The one-carbon metabolism as an underlying pathway for placental DNA methylation – a systematic review

Figures & data

Figure 1. Simplified overview of the one-carbon metabolism.

Dietary methionine is converted to homocysteine via SAM and SAH. SAM is a major methyl donor for biological processes, including DNA methylation. The remethylation of homocysteine to methionine depends on other moieties including vitamin B12 as cofactor and 5 methyl tetrahydrofolate as substrate. MTHFR = methylenetetrahydrofolate reductase, BHMT = betaine-homocysteine S-methyltransferase, MS = methionine synthase, SAM = s-adenosylmethionine, SAH = s-adenosylhomocysteine

Figure 2. The one-carbon metabolism as an underlying pathway affecting placental DNA methylation and lifelong health outcomes of the offspring.

Maternal exposures such as nutrition and the use of vitamin supplements, are involved in the one-carbon metabolism, which provides methyl groups for DNA methylation processes (left). DNA methyltransferases (DNMTs) transfer methyl groups to the cytosine-phosphate-guanine sides (CpGs). Derangement of the one-carbon metabolism are proposed mechanisms affecting DNA methylation processes (bottom). The placenta is the intermediate between the maternal environment and embryonic and foetal growth and development. Epigenetic derangements can affect (1) directly the functioning of the placenta and subsequently foetal growth and development, and (2) the foetal epigenome with potential consequences for foetal growth and development, and 1 and 2 both have consequences for lifelong health outcomes in the offspring.

Figure 3. Flowchart of included and excluded articles. 1 CM = one-carbon moieties.

Table 1. Study characteristics and main findings of included studies ordered according to the quality score.

