Regulation of birthweight by placenta-derived miRNAs: evidence from an arsenic-exposed birth cohort in Bangladesh

Figures & data

Table 1. Distributions of selected characteristics of the study participants by study phases. Data were expressed as Mean ± SD or Median (range) except where indicated.

	Phase 1
Characteristics	Upper extreme birthweight group (n = 77)	Lower extreme birthweight group (n = 77)	P value	Phase 2 (n = 364)
Birthweight (kg)	3.5 (3.3 – 3.9)	2.1 (0.8 – 2.2)	<0.001	2.9 (1.0−4.3)
Maternal age (years)	23.5 ± 4.3	23.3 ± 4.2	0.7	22.7 ± 3.7
Gestational age (weeks)	38.5 ± 1.4	35.1 ± 3.0	<0.001	37.9 ± 2.0
Enrollment BMI, n (%)			0.03
≤ 18.5 kg/m^2	12 (15.6)	23 (29.9)		92 (25.3)
> 18.5 to ≤ 25.0 kg/m^2	53 (68.8)	50 (64.9)		239 (65.7)
> 25.0 kg/m^2	12 (15.6)	4 (5.2)		33 (9.0)
Maternal Education, n (%)			0.01
No formal education	5 (6.5)	18 (23.4)		57 (25.7)
Primary education	21 (27.3)	20 (26.0)		127 (34.9)
Secondary or higher	51 (66.2)	39 (50.6)		180 (49.4)
Infant sex, n (%)			0.4
Female	43 (55.8)	38 (49.4)		179 (49.2)
Male	34 (44.2)	39 (50.6)		185 (50.8)
Study site, n (%)			<0.001
Sirajdikhan	33 (42.9)	10 (13.0)		173 (47.5)
Pabna	44 (57.1)	67 (87.0)		191 (52.5)
No. of past pregnancies, n (%)			0.5
0	27 (35.1)	26 (33.8)		143 (39.5)
1	28 (36.4)	23 (29.9)		118 (32.6)
> 1	22 (28.6)	28 (36.4)		101 (27.9)
Cord blood arsenic (μg/dl)	0.54 (0.1–7.1)	0.70 (0.09–6.0)	0.003	0.60 (0.1–15.5)

Table 2. The effects of a 1-SD increment in placental expression of miRNA on gestational age and birthweight in Phase 2 study (n = 364). The indirect, direct, and total effects of miRNA expressions on birthweight were estimated using causal mediation analysis considering gestational age a mediator.

