Adverse Birth Outcome Among Mothers With Low Serum Cholesterol

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ARTICLE

Adverse Birth Outcome Among Mothers With Low

Serum Cholesterol

Robin J. Edison, MD, MPHa_{, Kate Berg, PhD}a_{, Alan Remaley, MD}b_{, Richard Kelley, MD, PhD}c_{, Charles Rotimi, PhD}d_{, Roger E. Stevenson, MD}e_, Maximilian Muenke, MDa

a_{Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland;}b_{National Institutes of Health Clinical Center,}

Bethesda, Maryland;c_{Kennedy Krieger Institute, Baltimore, Maryland;}d_{National Human Genome Research Center, Howard University, Washington, DC;}e_Greenwood

Genetic Center, Greenwood, South Carolina

The authors have indicated they have no ﬁnancial relationships relevant to this article to disclose.

ABSTRACT

OBJECTIVE.The objective of this study was to assess whether low maternal serum cholesterol during pregnancy is associated with preterm delivery, impaired fetal growth, or congenital anomalies in women without identified major risk factors for adverse pregnancy outcome.

METHODS.Mother-infant pairs were retrospectively ascertained from among a cohort of 9938 women who were referred to South Carolina prenatal clinics for routine second-trimester serum screening. Banked sera were assayed for total cholesterol;

⬍10th percentile of assayed values (159 mg/dL at mean gestational age of 17.6

weeks) defined a “low total cholesterol” prenatal risk category. Eligible women were aged 21 to 34 years and nonsmoking and did not have diabetes; neonates were liveborn after singleton gestations. Total cholesterol values of eligible moth-ers were adjusted for gestational age at screening before risk group assignment. The study population included 118 women with low total cholesterol and 940 women with higher total cholesterol. Primary analyses used multivariate regres-sion models to compare rates of preterm delivery, fetal growth parameters, and congenital anomalies between women with low total cholesterol and control

subjects with mid–total cholesterol values⬎10th percentile but⬍90th percentile.

RESULTS.Prevalence of preterm delivery among mothers with low total cholesterol was 12.7%, compared with 5.0% among control subjects with mid–total choles-terol. The association of low maternal serum cholesterol with preterm birth was observed only among white mothers. Term infants of mothers with low total cholesterol weighed on average 150 g less than those who were born to control mothers. A trend of increased microcephaly risk among neonates of mothers with low total cholesterol was found. Low maternal serum cholesterol was unassociated with risk for congenital anomalies.

CONCLUSIONS.Total serum cholesterol⬍10th population percentile was strongly as-sociated with preterm delivery among otherwise low-risk white mothers in this pilot study population. Term infants of mothers with low total cholesterol weighed less than control infants among both racial groups.

www.pediatrics.org/cgi/doi/10.1542/ peds.2006-1939

doi:10.1542/peds.2006-1939

Key Words

cholesterol, preterm birth, birth defects, congenital defects, birth weight, embryonic and fetal development, lipoproteins

Abbreviations

LDL-C—low-density lipoprotein cholesterol

TC—total cholesterol

IUGR—intrauterine growth restriction LMSC—low maternal serum cholesterol GA— gestational age

GGC—Greenwood Genetic Center NIH—National Institutes of Health OR— odds ratio

CI— conﬁdence interval

Accepted for publication Mar 14, 2007

Address correspondence to Maximilian Muenke, MD, Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Department of Health and Human Services, 35 Convent Dr, MSC 3717, Building 35, Room 1B-203, Bethesda, MD 20892-3717. E-mail: mmuenke@nhgri.nih.gov

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M

ATERNAL CHOLESTEROL IS essential for both the hormonal and physical changes of early

pregnan-cy.1–8 _{Circulating low-density lipoprotein cholesterol}

(LDL-C) is the chief substrate for placental progesterone

biosynthesis.9,10 _{Subclasses of high-density lipoprotein}

cholesterol also participate in placental cholesterol

bal-ance.11_{Cholesterol in plasma membranes is a bulk}

con-stituent of decidual tissue critical to implantation and

uteroplacental vascularization.12–15_{Alterations in}

placen-tal cholesterol concentrations have been associated with changes in placental transport functions during

gesta-tion.16 _{Gestational disorders such as preeclampsia and}

gestational diabetes, as well as maternal alcohol con-sumption, have been associated with lipoprotein chang-es.11,17–21

Maternal cholesterol is probably transported to the early embryo and would be available for development of

both embryonic and placental tissues.22–28 _Longitudinal

studies have documented specific changes in total and fractionated cholesterol levels in maternal serum as ges-tation progresses; total cholesterol increases substantially

during the second and third trimesters.29–35_This

physio-logic hypercholesterolemia of later pregnancy suggests an adaptive function for pregnancy maintenance or fetal

