Phytoestrogen Content Research Articles

Phytoestrogen content of subterranean clover related to ewe growth and reproduction

Phytoestrogen content of subterranean clover related to ewe growth and reproduction

Impact of the Phytoestrogen Content of Laboratory Animal Feed on the Gene Expression Profile of the Reproductive System in the Immature Female Rat

The effect of the dietary background of phytoestrogens on the outcome of rodent bioassays used to identify and assess the reproductive hazard of endocrine-disrupting chemicals is controversial. Phytoestrogens, including genistein, daidzein, and coumestrol, are fairly abundant in soybeans and alfalfa, common ingredients of laboratory animal diets. These compounds are weak agonists for the estrogen receptor (ER) and, when administered at sufficient doses, elicit an estrogenic response in vivo. In this study, we assessed the potential estrogenic effects of dietary phytoestrogens at the gene expression level, together with traditional biologic end points, using estrogen-responsive tissues of the immature female rat. We compared the gene expression profile of the uterus and ovaries, as a pool, obtained using a uterotrophic assay protocol, from intact prepubertal rats fed a casein-based diet (free from soy and alfalfa) or a regular rodent diet (Purina 5001) containing soy and alfalfa. Estrogenic potency of the phytoestrogen-containing diet was determined by analyzing uterine wet weight gain, luminal epithelial cell height, and gene expression profile in the uterus and ovaries. These were compared with the same parameters evaluated in animals exposed to a low dose of a potent ER agonist [0.1 μg/kg/day 17α-ethynyl estradiol (EE) for 4 days]. Exposure to dietary phytoestrogens or to a low dose of EE did not advance vaginal opening, increase uterine wet weight, or increase luminal epithelial cell height in animals fed either diet. Although there are genes whose expression differs in animals fed the soy/alfalfa-based diet versus the casein diet, those genes are not associated with estrogenic stimulation. The expression of genes well known to be estrogen regulated, such as progesterone receptor, intestinal calcium-binding protein, and complement component 3, is not affected by consumption of the soy/alfalfa-based diet when assessed by microarray or quantitative reverse transcriptase–polymerase chain reaction analysis. Our results indicate that although diet composition has an impact on gene expression in uterus and ovaries, it does not contribute to the effects of an ER agonist.

Determination of phytoestrogens in traditional medicinal herbs using gas chromatography–mass spectrometry

Quantitative analytical methods for the 17 phytoestrogens containing isoflavonoids, lignans, and mycoestrogens in herbs were developed, and the amount of phytoestrogens was determined in 22 traditional medicinal herbs. The focus of this study was to simplify the purification procedure for removing many kinds of interferences in herbs, and to select adequate derivatization reagent for getting desirable selectivity and sensitivity in the quantitative determination of phytoestrogens. To satisfy these goals, we performed a solid-phase extraction with Oasis HLB cartridges following enzymatic and acidic hydrolysis, and we used the mixture of MSTFA/NH 4I/DTE (1000:4:5, v/w/w) to form TMS derivatives of phytoestrogens. Overall recovery was more than 84% in all of the phytoestrogens, and the limit of quantification for phytoestrogens in herbs were set at 0.2 μg/g. Coefficient of variation percentages were in the range of 0.18–15.68% (within-day) and 0.23–16.61% (day-to-day), respectively. Most of the isoflavonoids and lignans were found in all of the herbs, but mycoestrogens were not detected at all. The Leguminosae family proved to be the richest source of isoflavonoids. Lignans such as enterodiol and enterolactone were detected at low concentration in most of the herbs. These results indicate that this assay is accurate and reliable for the determination of phytoestrogens in herbs. Also, information regarding the phytoestrogen contents in traditional medicinal herbs is useful in the prevention and treatment of chronic diseases such as cancer, osteoporosis, dementia, and cardiovascular disease.

