The hypothesis that perinatal factors affect breast cancer risk was formally articulated in the early 1990s . Since then, the accumulated evidence has linked two important perinatal factors with this risk, namely birth size [2–5] and pregnancy toxemia . The underlying biological mechanisms are not known, but the endocrine environment during the perinatal period is thought to influence the risk of breast cancer in adulthood [6–10]. Our goal was to harvest information on early life endocrine factors which could account for the sharply higher incidence of breast cancer among Caucasian women in United States compared with Chinese women in China. To that end, we have determined levels of hormones with mammotropic potential in the maternal serum as well as the cord blood, in pregnancies of Caucasian women in Boston, USA and Chinese women in Shanghai, China. Although some results from this project have been previously reported [6, 11, 12], this paper presents new data on several additional hormones as well as an integrated picture of maternal and cord blood hormone levels in the two populations with contrasting breast cancer incidence.
From March 1994 to October 1995, all pregnant women coming for their first routine prenatal visit to the collaborating maternity clinics of the Beth Israel hospital in Boston, USA, and the Shanghai Medical University in China were met by authorized health professionals, who ascertained the woman’s eligibility to participate, explained to her the objectives of the study and obtained informed consent . A total of 402 Caucasian women in Boston, USA, and 424 Asian women in Shanghai, China, were identified. The study was approved by the Institutional Review Boards of the Beth Israel Hospital, Shanghai Medical University and Harvard School of Public Health. Eligibility criteria included age <40 years old, a maximum parity of two, absence of a prior diagnosis of diabetes mellitus or thyroid disease, no hormonal medication during the index pregnancy and no known fetal abnormality. The maximum parity of two criterion was imposed by the one-child policy implemented in China and the need for comparability of the two cohorts.
Of the 402 eligible women in Boston, 77 refused to participate in one or more aspects of the study, 9 were subsequently excluded because of a spontaneous or induced abortion in the index pregnancy, 2 were excluded because of twin birth, whereas 10 were lost to follow-up after the initial meeting. Of the remaining 304 women, we excluded 35 women with gestational age below 37 or above 42 weeks, 16 additional women with pregnancy toxemia and another 12 women with missing information. Eventually, 241 Caucasian women were considered in the present analysis. Additional exclusions were necessary due to limited availability of biological samples for some pregnancies.
Of the 424 eligible women in Shanghai, 15 refused to participate in one or more aspects of the study, 2 women were excluded owing to induced abortion in the index pregnancy and another two because of twin birth, whereas 7 women were lost to follow-up leaving a total of 398 women. For 59 of those, no blood collection was accomplished. Of the remaining 339 women, we excluded 44 women who had gestation duration outside the range of 37–42 weeks inclusive. There were no Asian women with preeclampsia. Eventually, 295 Asian women were considered in the present analysis. Additional exclusions were necessary due to limited availability of biological samples for some pregnancies. It is noteworthy that, although exclusions were numerous, they were done for technical or administrative reasons, so their consequence is reduction of statistical power but not introduction of bias. Exclusions that could theoretically affect validity were those generated by losses to follow-up and these were minimal (2.5% for women in Boston and 1.7% for women in Shanghai).
Baseline sociodemographic and lifestyle information was recorded in interviews at the 16th and the 27th gestational week visit of the women to the clinic. Information about medical conditions was abstracted from the women’s medical records. At delivery, additional information concerning the newborn, including duration of gestation and birth size parameters, was recorded. Detailed information concerning the study protocol and implementation has been published .
During the visits to the maternity clinics at the 16th and the 27th gestational week, 10 ml of venous blood was drawn from every woman at each visit. At delivery, cord blood was also collected. All blood samples were collected into sterile tubes without preservatives and stored at −4°C for up to 24 h until centrifugation. Samples were then centrifuged, and the serum was aliquoted and stored for hormonal assays at −80°C. In Shanghai, blood samples were transported in a cooler to a laboratory near Shanghai Medical University. Serum aliquots were stored at −20°C for 5–7 days in the laboratory before being transported to Shanghai Medical University and stored at −80°C. All samples were shipped by air on dry ice to Boston where they were stored at −80°C together with the samples from Boston.