growth.36_{Decreased levels of maternal total cholesterol}

(TC) and LDL-C have been reported in association with

intrauterine growth restriction (IUGR).37,38 _Conversely,

maternal hypercholesterolemia is suspected to be

inju-rious, because concentrations ⬎300 mg/dL have been

linked to increased cholesterol deposition in the fetal

aorta39_{; the “fetal origins hypothesis” links this}

phenom-enon to subsequently increased risk for cardiovascular

disease in the adult offspring.40–42 _{It is not known}

whether optimal levels of maternal serum cholesterol during pregnancy can be defined. We are not aware of data addressing any potential risk to the pregnancy when maternal serum cholesterol falls below a lower bound. This pilot study investigated the premise that low maternal serum cholesterol (LMSC) during gestation may be associated with adverse pregnancy outcomes. In view of the evidence for maternal transport of choles-terol to the embryo in early pregnancy, we reasoned that maternal cholesterol may have a disproportionate im-pact during critical periods for placentation and early

neuroepithelial expansion.5,6,43–48 _{Specifically, we}

hy-pothesized a priori that LMSC may increase the risk for preterm delivery, microcephaly, or other central

ner-vous system anomalies.7,49–53 _{Secondarily, we}

hypothe-sized an increased risk for fetal growth restriction and

congenital anomalies.7,50,54

Our study population was retrospectively sampled from a population-based cohort of 9938 women whose blood had been banked concurrently with routine serum screening performed at prenatal clinics that serve west-ern South Carolina. This screening occurred over a broad spectrum of gestational ages (GAs) throughout the

sec-ond trimester (range: 13–23 weeks). We derived the population distribution of maternal TC from all banked

sera and defined a low-TC risk exposure as TC ⬍10th

percentile of this screened population (159 mg/dL at a mean GA 17.6 weeks, or 4.11 mmol/L at that date). For facilitation of comparisons among cholesterol values sampled at different GAs, all TC values in the study population were adjusted for GA at date of screening using linear regression methods. Women with higher levels of TC formed the pool of potential control subjects; their adjusted TC values were separated into mid-TC and high-TC categories encompassing the 10th to 90th and

⬎90th population percentiles, respectively (90th

popu-lation percentile at 17.6 weeks’ GA was 261 mg/dL, or 6.75 mmol/L). Eligibility criteria were designed to min-imize identifiable pregnancy risk factors or potential confounders; among other characteristics, participants were aged 21 to 34; had no history of smoking, type 1 diabetes, or other medical or gestational risk conditions; and had ultrasound-dated singleton pregnancies that re-sulted in a live birth. The study hypotheses were tested by assessing risk ratios for specific adverse outcomes in infants of mothers with low TC compared with mid-TC control mothers, using multivariate regression models.

METHODS

Study Population Ascertainment

The Greenwood Genetic Center (GGC) in Greenwood, South Carolina, has an established protocol for collecting baseline maternal data and banking serum samples from all consenting women who are referred for routine sec-ond-trimester serum screening to clinics that serve urban and rural areas of western South Carolina. Insti-tutional review boards of participating institutions ap-proved this study protocol. Serum samples and clinical data were identified to National Institutes of Health (NIH) investigators only by a study number assigned at GGC. Banked sera from all women who were consecu-tively screened between 13 and 23 weeks’ gestation (mean: 17.6 weeks; SD: 1.5 weeks) during 1996 –2001

were assayed for TC (n ⫽ 9938). Although women in

this cohort delivered primarily at 2 hospitals, for this pilot investigation, ascertainment of hospital charts was limited to women who delivered at the hospital closest

to GGC (composing⬃40% of the total screened cohort).

We defined LMSC pregnancies as those in which mater-nal TC fell below the 10th percentile of the screened

cohort (⬍159 mg/dL, after adjusting the assayed TC

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groups. The majority of exclusions in each group were

because of age ⬍21 years, positive smoking history,

and/or pregnancy dating by last menstrual period rather than by ultrasound. The final cohort available for anal-ysis included 118 mothers with low-TC and 940 com-parison mothers classified as having mid-TC or high-TC.

Serum Cholesterol Assays

Frozen sera (⫺80°C) were shipped on dry ice from GGC

to the NIH. TC in serum was analyzed enzymatically by the cholesterol oxidase method on a Hitachi 917 ana-lyzer using reagents from the manufacturer (Roche, In-dianapolis, IN). Both the interassay and intra-assay

co-efficients of variation were⬍5%.

Determination of Eligibility

Baseline data recorded by participants and clinic staff at the time of serum screening were sent to the NIH in anonymized files identified by study codes linked to the serum samples. Eligibility criteria were designed to in-crease uniformity among the study population and to exclude those who were at elevated risk for adverse

pregnancy outcome: we selected participants who were aged 21 to 34 years at screening and had a singleton gestation, race/ethnicity identified as black or white non-Hispanic only, no history of type 1 diabetes, no current or previous smoking, no other described sub-stance use, and no reports of previous abnormal preg-nancy history. Medical charts for the index pregpreg-nancy and its outcome were sought for potentially eligible

women. Mothers who were evaluated for ⬎1 eligible

pregnancy during the screening period had data

in-cluded for 1 randomly selected pregnancy only (n ⫽

112).

As clinical data were reviewed at the NIH, additional patients were excluded when charts indicated significant intercurrent infections or other illness, preeclampsia or other gestational disorders except idiopathic IUGR, sub-stance use, or a genetic syndrome diagnosed in the ne-onate. We also excluded patients with conflicting data regarding GA at delivery or other key outcome variables. Posthoc, we excluded neonates with a structural cardiac defect, because such infants had extremely high rates of IUGR and preterm delivery.

