
==== Front
Hum Reprod
Hum Reprod
humrep
Human Reproduction (Oxford, England)
0268-1161
1460-2350
Oxford University Press

39025484
10.1093/humrep/deae168
deae168
Original Article
Early Pregnancy
AcademicSubjects/MED00905
Periconceptional maternal supplement intake and human embryonic growth, development, and birth outcomes: the Rotterdam Periconception Cohort
https://orcid.org/0000-0002-3498-5579
Schenkelaars N Department of Obstetrics and Gynaecology, Erasmus MC, University Medical Center, Rotterdam, The Netherlands

https://orcid.org/0000-0002-0316-6159
Schoenmakers S Department of Obstetrics and Gynaecology, Erasmus MC, University Medical Center, Rotterdam, The Netherlands

https://orcid.org/0000-0002-3008-2567
Rousian M Department of Obstetrics and Gynaecology, Erasmus MC, University Medical Center, Rotterdam, The Netherlands

https://orcid.org/0000-0002-1675-2931
Willemsen S P Department of Biostatistics, Erasmus MC, University Medical Center, Rotterdam, The Netherlands

https://orcid.org/0000-0002-5860-3993
Faas M M Department of Pathology and Medical Biology, University of Groningen and University Medical Center Groningen, Groningen, The Netherlands

https://orcid.org/0000-0002-4353-5756
Steegers-Theunissen R P M Department of Obstetrics and Gynaecology, Erasmus MC, University Medical Center, Rotterdam, The Netherlands

Correspondence address. Department of Obstetrics and Gynaecology, Erasmus MC, University Medical Center, Rotterdam, Dr Molewaterplein 40, 3015 GD Rotterdam, The Netherlands. Tel: +31-6-12472643; E-mail: r.steegers@erasmusmc.nl
9 2024
18 7 2024
18 7 2024
39 9 19251933
21 12 2023
13 6 2024
5 7 2024
© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology.
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Abstract

STUDY QUESTION

Is periconceptional multiple-micronutrient supplement (MMS) use including folic acid (FA) compared to FA use only associated with increased embryonic growth, development, and birth weight in a high-risk population?

SUMMARY ANSWER

Women with MMS intake show no significant differences in first-trimester morphological embryo development, but increased first-trimester embryonic growth trajectories and fewer neonates born small for gestational age (SGA), less than the 3rd percentile (<p3), compared to women using only FA.

WHAT IS KNOWN ALREADY

Periconceptional maternal FA intake in the general population is associated with increased embryonic and fetal growth, and reduced risks of neural tube defects, other congenital malformations, low birth weight, and neonates born SGA.

STUDY DESIGN, SIZE, DURATION

A prospective tertiary hospital-based cohort study (the Rotterdam Periconceptional Cohort) was conducted from January 2010 to December 2020.

PARTICIPANTS/MATERIALS, SETTING, METHODS

We included 1076 women from the Rotterdam Periconceptional Cohort, before 10 weeks of pregnancy with follow-up until delivery. Embryonic growth was assessed by measurement of crown-rump length (CRL) and embryonic volume (EV), and embryonic morphology was described by Carnegie stages using longitudinal three-dimensional ultrasound scans and virtual reality techniques. Birth outcomes were extracted from medical records. General characteristics and supplement use were extracted from research questionnaires.

MAIN RESULTS AND THE ROLE OF CHANCE

This study showed increased embryonic growth trajectories (adjusted models, CRL: β = 0.052, 95% CI 0.012–0.090, EV: β = 0.022, 95% CI 0.002–0.042) in women using MMS compared to those using only FA. Moreover, a 45% reduced risk of a neonate-born SGA (<p3) was shown in women using MMS compared to FA users (adjusted OR = 0.546, 95% CI 0.308, 0.969). Embryonic morphological development (Carnegie stages) and the occurrence of miscarriages did not differ between women using MMS or solely FA.

LIMITATIONS, REASONS FOR CAUTION

Following the heterogeneity of the composition and dose of MMS preparations, it is unclear which specific micronutrient, combination, or dose explains the increased embryonic growth trajectory and reduction in risk for SGA. This also hampers the possibility of differentiating between the effects of FA alone or as a component of MMS.