Author (year) – Country	Study design	Population and sample size	Exposure and timing	DNA methylation technique	Placental tissue	Main outcomes	Quality Score
Devi (2017) – India [30]	Randomized controlled trial	Healthy pregnant women with low serum vitamin B12 (<200 pmol/L) in first trimester (n = 66)	Group 1: 500 mL milk +10 mg vitamin B12/d (n = 23). Group 2: 500 mL milk + placebo (n = 20). Group 3: placebo (n = 23). Late first trimester till delivery.	Global methylation: LINE-1 methylation – MethyLight Gene specific: VEGF promoter — MS-HRM	Foetal side	Use of milk with or without vitamin B12 did not change VEGF promoter or LINE-1 methylation.	8
Dou (2022) – USA [31]	(Subgroup of) 2 prospective cohorts	Couples at increased risk for offspring with ASD. N = 70 (MARBLES cohort) + N = 88 (EARLI cohort)	Maternal multivitamin use in first month of pregnancy	Global methylation: Mean array methylation – MARBLES: 450K array EARLI: EPIC array Genome wide: individual CpGs – MARBLES: 450K array, EARLI: EPIC array	Foetal side (MARBLES) Central full thickness biopsy (EARLI)	Negative association between mean array methylation and vitamin use: −0.60% (CI −1.08, − 0.13; MARBLES); −0.52% (CI −1.04, 0.01; EARLI). No associations with individual CpGs after FDR correction. Using a less stringent threshold (of p<0.01), 9216 (MARBLES) and 3442 (EARLI) differentially methylated CpGs of which ±95% were hypomethylated in women using vitamins (average −4%). Identified CpGs were enriched in neuronal developmental pathways.	8
Lecorguille (2022) — France [32]	Prospective cohort	Healthy pregnant women (n=573)	1. Dietary intake in year before pregnancy reflecting differences in one-carbon moieties 2. Dietary supplements 3 months before and during pregnancy	Global methylation: LINE-1 and Alu repetitive elements methylation. —Pyrosequencing Genome wide: DMRs and individual CpGs —450K array	Foetal side, central	No associations between dietary patterns and individual CpGs or global methylation. ‘Varied and balanced’ diet associated with a DMR related to NPDC1 (p=0.02). ‘Vegetarian tendency’ associated negatively with 2 DMRs related to DLL1 (p=5.3.10^−7, p=7.0.10^−9) and FAR1 (p=3.7.10^−9, p=7.8.10^−3). ‘Bread and starchy food’ associated negatively with AS3MT (p=0.02) and GSDMD (p=0.004) and positively with SLC25A46 (p=0.01) and ZNF175 (p =0.006) DMRs. Vitamin use associated with Alu repetitive elements methylation (β=0.40, p=0.005) compared to no vitamins. No associations for vitamin use only before or during pregnancy or with LINE-1 methylation.	7
Castillo-Castrejon (2021) — Guatemale & Pakistan [33]	Randomized controlled trial	Preconceptional women. Q1 and Q4 based on newborns’ birth length (n=24 in both Guatemale and Pakistan, n=48)	Group 1: Daily SQLNS ≥3 months before conception until delivery Group 2: No supplement use.	Gene specific: P2 promoter region of IGF1 —Pyrosequencing	Four representative biopsies	SQLNS use did not change IGF1 promoter methylation.	6
Jiang (2012) – USA [34]	Randomized controlled trial	Healthy women in third trimester (n=24)	Group 1: 480mg choline/d (n=12). Group 2: 930mg choline/d (n=12). 12 weeks trial, supplements till delivery	Global DNA methylation —LC-MS/MS Gene specific: CRH, GNAS-AS1, LEP, IL10 promoters; the 5= untranslated exon 1F (and flanking regions) of NR3C1, DMR0 of IGF2 —Base-specific cleavage and MS	Full-thickness biopsies from centre of each quadrant	Higher choline intake (930mg/d) increased global DNA methylation (4.4 ±0.2% VS 3.6 ± 0.2%, p=0.02). Higher choline intake (930mg/d) increased methylation of CRH (p=0.05) and NR3C1 (p=0.002). Higher methylation was found for 4/15 NR3C1 CpGs and no individual CRH CpG reached significance. Higher choline intake (930mg/d) decreased methylation of 1/11 GNAS-AS1 CpG (p=0.02), average GNAS-AS1 methylation was not changed. Higher choline intake did not change methylation of DMR0 of IGF2 or promoter regions of IL10 or LEP.	6
