Altered thyroid hormone profile in offspring after exposure to high estradiol environment during the first trimester of pregnancy: a cross-sectional study

A cross-sectional survey was undertaken at the ART Unit in the Women’s Hospital, School of Medicine, Zhejiang University, China. The study was approved by the Ethical Committee of Women’s Hospital, School of Medicine, Zhejiang University and was registered in the Chinese Clinical Trial Registry (ChicCTR-OCC-14004682). The ART unit is one of the earliest licensed by the Health Ministry of People’s Republic of China and is also one of the largest in the country. A total of 949 Asian singletons aged 3 to 10 years old delivered from January 2003 to March 2011 were included in this study of which 357 cases were after fresh ET, 212 cases after frozen ET, and 380 after NC. Face-to-face interviews were carried out and the children were examined thoroughly. Interviews took place in the visit room individually and lasted approximately 30 minutes. Written consent was obtained in every case prior to assessment.

All NC cases were randomly selected and matched for age and gender to cases in the fresh ET group and frozen ET group. These patients had been referred to the Physical Examination Center for a regular health examination and were recruited when they agreed to participate. All cases were healthy, did not take any medication, and lived in iodine-replete areas. None of the NCs had congenital hypothyroidism. Mothers with thyroiditis, hyperthyroidism, or hypothyroidism during pregnancy were excluded.

Additionally, we studied 183 full-term singleton infants, including 55 fresh ET, 48 frozen ET, and 80 NC born in our hospital. They were matched according to maternal age, gestational age, and birth weight and all IVF babies were conceived after traditional IVF, whereas infants conceived after intracytoplasmic sperm injection were excluded. All newborns selected above were free of obstetric complications during pregnancy such as pregnancy-induced hypertension, gestational diabetes mellitus, placenta previa, and intrahepatic cholestasis. Mothers with thyroiditis, hyperthyroidism, or hypothyroidism during pregnancy were also excluded.

During all the fresh ET cycles, ovarian stimulations were performed by the standard long or short protocols as previously described [17]. Oocyte retrieval was carried out 34 to 36 h after hCG injection. Fresh and frozen ET were performed on Day 2 or Day 3. Cryopreserved embryos were thawed on the day of the transfer and all the frozen ETs proceeded in natural cycles without COH. Children and babies from the fresh or frozen ET groups were excluded if the women had received donated oocytes or sperm, or if they had undergone pre-implantation genetic diagnosis.

On the other hand, maternal serum E₂ levels on hCG administration day of 55 fresh ET newborns were obtained from database of the hospital. In order to detect the maternal E₂ level, blood samples of another 530 women with singleton conceptions at 4 to 12 weeks of gestation were obtained from October 2012 to June 2013, including 206 fresh ET, 124 frozen ET, and 200 NC and their clinical gestational data were also obtained from the database.

A detailed medical history was obtained from all participants. Data recorded included the maternal age, sex, ART methods, infant birth weight, birth length, gestational age, and mode of feeding. Gestational age was calculated based from the date of ET and the date of birth. The same trained female nurse examined all children. Physical examination included height, weight, body mass index, systolic blood pressure, diastolic blood pressure, and heart rate. We had access to complete clinical data for each individual.

Samples were collected from umbilical cord blood of newborns at birth and peripheral blood of 3- to 10-year-old children at 8:00 am after overnight fasting. Serum T3, FT3, T4, FT4, and TSH were assayed with the Abbott Architect i2000 assay (Abbot Diagnostics) in the local laboratory. The intra- and inter-assay coefficients of variation for the determination of all biochemical variables were less than 5%.

Characteristics and cycle parameters of patients were obtained from databases of the Department of Reproductive Endocrinology, Women’s Hospital, School of Medicine, Zhejiang University. To explore the correlation between maternal E₂ level and thyroid hormones in offspring, we analyzed serum levels of T4 and FT4 of 55 fresh ET newborns and gestational E₂ levels on the day that their mothers received hCG.

Either the two-tailed Student’s t-test or ANOVA were used to evaluate continuous parametric data. Relationships between type of conception and thyroid function was analyzed by a regression model with adjustment for age of child, type of assisted reproduction, and sex of child or maternal variables (pregnancy-induced hypertension, gestational diabetes mellitus, placenta previa, or intrahepatic cholestasis); the χ² test was used to compare categorical data. Pearson’s correlation coefficient was applied to explore the association of T4 and FT4 with E₂ levels. Log transformations were conducted when the normality assumption was not satisfied. Analyses were conducted using SPSS statistical software (version 16.0) and P <0.05 indicated statistical significance.

Table 1 presents physical examination and medical history characteristics of children conceived by fresh ET, frozen ET, and NC. All groups were similar according to age, gender, birth length, mode of feeding, current weight/length, rate of preterm delivery, blood pressure, heart rate, and maternal age. Children conceived by fresh ET had significantly lower gestational age and birth weight compared with NC, whereas no statistically significant differences were observed between frozen ET and NC. Maternal age in the NC group was significantly lower than in the fresh ET and frozen ET groups. Table 2 presents the physical examination and medical history characteristics of newborns born after fresh ET, frozen ET, and NC. All groups were similar according to gender, birth weight/length, gestational age, and maternal age.

