Associations between infant sex and DNA methylation across umbilical cord blood, artery, and placenta samples

Figures & data

Table 1. PRISM participant characteristics by tissue and associations with infant sex.

	Cord blood (N = 179)			Placenta (N = 229)			Artery (N = 229)
	N	%	p, association with sex ^a	N	%	p, association with sex ^a	N	%	p, association with sex ^a	p, difference between tissues ^b,c
Maternal characteristics
Age at delivery (yrs), median (IQR)	32.0	(28.6, 34.8)	0.37	31.3	(26.6, 34.6)	0.66	31.5	(26.7, 34.7)	0.77	0.42
Education ^d
≤ High school diploma/GED	34	19%	0.33	61	27%	0.14	61	27%	0.20	0.12
> High school diploma/GED	143	81%		164	73%		164	73%
Race/ethnicity ^e
White	68	38%	0.67	75	33%	0.38	78	34%	0.85	0.67
Black	69	39%		91	40%		91	40%
Hispanic	23	13%		43	19%		39	17%
Other	19	11%		18	8%		19	9%
Infant characteristics
Sex
Male	96	54%	-	126	55%	-	124	54%	-	0.96
Female	83	46%		103	45%		105	46%
Gestational age (wks), median (IQR)	39.1	(38.2, 40.0)	0.91	39.1	(38.1, 40.0)	0.35	39.1	(38.1, 40.0)	0.64	0.81
Preterm ^f	13	7%	0.76	21	9%	0.37	19	8%	0.56	0.79
Birth weight (g), median, (IQR)	3,380	(3,073, 3,620)	0.87	3,315	(3,010, 3,580)	0.66	3,330	(3,050, 3,610)	0.39	0.67
Low birth weight ^g	11	6%	0.32	15	7%	0.50	14	6%	0.61	0.98

a. P-value for associations with infant sex; Mann–Whitney test for continuous variables; χ² test for categorical variables. b. Kruskal–Wallis Test for continuous variables; χ² test for categorical variables. c. Participants with cord blood, placenta, and artery samples; N = 159; participants with cord blood and placenta samples: N = 165; participants with cord blood and artery samples: N = 169; participants with placenta and artery samples: N = 218. d. Cord blood: N = 177; placenta: N = 225; artery: N = 225. e. Placenta: N = 227; artery: N = 227. f. < 37 weeks gestation. g. < 2,500 g.

Table 2. Summary of differentially methylation regions (DMRs) and positions (DMPs) associated with infant sex. DMRs identified using DMRcate with the FDR cut-off = 0.01, λ = 1,000, and the minimum number of CpGs = 10. DMPs identified using limma applied to M-values. All models adjusted for cellular heterogeneity using ReFactor components.

	# of DMRs	# (%) of DMRs with higher mean methylation among females	# of DMPs	# (%) of DMPs with higher methylation among females
Cord blood ^a	183	163 (89%)	2,119	1,948 (92%)
Placenta ^b	257	100 (39%)	2,281	291 (13%)
Artery ^c	419	391 (93%)	3,405	3,179 (93%)

DMR: differentially methylated region. a. N = 179. b. N = 229. c. N = 229.

Table 3. Ranking of top-10 DMRs associated with infant sex. DMRs identified using DMRcate modelling females vs. males adjusting for ReFactor components with the FDR cut-off = 0.01, λ = 1,000, and the minimum number of CpGs = 10. DMRs ordered by Fisher combined probability transform of the individual CpG FDRs.