Liu (2019) — China [35]	Case-controls from prospective cohort	34 SGA cases, 62 AGA controls	Preconceptional folic acid supplement use	Gene specific: 2 ANKRD20B CpGs, 9 FAM198A CpGs; both were top ranked variable positions between 3 SGA and 3 AGA (450K array) – Pyrosequencing	Foetal side, central	In male offspring, lower methylation of 5/9 sites within FAM198A associated with preconceptional folic acid supplement use (p=0.047, p=0.050, p=0.039, p=0.026, p=0.043). No associations in female offspring or for ANKRD20B.	6
Loke (2013) – Australia [36]	(Subgroup of) prospective twin cohort	Monozygotic (n=34) and dizygotic (n=33) twin pairs	1. Folic acid supplements <12 weeks of gestation 2. Serum vitamin B12 + homocysteine at 28 weeks of gestation	Gene specific: 4 IGF2/H19 DMRs —EpiTYPER MassARRAY	Unclear	Folic acid supplement use associated with lower methylation (−4%) of H19 promoter DMR. No associations between folic acid supplement use and methylation of IGF2/H19 ICR, DMR0 and DMR2 of IGF2. No associations between vitamin B12 or homocysteine and methylation of 4 IGF2/H19 DMRs.	6
Nakanishi (2021) – Japan [37]	Prospective cohort	Healthy pregnant women (n=124)	Maternal plasma choline levels in first trimester, third trimester and at term, choline levels in cord blood.	Global methylation: LINE-1 methylation – Pyrosequencing Gene specific: CpGs sites in MEST, MEG, H19, PPARA, PPARG, ADIPOQ, LEP, NADH, NDUFb6, NR3C1, RXRA, HSD11β2 —Pyrosequencing	Full-thickness biopsy, central	Negative associations between maternal choline and PPARG1A, NR3C1, HSD11β2, PPARA and RXRA methylation at all three time points (range: r=−0.188 — r=−0.452) and between cord blood choline and NR3C1 (r=−0.20, p=0.033) and PPARA (r=0.223, p=0.017) methylation. Maternal choline in 1^st trimester associated negatively with AdipoQ methylation (r=−0.307, p=0.001) and 3^th trimester choline associated negatively with AdipoQ (r=−0.201, p=0.030) and NDUFB6 (r=−0.214, p=0.021) methylation. No associations with MEG3, H19, MEST and LEP. LINE-1 methylation associated negatively with choline in 1^st and 3^th trimester (r= −0.205, p=0.022; r=−0.207, p=0.026) and in cord blood (r=−0.248, p=0.008).	6
Schmidt (2016) – USA [38]	Case-controls from prospective cohort	Couples at increased risk for offspring with ASD. ASD (n=24) and controls (n=23).	Food folate intake during pregnancy and vitamin use in first month of pregnancy	Global methylation: predefined partially- or highly methylated domains —MethylC-seq	Foetal side, midway cord insertion and periphery	No associations between folate intake (after FDR correction) or vitamin use and methylation of predefined partially- or highly methylated domains.	6
Heil (2019) – The Netherlands [39]	Case-control study	Pregnancies with PE (early onset n=11, late onset n=11), FGR (n=20), PTB (n=15) and controls (n=25)	SAM and SAH concentrations in placental tissue	Gene specific: 1 CpG in the 5’flanking region of the PIGF gene —EpiTYPER MassARRAY	Foetal side, 4 biopsies 3cm around cord insertion, removal 2mm of top layer.	SAM and SAM:SAH ratio were positively associated with PIGF methylation (r=0.31, p=0.006; r=0.24, p=0.04, respectively).	5
Khot (2017) – India [40]	Case-control study	Term (n=69) and preterm (n=71) deliveries in healthy women	Plasma vitamin B12, folate and homocysteine just after delivery	Gene specific: MTR promoter — Pyrosequencing	Random biopsies from decidual side	Vitamin B12 associated negatively with methylation of 3/7 investigated CpGs in the MTR promoter (r =-0.208, p=0.049; r =-0.248, p=0.017; r=−0.298, p=0.004). Women delivering preterm had lower plasma folate and higher homocysteine compared to term controls. Mean MTR promoter methylation was 0.5% higher (p< 0.01) in preterm births. 5/7 studied CpGs had higher methylation in preterm placentas only after vaginal deliveries. No associations between homocysteine or folate and DNA methylation were investigated.	5