WIDER IMPLICATIONS OF THE FINDINGS

Our findings emphasize the importance of periconceptional maternal MMS use as a potential preventative intervention against reduced embryonic growth and neonates born SGA. Therefore, we recommend the periconceptional use of MMS in women at risk of inadequate micronutrient intake. However, awareness of potentially harmful side effects of high doses and combinations of micronutrients is essential, therefore the optimal composition and dose need to be investigated, and careful surveillance is recommended.

STUDY FUNDING/COMPETING INTEREST(S)

This research was funded by the Department of Obstetrics and Gynaecology, Erasmus MC, University Medical Center, Rotterdam, The Netherlands, and the ZonMw grant Open Competition 2018 (09120011910046). The authors declare that they have no conflict of interest.

TRIAL REGISTRATION NUMBER

NTR4356.

maternal supplement intake
multivitamins
embryo
pregnancy
birth weight
Department of Obstetrics and Gynecology, Erasmus MC, University Medical Center 09120011910046
==== Body
pmcIntroduction

During pregnancy, the maternal nutrient metabolism is affected by a series of continuous physiological adaptations, while simultaneously energy and nutritional demands increase (King, 2000). In the periconceptional period, 14 weeks before until 10 weeks after conception, vitamins B6, B9 (folate), and B12 are required for the biosynthesis of nucleotides and DNA as part of the one-carbon metabolism (1-CM). Deficiencies in 1-CM nutrients disturb cell division, multiplication, and differentiation and affect gametogenesis, fertilization, and embryonic development before and after implantation (Steegers-Theunissen et al., 2013; Parisi et al., 2017). In addition to an increased need for these vitamins in early pregnancy, micronutrients, such as vitamin A and E, copper, iron, and zinc (Gernand et al., 2016) are also required. At the end of the first trimester, neurological development, tissue deposition, and body composition require also vitamin D and iodine (Fig. 1) (Gernand et al., 2016).

Figure 1. Required multiple micronutrients in the periconceptional period and in prenatal development. (Information from Steegers-Theunissen et al., 2013; Gernand et al., 2016.)

To increase awareness of the need for these essential biological processes, the World Health Organization (WHO) guideline on antenatal care recommends a daily healthy diet and use of oral supplementation of 30–60 mg elemental iron and 400 µg folic acid (FA) (World Health Organization, 2016). However, no general guidelines or recommendations for the use of multiple-micronutrient supplements (MMS) during the periconceptional period have been developed so far. Given the high prevalence of micronutrient deficiencies in many developing and developed countries (Blumfield et al., 2013; Mannar et al., 2020), the current general nutrient intake appears to be inadequate (Parisi et al., 2014). A potential explanation is the increasing widespread adherence to a Western dietary pattern, which is characterized by the ‘double burden’ of malnutrition: a low intake of micronutrients and a high intake of ‘empty’ calories and macronutrients (Bouwland-Both et al., 2013; Huijgen et al., 2014).

Adequate periconceptional FA supplement use in the general population is associated with increased embryonic and fetal growth and reduced risks of neural tube defects, congenital malformations, and neonates born small for gestational age (SGA) or with a low birth weight (LBW) (Timmermans et al., 2009; van Uitert et al., 2013). Despite the fact that micronutrients are known to be equally important before conception, to support gametogenesis and optimize the maternal environment, previous studies regarding MMS have ignored the periconceptional period (Steegers-Theunissen et al., 2013; Gernand et al., 2016). Studies mainly target MMS intake initiated from the first trimester onwards and investigate the associations with late fetal growth and birth outcomes (Steegers-Theunissen and Steegers, 2003). A recent Cochrane review demonstrated that MMS intake during pregnancy reduces the occurrence of neonates born with LBW and/or SGA (Keats et al., 2019).

To address the knowledge gap, we aim to investigate whether periconceptional intake of MMS in addition to FA impacts embryonic growth and development, rate of miscarriages, and subsequently birth outcomes. If advantages in terms of early growth and subsequent outcomes are shown, periconceptional intake of MMS might be considered as a general recommendation to all women who are trying to conceive.

Materials and methods

Study population and design

The selected study population participated in the Rotterdam Periconceptional Cohort (Predict study) between January 2010 and December 2020. This is an ongoing prospective tertiary hospital-based cohort, embedded within the obstetric outpatient clinic of the Department of Obstetrics and Gynaecology, Erasmus MC, University Medical Center, Rotterdam, The Netherlands. The recruitment of the participants is described in the Cohort Profile papers (Steegers-Theunissen et al., 2016; Rousian et al., 2021). Eligible participants were at less than 13 weeks gestation of a singleton pregnancy and provided written informed consent prior to participation. Ethical approval for this study was obtained from the Medical Ethical and Institutional Review Board of the Erasmus MC, Rotterdam, The Netherlands (MEC-2004-277). A total of 2051 pregnancies were initially included in the Predict study between 2010 and 2020; a flowchart of the study population selection for analysis is shown in Fig. 2.