Chr	Start position ^a	End position	# of CpGs	Fisher	Mean difference in % methylation ^b	Overlapping genes
Cord blood (N = 179)
6	32,116,841	32,122,436	101	5.36E-77	1.47	PPT2, PPT2-EGFL8, PRRT1
20	30,071,726	30,074,596	12	9.20E-68	5.75	REM1, LINC00028
15	75,018,150	75,019,376	27	6.09E-62	2.39
11	2,292,000	2,293,665	37	3.97E-61	2.67	ASCL2
1	2,344,133	2,345,894	22	1.29E-60	1.79	PEX10
3	195,942,717	195,943,713	10	1.83E-60	3.38	SLC51A, PCYT1A
6	28,583,655	28,584,464	18	1.85E-57	3.05	SCAND3
17	48,911,410	48,913,424	21	3.15E-57	2.63	WFIKKN2
1	78,511,140	78,512,129	12	2.74E-50	1.78	GIPC2
6	28,601,269	28,601,519	13	1.08E-45	6.99	RP11-373N24.2
Placenta (N = 229)
1	3,619,574	3,624,292	18	5.58E-77	−3.79	TP73
20	61,146,941	61,149,399	22	4.71E-70	−3.01	C20orf166, C20orf166-AS1
12	130,501,516	130,502,586	12	9.48E-48	−4.55
1	53,904,065	53,905,934	15	8.66E-46	2.46	SLC25A3P1
21	27,106,440	27,108,257	13	1.61E-45	−0.67	GABPA, ATP5J
11	45,713,046	45,719,826	31	2.15E-43	−2.30
8	146,227,573	146,229,440	20	3.99E-43	−4.53	ZNF252P-AS1, ZNF252P
12	53,241,452	53,243,489	10	4.33E-40	−2.49	KRT78
1	174,967,663	174,968,980	18	1.91E-39	1.26	CACYBP
6	33,864,689	33,869,169	23	2.20E-38	−2.81	RP3-468B3.2, LINC01016
Artery (N = 229)
17	46,668,715	46,680,461	52	7.78E-231	3.86	HOXB-AS3, HOXB3, HOXB5, HOXB6
2	119,602,212	119,617,128	70	1.28E-191	2.75	EN1
1	2,344,089	2,345,894	24	3.15E-166	2.75	PEX10
15	83,951,663	83,954,849	31	2.44E-143	2.20	RP11-382A20.4, BNC1
2	177,012,117	177,017,797	35	2.09E-127	4.95	HOXD3, MIR10B, HOXD4
13	49,792,685	49,796,489	14	1.58E-111	3.02	MLNR
17	46,799,479	46,805,563	30	1.35E-87	1.90	PRAC2, PRAC1, MIR3185, HOXB13
17	46,655,796	46,660,940	23	3.83E-87	4.37	HOXB-AS3, HOXB3, HOXB4, MIR10A
6	144,385,261	144,386,848	24	8.61E-80	1.77	PLAGL1
15	75,018,150	75,019,376	27	2.93E-77	1.88

Chr = chromosome. a. GRCh37/hg19 assembly. b. Mean difference in % methylation for females vs. males.

Figure 1. Overlap between DMRs in cord blood, placenta, and artery samples associated with infant sex. DMRs identified using DMRcate modelling females vs. males and adjusting for ReFactor components with the FDR cut-off = 0.01, λ = 1,000, and the minimum number of CpGs = 10. DMRs with consistently higher mean methylation among females are represented in Orange, DMRs with consistently higher mean methylation among males are represented in purple, and DMRs with different mean directions of association between tissues are represented in grey. Mean differences in % methylation for each DMR are displayed in the scatter plot. Cord blood: N = 179; placenta: N = 229; artery: N = 229.

Table 4. Overlapping differentially methylated regions (DMRs) associated with infant sex in cord blood, placenta, and artery samples. DMRs identified using DMRcate modelling females vs. males adjusting for ReFactor components with the FDR cut-off = 0.01, λ = 1,000, and the minimum number of CpGs = 10. DMRs ordered by chromosomal coordinate.

