DNA methylation in promoter regions of genes involved in the reproductive and metabolic function of children born to women with PCOS

Figures & data

Table 1. Clinical and biochemical characteristics of control and PCOS pregnant women with (PCOS+M) and without metformin (PCOS)

	Control (n = 24)	PCOS (n = 12)	PCOS+M (n = 12)	P-values
Pre-pregnancy
Age (years)	23.5 (20.2–30.7)	29.5 (26.0–31.0)	30.5 (30.0–35.2) ^b	0.013
Weight (kg)	59.5 (52.8–66.0)	69.0 (56.0–80.0)	72.0 (68.0–76.8)^b	0.002
BMI (Kg/m^2)	22.9 (21.1–26.0)	26.1 (23.7–29.1)	29.3 (27.1–30.9) ^b	<0.001
Overweight/obesity (%)	29.2	58.3	83.3^b	0.007
At beginning
Weight (kg)	59.0 (52.5–66.0)	69.0 (57.0–80.0)	70.8 (68.0–77.0)^b	0.002
BMI (Kg/m^2)	23.7 (21.1–25.7)	27.6 (24.4–29.9)^a	28.8 (27.1–30.9) ^b	<0.001
At third trimester
Weight (Kg)	71.0 (65.8–77.8)	78.0 (69.0–90.0)^a	78.8 (70.9–81.5)	0.096
BMI	29.1 (26.7 – 31.3)	30.2 (28.5 – 36.1)^a	31.9 (28.2 – 32.4)	0.026
SBP (mm Hg)	120 (110–120)	110 (105–110)	120 (110–120)	0.087
DBP (mm Hg)	70 (60–77)	62 (60–70)	70 (70 – 70)	0.448
GWG (Kg)	12.9 (8.2–16.5)	11.0 (6.5–15.8)	5.0 (2.8–7.9)^b, c	0.001
PIH (%) (n)	0	8.3 (1/12)	0 (0/12)	0.216
GDM (%) (n)	0	16.7 (2/12)^a	66.7 (8/12) ^b,c	<0.001

BMI: Body mass index; SBP: Systolic blood pressure; DBP: Diastolic blood pressure; GWG: gestational weight gain; PIH: pregnancy induced-hypertension; GDM: gestational diabetes mellitus. Data are expressed as median and interquartile range for continuous variables and percentage for categorical variables. For continuous variables differences were calculated by one way ANOVA test followed by Bonferroni test or Kruskal Wallis followed by Dunn tests according to the normal distribution of data. Categorical variables were analysed by chi-square test. ^aP < 0.05 between control and PCOS; ^bP < 0.05 between control and PCOS+M; ^cP < 0.05 between PCOS and PCOS+M.

Table 2. Clinical characteristics of control and PCOS infants (daughters and sons) born to control and PCOS mothers treated (PCOS+M) and non-treated (PCOS) with metformin during pregnancy

	Daughters				Sons
	Control (n = 12)	PCOS (n = 6)	PCOS+M (n = 6)	P-values	Control (n = 12)	PCOS (n = 6)	PCOS+M (n = 6)	P-values
At birth
Gestational age (wk)	39.0 (39.0–40.0)	39.0 (38.0–40.0)	38.0 (36.0–38.0)^b	0.005	39.5 (38.2–40.0)	39.5 (38.7–41.0)	39.0 (37.7–39.5)	0.474
Weight (g)	3545 (3257–3750)	3275 (2690–3905)	3285 (2895–3515)	0.346	3575 (3402–4010)	3588 (2965–3875)	3417 (3015–3692)	0.534
Z-score weight	0.76 (0.15–1.06)	0.04 (−1.26–1.87)	0.34 (−0.25–0.93)	0.601	1.11 (0.55–1.93)	1.03 (−0.73–1.58)	0.57 (−0.25–1.34)	0.631
Length (cm)	50.0 (49.0–51.0)	47.2 (44.7–50.4)	47.7 (45.9–49.2)	0.046	50.0 (49.2–51.7)	50.0 (49.2–51.0)	49.5 (48.2–51.4)	0.659
Z-score length	0.22 (−0.27–0.68)	−0.39 (−1.38–0.60)	−0.12 (−0.83–0.17)	0.381	0.22 (0.06–0.85)	0.30 -(0.25–0.54)	0.13 -(0.70–0.88)	0.775
SGA (%) (n)	0 (0/12)	16.7 (1/6)	0 (0/6)	0.209	8.3 (1/12)	16.7 (1/6)	0 (0/6)	0.579
LGA (%) (n)	16.7 (2/12)	33.3 (2/6)	0 (0/6)	0.301	33.3 (4/12)	33.3 (2/6)	16.7 (1/6)	0.739
At study
Age (years)	0.17 (0.17–0.23)	0.25 (0.17–0.25)	0.25 (0.23–0.25)^b	0.006	0.17 (0.17–0.25)	0.21 (0.17–0.27)	0.25 (0.17–0.33)	0.077
Weight (g)	6115 (5327–6650)	6335 (5425–6605)	5175 (4747–6337)	0.336	5600 (5400–8160)	6400 (6050–7540)	6375 (5587–7012)	0.040
Z-score weight	0.94 (0.18–1.48)	0.73 (−0.04–1.28)	−1.02 (−1.52–0.42)^b	0.032	−0.29 (−0.95–0.48)	0.49 (−0.04–1.79)	0.68 (−0.79–2.06)	0.080
Length (cm)	58.0 (57.0–60.7)	57.5 (55.5–59.0)	57.5 (55.2–60.1)	0.419	59.0 (58.0–60.9)	60.0 (59.0–62.5)	57.5 (57.0–61.0)	0.267
Z-score length	−0.13 (−0.59–1.45)	−0.73 (−1.56 – −0.08)	−1.30 (−2.17 – −0.20)^b	0.008	−0.24 (−0.71–0.62)	0.03 (−0.57–1.43)	−0.33 (−1.71–1.36)	0.789
Weight gain (g)	2735 (1820–3212)	2620 (2390–3170)	2485 (1480–3327)	0.886	2145 (1652–2732)	2967 (2270–4147)	3615 (2881–4510)^b	0.020
EMB (%) (n)	80 (8/10)	83.3 (5/6)	50.0 (3/6)	0.338	77.8 (7/9)	25.0 (1/4)	66.7 (4/6)	0.186