				Associations with Birthweight
	Associations with Gestational Age			Indirect Effects			Direct Effects			Total Effects
miRNA	Coefficient (95% CI)	P value	FDR	Coefficient (95% CI)	P value	FDR	Coefficient (95% CI)	P value	FDR	Coefficient (95% CI)	P value	FDR	PM ^b %
let7g	−0.46 (−0.78, −0.14)	0.005	0.042	−37.5 (−77.4, −1.8)	0.044	0.132	18 (−86.5, 115.3)	0.798	0.582	−19.5 (−145.6, 79.8)	0.716	0.676
miR-124a	0.1 (−0.49, 0.69)	0.736	0.581	1.6 (−66, 74.4)	0.910	0.454	−46.3 (−212.2, 123.3)	0.650	0.547	−44.7 (−224.2, 137.2)	0.674	0.676
miR-127	−0.03 (−0.25, 0.18)	0.774	0.581	−1.7 (−23.5, 15.6)	0.832	0.453	27.3 (−25.7, 75.4)	0.300	0.453	25.7 (−32, 78.9)	0.360	0.539
miR-1290^a	−0.67 (−0.96, −0.38)	<0.001	<0.001	−52.9 (−95.4, −22.6)	0.000	0.003	−107.1 (−174.3, −31.8)	0.005	0.080	−160.1 (−235.6, −74)	<0.001	0.009	33.1
miR-130a	0.02 (−0.33, 0.37)	0.910	0.608	7.7 (−17.7, 41.4)	0.530	0.413	−59.8 (−150.2, 27)	0.194	0.443	−52.1 (−148.4, 37.1)	0.278	0.531
miR-135b	0.1 (−0.24, 0.43)	0.569	0.554	−6.4 (−44.5, 26.7)	0.764	0.444	−12.7 (−105, 71)	0.748	0.570	−19.1 (−111, 74.7)	0.686	0.676	33.6
miR-138	−0.2 (−0.51, 0.11)	0.210	0.385	−13.5 (−39.5, 2.6)	0.152	0.302	−50.1 (−102.2, −5.8)	0.042	0.363	−63.6 (−124.9, −13.9)	0.018	0.216	21.2
miR-149	−0.04 (−0.44, 0.35)	0.832	0.601	−9 (−42.6, 21.7)	0.586	0.413	−44.6 (−126.3, 46.2)	0.324	0.453	−53.6 (−136.6, 50.8)	0.270	0.531	16.8
miR-15a	−0.25 (−0.56, 0.07)	0.128	0.328	−15.1 (−43.4, 13.3)	0.296	0.327	3.2 (−79.3, 75.2)	0.984	0.642	−11.9 (−95.2, 65.3)	0.736	0.676
miR-16	−0.14 (−0.42, 0.15)	0.342	0.440	−11.1 (−34.5, 8.8)	0.300	0.327	42.2 (−13.6, 101.6)	0.140	0.413	31 (−24.9, 95.6)	0.306	0.531
miR-181a	−0.02 (−0.28, 0.23)	0.848	0.601	−3.8 (−23.7, 9.5)	0.562	0.413	−41.3 (−88, −0.4)	0.054	0.363	−45.1 (−98.3, −4.6)	0.040	0.240	8.3
miR-181c	−0.16 (−0.52, 0.19)	0.373	0.440	−7.5 (−33.2, 10.3)	0.450	0.387	−56.3 (−121, −0.6)	0.058	0.363	−63.8 (−135.8, −3.5)	0.038	0.240	11.7
miR-185	−0.42 (−0.93, 0.09)	0.106	0.300	−20.2 (−73.1, 26.1)	0.468	0.387	−8.4 (−130.2, 123.3)	0.878	0.611	−28.6 (−162.5, 107.7)	0.708	0.676	70.7
miR-195	−0.56 (−0.82, −0.3)	<0.001	<0.001	−40.9 (−78.3, −13.8)	0.002	0.024	−47.1 (−120.9, 34.2)	0.278	0.453	−87.9 (−170.4, 0.8)	0.053	0.271	46.5
miR-199a	0.27 (−0.11, 0.64)	0.164	0.366	35.1 (8, 70)	0.014	0.056	−70 (−167.3, 10.9)	0.112	0.413	−34.9 (−139.3, 49.8)	0.520	0.623
