Fetal exposure to phytoestrogens—The difference in phytoestrogen status between mother and fetus

Abstract

The goal of this study was to investigate fetal exposure to phytoestrogens, estrogenic compounds derived from plants, by measuring serum concentrations of phytoestrogens in maternal and cord blood. This study included 51 mothers scheduled for cesarean section (C-section), to obtain the serum of mother and fetus at almost the same time. Serum concentrations of phytoestrogens, including genistein (Gen), daidzein (Dai), coumestrol (Cou), equol (Equ; a metabolite of Dai), and sulfate-conjugated Gen, were measured in maternal and cord blood samples by LC-MS/MS and HPLC. It was found that phytoestrogens were transferred from mother to fetus. The detection rates of Gen, Dai, Equ, and Cou in cord serum were 100%, 80%, 35%, and 0%, respectively. Levels of Gen and Dai were higher in cord than in maternal serum (mean=19.4 ng/ml vs.7.2 ng/ml and 4.3 ng/ml vs.1.8 ng/ml for Gen and Dai, respectively). However, a reverse pattern was seen for Equ (cord mean=0.9 ng/ml, maternal mean=2.0 ng/ml). The correlations were significant between the concentration levels of Gen and Dai, Gen and Equ, and Gen and Dai plus Equ in cord serum. However, in maternal serum, the correlations were weak. Also, in 8 of 10 cord serum samples, sulfate-conjugated Gen was detected (mean=5.2 ng/ml, standard deviation=4.7), but it was detected from only one maternal serum (8.7 ng/ml). This study demonstrates placental transfer of phytoestrogens from mother to fetus. It is suggested that the metabolic and/or excretion rates of phytoestrogens are different between mother and fetus and once phytoestrogens are transferred to the fetus, they tend to stay in the fetal side longer than in the maternal side. While the implications for the health and development of the newborn are not known, these data suggest that the effects of fetal exposure to phytoestrogens should be studied further.

Introduction

It is well known that many plants contain natural estrogenic compounds, called phytoestrogens (Price and Fenwick, 1985). Soy is one of the major sources of phytoestrogens and it is also an important part of the food culture in Asian countries. Traditional Asian foods, rich in vegetables and legumes, have been gaining favor in the West and an increasing number of Westerners are accepting vegetable proteins instead of animal proteins (Messina and Messina, 1991; North et al., 2000; Price and Fenwick, 1985). Isoflavone tablets are taken as a supplement by young women at reproductive age, and in the United States, 20–25% of newborns are fed soy formula milk at some time during infancy (Badger et al., 2002; Barrett, 2002).

There are several reports on the beneficial effects of phytoestrogens on human health, such as preventing breast, prostate, colon, and endometrial cancer (Adlercreutz, 1998, Adlercreutz, 2002; Adlercreutz et al., 1991; Fritz et al., 1998; Horn-Ross et al., 2003; Lamartiniere, 2000; Lamartiniere et al., 1998; Mazur and Adlercreutz, 2000; Sarkar and Li, 2003; Wang et al., 2002) or lowering cholesterol and reducing blood pressure (Rivas et al., 2002; Sirtori et al., 1977) and improving menopausal symptoms such as hot flashes (Adlercreutz et al., 1992). However, other authors argue that there is actually little beneficial effect of phytoestrogens (Keinan-Boker et al., 2004; Nikander et al., 2003; Register et al., 2003).