Figure 2. Flowchart of study inclusions and exclusions.

Data collection

General characteristics

Data on maternal characteristics were extracted from the self-reported questionnaires and included: age, tobacco, and alcohol use between 4 weeks prior to and 8 weeks after conception, geographical origin categorized as Western or non-Western according to the classification of the Dutch Central Bureau of Statistics (CBS) (Alders, 2001), educational level classified as low, intermediate, or high according to the CBS (Statistics CBo, 2021), parity categorized as nulliparous or multiparous, and mode of conception categorized in natural conception and in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI). At the first study visit, anthropometrics were measured, including the body mass index (BMI). Venous blood was withdrawn at study enrolment (<10 weeks of gestation), from women included up until December 2016. The Elecsys Folate III with a Cobas 8000 system, by Cobas® Roche Diagnostics, was used for in vitro quantitative determination of human serum folate. Extensive protocols can be assessed online at www.diagnostics.roche.com (assessed 9 October 2023).

Supplement use

MMS and FA use were extracted from the questionnaires. Based on this, participants were assigned to one of the following groups of supplement use: MMS which includes FA, and FA use only. It was specified whether they started prior to or after conception (<10 weeks gestation). For the FA group, the dosage of 0.4–0.5 or 5 mg/per day was specified. Information on which specific brand or content MMS was used is available, however, due to heterogeneity of the content and dose of the micronutrients, it was not possible to further analyze this information with regard to our specific research question.

Embryonic outcomes

Embryonic growth was longitudinally assessed using the crown-rump length (CRL) and embryonic volume (EV), while embryonic morphology development was assessed using the Carnegie stages. These measurements were performed using three-dimensional (3D) ultrasound data, which were collected by trained medical doctors. The 3D ultrasound examinations were performed longitudinally from enrolment until the end of the first trimester, with a maximum of six ultrasounds per pregnancy. The 3D ultrasound examinations were acquired using a 6–12-MHz transvaginal ultrasound probe of the Voluson E8 or E10 system, and the acquired 3D data were visualized and analyzed as 3D holograms using virtual reality (VR) systems and V-scope software, developed in the Erasmus MC (Baken et al., 2015). CRL and EV measurements were performed by trained researchers using the VR application, enabling depth perception, examination in all dimensions, and semi-automated analyses of volumetric measurements. An extensive description of the 3D ultrasound scans and the VR application can be found in the papers by Rousian et al. (2010, 2021).

The Carnegie classification consists of 23 stages, representing the morphological development of the embryo in the first 8 weeks after fertilization (O’Rahilly and Muller, 2010). For the embryonic staging, the external (limb development and curvature of the developing embryo) and internal (brain ventricle development) morphological criteria were primarily used (Rousian et al., 2013). Miscarriages included early miscarriage (intrauterine pregnancy loss <10 weeks’ size on ultrasound) and fetal miscarriage (pregnancy loss ≥10 weeks’ size with a fetus (≥33 mm) and <16 + 0 weeks of gestation on ultrasound) (Kolte et al., 2015).

In naturally conceived pregnancies with regular menstrual cycles, the gestational age (GA) was calculated based on the first day of the last menstrual period (LMP). The GA of IVF/ICSI pregnancies was calculated based on the date of oocyte retrieval. In this study, CRL was not used to calculate the GA, because it is one of the major outcome measures. Regular menstrual cycles were defined as between 21 and 35 days. For menstrual cycles with a fixed length between 21–24 or 32–35 days, the GA was adjusted for the length of the cycle. GA was considered unreliable for irregular cycles, for absent LMP information, or when the calculated GA deviated by more than 7 days from the expected GA based on the CRL.

Birth weight outcomes

Birth weights were extracted from medical birth records. Birth weight classification was based on the curves of Hoftiezer et al. (2019), which represent the optimal weight of children born at the given GA, where a distinction is made for fetal sex. The Hoftiezer curves are based on data from the Perinatal Registry of Dutch newborns between 2000 and 2014, of which all newborns with risk factors for abnormal fetal growth were excluded (Hoftiezer et al., 2019). A neonate born with a birth weight below the 10th Hoftiezer percentile was defined as SGA, where we distinguish for neonates born below the 3rd percentile and those above the 95th percentile were defined as large for gestational age (LGA).