Plasma Phytoestrogens Are Not Altered by Probiotic Consumption in Postmenopausal Women with and without a History of Breast Cancer

Soy phytoestrogens were suggested to reduce the risk of a number of diseases including breast cancer. Given that these compounds are metabolized by bacteria, alteration of intestinal bacteria and enzymes may affect phytoestrogen metabolism. We hypothesized that probiotics, when consumed with soy protein, would increase plasma isoflavones, as well as equol producer frequency, in postmenopausal women. We further hypothesized that these effects would differ between women who have had breast cancer and women who have not. To test these hypotheses, 20 breast cancer survivors and 20 controls completed four 6-wk treatments in a randomized, crossover design: supplementation with soy protein (S) (26.6 +/- 4.5 g protein, 44.4 +/- 7.5 mg isoflavones/d); soy + probiotics (S+P) (10(9) colony-forming units Lactobacillus acidophilus DDS+1 and Bifidobacterium longum, 15-30 mg fructooligosaccharide/d); milk protein (M) (26.6 +/- 4.5 g protein/d); and milk + probiotics (M+P). Plasma phytoestrogen concentrations did not differ between controls and survivors, although genistein tended to be lower in survivors at baseline (P = 0.15), and during soy (P = 0.16) and milk protein (P = 0.16) consumption. As expected, soy consumption increased plasma phytoestrogen concentrations (P < 0.0001). Plasma phytoestrogen concentrations and the number of equol producers did not differ between the S and S+P diets. At the same time, plasma equol concentrations as well as urinary equol excretion in 2 subjects were more than 7-fold different between the 2 diets. These results indicate that this particular probiotic supplement does not generally affect plasma isoflavones, although the large differences between plasma and urinary equol in some subjects suggest that equol producer status may be modifiable in some individuals.

Cumulative Dietary Energy Intake Determines the Onset of Puberty in Female Rats

Laboratory animal diets for studies to determine the endocrine-disrupting potential of chemicals are under scrutiny because they can affect both assay control values and assay sensitivity. Although phytoestrogen content is important, we have previously shown that a phytoestrogen-rich diet and a phytoestrogen-free diet were equally uterotrophic to rats and advanced vaginal opening (VO) when compared with the standard diet RM1. Abolition of the effects by the gonadotrophin-releasing hormone antagonist Antarelix indicated that these effects were mediated through the hypothalamus–pituitary–reproductive organ axis. In the present study, we investigated the relationship between cumulative energy intake and sexual maturation in female rats. Infant formula (IF) at different concentrations and synthetic diets, with a wide range of metabolizable energy (ME) values, were used to modulate energy intake. Increasing energy intake was associated with an increase in uterine weight (absolute and adjusted for body weight) for both IF and the synthetic diets. In both cases, the increased uterine weight was directly proportional to energy intake. Body weight was unaffected by IF consumption but, in the case of the diets, was increased proportionally with energy consumption. Antarelix abolished the uterine weight increases with both formula and the diets, whereas body weight was unaffected. The mean day of VO was also advanced by high-ME diets and IF, whereas body weight at VO was unaffected. VO occurred at an energy intake of approximately 2,300 kJ/rat determined by measuring total food intake from weaning to VO, indicating that this cumulative energy intake was the trigger for puberty. ME is therefore a critical factor in the choice of diets for endocrine disruption studies.

Phytoestrogen Concentrations in Serum and Spot Urine as Biomarkers for Dietary Phytoestrogen Intake and Their Relation to Breast Cancer Risk in European Prospective Investigation of Cancer and Nutrition-Norfolk

