Annals of Internal Medicine
Periconception Red Blood Cell Folate and Offspring Congenital
Heart Disease
Nested Case–Control and Mendelian Randomization Studies
Hongyan Chen, PhD*; Yi Zhang, MPH*; Dingmei Wang, MD*; Xiaotian Chen, Msc; Mengru Li, MD; Xiangyuan Huang, Msc;
Yuan Jiang, Msc; Yalan Dou, PhD; Yin Wang, PhD; Xiaojing Ma, MD; Wei Sheng, PhD; Bing Jia, MD; Weili Yan, PhD; and
Guoying Huang, MD; for the SPCC (Shanghai Preconception Cohort) Group†
Background: Periconception folic acid supplementation has
been suggested to protect against congenital heart disease
(CHD), but the association between maternal red blood cell
(RBC) folate, the gold-standard biomarker of folate expo-
sure, and subsequent offspring CHD risk is lacking.
Objective: To quantify the association between periconception
maternal RBC folate and offspring CHD risk.
Design: Prospective, nested, case–control study and 1-sample
Mendelian randomization. (ClinicalTrials.gov: NCT02737644)
Setting: 29 maternity institutions in 12 districts of Greater
Shanghai, China.
Participants: All 197 mothers of offspring with CHD and
788 individually matched mothers of unaffected offspring
from the SPCC (Shanghai Preconception Cohort).
Measurements: Maternal RBC folate was measured before
or at early pregnancy. Odds ratios [ORs] were estimated using
conditional logistic regression after adjustment for covariates.
Mendelian randomization was done using the methylenetetrahy-
drofolate reductase (MTHFR) C677T as the genetic instrument.
Results: Case patients had lower median maternal RBC fo-
late concentrations than control participants (714 nmol/L
[interquartile range, 482 to 1008 nmol/L] vs. 788 nmol/L
[557 to 1094 nmol/L]). Maternal RBC folate concentrations
were inversely associated with offspring CHD (adjusted OR
C ongenital heart disease (CHD) is the most common
congenital defect worldwide, affecting about 9 in
1000 newborns (1). Prenatal and postnatal screening, di-
agnosis, and surgery have been fairly successful in man-
aging CHD, but strategies for primary prevention are
limited; substantial efforts are required to identify major
modifiable risk factors. Folate is a crucial nutrient required
for embryonic development, and maternal folate supple-
mentation is well established to protect against neural
tube defects (NTDs) (2, 3). Such protective effects seen in
clinical trials and food-fortification programs for NTDs (4–
7) may extend to CHD; this is biologically plausible as neu-
ral crest cells with a high demand for folate also contribute
See also:
Summary for Patients
Web-Only
Supplement
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ORIGINAL RESEARCH
per 100 nmol/L, 0.93 [95% CI, 0.89 to 0.99]). The adjusted
OR for mothers with periconception RBC folate of 906 nmol/L
or more (vs. <906 nmol/L) was 0.61 (CI, 0.40 to 0.93).
Mendelian randomization showed that each 100-nmol increase
in maternal RBC folate concentrations was significantly associ-
ated with reduced offspring CHD risk (OR, 0.75 [CI, 0.61 to
0.92]).
Limitation: Potential confounding due to unmeasured cova-
riates in the nested case–control study.
Conclusion: Higher maternal RBC folate is associated with
reduced offspring CHD risk. For primary CHD prevention,
higher target RBC folate levels than currently recommended
for neural tube defect prevention may be needed and war-
rant further study.
Primary Funding Source: National Key Research and
Development Program of China, National Natural Science
Foundation of China, China Postdoctoral Science Foundation,
and Chinese Academy of Medical Sciences Innovation Fund
for Medical Sciences.
Ann Intern Med. doi:10.7326/M22-0741 Annals.org
For author, article, and disclosure information, see end of text.
This article was published at Annals.org on 23 August 2022.
* Dr. H. Chen, Mr. Zhang, and Dr. D. Wang contributed equally.
† For members of the SPCC Group, see the Supplement (available at
Annals.org).
to the outflow tract septation of the heart (8). However, evi-
dence from epidemiologic studies on CHD is conflicting.
Some studies have found significant associations between
folic acid supplementation and CHD (9–13), but none
have reported a significant association for serum folate
(14–17). Methodological limitations, including the timing
and accuracy in folate status measurements, may contrib-
ute to the controversial findings.
Red blood cell (RBC) folate reflects the long-term fo-
late status and is the gold-standard measure recom-
mended by the World Health Organization (WHO) (18).
However, no prior studies have measured this biomarker
in the critical time window before or close to fetal heart
development to quantify the association between mater-
nal folate and offspring CHD. Consequently, whether
and to what extent maternal folate is associated with off-
spring CHD risk remains unclear. In this study, we investi-
gated the association between periconception maternal
RBC folate and offspring CHD risk using a nested case–
control study within the SPCC (Shanghai Preconception
Cohort) (19). Considering that ethical considerations limit
Annals of Internal Medicine © 2022 American College of Physicians 1
ORIGINAL RESEARCH
randomized controlled trials of folic acid supplementation
on CHD as the primary outcome (3), we attempted to test
the causality of the maternal folate–CHD association using
the Mendelian randomization approach (20, 21). This
approach has been accepted as an alternative method
using genetic variants as instrumental variables to assess
causal relationships. Similar to random allocation of inter-
vention in randomized controlled trials, Mendelian ran-
domization uses a person's naturally occurring randomized
exposure to genetic variants that are highly associated with