Moeini (2022) – Iran [41]	Case-control study	Term (n=25) and preterm (n=25) deliveries in healthy women	Serum folate and vitamin B12, timing unclear	Gene specific: MMP-9 promoter —Epitect Methyl-II PCR assay kit	Unclear	Folate and vitamin B12 associated with MMP-9 promoter methylation (r=0.58, p<0.001; r=0.68, p <0.001, respectively).	5
van Otterdijk (2023) – USA [42]	Subgroup of cohort study	Mother-infants dyads homozygous for the C or T allele of MTHFR C677T polymorphism (both n=45)	Red blood cell folate, vitamin B12, SAM, SAH in cord blood.	Global methylation: Average methylation of the following regions: CpG islands, shores, shelves and open sea —450K array Genome wide: individual CpGs —450K array	Foetal side, near umbilical cord	RBC folate correlated positively with methylation of CpG islands and ‘shelves’ for the total study population and in ‘open sea’ and ‘shores’ only for the TT genotype. No significant associations between individual CpGs and RBC folate after multiple testing correction. SAM:SAH ratio correlated positively with methylation of ‘open sea’ and ‘shore’ regions and with individual loci located in RAD54L2, ETS2, ZNF836 and ABAT. No significant associations between SAM or SAH and region-specific DNA methylation patterns. SAM significantly associated with 2 loci located in LOXL3 and GNL1 gene bodies and SAH with 1 CpG in ZC3H12D CpG island. No associations between vitamin B12 and regional methylation or methylation of individual loci.	5
Rahat (2022)* — India [43]	Case-control study	Uncomplicated terminations in 1^st/2^nd trimester (both n=30), term and PE cases (both n=30)	Folate levels in placental villi	Global methylation: —ELISA-based kit and LINE-1 methylation —MS-HRM	Placenta villi	No significant associations between folate levels in placental villi and global methylation.	5
Sletner (2021) – Norway [44]	(Subgroup of) prospective cohort	Healthy pregnant women, ethnic south Asian (N=40) or European (n=40)	Serum folate and vitamin B12 at enrolment (<20 weeks of gestation)	Gene specific: 13 CpGs in the Transcription start site of LEP — Pyrosequencing	Full thickness biopsy, central	Folate associated with methylation of 9/13 CpGs in a multivariate model (range: β=0.16—0.26, p=0.003—p=0.03) but not in univariate models. No associations between vitamin B12 and methylation of 13 LEP CpGs in univariate analyses, negative associations with 2/13 CpGs in multivariate models (β=-0.8, p=0.02; β=-0.02, p=0.02).	5
Ge (2015) — China [45]	Case-control study	Pregnancies with PE (n=127) and controls (n=132)	Homocysteine in maternal plasma on admission	Gene specific: MTHFR promoter — Methylation specific PCR	Central biopsy and 1 from either side of it	MTHFR methylation indices were higher in PE (26.8% VS 15.2%, p<0.05) and homocysteine was higher in PE (p<0.05). No association between homocysteine and methylation was investigated.	4
Pinunuri (2020) – Chile [46]	Case-controls from prospective cohort	Term (n=16) and preterm (gestational age 32–36 weeks, n=23) deliveries in healthy women	Folate and vitamin B12 in umbilical cord blood	Gene specific: CpG island upstream of transcription initiation site FOLR1 —MS-HRM	Chorionic and basal plates biopsies near cord insertion	Cord blood folate associated with FOLR1 methylation in the chorionic plate (Rho =0.665, p≤0.05) but not in the basal plate. There were no associations between cord blood vitamin B12 and FOLR1 methylation.	4