Abstract Subjects of this study consisted of 333 women (aged 45–75 years) drawn from a large United Kingdom prospective study of diet and cancer, the European Prospective Investigation of Cancer and Nutrition-Norfolk study. Using newly developed gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry methods incorporating triply 13C-labeled standards, seven phytoestrogens (daidzein, genistein, glycitein, O-desmethylangolensin, equol, enterodiol, and enterolactone) were measured in 114 spot urines and 97 available serum samples from women who later developed breast cancer. Results were compared with those from 219 urines and 187 serum samples from healthy controls matched by age and date of recruitment. Dietary levels were low, but even so, mean serum levels of phytoestrogens were up to 600 times greater than postmenopausal estradiol levels. Phytoestrogen concentrations in spot urine (adjusted for urinary creatinine) correlated strongly with that in serum, with Pearson correlation coefficients &amp;gt; 0.8. There were significant relationships (P &amp;lt; 0.02) between both urinary and serum concentrations of isoflavones across increasing tertiles of dietary intakes. Urinary enterodiol and enterolactone and serum enterolactone were significantly correlated with dietary fiber intake (r = 0.13–0.29). Exposure to all isoflavones was associated with increased breast cancer risk, significantly so for equol and daidzein. For a doubling of levels, odds ratios increased by 20–45% [log2 odds ratio = 1.34 (1.06–1.70; P = 0.013) for urine equol, 1.46 (1.05–2.02; P = 0.024) for serum equol, and 1.22 (1.01–1.48; P = 0.044) for serum daidzein]. These estimates of risk are similar to those established for estrogens and androgens in postmenopausal breast cancer but need confirmation in larger studies.

A pilot study of phytoestrogen content of soy foods and traditional Chinese medicines for women's health in Hong Kong

In view of the possible health benefits of phytoestrogens, a pilot study was carried out to quantitate the phytoestrogen content of soy foods and tea commonly consumed in Hong Kong, and also of traditional Chinese medicinal (TCM) products that are prescribed for menopausal symptoms and diseases relating to the menopause. Assays of daidzein and genistein were carried out using high-performance liquid chromatography, after extraction procedures. The TCM products were found to contain phytoestrogen in quantities comparable with soy products. Moreover, certain types of Chinese tea contained large quantities of phytoestrogens in the leaves, but also yielded comparable quantities in the infusion for drinking. The phytoestrogen content of these TCM may provide a scientific basis for their actions. However, clinical efficacy can only be determined by clinical trials.

Identification and concentration of soy phytoestrogens in commercial dog foods.

To identify and determine the concentrations of phytoestrogens in commercial dog foods. 24 commercial dog foods, including 12 moist or dry extruded commercial dog foods that contained soybeans or soybean fractions and 12 foods without any soybean-related ingredients listed on the label. Foods were analyzed for phytoestrogen content, including 4 isoflavones (genistein, glycitein, daidzein, and biochanin A), 1 coumestan (coumestrol), and 2 lignans (secoisolariciresinol and matairesinol) by use of acid-methanol hydrolysis and high-pressure liquid chromatography with UV-absorbance detection. Phytoestrogens were identified and quantified by reference to authentic standards. Isoflavones, coumestans, and lignans were undetectable in diets that did not list soybean-related ingredients on the label. Only 1 of the 12 diets that included soybean or soybean fractions had undetectable concentrations of phytoestrogens and that product contained soy fiber. The major phytoestrogens were the isoflavones daidzein (24 to 615 microg/g of dry matter) and genistein (4 to 238 microg/g of dry matter). Soybean and soybean fractions are commonly used ingredients in commercial dog foods. Dietary intake of phytoestrogens may have both beneficial and deleterious health effects. Our results indicated that certain commercial dog foods contain phytoestrogens in amounts that could have biological effects when ingested long-term.

New data regarding phytoestrogens content in bovine milk

This study investigates the occurrence of the main phytoestrogens in various commercial bovine milk samples, representing the main foodstuff of a critical population. The results show the presence of isoflavones at trace levels (i.e. in the 0.1–5.0 μg l −1 range) in most of the samples, and the presence of equol and enterolactone in all analyzed samples at relatively high concentrations (14.1–293 and 14.3–94.4 μg l −1, respectively). The consequent potential estrogenic effect on specific populations of children could be questioned. New data are also provided regarding first the correlation between the concentrations of the different isoflavones and second the influence of the animal feedstuff on the milk phytoestrogen content.