TABLE 1 Ascertainment of Low-TC Risk Group and Higher-TC Control Group

Parameter Maternal Sera Assayed for TC (N⫽9938)

Low-TC Risk Group Selection (975 Pregnancies With Screened Maternal TC

⬍159 mg/dL)

Higher-TC Control Group Selection (8963 Pregnancies With Screened Maternal TC

ⱖ159 mg/dL)

Pa

Retained,

n(%)

Excluded,

n(%)

Retained,

n(%)

Excluded,

n(%)

Maternal chart not retrievedb ₅₁₉ ₅₈₁₄ ⬍_.001

Maternal charts reviewed 476 (48.8) 3149 (35.1) ⬍.001

Pregnancies excluded at baseline 313 (65.8) 1940 (61.6) .08

Frequency of individual exclusion

criteriac

Age⬍21 y 183 (38.4) 872 (27.7) ⬍.001

Age⬎34 y 21 (4.4) 144 (4.6) .88

Not dated by ultrasound 89 (18.7) 414 (13.1) .001

Smoking 142 (29.8) 390 (12.4) ⬍.001

Race/ethnicity Hispanic, Asian, or Other 35 (7.4) 69 (2.2) ⬍.001

Pregestational diabetes 15 (3.2) 16 (0.5) ⬍.001

Twin gestation 4 (0.8) 54 (1.7) ⬍.001

⬎1 eligible pregnancy to same

mother

9 (1.9) 45 (1.4) .44

Pregnancy lost to follow-up 47 (9.9) 233 (7.4) .06

Total baseline exclusion criteria 545 (1.7 per excluded individual) 2237 (1.2 per excluded individual) ⬍.001

Maternal and neonatal charts reviewed 163 (34.2) 1209 (38.4) .08

Pregnancies excluded after data review

45 (27.6) 269 (22.2) .13

Substance use, intercurrent illnessd _{19 (11.7)} _{102 (8.4)} _.17

Fetal deathe _{3 (1.8)} _{24 (2.0)} _.90

Cardiac malformation 1 (0.6) 7 (0.6) .96

Missing or conﬂicting data 22 (13.5) 136 (11.2) .40

Total analyzed mother-infant pairs 118 (12.1) 940 (10.5) .12

a_{Assessed between groups by}␹2_analysis.

b_{Medical charts were retrieved for mothers who delivered at a single hospital convenient to GGC.}

c_{Each exclusion criterion listed separately; total count of exclusion criteria exceeds number of excluded pregnancies.} d_{Preeclamptic pregnancies excluded.}

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Predictor, Covariate, and Outcome Variables

In addition to analyzing primary data, we generated specific predictor, covariate, and outcome variables to model our results. Because our chief hypothesis con-cerned LMSC as a predictor of adverse pregnancy out-come, we sought to isolate intrinsic differences in ma-ternal TC levels from the introduced component of TC attributable to GA when samples were drawn. It is well established that, in general, maternal TC rises progres-sively with GA from the second trimester forward in pregnancy, with various percentage increases of LDL-C and TC described during the second and third trimesters

by several authors.33,34,55,56_{For investigation of whether}

the increase of TC with GA in our cohort could be expressed as a percentage rise in TC per unit of increase in GA, a model would be required to use logarithms of TC rather than arithmetic values themselves in a linear regression equation. Such log transformations are com-monly applicable to biological variables and have been used in other applications of human TC data, generally

to correct for the nonnormal distribution of TC.57–60_The

model that best fit our study data is as follows:

logTC-(adjusted)⫽logTC(raw)⫹slope(17.6⫺GA). This

equa-tion yields a downward adjustment of raw TC values

where GA⬎17.6 and an upward adjustment where GA

⬍ 17.6. The slope values for GA (0.026 for white

women, 0.032 for black women) were significant atP⬍

.0001 in the linear regression model. All TC values that were adjusted via this model represent the expected value of an individual’s TC if it had been sampled at the mean GA of 17.6 (Table 2 gives distributions of raw and adjusted TC). Adjusted TC was examined as a continu-ous and an ordinal variable; however, on the basis of our a priori definition of a low-cholesterol exposure,

most of our analyses used TC ⬍10th percentile as the

risk variable. The outcome of preterm delivery was defined as delivery before 37 completed weeks of ges-tation and term delivery as 37 to 41 completed weeks.

Inspection of the relationship between preterm birth and maternal cholesterol on an ordinal scale (as in Fig 1) revealed a nonlinear relationship with increased prevalence at both tails of the cholesterol distribution. Because previous reports suggested a possibly in-creased risk for prematurity associated with very high

maternal cholesterol,61 _{we dichotomized cholesterol}

risk as ⬍10th percentile versus 10th to 90th

percen-tiles (159 –261 mg/dL). We further defined a variable

dichotomizing cholesterol values ⬎90th percentile

(261 mg/dL) versus values in the 10th to 90th percen-tile range to characterize this posthoc association of elevated serum cholesterol with preterm delivery. The low-TC risk group for prematurity was subdivided into progressively lower levels of TC (less than fifth per-centile, less than third percentile).