Results are expressed as median and interquartile range. SGA: small for gestational age; LGA: large for gestational age and EMB: exclusive maternal breastfeeding. Data are expressed as median and interquartile range for continuous variables and percentage for categorical variables. For continuous variables differences were calculated by one way ANOVA test followed by Bonferroni test or Kruskal Wallis followed by Dunn tests according to the normal distribution of data. Categorical variables were analysed by chi-square test. ^aP < 0.05 between control and PCOS; ^bP < 0.05 between control and PCOS+M; ^cP < 0.05 between PCOS and PCOS+M.

Figure 1. Methylation levels (β-value) in the promoter regions of the leptin receptor (LEPR) (a), leptin (LEP) (b), adiponectin receptor 2 (ADIPOR2) (c) and androgen receptor (AR) genes (d) in daughters of control (control, n = 12), PCOS women (PCOS, n = 6) and PCOS women treated with metformin during pregnancy (PCOS+M, n = 6). Data are shown as median ± SEM. Dots indicate the cases in each group. Differences were calculated by one-way ANOVA followed by Bonferroni test or Kruskal-Wallis test followed by Dunn test. ^aP < 0.05 between control and PCOS; ^bP < 0.05 between control and PCOS+M; ^cP < 0.05 between PCOS and PCOS+M

Figure 2. Methylation levels (β-value) in the promoter regions of the leptin (LEP) (a), antimüllerian hormone (AMH) (b) and androgen receptor (AR) genes (c) in sons of control (control, n = 12), PCOS women (PCOS, n = 6) and PCOS women treated with metformin during pregnancy (PCOS+M, n = 6). Data are shown as median ± SEM. Dots indicate the cases in each group. Differences were calculated by one-way ANOVA followed by Bonferroni test or Kruskal-Wallis test followed by Dunn test. ^aP < 0.05 between control and PCOS; ^bP < 0.05 between control and PCOS+M; ^cP < 0.05 between PCOS and PCOS+M

Figure 3. Z-Score of the promoter regions of LEP, LEPR, ADIPOQ, ADIPOR1, ADIPOR2, AMH and AR in daughters and sons of control (control, n = 12/12), PCOS women (PCOS, n = 6/6) and PCOS women treated with metformin during pregnancy (PCOS+M, n = 6/6). Data are shown as median ± SEM. Dots indicate the cases in each group. Differences were calculated by Kruskal-Wallis test followed by Dunn test. ^aP < 0.05 between control and PCOS; ^bP < 0.05 between control and PCOS+M

Figure 4. Correlation analysis between the methylation levels (β-value) of the CpG sites of the promoter regions of the AR (chromosome X, ChrX), and LEP (chromosome 7, Chr7) genes (and Z-Score of the analysed promoter regions), and the maternal characteristics and anthropometric parameters at birth and postnatal age in daughters and sons. Dots indicate the cases in each group. The associations between variables were calculated by Spearman’s rank correlation analysis. P < 0.05 was considered as significant level