Selecting the appropriate rodent diet for endocrine disruptor research and testing studies.

Selecting the optimum diet for endocrine disruptor (ED) research and testing studies in rodents is critical because the diet may determine the sensitivity to detect or properly evaluate an ED compound. Dietary estrogens can profoundly influence many molecular and cellular event actions on estrogen receptors and estrogen-sensitive genes. The source, concentration, relative potency, and significance of dietary estrogens in rodent diets are reviewed, including dietary factors that focus specifically on total metabolizable energy and phytoestrogen content, which potentially affect ED studies in rodents. Research efforts to determine dietary factors in commercially available rodent diets that affect uterotrophic assays and the time of vaginal opening in immature CD-1 mice are summarized. A checklist is provided of important factors to consider when selecting diets for ED research and testing studies in rodents. Specific metabolizable energy levels are recommended for particular bioassays. Discussions include the between-batch variation in content of the phytoestrogens daidzein and genistein, the effects of total metabolizable energy and phytoestrogens on the timing (i.e., acceleration) of vaginal opening, and increased uterine weight in immature CD-1 mice. It is concluded that rodent diets differ significantly in estrogenic activity primarily due to the large variations in phytoestrogen content; therefore animal diets used in all ED studies should ideally be free of endocrine-modulating compounds.

Serum phytoestrogens and prostate cancer risk in a nested case-control study among Japanese men.

The purpose of this study was to examine whether a high serum concentration of phytoestrogens reduces the risk of prostate cancer in a case-control study nested in a community-based cohort in Japan (Japan Collaborative Cohort (JACC) Study). Information on lifestyles and sera of the subjects were collected in 1988-90, and they were followed up to 1999. Incident and dead cases of prostate cancer and controls were matched for study area and age. Phytoestrogens and sex hormones in sera stored at - 80 degrees C were measured in 2002. Of 14,105 male subjects of the cohort who donated their sera, 52 cases and 151 controls were identified. Three datasets were analyzed; 1) all subjects, 2) 40 cases and 101 controls after excluding subjects with low testosterone levels who were suspected of having had medical intervention, and 3) 28 cases and 69 controls with prostate specific antigen level of </= 10.0 ng/ml. The odds ratio (OR) for the highest level to the lowest was 0.38 (95% confidence interval (CI); 0.13, 1.13) for genistein, 0.41 (0.15, 1.11) for daidzein, and 0.34 (0.11, 1.10) for equol for the second dataset. Genistein and daidzein showed similar findings in the third one. Equol and equol/daidzein ratio showed consistent findings in all three datasets (OR = 0.39, 95% CI; 0.13, 0.89, trend P = 0.02 for the first dataset). Their effects seemed to be independent of serum sex hormones. In conclusion, serum genistein, daidzein, and equol seemed to dose-dependently reduce prostate cancer risk.

Dietary influence on the impact of ethinylestradiol-induced alterations in the endocrine/reproductive system with perinatal maternal exposure

We investigated the effects of two diets, differing in phytoestrogen contents, on the phenotypic changes induced in the endocrine/reproductive system by perinatal exposure to an estrogen agonist during a critical period for brain sexual differentiation in rats. Ethinylestradiol (EE) was mixed at a concentration of 0.5 ppm into two diets: CRF-1, a standard rodent diet containing soybean-derived phytoestrogens; and a soy-free (SF) diet. These diets were provided to maternal Sprague–Dawley rats during gestational day 15 to postnatal day 10. Growth suppression of offspring was evident with EE especially during the exposure period and was slightly enhanced with the SF diet. On the other hand, most of the female offspring exposed to EE with CRF-1 showed early onset of vaginal opening, strong irregularity in estrous cycle (persistent estrus) and profound histopathological alterations, such as multifollicular ovaries, endometrial hypertrophy, and diffuse hyperplasia of the anterior pituitary. These EE-induced changes were much less pronounced with the SF diet. The results thus demonstrated differential effects of perinatal EE depending on the basal diet used, with enhancement of typical estrogenic responses in females by potential soybean-derived factor(s).