We tested potential covariates on the basis of the literature of prematurity and availability of data. Season and year of delivery were evaluated in initial models. Maternal weight at screening was used as a surrogate adjustment for effects that are known to be associated with extremes of BMI, prepregnancy weight, and/or

weight gain in pregnancy.62,63_{Maternal weight was not}

correlated with GA at screening (P⫽.56), so the

mea-sured value at screening was used. Maternal age at screening was used in adjusted models as a categorical variable (ages 21–24, 25–29, and 30 –34). Gender of infant and maternal race were covariates in many mod-els.

We dichotomized outcome variables reflecting fetal growth. Infants who were born at each GA were classi-fied as less than or more than the 5th or 10th percentile

for weight, length, and head circumference.64–66_{We then}

defined IUGR as weight and length both⬍10th

percen-tile for GA, regardless of head circumference. Infants were classified as having microcephaly when the head

circumference was⬎2 SD below the mean for GA (less

TABLE 2 Distribution of Maternal Serum Cholesterol Values According to Percentile Ranges, as Derived From Screened Pregnant Population and Applied to Study Population

Parameter N Screened Population Percentiles Total, %

Low-TC Groups Mid-TC Group High-TC Group

⬍3rd 3rd–5th 5th–10th 10th–50th 50th–90th 90th–95th ⱖ95th

Screened population TC, mg/dL 9938 ⬍138 138 to⬍147 147 to⬍159 159 to⬍204 204 to⬍261 261 to⬍280 ⱖ280

Screened populationa

TC not adjusted for GA,n(%)b ₉₉₃₈ _{276 (2.8)} _{219 (2.2)} _{480 (4.8)} _{3918 (39.4)} _{4049 (40.7)} _{483 (4.9)} _{513 (5.2)} ₁₀₀

Study population

TC not adjusted for GA,n(%)b ₁₀₅₈ _{48 (4.5)} _{30 (2.8)} _{43 (4.1)} _{363 (34.3)} _{446 (42.2)} _{60 (5.7)} _{68 (6.4)} ₁₀₀

TC adjusted for GA,n(%)c,d ₁₀₅₈ _{37 (3.5)} _{35 (3.3)} _{46 (4.3)} _{378 (35.7)} _{444 (42.0)} _{64 (6.0)} _{54 (5.1)} ₁₀₀

a_{TC risk groups: low-TC,}_n_⫽_{975, 9.8%; mid-TC,}_n_⫽_{7967, 80.1%; high-TC,}_n_⫽_{996, 10.1%.}

b_{These lines represent the distribution of TC values as assayed directly (raw values). The GA at which they were obtained range from weeks 13 to 23 (mean: 17.6; SD: 1.5) for the screened population}

and from weeks 15 to 23 (mean: 17.7; SD: 1.3) for the study population.

c_{TC risk groups (by adjusted TC): low-TC,}_n_⫽_{118, 11.1%; mid-TC,}_n_⫽_{822, 77.7%; high-TC,}_n_⫽_{118, 11.1%.}

d_{Values in this line result from adjusting the assayed TC values for GA at which serum was obtained and enumerating the numbers and percentages of mothers in the study population whose}

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than fifth percentile), whereas the length was ⬎10th percentile.

Analysis

We performed 3 levels of analysis. First, we directly tested our a priori hypotheses, with adjustment for an-ticipated covariates. Results are reported from models that fit the data best. Next, we performed supplemental testing of alternative risk, covariate, and outcome vari-ables to evaluate robustness of the original findings against potential sources of classification error and par-ticularities of model selection. Finally, we conducted posthoc analyses to characterize patterns that were evi-dent in the data but had not been anticipated. All anal-yses were performed using procedures from SAS 8.2

(SAS Institute, Inc, Cary, NC).67 _{Statistical significance}

was assessed by nominalPvalue for a priori hypotheses.

Supplemental testing was not designed to test alternative

hypotheses, so these confidence intervals and P values

were interpreted as descriptive measures characterizing patterns in our data and robustness of primary hypoth-eses.

Relationship of maternal cholesterol to preterm de-livery was assessed by logistic regression models that estimated the odds ratios (ORs), 95% confidence

inter-vals (CIs), and P values associated with preterm birth

among infants of low-TC mothers compared with those with maternal cholesterol values in the mid-TC 10th to 90th percentile range. Models were adjusted for mater-nal race, age category, weight in pounds, infant gender, and presence of IUGR. Secondary analyses tested robust-ness of OR estimates by modifying definitions of key variables. These measures included using as controls only pregnancies with maternal TC between 25th and 75th percentiles (179 –231 mg/dL), examining risks for

delivery atⱕ35 weeks compared with the more

homo-geneous term weeks 39 to 41, replicating key analyses with unadjusted cholesterol values, and incorporating

different covariates. We further characterized maternal subgroups with TC less than fifth and less than third population percentiles (147 and 138 mg/dL, respec-tively). Postterm deliveries were excluded from analyses. Direct measurements of fetal growth at term were tabulated, comparing both low-TC and high-TC groups with the mid-TC reference group. Both univariate and multivariate adjusted models were used to compare size of term infants, excluding those who were classified as

growth-restricted or macrosomic (weight ⬍10th or

⬎90th percentile, examined separately). Multivariate

lo-gistic regression models were used to estimate ORs

as-sociated with microcephaly, IUGR, or size ⬍10th

per-centile for GA.