miR-200c	0.42 (0.03, 0.82)	0.038	0.131	19.8 (−8.7, 51.8)	0.230	0.327	−26.9 (−129, 82.7)	0.716	0.570	−7 (−114.9, 114.7)	0.958	0.702
miR-205	0.15 (−0.33, 0.63)	0.530	0.538	17.9 (−13.4, 53.2)	0.274	0.327	−37.9 (−127.5, 56.8)	0.448	0.541	−20.1 (−115.2, 77.6)	0.720	0.676
miR-20b	−0.23 (−0.52, 0.07)	0.137	0.328	−11.4 (−48.6, 17.9)	0.464	0.387	−31.6 (−108, 44.9)	0.374	0.498	−43 (−129.1, 43.8)	0.332	0.531	26.5
miR-21	−0.06 (−0.4, 0.28)	0.736	0.581	−2.2 (−47.1, 25)	0.902	0.454	31.4 (−54.6, 122.5)	0.550	0.541	29.2 (−68.2, 119.4)	0.596	0.669
miR-222	0.16 (−0.16, 0.49)	0.322	0.440	15.6 (−5.9, 48.4)	0.226	0.327	−17.8 (−101, 61.3)	0.612	0.541	−2.2 (−83.7, 79.1)	0.902	0.702
miR-27a	−0.4 (−0.74, −0.06)	0.023	0.089	−36.1 (−69.5, −11.4)	0.010	0.048	27.6 (−77.5, 100.1)	0.582	0.541	−8.5 (−123.9, 64)	0.774	0.678
miR-296	0.26 (−0.13, 0.64)	0.189	0.385	13.8 (−7.9, 44.2)	0.272	0.327	−46.4 (−105.1, 17.1)	0.140	0.413	−32.6 (−91.9, 37.2)	0.320	0.531
miR-30c	0.32 (0.05, 0.58)	0.019	0.086	15.1 (0.7, 40.3)	0.070	0.168	15.6 (−40.4, 89.2)	0.586	0.541	30.7 (−22.4, 96.8)	0.276	0.531	49.3
miR-320b	−0.05 (−0.36, 0.26)	0.750	0.581	3.4 (−19.6, 33.6)	0.802	0.447	33.6 (−30.9, 107.5)	0.312	0.453	37 (−31.2, 117.2)	0.328	0.531	9.1
miR-3245p	0.32 (0.08, 0.57)	0.009	0.051	28 (9.2, 54.9)	0.004	0.032	−5.9 (−72.4, 48)	0.800	0.582	22 (−41.6, 74.8)	0.500	0.620
miR-328	0.45 (0.26, 0.64)	<0.001	<0.001	20.4 (5.8, 45.4)	0.007	0.040	11.1 (−31.7, 63.9)	0.551	0.541	31.4 (−7.7, 86.5)	0.123	0.489	64.8
miR-331	0.13 (−0.15, 0.42)	0.363	0.440	5.2 (−16.8, 32.2)	0.660	0.439	44.3 (−31.2, 107.7)	0.226	0.453	49.5 (−25.9, 116.7)	0.184	0.531	10.5
miR-3373p	−0.12 (−0.4, 0.15)	0.381	0.440	−4.9 (−37.5, 20)	0.726	0.444	1.2 (−57.9, 71.7)	0.966	0.642	−3.7 (−78.3, 68.8)	0.928	0.702
miR-34b	0.22 (−0.12, 0.56)	0.201	0.385	7 (−26.4, 39.3)	0.704	0.444	27.7 (−60.3, 109.5)	0.538	0.541	34.7 (−56.6, 118.1)	0.492	0.620	20.2
miR-34c	−0.07 (−0.31, 0.18)	0.592	0.560	−8.1 (−35.1, 10.9)	0.464	0.387	−29.4 (−74.8, 24.1)	0.326	0.453	−37.5 (−90.1, 19.9)	0.220	0.531	21.5
miR-373	0.05 (−0.29, 0.39)	0.782	0.581	1 (−27.8, 30.5)	0.908	0.454	6.6 (−78.4, 83.1)	0.942	0.641	7.6 (−83.2, 84.8)	0.920	0.702	13.5
miR-376b	−0.23 (−0.65, 0.2)	0.303	0.440	−8.6 (−47.2, 24.3)	0.652	0.439	18.3 (−64.6, 101)	0.620	0.541	9.7 (−85.4, 101.5)	0.752	0.676