Hormonal Profile And Histopathological Study On The Influence Of Silymarin On Both Female And Male Albino Rats

Silymarin is a mixture of flavonoids extracted from seeds of milk thistle (Silybum marianum [L] Gaertn ). Extracts of S. marianum have been used in the treatment of liver diseases for more than 2000 years. Flavonoids are a large group of polycyclic phenols of plant origin. Flavonoids displaying estrogenic effects (phytoestrogen). Phytoestrogens show an estrogenic activity owing to the structural similarity with the estrogen skeleton ,mimicking natural estrogens, it binds to estrogen receptor and modulates its activity . These effects cause complex changes inducing a shift in an overall hormonal balance of the individual. Long term intake of feeds with a higher content of phytoestrogens can induce transient or permanent infertility. Silybum marianum leaves have been traditionally used in Egypt for their antifertility effects .Also, the antifertility property of silybum marianum leaves was investigated in female albino rats. This property highlights the impotance to clarify the role of silymarin (420mg /day ) a human therapeutic dose on both female and male albino rats. Silymarin (151.2 mg/kg BW) prevented pregnancy in adult female albino rats on days 1-5 post coitum. In female rats the follicle stimulating hormone (FSH) displayed marked increase, while the luteinizing hormone (LH) and estradiol remained within normal levels. These results showed a distinct increase in number and size of ovarian growing follicles while the effect of silymarin on the uterine wall was in the form of hypertrophy of endometrial epithelium, also increase in the number of uterine glands. Ovaries taken from females treated with silymarin for one and two months contained a large number of growing follicles. In male rats treated with silymarin (151.2 mg/kg BW) for one month, testosterone and LH were increased significantly, meanwhile estradiol not changed. Testes of rats treated with silymarin for one month showed well developed spermatogenesis with lot of sperms. The epididymis of the same group contained a heavy number of sperms. While rats treated with silymarin for two months displayed significant decrease in serum testosterone levels, while LH and estradiol not changed. These rats displayed some tubules contained well developed spermatogenic layer and normal leyding cells, others appeared moderated by affected. The epididymis contained less number of sperms. These results indicated that silymarin (151.2mg /kgBW) a dose equivalent to human therapeutic dose (420mg/ day) prevented pregnancy in female rats and caused some histological changes in the ovary and uterus, while it has biological benefits for male rats during short treatment.

Hormonal Profile And Histopathological Study On The Influence Of Silymarin On Both Female And Male Albino Rats

Silymarin is a mixture of flavonoids extracted from seeds of milk thistle (Silybum marianum [L] Gaertn ). Extracts of S. marianum have been used in the treatment of liver diseases for more than 2000 years. Flavonoids are a large group of polycyclic phenols of plant origin. Flavonoids displaying estrogenic effects (phytoestrogen). Phytoestrogens show an estrogenic activity owing to the structural similarity with the estrogen skeleton ,mimicking natural estrogens, it binds to estrogen receptor and modulates its activity . These effects cause complex changes inducing a shift in an overall hormonal balance of the individual. Long term intake of feeds with a higher content of phytoestrogens can induce transient or permanent infertility. Silybum marianum leaves have been traditionally used in Egypt for their antifertility effects .Also, the antifertility property of silybum marianum leaves was investigated in female albino rats. This property highlights the impotance to clarify the role of silymarin (420mg /day ) a human therapeutic dose on both female and male albino rats. Silymarin (151.2 mg/kg BW) prevented pregnancy in adult female albino rats on days 1-5 post coitum. In female rats the follicle stimulating hormone (FSH) displayed marked increase, while the luteinizing hormone (LH) and estradiol remained within normal levels. These results showed a distinct increase in number and size of ovarian growing follicles while the effect of silymarin on the uterine wall was in the form of hypertrophy of endometrial epithelium, also increase in the number of uterine glands. Ovaries taken from females treated with silymarin for one and two months contained a large number of growing follicles. In male rats treated with silymarin (151.2 mg/kg BW) for one month, testosterone and LH were increased significantly, meanwhile estradiol not changed. Testes of rats treated with silymarin for one month showed well developed spermatogenesis with lot of sperms. The epididymis of the same group contained a heavy number of sperms. While rats treated with silymarin for two months displayed significant decrease in serum testosterone levels, while LH and estradiol not changed. These rats displayed some tubules contained well developed spermatogenic layer and normal leyding cells, others appeared moderated by affected. The epididymis contained less number of sperms. These results indicated that silymarin (151.2mg /kgBW) a dose equivalent to human therapeutic dose (420mg/ day) prevented pregnancy in female rats and caused some histological changes in the ovary and uterus, while it has biological benefits for male rats during short treatment.