lifetime RBC folate and can thus overcome the problems of
reverse causation and confounding that are typical of con-
ventional observational studies (22).
METHODS
Study Population
Initiated in March 2016, the SPCC is an ongoing pro-
spective cohort study designed to investigate the associ-
ations between periconception essential nutrients and
offspring CHD risk. It recruited participants from 29 mater-
nity institutions in 12 districts of Greater Shanghai, China.
The rationale and methods of the SPCC have been pub-
lished previously (19). Briefly, couples who attended the
preconception clinics and intended to conceive within 1
year and early pregnant women who attended the first
prenatal examination within 14 weeks of gestation were
recruited. Information on lifestyle, dietary supplementa-
tion, pregnancy history, and anthropometric data was
collected from standardized questionnaires (see the
Supplement Methods [available at Annals.org] for a
detailed description), and blood samples were drawn at
recruitments. All of the pregnancies were followed up
with routine prenatal health care procedures. Blood
samples were treated according to the requirements of
RBC folate examinations and stored at the central labo-
ratory in 80  C freezers until analysis. As of December
2021, delivery outcomes were available for 22 753 of
35 166 enrolled women. The Ethics Committee of the
Children's Hospital of Fudan University approved the
study protocol (institutional review board number
201649). All participants provided written informed
consent before enrollment. This study followed the
STROBE (Strengthening the Reporting of Observational
Studies in Epidemiology) reporting guideline.
Identification of Case Patients and Match of
Control Participants
Case patients were defined as mothers of offspring
with CHD, including live births, stillbirths, and abortions
because of fetal cardiac anomalies. All pregnancies had
prenatal ultrasound scans in the second trimester (around
20 to 24 weeks of gestation) performed by trained ultraso-
nographers; abnormal findings were confirmed by author-
ized tertiary fetal medical centers. All newborns had routine
neonatal CHD screening, and those screened positive were
subsequently sent for further echocardiographic confirma-
tion (23, 24). Those who screened negative attended rou-
tine child care visits at 42 days, 3 months, 6 months, and
1 year after birth; those with late-presenting cardiac symp-
toms (such as cyanosis, tachypnea, and feeding difficulty)
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RBC Folate and Offspring Congenital Heart Disease
and with a CHD diagnosis from any other hospitals after
birth were further identified. Telephone calls by trained
staff were subsequently made to confirm the informa-
tion from these parents about the CHD diagnosis of their
children (see the Supplement Methods for a detailed
description).
The nomenclature of the International Paediatric and
Congenital Cardiac Code (of the Nomenclature Working
Group of the International Society for Nomenclature of
Paediatric and Congenital Heart Disease) was used to
define CHD (25). We excluded the following conditions:
simple patent foramen ovale, patent ductus arteriosus
closed within 3 months of age, physiologic pulmonary
branch stenosis resolved at postnatal follow-up, and
atrial septal defect closed within 3 months of age. In
combined cardiac defects, either the most clinically sig-
nificant structural anomaly or the defect necessitating
the earliest intervention was considered the predomi-
nant diagnosis.
Eligible control participants were mothers of unaf-
fected offspring randomly selected from the cohort.
Each confirmed case was matched to 4 control partici-
pants by maternal age (±2 years), blood collection time
(±3 months), participating site, and enrollment status
(before conception or at early pregnancy).
Biomarker Measurement
Levels of RBC folate, serum folate, and serum vitamin
B12 were measured by using a chemiluminescence
microparticle immunoassay (ARCHITECT i2000SR Analyzer;
Abbott Laboratories) as previously described (19). The
interassay coefficient of variations of QC1-QC3 for RBC
folate, serum folate, and vitamin B12 was less than 7.5%.
The intraassay coefficient of variations was less than 6.5%
for the study population. The methylenetetrahydrofolate
reductase (MTHFR) C677T genetic variant was geno-
typed using the TaqMan allelic discrimination assay on
the platform of QuantStudio Real-Time PCR software
(Applied Biosystems) with standard quality control. The
CC, CT, and TT genotypes were recoded as 0, 1, and 2.
Statistical Analysis
We assumed a 2-sided a of 0.05, an SD for estimated
RBC folate concentrations scaled to a 100-nmol/L incre-
ment of 3.75, and an r2 of 0.06. The r2 value represents
the relationship between estimated RBC folate concen-
tration per 100-nmol/L increment and other analyzed
covariates estimated from a previous cohort study (26).
A 100-nmol/L increment was chosen because it
roughly corresponds to an average monthly increment in
RBC folate concentrations with 400 mcg per day folic
acid supplementation within 3 months (27). Based on a
conditional logistic regression model with a case to con-
trol ratio of 1:4, our sample size with 197 case patients
would have 80% power to detect an odds ratio (OR) of
smaller than 0.94 or larger than 1.06. Power calculation
was conducted using PASS 16.0 software (NCSS).
Our primary analysis evaluated the association of
continuous maternal RBC folate concentrations (per 100
nmol/L) with subsequent offspring CHD risk. Odds ratios
and 95% CIs were estimated by conditional logistic
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RBC Folate and Offspring Congenital Heart Disease ORIGINAL RESEARCH