miR-424	0.33 (0.08, 0.57)	0.010	0.051	25.1 (5.5, 53.6)	0.024	0.082	−17.3 (−77.1, 45.2)	0.554	0.541	7.8 (−59.5, 69.3)	0.828	0.692
miR-454	−0.02 (−0.37, 0.32)	0.894	0.608	15.4 (−23.2, 56.8)	0.442	0.387	−16.8 (−128.2, 80.5)	0.674	0.553	−1.4 (−127.6, 106.1)	0.896	0.702
miR-455	0.13 (−0.29, 0.55)	0.534	0.538	19.1 (−15, 58.3)	0.264	0.327	45.1 (−33.7, 134.5)	0.274	0.453	64.3 (−31.3, 159.4)	0.148	0.531	29.7
miR-489	−0.21 (−0.45, 0.03)	0.084	0.262	−13.5 (−32.5, 4.9)	0.164	0.302	−57.5 (−109.4, 2.8)	0.068	0.363	−71 (−129.2, −3)	0.034	0.240	19.0
miR-518e	−0.21 (−0.56, 0.14)	0.241	0.417	−19.9 (−50.9, 0.9)	0.092	0.200	44.4 (−48.5, 136.4)	0.296	0.453	24.6 (−73.3, 118.8)	0.538	0.624
miR-518f	0 (−0.31, 0.31)	0.989	0.630	−4.9 (−37.7, 26.8)	0.778	0.444	−44.3 (−139.6, 34.2)	0.324	0.453	−49.1 (−146.8, 39.4)	0.330	0.531	10.0
miR-522	0.07 (−0.26, 0.4)	0.682	0.581	−2.8 (−25.1, 18.5)	0.754	0.444	52.3 (−16.1, 130.9)	0.142	0.413	49.5 (−32.1, 125.6)	0.198	0.531
miR-524	0.01 (−0.33, 0.36)	0.936	0.608	−5.4 (−31.8, 23.8)	0.728	0.444	17.5 (−71, 111.3)	0.740	0.570	12.1 (−84.1, 111.5)	0.828	0.692
miR-5423p	0.13 (−0.15, 0.41)	0.372	0.440	13.4 (−13.1, 39.9)	0.346	0.360	21 (−60.6, 85.9)	0.626	0.541	34.4 (−59.3, 105)	0.472	0.620	39.0
miR-625	−0.13 (−0.44, 0.19)	0.438	0.472	−15.8 (−50.9, 10.9)	0.274	0.327	46.1 (−29.3, 119.4)	0.174	0.443	30.3 (−53.6, 112.5)	0.384	0.552
miR-629	0.01 (−0.32, 0.35)	0.932	0.608	−5.7 (−27.8, 18.5)	0.580	0.413	45.5 (−22.7, 114.5)	0.194	0.443	39.8 (−37.7, 113.8)	0.308	0.531
miR-720	0.13 (−0.1, 0.35)	0.274	0.440	9.2 (−6.9, 27.3)	0.284	0.327	75.1 (26, 116.1)	0.002	0.070	84.2 (31.4, 128.1)	0.001	0.022	10.9
miR-8863p	0.07 (−0.31, 0.46)	0.718	0.581	−1.9 (−35.8, 22.2)	0.860	0.454	24.2 (−57.9, 106.2)	0.622	0.541	22.3 (−71.1, 108)	0.682	0.676
miR-9	0.11 (−0.17, 0.39)	0.439	0.472	6.7 (−12.6, 34.8)	0.542	0.413	−5.2 (−65.4, 56.4)	0.868	0.611	1.5 (−53.1, 62.7)	0.944	0.702
miR-99a	−0.1 (−0.53, 0.33)	0.648	0.577	−27 (−61.2, 0.9)	0.064	0.168	−27 (−119.4, 80.3)	0.604	0.541	−54 (−157.6, 64)	0.340	0.531	50.0
miR-99b	0.17 (−0.15, 0.48)	0.299	0.440	9.4 (−8, 37.2)	0.412	0.387	61.8 (−15, 117.7)	0.098	0.413	71.2 (−6.1, 128.7)	0.066	0.296	13.2
miR-99b*	−0.16 (−0.81, 0.48)	0.618	0.567	1.8 (−40.1, 45.7)	0.940	0.460	46.2 (−68.3, 154)	0.412	0.527	48 (−73.2, 159.6)	0.422	0.583	3.7