Phytoœstrogènes chez la femme ménopausée

Many women seek alternatives to hormone replacement therapy (HRT). Phytoestrogens are nonsteroidal compounds with estrogenic or antiestrogenic properties. Six of the 16 clinical trials suggest a significant reduction in alleviating symptoms but to a lesser degree than HRT. A meta-analysis showed that phytoestrogens improve lipid profile. The 14 recent clinical trials led, however, to divergent findings. Small clinical trials suggested a protective effect of phytoestrogens on bone metabolism. High concentration of phytoestrogens was associated with a reduction in breast cancer risk in case-control studies.

A model to estimate the oestrogen receptor mediated effects from exposure to soy isoflavones in food

The advantages that regular consumption of a diet containing soy may have on human health have been enshrined in a major health claim that has been approved by the Food and Drug Administration in the USA, regarding potential protection from heart disease by soy. This could have a major influence on the dietary consumption patterns of soy for consumers and lead to the development of soy enriched foods to enable consumers to achieve the benefits thought to be associated with increased soy consumption in a Western diet. If an increase in soy consumption is beneficial to particular disease conditions, there is always the possibility that there will be effects other than those that are desirable. For soy-containing foods there has been concern that the phytoestrogen content of soy, which is composed of several isoflavones, could be a separate health issue, due to the oestrogen-like activity of isoflavones. To address this, a method has been developed to estimate, relative to 17-β oestradiol, the activity of the common isoflavones present in soy phytoestrogens, based on their binding to and transcriptional activation of the major oestrogen receptor sub-types alpha and beta. Using this approach, the additional oestrogen-like activity that would be expected from inclusion of soy supplemented foodstuffs in a Western diet, can be determined for different sub-populations, who may have different susceptibilities to the potential for the unwanted biological effects occurring with consumption of soy enriched foods. Because of the theoretical nature of this model, and the controversy over the nature of whether some of the oestrogen-like effects of phytoestrogens are adverse, the biological effects of soy isoflavones and their potential for adverse effects in man, is also reviewed. The question that is critical to the long term safe use of foods enriched in soy is, which observed biological effects in animal studies are likely to also occur in man and whether these would have an adverse effect on human health.

Influence of Prenatal Diethylstilbestrol (DES) and dietary phytoestrogens on sperm function and reproductive organ development in male mice