Figure 1. Flow chart of the study population in the nested case–control study within the SPPC.

SPCC, March 2016 to December 2021

Preconception women of Early pregnancies, <14 weeks

reproductive age (n = 21 115) of gestation (n = 15 615)

Delivery outcomes available (n = 8732) Delivery outcomes available (n = 14 021)

Case patients with confirmed CHD (n = 197) Matched control participants (n = 788)
With valid maternal blood sample: 172 With valid maternal blood sample: 788
Without valid maternal blood sample: 25 Without valid maternal blood sample: 0

CHD = congenital heart disease; SPPC = Shanghai Preconception Cohort.

regression models, adjusting for potential confounding recommended by the WHO for primary NTD prevention
factors, including the duration between enrollment and (18). In a secondary analysis, we categorized RBC fo-
conception (months; with preconception accruals as late concentrations into 7 groups in a per 226-nmol/L
negative numbers and postconception accruals as posi- (100-ng/mL) interval to inspect changes in CHD risk to
tive numbers), prepregnancy body mass index (kg/m2), determine whether there would be a dose–response rela-
history of abortion (yes or no), smoking exposure (yes or tionship: less than 226 nmol/L, 226 to 452 nmol/L, 453 to
no), alcohol drinking (yes or no), education levels (high 679 nmol/L, 680 to 905 nmol/L, 906 to 1131 nmol/L, 1132
school or lower, college, or postgraduate), and vitamin to 1359 nmol/L, and 1360 nmol/L or more. We performed
B12 levels (per 100 pmol/L). To aid in clinical interpreta- a trend test by treating the dose group with the 7 cate-
tion and comparison with other studies, we dichotom- gories (coded as 1 to 7) as a continuous variable in the
ized RBC folate levels by the threshold of 906 nmol/L conditional logistic regression model. We estimated the
population risk in the 7 categories of RBC folate concen-
trations, assuming that the control participants would be
Table 1. Spectrum of the 197 CHD Cases
representative of the nonaffected offspring and using the
overall CHD rate of 8.98 per 1000 births in Shanghai (28).
Predominant Defect Case Patients With Risks were derived from the odds of CHD in each group
CHD, n (%)*
by dividing the number of case patients by the weighted
Simple defects 161 (82)
number of control participants, and CIs were calculated
Ventricular septal defect 82 (42)
Atrial septal defect 51 (26) assuming a Poisson distribution (29, 30) (see details
Patent ductus arteriosus 21 (11) in the Supplement Methods).
Pulmonary stenosis 7 (4) To test the causality of the association, we performed
1-sample Mendelian randomization using the MTHFR
Complex defects 36 (18)
Tetralogy of Fallot 13 (7)
C677T variant as the instrument variable. This variant is a
Vascular ring† 6 (3) well-established functional polymorphism related to fo-
Hypoplastic left heart syndrome 4 (2) late metabolism that is associated with a 10% to 35%
Double outlet right ventricle 3 (2) reduction in circulating folate and RBC folate (31, 32)
Functionally univentricular heart 3 (2)
Total anomalous pulmonary venous connection 2 (1)
and has been reported to be a risk factor for offspring
Hypoplastic right heart syndrome 2 (1) CHD (9). As an instrumental variable, the MTHFR C677T
Tricuspid atresia 1 (1) satisfied the 3 main assumptions for Mendelian random-
Persistent truncus arteriosus 1 (1) ization analysis (22): the genotype should be robustly
Tricuspid stenosis 1 (1)
associated with the intermediate phenotype, should not
Total 197 (100) be associated with confounding factors, and should
exert its effect on the outcome only through the specific
CHD = congenital heart disease. intermediate phenotype (see the detailed description in
* Because of rounding, the percentages may not total to 100.
† Including 3 cases with double aortic arch, 2 right aortic arch with the Supplement Methods).
aberrant left subclavian, and 1 pulmonary sling, all of which were CHD The Mendelian randomization estimate was com-
cases of clinical significance. puted using the adjusted 2-stage method (33). Given the
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ORIGINAL RESEARCH RBC Folate and Offspring Congenital Heart Disease