Levels of estradiol (E2), estriol (E3), progesterone, sex hormone-binding globulin (SHBG), testosterone, adiponectin, insulin-like growth factor 1 (IGF-1) and insulin-like growth factor-binding protein 3 (IGFBP3) were measured in both the maternal sera and the cord blood. Prolactin was measured only in maternal sera and insulin-like growth factor 2 (IGF-2) only in cord blood.
Measurements were conducted in two time periods. Maternal levels of E2, E3, SHBG, progesterone and prolactin were measured in the late 1990s at the Department of Clinical Chemistry of the Uppsala University Hospital in Sweden , whereas maternal levels of testosterone, adiponectin, IGF-1 and IGFBP3 as well as all hormone levels in cord blood were measured in 2006 at the ILAT Steroid RIA Laboratory of the University of Massachusetts Medical School. Measurements per each hormone were conducted simultaneously for samples from Boston and Shanghai.
Maternal estradiol-17b was measured with a time-resolved competitive solid-phase fluoroimmunoassay (AutoDELFIA Estradiol Kit; Wallac Oy, Turku, Finland), with laboratory imprecision 4.6% ± 0.8%. Maternal unconjugated E3 was measured with a similar time-resolved competitive solid-phase fluoroimmunoassay method (AutoDELFIA Unconjugated Oestriol Kit; Wallac Oy), with laboratory imprecision 8.0% ± 1.8%. Maternal SHBG was measured with a time-resolved noncompetitive solid-phase sandwich fluoroimmunoassay (AutoDELFIA SHBG Kit; Wallac Oy), with laboratory imprecision 4.8% ± 1.3%. Maternal progesterone was measured with a time-resolved competitive solid-phase fluoroimmunoassay (AutoDELFIA Progesterone Kit; Wallac Oy), with laboratory imprecision 1.7% ± 0.9%. Maternal prolactin was measured with a time-resolved noncompetitive solid-phase sandwich fluoroimmunoassay (AutoDELFIA Prolactin Kit; Wallac Oy), with laboratory imprecision 3.0% ± 0.3%. Maternal and cord blood testosterone was measured by radioimmunoassay kits from Diagnostic Products Corporation (DPC, Los Angeles, CA), with inter- and intra-assay coefficients of variation (CVs) of 8.9% and 5.6%, respectively. Maternal and cord blood adiponectin was measured by radioimmunoassay (Linco Research, St. Charles, MO) with inter- and intra-assay CV of 7.2% and 4.7%, respectively. Maternal and cord blood IGF-1 and IGFBP3 as well as cord blood IGF-2 were measured by coated-tube immunoradiometric assay kits (Diagnostic System Laboratories, Inc., Webster, TX). The laboratory-estimated inter- and intra-assay CVs were, respectively, 9.0% and 3.3% for IGF-1; 8.0% and 4.8% for IGFBP3 and 5.9% and 3.4% for IGF-2. There was no detectable cross-reactivity of the IGF-1 assay with IGF-2 according to the manufacturer’s specificity assessment. Cord blood E2 was measured by radioimmunoassay using kits from DPC (Los Angeles, CA). The inter-assay CV was 6.8% and the intra-assay CV was 3.4%. Cord blood unconjugated E3, progesterone and SHBG were measured using chemiluminescent immunoassay methodologies from DPC (Los Angeles, CA). The inter-assay CVs were 9.2%, 7.9% and 4.8% and the intra-assay CVs were 6.6%, 6.3% and 2.0%, respectively.
The characteristics of women and their singleton offspring in Boston, MA, USA, and Shanghai, China are shown in Table 1. In comparison to women in Boston, women in Shanghai were younger, of shorter stature, with lower prepregnancy BMI and generally primiparous (on account of the one-child policy in China), whereas their offspring were more frequently boys and of lower birth weight. These factors were accounted for in comparisons of hormones between centers.