Objective: The influence of exogenous estrogens on male reproductive parameters remains controversial. Prenatal estrogen exposure may influence male organ development and dietary phytoestrogens have been shown to exert physiologic estrogen activity, with the possible consequence of endocrine disruption. We sought to determine the effects of both in utero exogenous estrogen exposure and dietary phytoestrogen intake on male reproductive organ development and sperm physiology in mice.Design: Experimental animal study.Materials and Methods: In utero exposure of males was performed on pregnant mice fed corn oil with no DES (control), 0.1μg/kg/day DES (low-dose), and 50μg/kg/day DES (high-dose) on gestation days 11–17. Subsequent male pups (n=36) were then fed Purina 5001 (Diet A, 491mg/g diet phytoestrogens) or Purina 5002 (Diet B, 159 mg/g diet phytoestrogens) until sacrifice on postnatal day 90. Testis and epididymal blotted weights were obtained prior to sperm isolation from the cauda epididymis by swim-out after mincing. Morphology, sperm capacitation and acrosome reaction (AR) was evaluated. Cumulus masses from gonadotropin hyperstimulated females were inseminated with sperm at a concentration of 1 million motile sperm per ml for in vitro fertilization (IVF). Percent fertilization, defined as development to the 2-cell stage, and percent embryo development to blastocyst was recorded for each male. Outcome means were analyzed with ANOVA, as well as 2-way ANOVA to detect interactive effects, with p<0.05 considered significant.Results: In utero high-dose DES resulted in decreased testis and epididymal weight, but was not statistically significant unless combined testis/epididymal weight was analyzed (p=0.023). No significant DES effect was seen on sperm morphology, sperm capacitation or acrosome reaction (all p0.05). Fertilization (p=0.477) and embryo development (p=0.410) with IVF was also not adversely affected by in utero DES exposure. No diet effect was seen on epididymal or testis weights or sperm morphology (all p0.05). A decrease in spontaneous capacitation (p<0.001) and AR (p=0.012) was found in the higher phytoestrogen diet. Higher dietary phytoestrogen content also resulted in increased fertilization rates (p=0.014) but lower development to blastocyst (p=0.007). Two-way ANOVA analysis showed no interaction between DES exposure and dietary phytoestrogen content among measured outcomes.Conclusion: In utero high-dose DES exposure decreased combined epididymal/testis weight but had no influence on sperm function or in vitro fertilizing ability. Higher dietary phytoestrogen intake resulted in desirable differences in sperm capacitation and acrosome reaction, as well as improved rates of fertilization with IVF. No interaction between these two exposures existed. Objective: The influence of exogenous estrogens on male reproductive parameters remains controversial. Prenatal estrogen exposure may influence male organ development and dietary phytoestrogens have been shown to exert physiologic estrogen activity, with the possible consequence of endocrine disruption. We sought to determine the effects of both in utero exogenous estrogen exposure and dietary phytoestrogen intake on male reproductive organ development and sperm physiology in mice. Design: Experimental animal study. Materials and Methods: In utero exposure of males was performed on pregnant mice fed corn oil with no DES (control), 0.1μg/kg/day DES (low-dose), and 50μg/kg/day DES (high-dose) on gestation days 11–17. Subsequent male pups (n=36) were then fed Purina 5001 (Diet A, 491mg/g diet phytoestrogens) or Purina 5002 (Diet B, 159 mg/g diet phytoestrogens) until sacrifice on postnatal day 90. Testis and epididymal blotted weights were obtained prior to sperm isolation from the cauda epididymis by swim-out after mincing. Morphology, sperm capacitation and acrosome reaction (AR) was evaluated. Cumulus masses from gonadotropin hyperstimulated females were inseminated with sperm at a concentration of 1 million motile sperm per ml for in vitro fertilization (IVF). Percent fertilization, defined as development to the 2-cell stage, and percent embryo development to blastocyst was recorded for each male. Outcome means were analyzed with ANOVA, as well as 2-way ANOVA to detect interactive effects, with p<0.05 considered significant. Results: In utero high-dose DES resulted in decreased testis and epididymal weight, but was not statistically significant unless combined testis/epididymal weight was analyzed (p=0.023). No significant DES effect was seen on sperm morphology, sperm capacitation or acrosome reaction (all p0.05). Fertilization (p=0.477) and embryo development (p=0.410) with IVF was also not adversely affected by in utero DES exposure. No diet effect was seen on epididymal or testis weights or sperm morphology (all p0.05). A decrease in spontaneous capacitation (p<0.001) and AR (p=0.012) was found in the higher phytoestrogen diet. Higher dietary phytoestrogen content also resulted in increased fertilization rates (p=0.014) but lower development to blastocyst (p=0.007). Two-way ANOVA analysis showed no interaction between DES exposure and dietary phytoestrogen content among measured outcomes. Conclusion: In utero high-dose DES exposure decreased combined epididymal/testis weight but had no influence on sperm function or in vitro fertilizing ability. Higher dietary phytoestrogen intake resulted in desirable differences in sperm capacitation and acrosome reaction, as well as improved rates of fertilization with IVF. No interaction between these two exposures existed.