Statistical analysis

Baseline characteristics are shown for the total population and stratified according to the group of supplement intake (Table 1). For continuous variables with parametric data, Student’s t-test was used, and non-parametric data were assessed with the Mann–Whitney U-test. Dichotomous categorical variables were assessed by Fisher’s exact test and polychotomous variables were assessed by the Chi-square test.

Table 1. Baseline characteristics of the total population and stratified according to groups of supplement use.

	Total study population n = 1076	Multiple-micronutrients n = 730	Folic acid n = 346	P-value	
Age (years), mean [±SD]	32.5 [± 4.4]	32.8 [± 4.3]	31.8 [± 4.6]	0.001	
BMI (kg/m2), median [IQR]	24.2 [22.0–27.7]	23.9 [21.8–27.0]	25.1 [22.3–28.6]	0.001	
 Missing	29	17	12	
Parity, n (%)				0.118	
 Nulliparous	547 (54.8)	382 (56.5)	165 (51.2)	
 Multiparous	451 (45.2)	294 (43.5)	157 (48.8)	
 Missing	78	54	24	
Mode of conception, n (%)				0.628	
 Natural	570 (53.0)	383 (52.5)	187 (54.0)	
 IVF/ICSI	506 (47.0)	347 (47.5)	159 (46.0)	
Geographical origin, n (%)				<0.001	
 Western	937 (87.2)	659 (90.3)	278 (80.8)	
 Non-western	137 (12.8)	71 (9.7)	66 (19.2)	
 Missing	2	0	2	
Educational level, n (%)				0.005	
 Low	76 (7.1)	43 (5.9)	33 (9.5)	
 Middle	356 (33.1)	227 (31.2)	129 (37.3)	
 High	642 (59.8)	458 (62.9)	184 (53.2)	
 Missing	2	2	0	
Smoking, n (%)				0.219	
 Yes	145 (13.5)	92 (12.6)	53 (15.4)	
 No	929 (86.5)	637 (87.4)	292 (84.6)	
 Missing	2	1	1	
Alcohol use, n (%)				0.155	
 Yes	317 (29.5)	225 (30.9)	92 (26.7)	
 No	756 (70.5)	503 (69.1)	253 (73.3)	
 Missing	3	2	1	
Serum folate nmol/l, mean [±SD]	45.6 [±1.1]	46.2 [±1.1]	44.3 [±2.7]	0.219	
 Missing	460	302	158	
Dose FA use, n (%)				<0.001	
 0.4–0.5 mg/day	505 (47.1)	233 (32.0)	272 (78.8)	
 5 mg/day	24 (2.2)	16 (2.2)	8 (2.4)	
 Unknown	544 (50.7)	479 (65.8)	65 (18.8)	
 Missing	3	2	1	
Timing FA use, n (%)				0.001	
 Preconceptional	908 (84.8)	634 (87.2)	274 (79.7)	
 During pregnancy	163 (15.2)	93 (12.8)	70 (20.3)	
 Missing	5	3	2	
Timing MMS use, n (%)			NA	NA	
 Preconceptional	287 (60.4)	276 (63.4)	
 During pregnancy	188 (39.6)	159 (36.6)	
 No use	259	NA	
 Missing	295	295	
Hoftiezer percentile, median [IQR]	0.47 (0.23–0.74)	0.48 (0.23–0.74)	0.45 (0.23–0.75)	0.252	
Missing	66	45	21	
MMS, multiple micronutrient supplements; FA, folic acid; BMI, body mass index; IVF, in vitro fertilization; ICSI, intracytoplasmic sperm injection.

The associations between supplement use and embryonic growth (CRL and EV trajectories) were assessed by linear mixed-effect modelling. To approach linearity in association with GA, the square root of the CRL measurements and the cube root of the EV measurements were used. For embryonic morphologic development (Carnegie stages), a generalized continuation-ratio model was estimated. The selection of confounders used in the models is based on the correlation of the covariates of our study population and literature. Model 1 is only adjusted for GA. Model 2 is additionally adjusted for maternal age, BMI, tobacco use, geographical origin, educational level, mode of conception, parity, and fetal sex.