The most critical period of fetal thyroid development is the first trimester of pregnancy (Figure 1A). We analyzed the maternal serum levels of E₂ during the first trimester in 206 patients after fresh ET, 124 patients after frozen ET, and 200 after NC (Figure 1B). In addition to the much higher level of E₂ upon hCG administration, the mean levels of serum E₂ of mothers in the fresh ET group were 4,351.3 ± 250.4 pmol/L at 4 weeks, 6,027.7 ± 529.7 pmol/L at 5 to 7 weeks, 7,305.2 ± 420.1 pmol/L at 8 weeks, and 9,691.9 ± 903.7 pmol/L at 9 to 12 weeks of gestation, which were all significantly higher than those in the frozen ET group (1,586.2 ± 126.9 pmol/L at 4 weeks, 3,020.3 ± 227.7 pmol/L at 5 to 7 weeks, 4,168.1 ± 238.1 pmol/L at 8 weeks, and 6,748.0 ± 482.2 pmol/L at 9 to 12 weeks, P <0.01, respectively), and those in NC group (1,427.9 ± 63.4 pmol/L at 4 weeks, 2,911.5 ± 249.4 pmol/l at 5 to 7 weeks, 3,968.3 ± 151.1 pmol/L at 8 weeks, and 6,310.5 ± 309.7 pmol/L at 9 to 12 weeks, P <0.01, respectively, Figure 1B). There was no significant difference between E₂ levels in the frozen ET and NC groups during the first trimester (Figure 1B).

We compared the serum levels of thyroid hormone profile between children conceived by fresh ET, frozen ET, and NC. Except for gestational age, birth weight, and maternal age, there were no significant differences in physical examination characteristics and medical history among the NC, fresh ET, and frozen ET groups (Table 1). Interestingly, in 3- to 10-year-old children conceived by fresh ET, the levels of T4, FT4, and TSH were significantly higher than those of children conceived by NC (crude P <0.01, Table 3). Although there was no significant difference, the levels of T3 and FT3 tended to be higher in the fresh ET group. The observation that frozen ET can avoid exposure to high maternal E₂ environment in IVF led us to compare the thyroid hormone profiles between the children conceived by fresh ET and frozen ET. We found that levels of T4 and TSH in the frozen ET group were significantly lower (crude P <0.01, Table 3) and nearer to the values of the NC group. In order to solidify the results, we further analyzed the relationship between type of conception and thyroid function with adjustment for age of child, ART type, and sex of child or maternal variables (pregnancy-induced hypertension, gestational diabetes mellitus, placenta previa, or intrahepatic cholestasis) by a regression model adjusting. We found that levels of T4, FT4, and TSH were still significantly higher than those of children conceived by NC (Adjusted P <0.01, Table 3). However, the levels of T4 and TSH in the frozen ET group were significantly lower than fresh ET (Adjusted P <0.01, Table 3).

We further calculated and compared the rate of 3- to 10-year-old children whose serum T4, FT4, or TSH levels were beyond the standard values. Two out of 357 children conceived by fresh ET (0.6%) were found to have T4 levels exceeding 150.84 nmol/L, while none was detected in the NC group. FT4 exceeding 19.05 pmol/L was present in 26 of 357 children conceived by fresh ET (7.3%) and in none in the NC group (P <0.01, Table 4). TSH exceeding 5.05 mIU/L was present in 22 of 357 children conceived by fresh ET (6.2%) and in 3 of 380 of the NC group (0.08%) (P <0.01, Table 4). Further, one child showed both elevated FT4 (>19.05 nmol/L) and TSH (>5.05 mIU/L) in the fresh ET group, whereas none did so in either the frozen ET or NC groups. Interestingly, no significant difference was found between serum TSH levels in the 3- to 10-year-old children conceived by frozen ET and those by NC (2.4% vs. 0.08%). Although the incidence of elevated FT4 in the frozen ET group (4.7%) was still significantly higher than that of the NC group (P <0.01, Table 4), compared to fresh ET, a decreased tendency of risk was observed in the frozen ET group.

Additionally, we examined the levels of T4, FT4, T3, FT3, and TSH in umbilical blood in 183 newborns, including 55 fresh ET cases, 48 frozen ET cases, and 80 NC cases. There were no significant differences in physical examination characteristics and medical history among the three groups (Table 2). Surprisingly, in newborns conceived by fresh ET, the levels of T4, FT4, and TSH were significantly higher than those of children conceived by NC (crude P <0.01, P <0.05, and P <0.01, respectively, Table 5). However, the levels of T4 and TSH in the frozen ET group were significantly lower than in the fresh ET group (crude P <0.05, Table 5), which were similar to values in the NC group. No significant differences in the levels of T3 and FT3 were found among the three groups (crude P >0.05, respectively). Moreover, we further analyzed the relationship between type of conception and thyroid function with adjustment for sex. We found that levels of T4, FT4, and TSH were also significantly higher than those in newborns conceived by NC (Adjusted P <0.01, P <0.05, and P <0.05, respectively, Table 5). However, levels of T4 and TSH in the frozen ET group were significantly lower compared with fresh ET (Adjusted P <0.05, Table 5). There was no significant difference between frozen ET and NC.

To further explore the relationship between maternal serum E₂ levels and the thyroid hormone profile, a total cohort of 55 newborn singletons born after fresh ET and their mothers were investigated further. We examined the serum E₂ levels on the day of hCG administration in these patients. We adopted bivariate correlation analysis to investigate the correlation between thyroid hormones and E₂, and found that the maternal serum E₂ levels on the day of hCG administration positively correlated with levels of T4 and FT4 of their offspring (r = 0.52, P < 0.01 and r = 0.47, P < 0.01, respectively; Figure 2A,B).