Table 2. Characteristics of Participants in the Nested Case–Control Study Within the SPCC (n = 985)*
Characteristics Case Patients With Matched Control Participants
CHD (n = 197) (n = 788)
Median age (IQR), y 30.1 (28.3–33.1) 30.0 (27.8–32.6)
Median duration between enrollment and conception (IQR), mo†
Preconception accrual 6.1 (2.1–14.2) 5.3 (2.2–10.7)
Postconception accrual 2.8 (2.2–2.9) 2.8 (2.3–3.0)
Ethnicity, n (%)
Han 195 (99) 778 (98.7)
Others 2 (1) 10 (1.3)
Median body mass index (IQR), kg/m2‡ 20.9 (19.2–22.8) 20.7 (19.2–22.4)
History of abortion, n (%) 58 (30) 225 (30.9)
Education attainment, n (%)
High school education or lower 18 (9) 79 (10.0)
College education 141 (72) 571 (72.5)
Postgraduate education 38 (19) 138 (17.5)
Smoking exposure, n (%) 28 (14) 134 (17.0)
Alcohol drinking, n (%) 26 (13) 148 (18.8)
Folate supplement before pregnancy, n (%)§ 37 (47) 157 (47.6)
Folate supplement during early pregnancy, n (%)§ 95 (89) 409 (93.0)
Mean serum vitamin B12 (SD), pmol/L 455 (186) 490 (210)
Median serum folate (IQR), nmol/L 31 (21–38) 32 (21–39)
No folate supplement 24 (18–31) 21 (15–30)
With folate supplement 32 (25–39) 35 (29–41)
Median RBC folate (IQR), nmol/L 714 (482–1008) 788 (557–1094)
No folate supplement 490 (343–740) 566 (418–810)
With folate supplement 830 (577–1072) 877 (651–1179)
RBC folate ≥906 nmol/L, n (%) 50 (29) 303 (38.5)
CHD = congenital heart disease; IQR = interquartile range; RBC = red blood cell; SPCC = Shanghai Preconception Cohort.
* Data were missing for 2.4% (n = 24) of body mass index, 6.6% (n = 65) of history of abortion, 3.1% (n = 30) of folate supplement, 2.5% (n = 25) of
RBC folate, 5.7% (n = 56) of serum folate, and 5.3% (n = 52) of serum vitamin B12. Because of rounding, not all percentages total 100. To convert
the folate values from nmol/L to ng/mL, divide by 2.265.
† Duration between enrollment and conception was calculated as the enrolled date minus the conception date, which was a negative number for
the preconception accrual (n = 430) and a positive number for postconception accrual (n = 555). For the descriptive purpose only, we used the
absolute values with the preconception accrual indicating the month before conception.
‡ Body mass index is the weight in kilograms divided by height in meters squared.
§ Folate supplement before conception was available to 408 participants recruited before conception. Folate supplement during early pregnancy
was available to 547 participants recruited before 14 weeks of gestation.