Quantification of isoflavones and lignans in serum using isotope dilution liquid chromatography/tandem mass spectrometry.

Phytoestrogens (isoflavones and lignans) are receiving increasing attention due to a potential protective effect against a number of complex diseases. However, in order to investigate these associations, it is necessary to accurately quantify the levels of phytoestrogens in foods and biological fluids. We report an assay for three isoflavones (daidzein, genistein, and glycitein), two metabolites of daidzein (O-desmethylangolensin and equol), and two lignans (enterodiol and enterolactone) in human serum using electrospray ionisation liquid chromatography/mass spectrometry (LC/MS) with selective reaction monitoring. A simple, highly automated sample preparation procedure requires only 200 microL of sample and utilises one solid-phase extraction stage. Limits of detection are in the region of 10 pg/mL for all analytes except equol, which had a limit of detection of approximately 100 pg/mL. The method developed is suitable for measuring the concentrations of phytoestrogens in blood samples collected from large epidemiological studies. The results of the analysis of serum samples from 300 men and women living in the UK are reported.

Phytoestrogen Content of Birdsfoot Trefoil and Red Clover: Effects of Growth Stage and Ensiling Method

The phytoestrogen content of birdsfoot trefoil (Lotus corniculatus L.) and red clover (Trifolium pratense L.) was determined. In addition, the effects of growth stage, wilting and ensiling additives on content were studied. Birdsfoot trefoil raw material and silage contained only traces of phytoestrogens. In red clover raw material and silage, the phytoestrogen content varied from 0.8% to 1.1% of dry matter. Content was affected by growth stage of the plant and wilting. Formononetin concentration decreased as the plant matured from budding to flowering stage, and wilting from 25% to 40% of dry matter content decreased genistein and biochanin A content. The phytoestrogen content of red clover silage was 18% higher than that of raw material. Silage additives also affected the content. Silage ensiled with an additive containing Lactobacillus plantarum bacterial inoculate had higher concentrations of genistein and biochanin A than silage made with formic acid.

Nutritional and health-related compounds in sprouts and seeds of soybean (Glycine max), wheat (Triticum aestivum.L) and alfalfa (Medicago sativa) treated by a new drying method

Nutritional and health-related compounds of alfalfa, wheat and soybean seeds dried by new drying process called the DIC process (controlled instantaneous pressure release) were evaluated, before and after sprouting. Vitamins (A, B1, B2, B6, C and E), minerals (Fe, Mg, Ca, K, Mn, Na, Cu and Zn) and phytoestrogens (genistein and daidzein ) content were determined. Alfalfa, soybean and wheat seeds dried by DIC showed similar content of biological compounds to seeds dried by traditional processes. Sprouting DIC-seeds significantly increased the levels of vitamins, minerals and phytoestrogens, improving their nutrititional value and health quality compared to fresh products. The increase of 1250-fold and 10-fold of the initial vitamin A and vitamin C content, respectively, of alfalfa seeds due to sprouting is remarkable. Sprouts from DIC-seeds showed a significantly higher vitamin A content than sprouts obtained from seeds dried by other methods. Soybean sprouts obtained from DIC-seeds showed a significant increase in free phytoestrogens, quantified as genistein (70.34±5.8 mg kg–1 d.w.) and daidzein (109.17.3±17.3 mg kg–1 d.w.).