The rate of congenital anomalies among cohort in-fants was compared between low-TC and control groups

by univariate ␹2 _{and multivariate regression analysis.}

This was also done for category of anomaly by organ system.

RESULTS

The 1058 mother-infant pairs that were retained for data

analysis represent just ⬎10% each of all LMSC and

higher cholesterol pregnancies that were screened for the original cohort (Table 1). Mothers with LMSC were

more often excluded for age⬍21 years, were more likely

to smoke, and were less likely to have had sonographic gestational dating. Mothers with LMSC in the study group were somewhat younger, heavier, and more likely to be of black race than were control mothers (Table 3). The prevalence of preterm delivery in this highly selected study cohort (6.6%) was considerably less than that observed in the background South Carolina

single-ton population (⬃12% in the sampled counties).68 _All

analyses that tested the association of LMSC with pre-term delivery showed statistically significant elevation of risk in analyses that compared mothers with low-TC with mothers with mid-TC (Table 4). Subgroup analyses

<147

<5th 5th 10th to 10th to 25th 25th to 50th 50th to 75th 75th to 90th 90th to >95th95th 147–159 159–179 179–204 204–231 231–261 261–280 >280

Maternal TC percentile and TC values, mg/dL

Prevalence of preterm

b

irth,

%

White mothers Black mothers

0 5 10 15 20 25

Deliveries by cesarean section 7/35

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1/37 (2.7)

6/28 (21.4)

1/18 (5.6)

1/78 (1.3)

3/51 (5.9) 7/151

(4.6) 4/98_(4.1) 11/165

(6.7) 5/94 (5.3) 7/140_(5.0)

3/45 (6.7)

5/45 (11.1)

1/19 (5.3)

4/35 (11.4)

4/19 (21.1)

FIGURE 1

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indicated that this association was present exclusively

among white mothers; black mother-infant pairs

showed no association between LMSC and preterm de-livery in any model (Fig 1, Table 4). Black women showed an excess of preterm delivery exclusively among the high-TC group. This substantial qualitative differ-ence between the races in patterns of prematurity led us to perform additional analyses of this and other birth outcomes separately by race, as posthoc findings. The

unadjusted OR among white mothers with TC ⬍10th

population percentile was 5.22 (95% CI: 2.52–10.8;P⬍

.0001), which increased slightly to 5.63 after adjustment for infant IUGR and gender, maternal age subgroup, and

maternal weight ⬎90th percentile (Table 4). The same

regression model when used to compare the risk for

birth atⱕ35 gestational weeks against birth at weeks 39

to 41 gave similar results (adjusted OR: 6.77; 95% CI:

2.93–15.6;P⬍.0001). All secondary analyses that tested

robustness of preterm findings among white mother-infant pairs gave results comparable to the primary anal-yses. In addition, analyses consistently indicated that maternal cholesterol values less than the third percentile had higher associated risk ratios than the corresponding 5th or 10th percentile thresholds for LMSC (Table 4).

Infants who were born at 37 to 41 weeks to mothers with low TC weighed on average 124 g less than those

who were born to control mothers (P ⫽ .021, 1-way

analysis of variance; Table 5). This difference increased to 147 g with adjustment for specific GA within the term interval, race, infant gender, maternal age group, and

TABLE 3 Maternal Baseline Characteristics According to Serum Cholesterol Level and Race

Characteristic All Mothers Black Mothers White Mothers

n Mean (SD), Range

Pa _Pb _n _{Mean (SD),} Range

Pa _n _{Mean (SD),} Range

Pa

Gestational week serum screened 1058 17.7 (1.3), 15–23 .035 381 17.9 (1.4), 15–23 677 17.6 (1.3), 15–23

Mid-TC group 822 17.8 (1.3), 15–23 Referent .009 288 18.0 (1.3), 15–23 Referent 534 17.7 (1.3), 15–23 Referent

Low-TC group 118 17.4 (1.5), 15–22 .005 .37 55 17.6 (1.5), 15–22 .036 63 17.3 (1.4), 15–21 .033

High-TC group 118 17.6 (1.2), 15–21 .19 .34 38 17.5 (1.2), 15–21 .03 80 17.7 (1.2), 15–21 .99

Maternal age at screening, y 1058 26.4 (3.7), 21–34 ⬍.0001 381 25.6 (3.7), 21–34 677 26.9 (3.6), 21–34

Mid-TC group 822 26.5 (3.7), 21–34 Referent ⬍.0001 288 25.6 (3.7), 21–34 Referent 534 27.0 (3.6), 21–34 Referent

Low-TC group 118 25.1 (3.5), 21–34 .0001 .96 55 25.1 (3.4), 21–34 .36 63 25.2 (3.7), 21–33 .0002

High-TC group 118 27.2 (3.6), 21–34 .04 .04 38 26.2 (3.8), 21–33 .37 80 27.7 (3.3), 21–34 .09

Maternal weight at screening, lb 1058 165 (42), 87–389 ⬍.0001 381 176 (48), 91–389 677 159 (37), 87–358