skewed distribution (Supplement Figure 1, available at
Annals.org), in the first stage, log-transformed maternal
RBC folate per 100 nmol/L was regressed on MTHFR
C677T genotypes by a linear regression model in the
control group only, based on the assumption that the
distribution of the exposure in the control participants is
similar to that of the general population, which is true for
rare diseases (34). The P for trend across the genotypes
was estimated using an additive model with the geno-
type treated as a continuous variable. The strength of
the instrument was confirmed by F statistics from the
regression (F > 10 is considered acceptable). In the
second stage, the binary outcome of offspring CHD
was regressed on the back-transformed fitted RBC fo-
late (per 100 nmol/L) and the residuals from the first
stage, which served as independent variables in a logis-
tic regression model using the vce (robust) option in
Stata. We used the robust SEs to minimize the potential
of heteroscedasticity and misspecification of the equa-
tions in the model (33).
We performed subgroup analyses to assess whether
the associations varied by enrollment status (preconcep-
tion or during early pregnancy) and CHD complexity
(simple defects and complex defects) with their respec-
tive matched control participants. Multiple imputation by
chained equations was done using the Stata mi imputed
4 Annals of Internal Medicine
chained command to impute missing data of maternal
RBC folate (for 25 CHD cases) and missing values of
covariates based on the assumption that the data were
missing at random. We imputed the data by CHD-
affected status and included maternal RBC folate, all
covariates considered in the multivariable models, and
other variables available that we thought were associ-
ated with the missingness in the imputation models.
Because of the skewed distribution of RBC folate and
serum folate, these 2 variables were log-transformed
before imputation. Ten imputed data sets were gener-
ated, with the results combined using the Rubin rule
(35, 36) (see the detailed Supplement Methods). We
did a sensitivity analysis by repeating the main analyses
restricting to the 172 case patients with measured
maternal RBC folate. We also assessed the association
of maternal serum folate concentrations as a continuous
variable with offspring CHD risk in an additional analy-
sis. All of the 95% CIs that are presented for the second-
ary and subgroup analyses have not been corrected for
multiplicity as there was no prespecified hypothesis.
Analyses were done using Stata 16.0 (StataCorp) and R
version 4.0.2 (R Development Core Team) for the fig-
ures. A 2-sided P value of less than 0.050 was consid-
ered statistically significant.
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RBC Folate and Offspring Congenital Heart Disease
Role of the Funding Source
The funders had no role in the design and conduct
of the study; collection, management, analysis, or inter-
pretation of the data; preparation, review, or approval of
the manuscript; or decision to submit the manuscript for
publication.
RESULTS
Characteristics of the Participants
Among the 22 753 women with delivery outcomes,
197 CHD cases were confirmed, with 86 from mothers
recruited before pregnancy and 111 from mothers
recruited at early pregnancy (Figure 1). The most com-
mon subtypes of offspring CHD were ventricular septal
defect (42%), followed by atrial septal defect (26%), pat-
ent ductus arteriosus (11%), tetralogy of Fallot (7%), pul-
monary stenosis (4%), and vascular ring (3%) (Table 1).
The final study sample for the nested case–control study
comprised 985 participants, with 788 matched control
mothers.
Baseline characteristics, including maternal age, eth-
nicity, prepregnancy body mass index, history of abor-
tion, educational levels, smoking, alcohol drinking status,
and folic acid supplement status, were similar between
case patients and control participants (Table 2). The me-
dian duration between enrollment and conception for
the preconception accrual and postconception accrual
was 5.3 months (interquartile range [IQR], 2.2 to 11.2
months) and 2.8 months (2.3 to 3.0 months), respectively,
and was similar between case patients and control partic-
ipants. Periconception RBC folate concentrations were
lower in case mothers than control mothers (714 nmol/L
[482 to 1008 nmol/L] vs. 788 nmol/L [557 to 1094 nmol/L]),
ORIGINAL RESEARCH
with similar patterns seen when stratified by folate sup-
plementation status. Only 29.1% of case patients had
maternal RBC folate concentrations of 906 nmol/L or
higher, which was lower than that of the control partici-
pants (38.5%). Characteristics of persons with (n = 172)
and without (n = 25) measured RBC folate were, in gen-
eral, similar (Supplement Table 1, available at Annals.
org).
Maternal RBC Folate and Offspring CHD Risk
Each additional 100-nmol/L increase in maternal
RBC folate concentrations was associated with a reduc-
tion in offspring CHD (adjusted OR, 0.93 [95% CI, 0.88 to
0.99]), and having RBC folate concentrations of 906 nmol/L
or more was associated with an adjusted OR of 0.61 (CI,
0.40 to 0.93) (Figure 2). In our secondary analysis
exploring a potential dose–response relationship, the
odds of CHD progressively decreased as RBC folate
increased (P for trend = 0.007). However, the magni-
tude of reduction plateaued at levels approaching
1360 nmol/L (Figure 2), suggesting a potential thresh-
old effect. Compared with those with maternal folate
levels less than 226 nmol/L, the estimated population
CHD risk per 1000 births was less than half among
those with levels greater than 906 nmol/L (Table 3).
Sensitivity analyses restricted to 172 case patients with
measured maternal RBC folate showed similar findings
(Supplement Figure 2 and Supplement Table 2, avail-
able at Annals.org).
Mendelian Randomization
One-sample Mendelian randomization was done in
170 case patients with CHD and 771 control participants
with maternal MTHFR C677T genotypes. The T allele

Figure 2. Associations between maternal RBC folate concentrations and the risk for offspring CHD.

Maternal RBC Folate Case Patients/Control Participants, n/N OR (95% CI) P Value

Continuous, per 100 nmol/L 197/788 0.93 (0.88–0.99) 0.014

Dichotomized by WHO threshold, nmol/L
<906 141/486 1
≥906 56/302 0.61 (0.40–0.93) 0.022
Groupings, nmol/L (ng/mL)
<226 (<100) 6/16 1 0.007
227–452 (100–199) 39/103 1.06 (0.34–3.23)
453–679 (200–299) 50/180 0.67 (0.21–2.13)
680–905 (300–399) 46/187 0.55 (0.18–1.76)
906–1131 (400–499) 23/126 0.40 (0.12–1.34)
1132–1359 (500–599) 15/67 0.47 (0.13–1.72)
≥1360 (≥600) 18/109 0.35 (0.10–1.26)
Mendelian randomization 170/771 0.75 (0.61–0.92) 0.007

0.03 0.10 0.30 1.00 3.00

Odds ratios for higher maternal RBC folate were calculated using conditional logistic regression models adjusting for the duration between enrollment
and conception, prepregnancy body mass index, history of abortion, maternal smoking status, alcohol drinking, educational attainment, and serum vita-
min B12 concentrations (n = 985). RBC folate was dichotomized based on the threshold value of 906 nmol/L set for primary neural tube defect preven-
tion. RBC folate concentrations were categorized into 7 groups to inspect changes in CHD risk and determine whether there would be a dose–response
relationship by performing a trend test in which the dose group with the 7 categories having values from 1 to 7 was treated as a numerical variable in
the conditional logistic regression model. The log scale was used on the x-axis. To convert the folate values from nmol/L to ng/mL, divide by 2.265. OR
= odds ratio; RBC = red blood cell; WHO = World Health Organization.