Tserga (2017) – USA [47]	(Subgroup of) prospective cohort	Healthy Caucasian women homozygous for C or T allele of MTHFR C677T polymorphism in mother and child (n=48)	1. Folic acid supplements started before VS during pregnancy 2. Red blood cell folate, SAM, SAH, vitamin B12 in cord blood	Gene specific: H19ICR —Pyrosequencing	Foetal side near cord insertion	No associations between preconceptional start of FA, red blood cell folate, vitamin B12 or SAM levels in cord blood and H19ICR methylation. H19ICR methylation associated with SAM:SAH ratio (R=0.38) and negatively with log(SAH) (R=−0.40) in cord blood.	4
Zhu (2019) – USA [48]	(Subgroup of) prospective cohort	Couples at increased risk for offspring with ASD. ASD cases (n=20) and controls (n=21)	Vitamin use in first month of pregnancy	Gene specific: CYP2E1 and IRS2 DMRs (2 ASD-associated DMRs) — Pyrosequencing Genome wide: DMRs —WGBS	Foetal side	Vitamin use associated with 376 DMRs. Genes were enriched for neuron fate commitment, central nervous system development, regulation of transcription and of phosphatidylinositol 3-kinase activity functions. Vitamin use associated negatively with IRS2 DMR methylation (p=0.039) and positively but not significantly with CYP2E1 (p=0.118).	4
Kulkarni (2011) – India [49]	Case-control study	Pregnancies with term PE (n=30), preterm PE (n=27) and controls (n=30)	Vitamin B12 in maternal plasma at delivery, homocysteine in maternal plasma in subset (16 controls, 28 term PE, 19 preterm PE)	Global DNA methylation —Methylamp^TM Global DNA Methylation Quantification Kit	5 different villi biopsies	Vitamin B12 was higher in preterm PE (261 ng/mL) and term PE (191 ng/mL) compared to controls (145 ng/mL) and homocysteine was also higher in preterm PE (17.59 uM) and term PE (14.43 uM) compared to controls (11.01 uM). Mean global methylation was higher in term PE (0.68%) and preterm PE (0.72%) compared to controls (0.53%), p <0.05. No association between vitamin B12 or homocysteine and DNA methylation was studied.	3
In vitro studies using human placental cell lines
Jiang (2016) – USA [50]	In vitro study	BeWo cell line	Incubation for 96 hours with 1mM choline chloride	Global methylation —ELISA-based kit	NA	Choline did not change global DNA methylation.	NA
Rahat (2017) — India [51]	In vitro study	JEG-3 and HTR-8/SVneo cell lines, first trimester cytotrophoblasts	Incubation for 48 hours with folic acid (10^−7 M and 10^−4 M)	Gene specific: DMRs of SNRPN, PEG10 and MEST — MS-HRM	NA	Folic acid decreased SNRPN methylation in JEG-3, HTR-8/SVneo and in cytotrophoblasts. At 10^−7 M, methylation decreased respectively by 1.5 (p<0.01), 1.2 (p<0.05) and 1.2 (p<0.05) and at 10^−4 by 1.8 (p<0.001), 2.2 (p<0.001) and 1.4 (p<0.01) fold. Folic acid did not change PEG10 or MEST methylation.	NA
Rahat (2022)* — India [43]	In vitro study	JEG-3 and HTR-8/SVneo cell lines	Incubation for 48 hours with folic acid (10^−7 M and 10^−4 M)	Global methylation: —ELISA-based kit and LINE-1 methylation —MS-HRM Gene specific: RASSF1A, P16, RB1, PRKCDBP, APC, c-myc, c-jun, VEGF, EGFR, hTERT, MMP2, MMP-9, TIMP1, TIMP2 —MS-HRM	NA	Folic acid incubation did not change LINE-1 methylation. Global methylation decreased in dose-dependent manner. JEG-3 cells: − 3.7% and −4.9% at 10^−7 M and 10^−4 M. HTR-8/SVneo cells: − 4.3% and −5.7% at 10^−7 M and 10^−4 M (p<0.001). In JEG-3 cells, folic acid increased methylation of APC (1.14x at 10^−7 M; 1.28x at 10^−4 M) and P16 (5x at 10^−7 M) and decreased for c-jun (1.1x at 10^−7 M). In HTR-8/SVneo cells, methylation increased for APC (4.1x at 10^−7 M) and decreased for P16 (2.4x at 10^−7 M and 3.5x at 10^−4 M), c-myc (1.8x at 10^−7 M and 2.5x at 10^−4 M) and c-jun (2.14 x at 10^−7 M and 5.5x at 10^−4 M)	NA