Mid-TC group 822 164 (41), 87–389 Referent ⬍.0001 288 175 (47), 91–389 Referent 534 158 (37), 87–318 Referent

Low-TC group 118 174 (50), 104–358 .06 .02 55 185 (55), 104–327 .25 63 164 (44), 109–358 .40

High-TC group 118 164 (37), 97–280 .56 .21 38 172 (43), 109–250 .87 80 160 (34), 97–280 .27

TC at screening, unadjusted, mg/dL 1058 210 (44), 93–435 .006 381 205 (45), 105–412 677 213 (44), 93–435

Mid-TC group 809 208 (26), 159–260 .032 284 205 (26), 159–260 525 209 (26), 159–260

Low-TC group 121 140 (14), 93–158 .31 56 141 (11), 105–158 65 139 (16), 93–158

High-TC group 128 289 (30), 261–435 .85 41 291 (33), 261–412 87 288 (30), 261–435

TC at screening, adjusted, mg/dL 1058 209 (44), 89–453 .002 381 204 (45), 104–407 677 212 (44), 89–453

Mid-TC group 822 208 (26), 159–261 .93 .004 288 204 (26), 160–260 .39 534 210 (26), 159–261 .07

Low-TC group 118 141 (14), 89–159 .73 .41 55 142 (11), 104–159 .77 63 140 (16), 89–159 .48

High-TC group 118 290 (33), 261–453 .08 .42 38 293 (34), 263–407 .37 80 289 (33), 261–453 .09

a_{Versus referent, assessed by 1-way analysis of variance.}

b_{Value for difference between races, assessed by multivariate regression models.}

TABLE 4 Prevalence of Preterm Birth and Adjusted Associations of Preterm Birth With Maternal Serum Cholesterol Level According to Maternal Race

Parameter All Mothers Black Mothers White Mothers

n/N

(% Preterm)

OR (95% CI)a _Pa _n_/_N (% Preterm)

OR (95% CI)a _Pa

Preterm birthb _{70/1058 (6.6)} _{22/381 (5.8)} _{48/677 (7.1)}

Mid-TC group 41/822 (5.0) Referent 15/288 (5.2) Referent 26/534 (4.9) Referent

Low-TC group

TC⬍10th percentile 15/118 (12.7) 2.93 (1.54–5.56) .001 2/55 (3.6) 0.81 (0.18–3.74) .79 13/63 (20.6) 5.63 (2.58–12.3) ⬍.0001

TC⬍5th percentile 8/72 (11.1) 2.51 (1.11–5.67) .027 1/37 (2.7) 0.57 (0.07–4.51) .59 7/35 (20.0) 5.32 (1.97–14.3) .001

TC⬍3rd percentile 6/37 (16.2) 3.62 (1.38–9.38) .008 0/16 (0) c c _{6/21 (28.6)} _{7.69 (2.56–23.1)} _.0003

High-TC group 14/118 (11.9) 2.66 (1.39–5.09) .003 5/38 (13.2) 2.60 (0.84–8.00) .096 9/80 (11.8) 2.74 (1.22–6.18) .015

a_{Assessed by multivariate logistic regression adjusted for maternal race, maternal weight group (}_⬍_{10th, 10th–90th, and}_⬎_{90th cohort percentiles), age group (20 –24, 25–29, and 30 –34 years),}

infant gender, and presence of IUGR.

b_{Numbers and prevalence rates of delivery at}_⬍_{37 completed weeks’ GA.}

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maternal weight (P⫽.0006, linear regression, excluding outliers to the normal birth weight distribution). The distributions of infant length and head circumference did not differ in any comparisons of LMSC and control

groups. Macrosomia (weight⬎90th US population

per-centile for GA at term66_{) was not associated with}

mater-nal TC level (P⫽.83,␹2_{test for trend). Among preterm}

infants, GA was the only significant predictor of size. Infants in the left tail of the size distribution,

measur-ing⬍10th percentile for their GA in weight, length, or

head circumference or meeting the composite definition of IUGR were no more likely to be born to mothers with LMSC than to control mothers (Table 6). This was true whether the thresholds for small size were derived from

1 of several published distributions64,66,69_{or classified by}

internal cohort percentiles. However, maternal TC less than the third percentile was consistently associated with low birth weight. Low maternal weight was the most significant predictor of low birth weight for GA. Microcephaly was approximately twice as prevalent among infants of mothers with LMSC as among infants of control mothers with mid-TC; significance testing in-dicated a trend in adjusted logistic regression (Table 6). Low-TC pregnancies in our study were not more likely than control pregnancies to produce an infant with a congenital anomaly. Infants with anomalies did have markedly increased frequency of either very low or very high maternal TC or maternal weight, as well as all combinations of these extremes (7 of 8 infants with cardiac anomalies and 11 of 15 with other major

anom-alies were born to mothers in the⬍5% or⬎90% tails of

maternal cholesterol and/or maternal weight

distribu-tion [P⬍.0001 in posthoc␹2_{analysis]; 7 of these infants}

also weighed ⬎90th percentile for GA). There was no

trend toward preterm delivery or IUGR as maternal weight increased in this study group; however, neonatal

weight⬎4000 g was strongly associated with increasing

maternal weight (P⬍.0001,␹2_{test for trend).}

DISCUSSION

Our data indicate a substantially elevated risk for pre-term delivery among white women whose TC was among the lowest 10% of the background population as sampled at 17 to 18 weeks’ GA. This finding was statis-tically significant in the initial pooled analysis as well and so does not represent exclusively a posthoc sub-group result. Factors that support the validity of this finding include the exceptionally high point estimate for risk for prematurity among mothers with very low ma-ternal cholesterol (138 mg/dL, the third population per-centile of the original screened cohort) and the consis-tent finding that LMSC was the strongest predictor of preterm delivery in every model tested. We considered possible sources of both systematic and random error: confirmatory analyses were conducted to address possi-ble classification error in the assignment of GA, to test