			Cord blood	Placenta	Artery
Chr	Start position ^a	End position	Mean difference in % methylation ^b	Mean difference in % methylation ^b	Mean difference in % methylation ^b	Overlapping genes
1	26,232,706	26,234,015	1.34	0.54	1.26	STMN1, MIR3917
1	111,506,021	111,507,160	−0.04	0.04	−0.19	RP11-96K19.5, LRIF1
1	154,244,759	154,245,414	0.61	0.49	0.57	HAX1
1	174,967,663	174,968,980	1.29	1.26	1.12	CACYBP
2	21,265,912	21,267,334	3.32	−2.76	2.28	APOB
2	27,850,964	27,852,231	0.64	1.11	0.46	ZNF512, RP11-158I13.2, GPN1, CCDC121
2	73,461,068	73,461,773	−0.08	−0.13	−0.10	CCT7
3	46,758,813	46,759,698	−3.80	−1.88	−3.05	PRSS50
6	27,258,466	27,259,237	1.31	1.30	1.17	POM121L2
6	28,226,885	28,227,482	2.09	2.41	2.17	NKAPL, ZKSCAN4
6	28,601,269	28,601,519	6.99	1.80	6.58	RP11-373N24.2
6	28,972,348	28,973,828	0.96	2.77	1.21	ZNF311
6	30,094,947	30,095,802	1.67	−1.91	0.78
6	31,631,638	31,633,028	0.87	0.31	0.75	CSNK2B, GPANK1
6	32,116,841	32,122,436	1.47	0.78	0.95	PPT2, PPT2-EGFL8, PRRT1
6	33,244,976	33,246,488	0.68	−0.83	0.74	B3GALT4
7	24,796,981	24,798,175	1.94	0.99	1.09	DFNA5
7	27,182,493	27,185,136	−2.20	0.41	1.04	HOXA-AS3, HOXA3, RP1-170O19.22, HOXA5, HOXA6
7	50,517,714	50,518,921	1.78	1.53	1.36	FIGNL1
7	95,025,194	95,027,158	1.28	2.17	1.09	PON1, PON3
7	150,019,955	150,021,553	2.52	1.41	1.62	LRRC61, ACTR3C
8	22,132,563	22,133,076	2.90	1.27	3.85	PIWIL2, CTD-2530N21.4
10	131,263,962	131,265,796	0.77	0.03	0.96	MGMT
11	2,291,652	2,293,665	2.67	1.41	1.00	ASCL2
12	1,099,823	1,101,044	0.53	0.32	0.50	ERC1, RP11-359B12.2
13	36,871,646	36,872,346	1.28	1.36	1.18	SOHLH2, CCDC169-SOHLH2, CCDC169
13	114,291,977	114,292,869	−0.79	−0.70	−0.71	TFDP1
15	75,018,150	75,019,376	2.39	2.36	1.88
16	89,557,017	89,558,207	−0.12	−0.66	−0.01	SPG7
17	7,486,551	7,487,249	1.08	0.92	1.47	MPDU1, AC113189.5
17	33,913,953	33,914,828	0.72	0.51	0.66	AP2B1
17	37,123,638	37,124,558	4.56	0.59	4.31	FBXO47
17	48,911,410	48,913,424	2.63	−1.47	1.43	WFIKKN2
19	19,625,264	19,627,257	1.56	1.05	2.06	NDUFA13, CTC-260F20.3, YJEFN3, TSSK6
19	55,972,504	55,973,778	−1.36	−0.98	−2.16	ISOC2
19	57,630,034	57,631,478	1.66	1.52	1.78	USP29
19	57,741,325	57,742,444	3.28	3.56	4.74	AURKC
20	25,128,211	25,129,562	1.71	−1.94	1.78	RP4-738P15.1
21	27,106,440	27,108,257	−0.78	−0.67	−0.85	GABPA, ATP5J

Chr = chromosome. a. GRCh37/hg19 assembly. b. Mean difference in % methylation for females vs. males.

Figure 2. Scatter plot of DMR chr6: 28,601,269–28,601,519.