*Same study.

WGBS = Whole-Genome Bisulfite Sequencing, LINE-1 = Long Interspersed Nuclear Element-1, ASD = autism spectrum disorder, SGA = small for gestational age, AGA = appropriate growth for gestational age, SQLNS = small quantity lipid-based micronutrient, CpG = cytosine-phosphate-Guanine, DMR = Differentially Methylated Region, SAM = S-adenosylmethionine, SAH = S-adenosylhomocysteine, PE = preeclampsia, FGR = foetal growth restriction, PTB = preterm birth, NA = not applicable, LC-MS/MS = Liquid Chromatography with tandem mass spectrometry, MS-HRM = Methylation-sensitive high-resolution melt analysis, MS = mass spectrometry, PCR = polymerase chain reaction.

Table 2. Direct associations between one-carbon moieties and global methylation, genome-wide methylation or gene-specific methylation in included clinical studies with an ErasmusAGE quality score of at least 5 out of 10 points.

Outcome	Exposure	Methylation	Ref
Global methylation	Dietary folate	=	[38]
	Folate in placental villi	=	[43]
	Umbilical cord RBC folate	↑/=	[42]
	Vitamin B12 supplements	=	[30]
	Umbilical cord vitamin B12	=	[42]
	Choline supplements	↑	[34]
	Choline in maternal blood: first trimester/third trimester/term	↓ / ↓ / =	[37]
	Choline in cord blood	↓	[37]
	Umbilical cord SAM SAH SAM:SAH Dietary patterns: ↑B-vitamins, choline, methionine ↓betaine ↑B6, folate, betaine ↓ B12 ↑Betaine ↓B2, B6, folate	= = ↑/= = = =	[42][32]
	Vitamin use in first month of pregnancy	=	[38]
		↓/ =	[31]
	Vitamin use before and during pregnancy Vitamin use before or during pregnancy	↑ / = = / =	[32][32]
Genome-wide	Exposure	Genes linked to identified CpGs
	Umbilical cord RBC Folate	=	[42][32]
	Umbilical cord vitamin B12	=
	Umbilical cord SAM SAH SAM:SAH Dietary patterns ↑B-vitamins, choline, methionine ↓betaine ↑B6, folate, betaine ↓ B12 ↑Betaine ↓B2, B6, folate	LOXL3 GNL1 ZC3H12D RAD54L2 ETS2 ZNF836 ABAT Genes linked to identified DMRs NPDC1 DLL1 FAR1 AS3MT GSDMD SLC25A46 ZNF175
	Dietary patterns Vitamin use in first month of pregnancy	Individual CpGs = Individual CpGs = *	[31]
Gene-specific	Exposure	Gene of interest
	Start folic acid preconceptional compared to start during pregnancy	FAM198A ↓(boys)/= (girls) ANKRD20B =	[35]
	Folic acid use in first trimester	H19 promoter DMR↓ 2 IGF2 DMRs IGF2/H19ICR =	[36]
	Maternal serum folate	MMP-9 ↑	[41]
		LEP =/↑	[44]
	B12 supplements	VEGF =	[30]
	B12 in maternal blood	MMP-9 ↑	[41]
		MTR ↓	[40]
		4 IGF2/H19 DMRs =	[36]
	Homocysteine in maternal blood	4 IGF2/H19 DMRs =	[36]
	Choline supplements	CRH NR3C1 ↑ GNAS-AS1 ↓/= IGF2 IL-10 LEP =	[34]
	Choline in first trimester maternal blood	AdipoQ PPARG1A NR3C1, HSD11β2 PPARA RXRA ↓ MEG3 H19 MEST LEP NDUFB6 =	[37]
	Choline in third trimester maternal blood	AdipoQ PPARG1A NR3C1, HSD11β2 PPARA NDUFB6 RXRA ↓ MEG3 H19 MEST LEP =	[37]
	Choline in term maternal blood	PPARG1A NR3C1 HSD11β2, PPARA RXRA ↓ AdipoQ MEG3 H19 MEST LEP NDUFB6 =	[37]
	Choline in cord blood	NR3C1 PPARA ↓ AdipoQ PPARG1A HSD11β2 RXRA MEG3 H19 MEST LEP NDUFB6 =	[37]
	Placenta SAM and SAM:SAH	PIGF ↑	[39]
	Small quantity lipid-based micronutrient use	IGF1 =	[33]

* No associations with individual CpGs after FDR correction. Using a less stringent threshold of p<0.01, 9216 (MARBLES) and 3442 (EARLI) differentially methylated CpGs of which ±95% was hypomethylated in vitamin groups (average -4%) were identified. Identified CpGs were enriched in neuronal developmental pathways.

Abbreviations: DMR = differentially methylated region, CpG = Cytosine-phosphate-Guanine, RBC = Red Blood Cell, SAM = S-adenosylmethionine, SAH = S-adenosylhomocysteine, FDR = False Discovery Rate.

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Data availability statement

The authors hereby confirm that the data supporting the findings of this systematic review are available within the article.