TABLE 5 Comparative Measurements of Term Infants by Maternal Serum Cholesterol Level and Race Parameter All Term Infants ( N ⫽ 958) Black Infants ( N ⫽ 349) White Infants ( N ⫽ 609) n Mean (SD) Unadjusted Difference From Referent P a

Adjusted _Difference From Referent

b

P

b

n

Mean (SD) Unadjusted Difference _From

Referent

P

a

b

P

b

n

Mean (SD) Unadjusted Difference _From

Referent

P

a

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the robustness of low-TC and higher-TC definitions, and to examine multivariate models for sensitivity to

covari-ate selection and other factors.70–72 _{All such tests of}

ro-bustness yielded consistent, statistically significant risk estimates for prematurity among white mothers with LMSC. Whether this finding can be replicated or ex-tended to other populations of pregnant women will be essential to examine.

Our a priori hypothesis that risk for microcephaly would be increased among infants who are born to mothers with LMSC was not confirmed statistically; however, the presence of a statistical trend and an esti-mated twofold increase in risk is provocative and like-wise deserves additional scrutiny. These data did not support the hypothesized associations of LMSC with size

⬍10th percentile for GA, with the exception of an excess

of infants with low weight for GA among mothers with TC in the lowest subgroup (less than third percentile). There was also a shift toward lower birth weights still within the reference range among term infants who were born to mothers with LMSC. As a posthoc finding, this must be regarded with caution; the additional rela-tive decrease in birth weight among term infants of mothers with LMSC after adjustment for multiple factors that are known to affect birth weight does support its

potential validity. The hypothesized association of LMSC with congenital anomalies was not supported.

The observed association between LMSC and preterm delivery requires validation. If real, then such an asso-ciation could be mediated directly by restricting the availability of substrate for hormonal and nutritional support of early pregnancy, including an effect on

pla-centation.12,18_{LMSC could also indicate altered}

lipopro-tein fractions or apolipoprolipopro-tein profiles, which have been associated with altered pregnancy outcome in var-ious scenarios implicating both maternal and fetal

geno-types.3,23,24,45,73–76 _{It would be of great interest to know}

how commonly TC in the moderately low 5% to 10% range as measured in midpregnancy correlates with a substantially lower value in early pregnancy, which could have affected first-trimester events, or how com-monly LMSC represents a relatively normal periconcep-tional cholesterol level that failed to rise as expected in the second trimester, affecting later events, such as week of delivery. As pregnancy progresses, a reduction in physiologic gestational hypercholesterolemia could af-fect aspects of lipid homeostasis in pregnancy (eg, fetal erythrocyte membranes normally have altered choles-terol concentrations in later pregnancy, with concomi-tant alterations in the function of membrane-based ion

TABLE 6 Adjusted Associations of Small Birth Size for GA With Maternal Serum Cholesterol Level According to Race

Parameter All Infants Black Infants White Infants

n/N(%) OR (95% CI)a _Pa _n_/_N_(%) _{OR (95% CI)}a _Pa _n_/_N_(%) _{OR (95% CI)}a _Pa

Weight⬍10th percentile for GAb _{60/1057 (5.7)} _{29/381 (7.6)} _{31/676 (4.6)}

Low-TC group

TC⬍10th percentile 8/118 (6.8) 1.28 (0.58–2.84) .55 4/55 (7.3) 1.11 (0.36–3.45) .85 4/63 (6.4) 1.41 (0.44–4.44) .56

TC⬍5th percentile 6/72 (8.3) 1.65 (0.66–4.10) .28 3/37 (8.1) 1.35 (0.37–4.94) .65 3/35 (8.6) 1.92 (0.51–7.26) .34

TC⬍3rd percentile 5/37 (13.5) 2.86 (1.03–7.93) .044 2/16 (12.5) 2.00 (0.41–9.76) .39 3/21 (14.3) 3.42 (0.84–13.9) .085

High-TC group 6/118 (5.1) 0.90 (0.37–2.19 .82 3/38 (7.9) 1.14 (0.31–4.27) .84 3/80 (3.8) 0.83 (0.24–2.86) .77

Length⬍10th percentile for GAb _{59/1053 (5.6)} _{26/381 (6.8)} _{33/672 (4.9)}

Mid-TC group 45/820 (5.5) Referent . 21/288 (7.3) Referent 24/532 (4.5) Referent

Low-TC group 9/116 (7.8) 1.40 (0.65–3.03) .39 3/55 (5.5) 0.86 (0.24–3.07) .81 6/61 (9.8) 2.03 (0.74–5.52) .17