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ORIGINAL RESEARCH
Table 3. Distribution of Case Patients and Control Participants and Estimated Population Risk for CHD by Maternal RBC Folate
Concentrations*
RBC Folate, nmol/L (ng/mL) Case Patients,
n (%)
<226 (<100) 6 (3)
227–452 (100–199) 39 (20)
453–679 (200–299) 50 (25)
680–905 (300–399) 46 (23)
906–1131 (400–499) 23 (12)
1132–1359 (500–599) 15 (8)
≥1360 (≥600) 18 (9)
Total 197 (100)
CHD = congenital heart disease; RBC = red blood cell.
* We estimated the population risk in the 7 categories of RBC concentrations, assuming that the control participants would be representative of the
nonaffected offspring and using the overall CHD rate of 8.98 per 1000 births in Shanghai (28). Risks were derived from the odds of CHD in each
group by dividing the number of case patients by the weighted number of control participants, and CIs were calculated assuming a Poisson distri-
bution (29, 30).
frequency of the MTHFR C677T polymorphism was 45.7%
in the control participants. This variant explained 4.4% of
the variance in log maternal RBC folate with an F statistic
of 37.0 (P = 1.8  10 9), suggesting that it is a valid instru-
ment. Median RBC folate concentrations were 744 nmol/L
(IQR, 535 to 987 nmol/L), 714 nmol/L (508 to 988 nmol/L),
and 1052 nmol/L (735 to 1536 nmol/L) for the CC, CT,
and TT genotypes (P for trend <0.001) (Supplement
Figure 3, available at Annals.org). The risk for offspring
CHD was significantly lower in persons carrying the
MTHFR C677T risk allele (P = 0.005). In addition, known
covariates were generally evenly distributed across
MTHFR C677T genotypes (Supplement Table 3, available
at Annals.org). Each genetically associated 100-nmol
increase in maternal RBC folate concentrations was signifi-
cantly associated with a 25% reduction in the odds of off-
spring CHD (OR, 0.75 [CI, 0.61 to 0.92]) (Figure 2).
Other Analyses
The associations were consistent with RBC folate
measured before conception (adjusted OR, 0.90 [CI,
0.82 to 1.00]) or during early pregnancy (adjusted OR,
0.95 [CI, 0.89 to 1.01]) and similar between simple
defects (adjusted OR, 0.92 [CI, 0.87 to 0.99]) and complex
defects (adjusted OR, 0.96 [CI, 0.86 to 1.08]) (Supplement
Figure 4, available at Annals.org). Maternal serum folate
was not significantly associated with offspring CHD risk
(adjusted OR, 0.99 per 1-nmol/L increase [CI, 0.98 to 1.01]).
DISCUSSION
Our prospective nested case–control study found
that higher periconception maternal RBC folate was sig-
nificantly associated with a lower risk for offspring CHD.
When comparing women whose RBC folate levels were
below the threshold of clinical sufficiency (906 nmol/L),
we saw a significant decline of 39% fewer CHD cases
among women whose RBC folate levels were above this
threshold. Of note, the association showed an approxi-
mate threshold effect in that the reduction in CHD risk
seemed to flatten out at RBC folate levels approaching
1360 nmol/L (600 ng/mL). Mendelian randomization
supported a causal role of maternal folate in reducing
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RBC Folate and Offspring Congenital Heart Disease
Control Participants, Estimated Risk for CHD
n (%) per 1000 Births (95% CI)
16 (2.0) 13.5 (4.9–29.3)
103 (13.1) 13.6 (9.7–18.6)
180 (22.8) 10.0 (7.4–13.2)
187 (23.7) 8.8 (6.5–11.8)
126 (16.0) 6.6 (4.2–9.8)
67 (8.5) 8.0 (4.5–13.3)
109 (13.8) 5.9 (3.5–9.4)
788 (100) 9.0 (7.8–10.3)
offspring CHD risk. Our findings provide strong evidence
supporting the preventive effect of periconception folic
acid supplementation on CHD prevention.
The mechanism by which maternal folate exerts its
effect on offspring CHD is not well understood but may
be through affecting neural crest cells that have a
demand for folate, particularly during the first 12 weeks
after conception (8). Evidence suggests that the neural
crest cells have been traced to several other parts of the
developing heart, including the inlet of the heart, semi-
nary values, and distal aortic arch (37, 38). Limiting folate
transport to embryonic tissues through nutritional folate
deficiency or genetic alternations may thus result in the
development of CHD (8).
Prior observational studies have extensively investi-
gated the association of periconception folic acid intake
on offspring CHD risk but with inconsistent findings,
whereas the few studies using blood samples have shown
a null association (14–17, 39). Differences in methods,
especially measurements of maternal folate exposure and
the timing, could partly explain the discrepancy. Indirect
measurement through self-reported folic acid intake, and
a single serum folate measurement that is subject to day-
to-day variations in folate consumption, is prone to mea-
surement error. Because fetus heart formation occurs
around the eighth week of gestation (9) and folate levels
decline after early gestation (40), folate exposure exam-
ined after this time window or measured postpartum (39)
is less likely to detect an association.