Odds ratios were calculated for the birth weight outcomes conforming to the Hoftiezer percentiles SGA (<p3 and <p10) and LGA (>p95), using binary logistic regression (Hoftiezer et al., 2019) and were adjusted for maternal age, BMI, tobacco use and parity. A mediation analysis was performed, to investigate the contribution of first-trimester embryonic growth (CRL and EV) in the association between supplement intake and neonates born SGA. Direct and total effects were estimated, and the indirect effect was determined using the product method, multiplying the two indirect effects. To calculate the 95% confidence intervals, bootstrapping with 5000 replicates was used (VanderWeele, 2016). Additionally, a sub-analysis was conducted by stratifying the groups of supplement intake based on whether the pregnancy ended in SGA (<p3), and examined whether the first-trimester growth (CRL and EV) differed between these groups (Richiardi et al., 2013). Lastly, the association between MMS intake and the occurrence of a miscarriage compared to FA use was tested by binary logistic regression, where ORs were calculated. Selected confounders are risk factors for miscarriage and include maternal age, BMI, geographical origin, mode of conception, smoking, and alcohol use (Wang et al., 2004; Quenby et al., 2021).

All analyses were performed using SPSS (version 28.1) and R-studio (version 4.1.2). P-values <0.05 were considered statistically significant.

Results

Baseline characteristics

We included 1076 pregnancies for analysis: 730 (67.8%) women used MMS, whereas 346 (32.2%) women used only FA (Fig. 2). Table 1 shows the baseline characteristics of the included participants and stratification according to the two supplement use groups.

At study enrolment, the mean maternal age was 32.5 (±4.4) years, with a median BMI of 24.2 kg/m2 (22.0–27.7). Almost half of the pregnancies were conceived by IVF/ICSI (46.0%) whereas 54.0% were naturally conceived. More than half of the women were primiparous (54.8%). The majority of the participants were of Western geographic origin (87.2%), highly educated (59.8%), and did not use any tobacco (86.5%) or alcohol (70.5%) in the periconceptional period. FA was taken preconceptionally in 84.8% of the participants, with a median initiation date of 126 [IQR 56–365] days prior to conception, whereas 15.2% initiated FA at a median of 28 [IQR 21–35] days of GA. The periconceptional mean serum folate level was 45.6 (±1.1) nmol/l.

Differences in baseline characteristics were seen after stratification; participants using MMS were significantly older, had a lower BMI, were more often of Western geographical origin, and were more highly educated compared to participants using FA only. Furthermore, participants using MMS started more often preconceptionally (87.2%) compared to participants using FA (79.7%). No differences were seen in parity, mode of conception, smoking, alcohol use, serum folate levels, and birth weight (Hoftiezer percentiles) after stratification for the group of supplement use (Table 1).

Supplement intake and embryonic outcomes

In Table 2, the positive associations between supplement intake and embryonic growth, estimated by the trajectories of CRL and EV, are shown. Women using MMS showed significantly larger embryos, based on the parameters √CRL (adjusted model: β = 0.052, 95% CI 0.012–0.090, P = 0.012) and ∛EV (adjusted model: β = 0.022, 95% CI 0.002–0.042, P = 0.031), compared to women using FA. In the MMS group, there was a nearly 2-fold increased chance of entering the following Carnegie stage earlier compared to the group using only FA, albeit the difference was not significant (adjusted model: OR = 1.768, 95% CI 0.746–4.19, P = 0.195) (Table 2). In Fig. 3, we visualized the differences in embryonic growth for CRL (A) and EV (B) at given GAs in effect estimate plots. The difference in embryonic growth (both CRL and EV) between the groups of supplement use was larger in very early pregnancy, around 7 weeks of GA, compared to 11 weeks of GA (CRL and EV respectively, 3.8% vs. 1.6% and 25.0% vs. 3.5%). Additionally, a sub-analysis was conducted for the occurrence of miscarriage in the study population before the exclusion of women with irregular menstrual cycles and unavailable first-trimester ultrasounds (n = 1687). There were 86 (7.5%) women using MMS who had a miscarriage, compared to 31 (6.3%) women using FA. The risk of miscarriage did not differ between the groups of supplement intake (OR = 1.070, 95% CI 0.682–1.677, P = 0.768).

Figure 3. Effect plots of differences in embryonic outcomes. The effects of supplement use are shown for crown-rump length (A), the embryonic volume (B), and after stratification of the population for SGA/not SGA (C/D). Reported calculated percentages (in A and B) are the differences in estimated CRL or EV between the supplement groups at 7  and 11 weeks of gestational age. CRL, crown-rump length; EV, embryonic volume; FA, folic acid; MMS, multiple micronutrient supplements; SGA, small for gestational age.