High-TC group 5/117 (4.3) 0.71 (0.27–1.88) .49 2/38 (5.3) 0.56 (0.12–2.62) .46 3/79 (3.8) 0.83 (0.24–2.90) .78

HC⬍10th percentile for GAb _{198/1053 (18.8)} _{105/380 (27.6)} _{93/673 (13.8)}

Low-TC group 23/115 (20.0) 1.11 (0.66–1.87) .68 15/54 (27.8) 1.17 (0.59–2.32) .65 8/61 (13.1) 0.95 (0.42–2.16) .91

High-TC group 19/118 (16.1) 0.74 (0.42–1.29) .28 11/38 (29.0) 0.91 (0.41–1.99) .81 8/80 (10) 0.62 (0.27–1.42) .26

IUGRc _{22/1056 (2.1)} _{11/381 (2.9)} _{11/675 (1.6)}

Low-TC group 2/118 (1.7) 0.68 (0.15–3.09) .61 1/55 (1.8) 0.58 (0.07–4.90) .62 1/63 (1.6) 0.69 (0.07–6.37) .74

High-TC group 1/118 (0.9) 0.34 (0.04–2.59) .29 0/38 (0) —e _—e _{1/80 (1.3)} _{0.65 (0.08–5.43)} _.69

Microcephalyd _{23/1053 (2.2)} _{14/380 (3.7)} _{9/673 (1.3)}

Low-TC group 5/115 (4.4) 2.56 (0.87–7.50) .087 3/54 (5.6) 1.91 (0.48–7.58) .36 2/61 (3.3) 3.41 (0.59–19.7) .17

High-TC group 2/118 (1.7) 0.96 (0.21–4.40) .95 1/38 (2.6) 0.70 (0.08–6.02) .74 1/80 (1.3) 1.46 (0.16–13.5) .74

HC indicates head circumference.

a_{Assessed by multivariate logistic regression models adjusted for infant gender, preterm versus term delivery, maternal weight group, age group, and race (in pooled models). All analyses compare}

risk for indicated outcome for low-TC or high-TC mothers compared with the mid-TC reference group.

b_{As deﬁned by Fenton.}65

c_{Deﬁned as weight and length both}⬍_{10th percentile for GA, irrespective of head size.} d_{Deﬁned as HC}_⬍_{2 SD below mean for GA, with length}_ⱖ_{1 SD below mean.}55

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channels36_{). The complex pathways that are associated}

with onset of labor may also potentially be affected by

altered steroid balance.49

Although low serum cholesterol level is known to be correlated with poor nutritional status, there was no correlation between maternal TC and maternal weight in this cohort. However, micronutrient deficiencies may be more common among the low-TC risk group studied here and could account for the observed adverse out-comes. Many such nutritional deficiencies have been studied as predictors of preterm delivery or low birth

weight.77–80 _{Why any of these potential mechanisms}

might manifest among white but not black mothers would be critical to address should these findings be

replicated.81,82_{By contrast, the extremely high risk ratios}

observed among mothers with TC below the third pop-ulation percentile suggests a severe and persistent dys-lipidemia, which might exert complex effects

through-out pregnancy.4,83 _{Our data also indicate a significantly}

increased risk for preterm delivery among mothers with

TC ⬎ 90th population percentile (261 mg/dL at 17.6

weeks’ GA). Therefore, the concept of an optimal range for maternal serum cholesterol during pregnancy may have merit.

Limitations are inherent in our methods and findings. The specific numeric thresholds for low and high TC are based on a mean sampling GA of 17.6 weeks and cannot be generalized to other populations with differing pro-files of gestational risk. The ascertainment of potential study subjects was incomplete, and the LMSC group differed in baseline characteristics from the control group: the specific effects of any resulting selection bi-ases cannot be predicted. We did not have access to some sociodemographic variables that are known to correlate with birth outcome, and we collected a subset of rele-vant clinical data commensurate with the goal of deter-mining whether comprehensive prospective studies are indicated. This pilot study had several a priori hypothe-ses, reducing somewhat the statistical impact of the ob-served associations.

Our strict inclusion criteria preclude extrapolation of these results to the general population, yet this design strategy has allowed us to discern an important prelim-inary finding. The strength and consistency of the ele-vated risk for preterm delivery among mothers with LMSC in this study should spur the prospective, longi-tudinal characterization of maternal cholesterol and li-poprotein profiles in subsequent investigations of pre-term birth.

ACKNOWLEDGMENTS

This research was supported by the Division of Intramu-ral Research, National Human Genome Research Insti-tute, NIH, Department of Health and Human Services.

We thank our GGC colleagues Ericka Strickland, RN, for extraction of clinical data from hospital records, Kim

Stewart for preparation of coded serum samples, and Karen Buchanan for compilation of anonymized data sets. We thank our NIH colleagues Robert T. Long for maintenance of serum samples and testing logs, Mau-reen Sampson for performance of all TC assays, and Julia Fekecs for drafting the figure and tables.

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Adverse Birth Outcome Among Mothers With Low Serum Cholesterol

Adverse Birth Outcome Among Mothers With Low

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