Table 5. Summary of Gene Ontology (GO) analyses of differentially methylated regions (DMRs) and positions (DMPs). One, 3, and 39 GO terms were identified with an overrepresentation of DMRs associated with infant sex in cord blood, placenta, and artery samples, respectively (p_FDR < 0.05). Twenty-eight and 9 GO terms were identified with an overrepresentation of DMPs associated with infant sex in cord blood in placenta and artery samples, respectively (p_FDR < 0.05). No GO terms were associated to infant sex in cord blood (p_FDR > 0.05). Closely related GO terms have been combined; for a complete list of sex-associated GO terms, see Supplemental Tables S2 and S4.

	Cord blood	Placenta		Artery
	DMRs	DMRs	DMPs	DMRs	DMPs
Biological process	Homophilic cell adhesion via plasma membrane adhesion molecules	Organ morphogenesis, including skeletal system morphogenesis	Tissue development including epidermis development and keratinization; System development; Peptide cross-linking; Monocyte, lymphocyte, and eosinophil chemotaxis; Response to chemokine, including chemokine-mediated signalling pathway	Tissue development; Embryo development; Pattern-specific processes including regionalization; Organ morphogenesis, including skeletal system and eye morphogenesis; Cranial nerve development; Male meiotic nuclear division
Molecular function			Structural constituent of skin epidermis; Serine-type endopeptidase inhibitor activity; Chemokine receptor binding; Voltage-gated cation channel activity	Regulation of gene expression including regulation of transcription by RNA polymerase II; DNA binding transcription factor activity including RNA polymerase II-specific TAP1 binding	Transcription regulatory region and sequence-specific DNA binding; DNA binding transcription factor activity including RNA polymerase II-specific
Cellular component			Keratin filament and cornified envelope	Chromosome and chromatin

Table 6. Top-10 DMPs associated with infant sex ranked by p-value. DMPs identified using limma applied to M-values modelling females vs. males and adjusting for ReFactor components. Effect size estimates from limma applied to Beta-values. Bolded CpGs were identified in more than one tissue.