The models for gestational age (mediator model) were adjusted for maternal age, education, no. of past pregnancies, BMI, infant sex, study site, natural log of cord blood arsenic exposure, and surrogate variables. The models for birthweight were additionally adjusted for gestational age and included a miRNA-gestational age interaction term.

^a The outcome model for miR-1290 also included an interaction term between miR-1290 and natural log cord blood arsenic exposure.

^b PM = Proportion mediated. Calculated for miRNAs that showed same direction of associations via both indirect and direct effect pathways in relation to birthweight.

Figure 1. Directed acyclic graph (DAG) showing conceptual models for causal mediation analysis. We hypothesized that placenta-derived miRNAs will affect birthweight directly or indirectly via changing gestational age.

Figure 2. Controlled Direct Effects (CDEs) of selected placenta-derived miRNAs in relation to birthweight at gestational age ranging from 29 to 41 weeks. Panel A1, A2, and A3 illustrate the changes in mean birthweight [g] for a 1-SD increment in placental expression miR-1290 when cord blood arsenic exposure is at 25th, 50th, and 75th percentiles, respectively. Panel B, C, and D illustrate the changes in mean birthweight [g] for a 1-SD increment in placental expression of miR-195, miR-328, and miR-489, respectively. Solid black lines surrounded by shaded area represent the effect estimates and 95% confidence intervals. The horizontal blue dashed lines show the reference values and the vertical blue dashed lines represent the gestational age of 37 weeks. Models were adjusted for natural log cord blood arsenic exposure, maternal age, education, enrollment BMI, number of past pregnancies, infant sex, study site and surrogate variables.

Figure 3. Gene enrichment pathways for selected miRNAs with in silico predicted targets. The vertical axis represents a description of the pathways, and the horizontal axis represents the – Log₁₀(P value) of the significant enrichment pathways.

Figure 4. Networks of miR-1290 and miR-195 with experimentally validated target genes. The green lines connecting the nodes represent activation, whereas the red lines connecting the nodes represent inhibition. The arrow indicates the direction of the association.

Table 3. Effect estimates of gestational age and selected miRNAs in relation to birthweight.

	Effect estimates of gestational age		Effect estimates of miRNA expression
miRNAs	Model 5^a	Model 7^c	Model 6^b	Model 7^c
let7g	78.7	78.9	−28.0	9.0
miR-1290	78.7	74.0	−121.1	−68.7
miR-138	78.7	77.1	−63.1	−50.0
miR-181a	78.7	78.1	−50.2	−46.1
miR-181c	78.7	77.8	−64.7	−57.5
miR-195	78.7	74.5	−98.3	−56.1
miR-199a	78.7	80.9	−35.3	−69.7
miR-27a	78.7	79.7	−8.9	27.9
miR-324-5p	78.7	78.6	32.6	1.1
miR-328	78.7	76.7	49.1	16.1
miR-424	78.7	79.7	7.8	−19.3
miR-489	78.7	77.1	−56.8	−43.0
miR-720	78.7	76.7	81.8	71.9

^a Model 5: $E\left[Birthweight\right]={\theta }_0+{\theta }_1\left(GestationalAge\right)+\theta {\mathrm{\prime }}_{2}Covariates$

^b Model 6: $E\left[Birthweight\right]={\beta }_0+{\beta }_1\left(miRNA\right)+\beta {\mathrm{\prime }}_{2}Covariates$

^c Model 7: $E\left[Birthweight\right]={\alpha }_0+{\alpha }_1\left(miRNA\right)+{\alpha }_2\left(GestationalAge\right)+\alpha {\mathrm{\prime }}_{4}Covariates$

Coefficients were scaled for a 1-SD change in miRNA expression in the placenta. Covariates included in the models were natural log cord blood arsenic exposure, maternal age, education, enrollment BMI, the number of past pregnancies, study site, infant sex and surrogate variables.

Table 4. Study design and analysis work flow.

Phase 1 (n = 154)
Study Design	Extreme Phenotype Sampling: 77 samples with birthweight ranging 0.8 – 2.2 kg 77 samples with birthweight ranging 3.3 – 3.9 kg
Expression platform	TaqMan RT-qPCR OpenArray for 754 preconfigured human miRNAs
Normalizations	Endogenous control; 2) Quantile; 3) Global; 4) Raw Ct
Association analyses	Associations between miRNA expression and gestational age Associations between miRNA expression and the extreme birthweight groups with and without adjusting for gestational age
MicroRNA selection	Total 49 miRNAs were screened out for further analysis in Phase 2
Phase 2 (n = 364)
Study Design	364 participants randomly selected from the full cohort (n = 1141) 312 new participants; 52 duplicates also selected in from Phase 1
Expression platform	TaqMan RT-qPCR Customized OpenArray Platform 310 samples in triplicate; 54 samples in duplicate
Normalization	Endogenous control
Association analyses	Associations between miRNA expression and gestational age Direct, indirect, and total effect associations between miRNA expression and birthweight considering gestational age a mediator Assess interactions between miRNA expression and gestational age as well as miRNA expression and arsenic exposure miRNA gene enrichment analysis with in silico predicted mRNA targets