Table 2. Associations between multiple-micronutrient supplement use and embryonic growth and development compared to folic acid use.

Outcome	Model 1	Model 2	
	β (95% CI)	P-Value	n	β (95% CI)	P-Value	n	
√CRL (mm)							
MMS	0.050 (0.013, 0.087)	0.009	1075	0.052 (0.012, 0.090)	0.012	970	
∛ EV (cm3)							
MMS	0.022 (0.023, 0.040)	0.023	1046	0.022 (0.002, 0.042)	0.031	944	
	OR (95% CI)	P-Value	n	OR (95% CI)	P-Value	n	
Carnegie stages							
MMS	2.015 (0.865, 4.694)	0.105	645	1.768 (0.746, 4.190)	0.195	600	
Model 1 is adjusted for GA. Model 2 is adjusted for GA, maternal age and BMI, mode of conception, maternal smoking status, geographical origin, educational level, parity, and fetal sex.

GA, gestational age; BMI, body mass index, CRL, crown-rump length, EV, embryonic volume, FA, folic acid; MMS, multiple micronutrient supplements; OR, odds ratio.

Supplement intake and birth weight

No significant differences were observed for the outcomes SGA <p10 nor LGA in the women using MMS compared to FA. However, lower percentages of neonates born SGA (<p3) were observed (respectively, 4.2% vs. 7.4%), with a 45% risk reduction of neonates born SGA (<p3) in women using MMS compared to those using only FA (OR = 0.546, 95% CI 0.308–0.969) (Table 3). The mediation analysis showed that first-trimester embryonic growth, partially mediated the association between maternal supplement intake and neonates born SGA (<p3). CRL explained 22.4% of the total effect of the association between supplemental intake and SGA, whereas EV explained 17.2% (Total effect: β = −0.558, 95% CI −1.154 to −0.962, indirect effect of CRL: β = −0.125, 95% CI −0.220 to −0.016 and EV: β = −0.096, 95% CI −0.220 to −0.004), see Supplementary Table S1. An additional sub-analysis in the population stratified for supplement use and the outcome SGA (<p3) showed that women using MMS had larger embryos (CRL and EV) compared to women using FA alone, whether or not the pregnancy ended in a neonate born SGA (Fig. 3C and D).

Table 3. Birth outcomes according to the group of supplement use: small for gestational age (<p3 and <p10) and large for gestational age (>p95).

	SGA <p3	SGA <p10	LGA >p95	
	Yes	No	Adj. OR (95% CI)	Yes	No	Adj. OR (95% CI)	Yes	No	Adj. OR (95% CI)	
MMS	29 (4.2)	656 (95.8)	0.546 (0.308, 0.969)	75 (10.9)	610 (89.1)	0.731 (0.486, 1.099)	30 (4.4)	655 (95.6)	0.759 (0.410, 1.405)	
FA	24 (7.4)	301 (92.6)	46 (14.2)	279 (85.5)	20 (6.2)	305 (93.8)	
SGA, small for gestational age; LGA, large for gestational age; FA, folic acid; MMS, multiple micronutrient supplements. Odds ratios are adjusted for maternal age, BMI, tobacco use, and parity.

Discussion

Summary of findings

This study shows that the periconceptional intake of MMS compared with FA supplement intake is associated with increased embryonic growth trajectories (both CRL and EV), and a 45% reduced risk of neonates born SGA (<p3). The association between maternal supplement intake and SGA was partially mediated by first-trimester embryonic growth; CRL explained 22.4% and EV explained 17.2% of the total effect. No associations were found with morphological embryonic development, miscarriage rate, SGA (<p10) or LGA.

Interpretation and comparison with other studies

The majority of previous research regarding maternal supplement intake has mainly focused on second and third-trimester fetal growth (restriction), LBW and SGA (Keats et al., 2019). However, following Barker’s hypothesis, intra-uterine conditions can already have an impact on early fetal development, with subsequent risks for offspring also later in life (Barker et al., 2002). We translated this hypothesis to the periconception period and investigated the associations between MMS, FA, and embryonic growth and development to provide better insight into the timing of its initiation, because of the first-trimester onset of the investigated pregnancy complications.