The 120-day lifespan of RBC folate enables a more
comprehensive temporal data capture. It is recom-
mended by the WHO as the most accurate biomarker of
folate exposure (18), with the threshold of 906 nmol/L
used as an indicator of folate sufficiency status regardless
of the assay (41). Our study is unique in that we used
maternal RBC folate measured in the periconception pe-
riod to investigate the association between maternal fo-
late exposure and subsequent offspring CHD risk. We
also took advantage of the population-based identifica-
tion and an accurate diagnosis of CHD, largely minimiz-
ing case selection bias and outcome misclassification.
Overall, 8.65 per 1000 pregnancies presented with a
clinically confirmed CHD, similar to the 8.98 per 1000
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RBC Folate and Offspring Congenital Heart Disease
live births previously reported in our large multicenter
screening study (28).
Because randomly assigning women not to take folic
acid supplementation is unethical given the established
protective effects of maternal folate on primary NTD pre-
vention (3), we used the individual-level Mendelian ran-
domization to test the causality of the association. The
resulting estimate was consistent with our observational
estimate and supported a causal role of maternal folate
in preventing offspring CHD. Such methodological fea-
tures enhanced the internal validity of the association we
observed.
Our findings have important public health implica-
tions. Despite the unequivocal benefit of folic acid sup-
plementation, its preconceptional use has not increased
substantially. Our previous cross-sectional study showed
that only 42.6% of women took the recommended 400 mcg
of folic acid daily before pregnancy (42), and, more dis-
turbingly, the population RBC folate levels of women
preparing for pregnancy remain at an overall geometric
mean of 490 nmol/L (arithmetic mean, 554 nmol/L) and
90.1% below the 906-nmol/L cutoff (43). In the context
that the folic acid recommendation from the WHO aims
primarily at preventing NTDs, we provide direct empiri-
cal evidence supporting that this recommendation also
needs to consider CHD—the most frequent and serious
congenital defect in this era—as the second target.
Notably, our dose–response illustration is analogous to
Daly and colleagues’ study of the Irish population,
which assessed the association between RBC folate dur-
ing early pregnancy (29). In that study, RBC folate levels
of 906 nmol/L or more were associated with an 87.9%
reduction in the population risk for NTD (0.8 vs. 6.6
cases per 1000 births). They further postulated that RBC
folate levels higher than 1292 nmol/L might confer
additional protective effects. Our study on CHD found a
51.3% reduction in the estimated population risk for
CHD when women could reach RBC folate levels within
906 to 1132 nmol/L (vs. <226 nmol/L: 6.6 vs. 13.5 per
1000 births), with an additional 4.7% reduced risk with
RBC levels of 1360 nmol/L or more. The effect of RBC
folate seems to flatten out at higher levels, suggesting
an approximate threshold effect on offspring CHD risk.
This is, indeed, consistent with the association seen for
NTDs that RBC folate concentrations in excess of 1300
to 1500 nmol/L provide little additional benefit for
prevention (44). Expanding on the evidence base for
recommendations and guidelines about primary pre-
vention of birth defects, we propose that higher target
RBC folate levels than currently recommended might
be advisable for periconception women to achieve the
effect of primary CHD prevention. However, before any
changes to practice guidelines are implemented, the
totality of evidence on other adverse effects of exces-
sive folate levels or unabsorbed folic acid on mother
and fetus needs to be carefully considered (45).
Our study has limitations. First, we acknowledge that
missed diagnosis of CHD, especially asymptomatic or
minor CHD cases, could not be completely ruled out
with our screening approach, which may lead to selec-
tion bias. Second, RBC folate concentrations may not be
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ORIGINAL RESEARCH
directly comparable to previous studies using different
measurement assays. In addition, estimates of CHD per
1000 births may be biased if the RBC folate distribution
from the selected matched control participants is not rep-
resentative of the population at risk. Finally, unmeasured
and residual confounding cannot be ruled out in this
nested, case–control, association study. Nevertheless,
Mendelian randomization analysis helps mitigate such
limitations. Our findings are generalizable to other pop-
ulations from regions without food fortification or with
low population folate levels.
In conclusion, our prospective nested case–control
analysis combined with 1-sample Mendelian randomiza-
tion provides direct evidence that higher maternal RBC
folate is associated with reduced risk for offspring CHD.
We propose that target RBC folate levels higher than
