal Growth Studies-Singletons cohort, 10-40 weeks of gestational age. The 1st (lowest) tertile curve is in blue, the 2nd (middle) tertile curve is in red, and the 3rd (highest) tertile is in green; Supplementary Figure S3. Back-transformed geometric means of femur length and gestational weeks for tertiles of the sum of even-chain SFAs within the NICHD Fetal Growth Studies-Singletons cohort, 10-40 weeks of gestational age. The 1st (lowest) tertile curve is in blue, the 2nd (middle) tertile curve is in red, and the 3rd (highest) tertile is in green. The gray shaded area and the blown-up graph on the right indicate a significant decrement in femur length from 14 to 18 weeks of gestation in both the 2nd and 3rd tertiles, compared with the 1st tertile; Supplementary Figure S4. Back-transformed geometric means of abdominal circumference and head circumference by gestational weeks for tertiles of lignoceric acid (24:0) and very long even-chain SFAs, respectively, within the NICHD Fetal Growth Studies-Singletons cohort, 10-40 weeks of gestational age. The 1st (lowest) tertile curve is in blue, the 2nd (middle) tertile curve is in red, and the 3rd (highest) tertile is in green. The gray shaded area and the blown-up graph on the right indicate a significant decrement in abdominal circumference from 29 to 39 week of gestation ( Figure S4A) in both the 2nd and 3rd tertiles of lignoceric acid (24:0), compared with the 1st tertile. The gray shaded area and the blown-up graph on the right indicate a significant decrement in head circumference from 18 to 33 week of gestation ( Figure S4B) in both the 2nd and 3rd tertiles of very long even-chain SFAs, compared with the 1st tertile.  Institutional Review Board Statement: The institutional review boards at all participating sites approved both the primary study and sub-study. All participants provided written informed consent prior to data collection. The participating clinical centers entered study documents and data, including ultrasound measurements and images, into the Clinical Trial Management System, which were then electronically transferred to the Data Coordinating Center.

Informed Consent Statement: Written informed consent was obtained from all subjects involved in this study.
Data Availability Statement: Data described in the manuscript, code book and analytic code will be available upon request pending application and approval of a data-sharing agreement.