Previous research confirmed that CRL is associated with fetal growth parameters, including estimated fetal weight (EFW) and birth weight (van Uitert et al., 2013). The study of Timmermans et al. (2009) showed in data from 6353 pregnancies that periconceptional FA supplementation was positively associated with late pregnancy fetal growth (SD 0.10, 95% CI 0.02–0.19), birth weight (SD 0.16, 95% CI 0.09–0.23) and reduced risks of LBW (57%) and SGA (60%) compared to those who did not use FA. However, only late fetal growth was investigated in this study. Importantly, our study showed that already first-trimester growth was different in pregnancies ending in a neonate born SGA (<p3) compared to neonates with a normal birth weight. Two studies investigated maternal risk factors in relation to first-trimester growth and birth weight outcomes (Mook-Kanamori et al., 2010; van Uitert et al., 2013). Mook-Kanamori et al. (2010) showed shorter CRL in women (n = 1631) who did not use FA supplements and smoked compared to mothers who did not smoke and had optimal FA supplement use (difference: −3.84 mm, 95% CI −5.71 to −1.98). The study of van Uitert et al. (2013) found no associations between the periconceptional intake of FA and CRL in the first trimester, which possibly could be explained by the low number of included pregnancies (n = 87), and a homogenous population with a relatively high percentage of FA supplement use (72.1%). Unfortunately, none of these studies investigated the use of MMS.

The significantly lower risk for SGA (<p3) with MMS use is consistent with previously published literature. The recent Cochrane review of Keats et al. (2019) included 17 randomized controlled trials (RCTs) investigating MMS with iron and FA versus iron supplementation with or without FA and found a relative risk of 0.92 (95% CI 0.88–0.97) for a neonate born SGA. Interestingly, only one of the 17 included RCTs found a significant difference in the incidence of LBW (<2500 g), while almost all relative risks were found to be below one (Fawzi et al., 2007). The results combined emphasize the need for studies with larger sample sizes.

Strengths and limitations

The most important strength of this study is the combination of study size and extensive first-trimester longitudinal data collection, providing information on fetal growth trajectories and corresponding maternal characteristics. These characteristics provide the opportunity to adjust for potential confounders, which is important to approximate the estimates of the associations between supplement intake and embryonic growth, development, and birth outcomes.

Our study is the first to investigate the use of MMS in a high-income Western population, whereas almost all previous research on MMS was conducted in non-Western or low- and middle-income countries because undernourishment is a specific frequent problem in these countries (Black et al., 2013; Keats et al., 2019). In contrast, women using MMS in this study are 90.2% of Western origin and highly educated (63.2%). While there is no general recommendation in the Netherlands regarding MMS use during pregnancy since Dutch women are assumed to have a sufficient dietary intake of all the necessary vitamins and minerals, a remarkably high percentage (68%) of women used MMS during pregnancy (Bakker et al., 2021).

A main limitation to consider is the difference in baseline characteristics between the two groups of supplement intake; women using MMS were older, had a lower BMI, were more often highly educated, and more often started their FA preconceptionally. These findings may suggest that the population using MMS overall has a better preconceptional nutritional status, and are ‘healthier’, compared to the population using only FA. Therefore, we corrected for the following in our analyses (model 2): maternal age, BMI, geographical origin, and educational level (in addition to GA, mode of conception, smoking status, parity, and fetal sex). However, inherent to the observational cohort study design (observational cohort study), it cannot be fully excluded that our results are biased by residual confounding from an overall ‘more healthy’ population using MMS. A further limitation to address is the heterogeneity of the MMS used. As a result, it remains unclear which specific nutrient or combination may be responsible for the larger embryonic growth and reduction in dysmaturity. It can be argued that women do not consume (micro)nutrients and minerals separately, rather than in a combined preparation, which raises the question of whether it might be harmful to use MMS in addition to a diet high in micronutrients. Overall, limited information is available on pregnancy-specific risks from excessive multiple-micronutrient intake. Commercial supplements, especially aimed for pregnancy, are subject to strict safety requirements, and the micronutrients are added in doses that should not exceed the upper intake level (UL). Moreover, the prevalence of micronutrient deficiencies continues to overshadow any concern about excessive intake (Gernand et al., 2016; Gernand, 2019). Since little is known about the risks and effects of micronutrient intake above the UL in pregnancy, and data regarding specific types and doses of supplements were known for part of our population, we determined the proportion of our population who exceeded the UL by supplemental intake. Based on the literature, we selected and tested vitamins A, B3, B6, C, D, E, FA, selenium, and zinc, see Supplementary Table S2 (Gernand, 2019). We observed that few women exceeded the UL based on their supplemental intake, which however restricted us from performing analyses in relation to growth, pregnancy, or birth outcomes.