those currently recommended for NTD prevention be
considered for primary CHD prevention. Further studies
across different populations or ethnicities would be use-
ful to support the extrapolation of our findings outside of
the considered contexts.
From Department of Clinical Epidemiology and Clinical Trial
Unit, Children's Hospital of Fudan University, National Children's
Medical Center, Shanghai, and Shanghai Key Laboratory of Birth
Defects, Shanghai, China (H.C., Y.Z., X.C., Y.J., Y.D., Y.W.);
Pediatric Heart Center, Children's Hospital of Fudan University,
National Children's Medical Center, Shanghai, China (D.W.,
M.L.); Department of Clinical Epidemiology and Clinical Trial
Unit, Children's Hospital of Fudan University, National Children's
Medical Center, Shanghai, China (X.H.); Pediatric Heart Center,
Children's Hospital of Fudan University, National Children's
Medical Center, Shanghai, and Shanghai Key Laboratory of Birth
Defects, Shanghai, China (X.M., B.J.); Shanghai Key Laboratory of
Birth Defects, Shanghai, China (W.S.); Department of Clinical
Epidemiology and Clinical Trial Unit, Children's Hospital of
Fudan University, National Children's Medical Center, Shanghai,
Shanghai Key Laboratory of Birth Defects, Shanghai, and
Research Unit of Early Intervention of Genetically Related
Childhood Cardiovascular Diseases (2018RU002), Chinese
Academy of Medical Sciences, Shanghai, China (W.Y.); and
Pediatric Heart Center, Children's Hospital of Fudan University,
National Children's Medical Center, Shanghai, Shanghai Key
Laboratory of Birth Defects, Shanghai, and Research Unit of Early
Intervention of Genetically Related Childhood Cardiovascular
Diseases (2018RU002), Chinese Academy of Medical Sciences,
Shanghai, China (G.H.).
Acknowledgment: The authors would like to thank all partici-
pants, physicians, and nurses for their help in the recruitment,
and the whole Shanghai Preconception Cohort team, including
interviewers, computer and laboratory technicians, clerical
workers, research scientists, volunteers, managers, and recep-
tionists. The authors thank Taavi Tillmann (Institute of Family
Medicine and Public Health, University of Tartu, Estonia) for his
support in reviewing and editing the manuscript, and Duolao
Wang (Global Health Trials Unit, Department of Clinical Sciences,
Liverpool School of Tropical Medicine, United Kingdom) for his
critical review, especially addressing statistical issues in the revision
of our manuscript.
Annals of Internal Medicine 7
ORIGINAL RESEARCH
Grant Support: By the National Key Research and Development
Program of China (grant numbers 2021YFC2701004 and
2016YFC1000506), the National Natural Science Foundation of
China (grant numbers 82070323 and 81273168), the China
Postdoctoral Science Foundation (grant number 2020M671000),
and the Chinese Academy of Medical Sciences Innovation Fund
for Medical Sciences (2019-I2M-5-002).
Disclosures: Disclosures can be viewed at www.acponline.org/
authors/icmje/ConflictOfInterestForms.do?msNum=M22-0741.
Reproducible Research Statement: Study protocol: Available at
https://bmjopen.bmj.com/content/bmjopen/9/11/e031076.full.pdf.
Statistical code and data set: Available to interested readers
upon reasonable request by contacting Dr. Weili Yan at yanwl@
fudan.edu.cn or Dr. Guoying Huang at gyhuang@shmu.edu.cn.
Corresponding Author: Guoying Huang, MD, PhD, Pediatric
Heart Center, Children's Hospital of Fudan University, National
Children's Medical Center, 399 Wan Yuan Road, Shanghai
201102, China (e-mail, gyhuang@shmu.edu.cn); and Weili Yan,
PhD, Department of Clinical Epidemiology & Clinical Trial Unit,
Children's Hospital of Fudan University, National Children's
Medical Center, 399 Wan Yuan Road, Shanghai 201102, China
(e-mail, yanwl@fudan.edu.cn).
Author contributions are available at Annals.org.
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Annals of Internal Medicine 9
Author Contributions: Conception and design: W. Yan, G.
Huang.
Analysis and interpretation of the data: H. Chen, Y. Zhang, D.
Wang, X. Chen, M. Li, X. Huang, Y. Jiang, Y. Dou, Y. Wang, X.
Ma, W. Sheng, B. Jia, W. Yan, G. Huang.
Drafting of the article: H. Chen, Y. Zhang, D. Wang.
Critical revision of the article for important intellectual content:
H. Chen, Y. Zhang, D. Wang, X. Chen, M. Li, X. Ma, W. Sheng, B.
Jia, W. Yan, G. Huang.
Final approval of the article: H. Chen, Y. Zhang, D. Wang, X.
Chen, M. Li, X. Huang, Y. Jiang, Y. Dou, Y. Wang, X. Ma, W.
Annals of Internal Medicine
Downloaded from https://annals.org by Northeast Normal University on 08/24/2022.
Sheng, B. Jia, W. Yan, G. Huang.
Provision of study materials or patients: X. Ma, W. Sheng, B. Jia,
W. Yan, G. Huang.
Statistical expertise: H. Chen, W. Yan.
Obtaining of funding: H. Chen, W. Yan, G. Huang.
Administrative, technical, or logistic support: Y. Zhang, X. Ma,
W. Sheng, B. Jia, W. Yan, G. Huang.
Collection and assembly of data: H. Chen, Y. Zhang, D. Wang,
X. Chen, M. Li, X. Huang, Y. Jiang, Y. Dou, Y. Wang, W. Yan, G.
Huang.
Annals.org