CpG	Chr	Position ^a	Gene	Feature category	P	PBonferroni	Female	Male	Mean difference in % methylation (95% CI)^b
Cord blood (N = 179)
cg12691488	1	243,053,673			2.11 ×10^−120	8.75 ×10^−115	7.83	32.29	−24.09	(−24.98, −23.20)
cg11643285	3	16,411,667	RFTN1	Body	1.51 ×10^−114	6.27 ×10^−109	91.84	75.59	15.92	(15.37, 16.48)
cg03691818	12	53,085,038	KRT77	Body	1.09 ×10^−74	4.51 ×10^−69	12.97	2.58	9.50	(8.73, 10.26)
cg17238319	3	16,428,391	RFTN1	Body	1.31 ×10^−74	5.42 ×10^−69	85.14	70.45	13.65	(12.73, 14.56)
cg03618918	1	160,865,097			8.10 ×10^−72	3.36 ×10^−66	69.55	79.99	−10.19	(−10.82, −9.55)
cg25294185	11	65,487,814	RNASEH2C	Body	6.92 ×10^−71	2.87 ×10^−65	4.46	14.57	−10.06	(−10.92, −9.20)
cg26921482	16	2,570,283	AMDHD2	TSS200	1.45 ×10^−65	6.01 ×10^−60	46.69	17.98	27.43	(25.50, 29.36)
cg26355737	13	114,292,172	TFDP1	Body	2.90 ×10^−60	1.20 ×10^−54	81.42	89.04	−7.82	(−8.44, −7.20)
cg04946709	16	59,789,030	LOC644649	Body	3.45 ×10^−58	1.43 ×10^−52	70.86	82.86	−11.99	(−13.03, −10.94)
cg06710937	13	23,489,940			5.27 ×10^−58	2.19 ×10^−52	9.40	2.74	6.61	(5.88, 7.35)
Placenta (N = 229)
cg02325951	14	89,878,619	FOXN3	Body	1.41 ×10^−75	5.85 ×10^−70	60.36	70.44	−10.30	(−11.00, −9.61)
cg12691488	1	243,053,673			1.57 ×10^−67	6.50 ×10^−62	7.40	19.41	−12.57	(−13.79, −11.35)
cg17612569	21	27,107,221	GABPA;ATP5J;ATP5J;ATP5J	TSS200;5'UTR;1stExon;Body	6.47 ×10^−65	2.69 ×10^−59	2.97	9.82	−8.33	(−9.12, −7.54)
cg13192180	17	65,717,569	NOL11	Body	1.82 ×10^−63	7.57 ×10^−58	72.86	86.14	−12.88	(−13.96, −11.80)
cg07355069	5	43,289,541	HMGCS1	3ʹUTR	3.49 ×10^−62	1.45 ×10^−56	85.23	93.19	−7.49	(−8.12, −6.86)
cg03691818	12	53,085,038	KRT77	Body	3.81 ×10^−53	1.58 ×10^−47	12.11	4.16	6.67	(5.93, 7.40)
cg02606018	12	10,658,281			8.07 ×10^−53	3.35 ×10^−47	23.38	29.68	−6.65	(−7.29, −6.01)
cg12780322	4	57,334,163	SRP72	Body	1.08 ×10^−48	4.48 ×10^−43	8.80	5.34	3.27	(2.89, 3.65)
cg15817705	1	209,406,063			1.06 ×10^−43	4.41 ×10^−38	60.52	68.49	−7.50	(−8.33, −6.68)
cg03986557	1	202,430,474	PPP1R12B;PPP1R12B	Body;TSS1500	1.24 ×10^−43	5.16 ×10^−38	90.96	84.28	7.01	(6.21, 7.81)
Artery (N = 229)
cg12691488	1	243,053,673			7.34 ×10^−143	3.05 ×10^−137	5.74	30.84	−25.02	(−26.03, −24.00)
cg11643285	3	16,411,667	RFTN1	Body	1.32 ×10^−120	5.48 ×10^−115	85.31	70.11	15.05	(14.46, 15.65)
cg03691818	12	53,085,038	KRT77	Body	2.59 ×10^−101	1.08 ×10^−95	13.31	2.49	10.01	(9.26, 10.76)
cg03618918	1	160,865,097			1.39 ×10^−95	5.76 ×10^−90	68.63	78.35	−9.81	(−10.32, −9.30)
cg26921482	16	2,570,283	AMDHD2	TSS200	7.88 ×10^−92	3.27 ×10^−86	43.94	13.27	29.01	(27.31, 30.72)
cg02325951	14	89,878,619	FOXN3	Body	1.78 ×10^−87	7.37 ×10^−82	61.70	72.73	−11.08	(−11.74, −10.41)
cg17612569	21	27,107,221	GABPA;ATP5J;ATP5J;ATP5J	TSS200;5'UTR;1stExon;Body	3.97 ×10^−79	1.65 ×10^−73	3.10	12.16	−10.35	(−11.23, −9.48)
cg25294185	11	65,487,814	RNASEH2C	Body	6.43 ×10^−79	2.67 ×10^−73	5.13	14.88	−10.15	(−10.92, −9.39)
cg04946709	16	59,789,030	LOC644649	Body	4.55 ×10^−75	1.89 ×10^−69	69.59	80.20	−10.19	(−10.95, −9.42)
cg25568337	6	157,098,338	ARID1B	TSS1500	5.17 ×10^−73	2.15 ×10^−67	19.62	11.01	8.11	(7.51, 8.70)

a. GRCh37/hg19 assembly. b. Mean difference in % methylation for females vs. males.

Figure 3. Volcano and Manhattan plots for DMPs associated with infant sex.

Data Availability

R script for analysis of replication analyses using GEO Accession GSE129841 is available at https://github.com/annebozack/GEO_infant_sex_rep_analysis. Additional data available upon request to the corresponding author.