Furthermore, based on our dataset, no distinction could be made between the use of an additional FA to the MMS or the FA being a component of the MMS. Since most MMS also contain FA, we cannot exclude that people are taking double doses of FA. However, at baseline, serum folate was measured, and no difference was shown between the supplement groups. Therefore, it is more likely that the larger embryonic growth and lower percentages of dysmaturity are due to the effects of MMS rather than a possible double dose of FA. Ideally, adjustments for total dietary intake, in terms of specific multiple micronutrients, would be made as well. This further approximates reality and provides insight into the extent to which MMS serves as a supplement or replacement for dietary intake. Furthermore, investigating women using only MMS or no supplements at all would have been preferable. Unfortunately, in our population, we had to exclude these two groups due to small sample sizes.

Implications and recommendations for future research

Developing appropriate guidelines for vitamin and mineral consumption in the periconceptional period is challenging. Plasma volume expansion and other pregnancy-related modifications will have an impact on the micronutrient concentrations, which are frequently used to assess the physiological demands of an individual. As a result, pregnancy recommendations are frequently extrapolated from estimates of adult intake requirements and then modified to take into account fetal nutrient accumulation, increased maternal demands to support tissue accretion and metabolism, and changes in nutrient absorption efficiency that occur during pregnancy (Gernand et al., 2016). To implement dietary recommendations and determine the risk of deficiencies in individuals and across groups, micronutrient intake guidelines for pregnancy will need pregnancy-specific data and an improved unified interpretation. Therefore, it would be beneficial to conduct an RCT comparing the use of MMS with solely FA, while accounting for vascular and metabolic pregnancy-related adaptations. Each woman’s specific vitamin status prior to conception and during each trimester should be monitored, as should the maternal dietary intake of nutrients throughout pregnancy, to determine whether MMS serves as a supplement or a replacement. This will enable the development of patient-tailored, specialized supplement recommendations that consider dietary intake as a part of lifestyle care.

Conclusion

The results of this study showed that women with periconceptional MMS have increased embryonic growth trajectories and a 45% risk reduction in SGA (<p3), compared to women using only FA. This association with SGA is partially mediated by the effect of the first-trimester growth. Our findings highlight the importance of supplement use in the periconceptional period and its impact on prenatal growth and offspring outcomes. Due to an apparent insufficient dietary intake in general, our study is the first to investigate the use of MMS in a high-income Western population, whereas almost all previous research on MMS has been conducted in non-Western or low- and middle-income countries, due to undernourishment being a specific frequent problem in these countries; especially in high-income nations, we want to emphasize the use of supplements during pregnancy. Given this, it is highly worthwhile to consider advising women to take MMS to support sufficient embryonic and prenatal growth and development. Nevertheless, it should be kept in mind that the risks of exceeding UL in pregnancy have to be monitored and therefore caution in terms of doses is required.

Supplementary Material

deae168_Supplementary_Table_S1

deae168_Supplementary_Table_S2

Acknowledgements

We would like to thank all the women who participated in the Rotterdam Periconceptional cohort (Predict study). In addition, we thank the entire Predict research team for the recruitment of participants, collecting and handling data, and performing 3D ultrasounds. Lastly, we acknowledge the financial support of the Department of Obstetrics and Gynaecology in conducting the Predict study, and the ZonMw foundation for financially supporting this study.

Data availability

The data underlying this article will be shared upon reasonable request to the corresponding author.

Authors’ roles

Initiation of the study: R.P.M.S.-T.; further conceptualization: N.S., S.S., M.R., and R.P.M.S.-T.; data curation: N.S.; writing of the first draft: N.S. and S.S.; writing review and editing: S.S., M.R., S.P.W., M.M.F., and R.P.M.S.-T.; supervision: S.S. and R.P.M.S.-T.; project administration: M.M.F. and R.P.M.S.-T. All authors have read and agreed to the submitted version of the manuscript.

Funding

Department of Obstetrics and Gynecology, Erasmus MC, University Medical Center, Rotterdam, The Netherlands; the ZonMw grant Open Competition 2018 (09120011910046).

Conflict of interest

The authors declare that they have no conflict of interest.
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