Recently, even adverse health effects of phytoestrogen exposure during the fetal and/or postnatal period have been reported. North et al. reported a significantly higher incidence rate of hypospadias among male offspring of vegetarian women compared to those of nonvegetarian women (North et al., 2000). In Puerto Rico, premature thelarche was associated with soy-based formula (Freni-Titulaer et al., 1986). In animal experiments fetal exposure to phytoestrogen has been associated with uterine adenocarcinoma (Newbold et al., 2001) and multioocyte follicles in mice (Jefferson et al., 2002). Neonatal treatment of female rats with phytoestrogens caused defeminization of the brain (Kouki et al., 2003). Wisniewski et al. reported that perinatal exposure to genistein (Gen; a phytoestrogen derived mainly from soy bean) caused long-term dysfunction in reproductive behavior in rats (Wisniewski et al., 2003). Our recent study reported altered gene expression without morphological change in the testis after neonatal exposure to Gen in mice (Adachi et al., 2004), and neonatal exposure to Gen was reported to affect male reproductive organs at the molecular level in adulthood (Shibayama et al., 2001). Concerns have arisen over exposing fetuses and newborns to high levels of phytoestrogens, since they are more sensitive to estrogenic compounds than adults (Barrett, 2002; Bocquet et al., 2001; Hughes et al., 2004; Irvine et al., 1998; Setchell et al., 1997; Sheehan, 1998).

However, there are few studies assessing human fetal exposure to phytoestrogens from umbilical cord plasma (Adlercreutz et al., 1999) or amniotic fluid (Foster et al., 2002), and little is known about the correlation between the concentration levels of phytoestrogens in mother and fetus.

In the current study, we compared phytoestrogen levels in maternal and umbilical cord serum. Cord serum reflects phytoestrogen concentration levels in fetus because the umbilical cord is a part of the fetus and cord serum is a part of the fetal circulation. The phytoestrogens analyzed in this study were Gen, daidzein (Dai; a phytoestrogen derived mainly from soybean), equol (Equ; a metabolite of Dai), and coumestrol (Cou; a phytoestrogen derived mainly from sprouts).

Phytoestrogens exist as free or sulfate- or glucuronic acid-conjugated forms in human serum (Shelnutt et al., 2002). The free form has estrogenic activity and the sulfate-conjugated forms are easily deconjugated to the free form. Therefore, free and sulfate-conjugated forms are estimated to be estrogenic in human serum (Shelnutt et al., 2002). The glucuronic acid-conjugated form loses its estrogenic activity (Shelnutt et al., 2002; Zhang et al., 1999). In comparison with estradiol, several studies using competition binding assays or a gene expression assay with a yeast system show that the free form of genistein has an affinity 10–1000-fold lower than that of estradiol (Kuiper et al., 1998; Morito et al., 2001, Morito et al., 2002).

To estimate how much estrogenically active phytoestrogen can be transferred from mother to fetus, sulfate-conjugated Gen was analyzed and measured from the cord and maternal serum of partial subjects. There are two conjugation sites on Gen, which are the 4′ and 7 positions (Shelnutt et al., 2002), hence these two sulfate-conjugated forms were measured in this study.

The first objective of the present study was to investigate whether phytoestrogens are transferred from mother to fetus. If so, the second and third objectives were to determine how much of the phytoestrogen was transferred and what would be the correlation between concentrations in maternal and fetal serum. Further, the fourth objective was to examine if there was a difference in the concentration levels of phytoestrogens between male and female newborns, as was reported for adults (Lu and Anderson, 1998). The fifth objective was to analyze if there was a correlation between the concentration level and the birth weight of the newborns, as was reported for bisphenol A and birth weight (Schonfelder et al., 2002). The sixth objective was to estimate the amounts of estrogenic-active free and sulfate-conjugated forms in the fetus. Also, there are two groups with regard to Equ production. One is the “equol producers” and the other is the “non-equol producers” (Rowland et al., 2000; Setchell et al., 2002; Watanabe et al., 1998). The last objective was to analyze how much equol was transferred to the fetus.

Only women delivering by scheduled cesarean section (C-section) were chosen as subjects in this study in order to obtain blood of mothers and fetuses at almost the same time. However, since all subjects were requested to fast for at least 15 h before C-section (and hence sampling), the concentration levels of food-derived phytoestrogens were low. Therefore, to estimate the concentration of phytoestrogens in Japanese adults, the serum of adult volunteers were also analyzed.