Soy, phyto-oestrogens and male reproductive function: a review

international journal of andrology ISSN 0105-6263 REVIEW ARTICLE Soy, phyto-oestrogens and male reproductive function: a review Christopher R. Cederroth,* Jacques Auger, Céline Zimmermann,* Florence Eustacheà and Serge Nef* *Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland, Service d’HistologieEmbryologie, Biologie de la Reproduction ⁄ CECOS, Hôpital Cochin, Paris, and àService d’Histologie-Embryologie-Cytogénétique, Biologie de la Reproduction ⁄ CECOS, Hôpital Jean Verdier, Bondy, France Summary Keywords: androgen, dietary soy, endocrine disruptors, genistein, isoflavones, phyto-oestrogens, reproduction, spermiogenesis, spermatogenesis Correspondence: Serge Nef, Department of Genetic Medicine and Development, University of Geneva Medical School, 1211 Geneva 4, Switzerland. E-mail: serge.nef@unige.ch Received 20 July 2009; revised 11 September 2009; accepted 25 September 2009 doi:10.1111/j.1365-2605.2009.01011.x There is growing interest in the possible health threat posed by the effects of endocrine disruptors on reproduction. Soy and soy-derived products contain isoflavones that mimic the actions of oestrogens and may exert adverse effects on male fertility. The purpose of this review was to examine the evidence regarding the potential detrimental effects of soy and phyto-oestrogens on male reproductive function and fertility in humans and animals. Overall, there are some indications that phyto-oestrogens, alone or in combination with other endocrine disruptors, may alter reproductive hormones, spermatogenesis, sperm capacitation and fertility. However, these results must be interpreted with care, as a result of the paucity of human studies and as numerous reports did not reveal any adverse effects on male reproductive physiology. Further investigation is needed before a firm conclusion can be drawn. In the meantime, caution would suggest that perinatal phyto-oestrogen exposure, such as that found in infants feeding on soy-based formula, should be avoided. Introduction to endocrine-disrupting (EDCs) and phyto-oestrogens chemical Epidemiological studies conducted over the last 50 years report an increased incidence of human male reproductive disorders. These health problems include failure of the testis to descend into the scrotum (cryptorchidism), hypospadias, increased incidence of testicular cancer and low semen quality. These disorders have been regrouped under the term Testis Dysgenesis Syndrome (TDS) (Skakkebaek et al., 2001). In northern America, the frequency of cryptorchidism, hypospadias and testicular cancer has almost doubled between the 1970s and 1990s (Toppari, 2002). This rapid increase of reproductive disorders suggests that environmental and ⁄ or life-style factors, such as exposure to endocrine disruptors, are the most likely causes. Endocrine disrupting chemicals were defined by the U.S. Environmental Protection Agency as ‘exogenous agents that interfere with synthesis, secretion, transport, metabolism, binding action or elimination of natural blood-borne hormones that are present in the body and are responsible for homeostasis, reproduction and developmental processes.’ EDCs are highly hetero304 geneous in structure and widespread in our environment. They include synthetic organic compounds such as pesticides (e.g. methoxychlor, dichloro-diphenyl-trichloroethane, DDT), fungicides (vinclozolin), pharmaceutical agents (e.g. diethylstilbestrol, DES), chemicals used as industrial solvents or lubricants and their byproducts (polychlorinated biphenyls, PCBs, dioxins), plastics (bisphenol A, BPA), plasticizers (phthalates), butyltins and flame-retardant polybrominated diphenyl ether (PBDE), to name a few. Less attention has been paid to the action of natural plant-derived endocrine disruptors, termed phyto-oestrogens. Phyto-oestrogens are non-steroidal compounds that can bind to both oestrogen receptor (ER)-a and ER-b because of their ability to mimic the conformational structure of oestradiol (Kuiper et al., 1997, 1998). Phytooestrogens are found in many vegetables and are particularly abundant in soy products. Genistin and daidzin, two major soy isoflavone glucosides, are present at high concentrations in soybeans and soybean-derived products and are a major source of xeno-oestrogen exposure in both humans (e.g. soy-based formula for infants; tofu) and animals (most commercially available diets). The two ª 2009 The Authors Journal compilation ª 2010 European Academy of Andrology • International Journal of Andrology 33 (2010), 304–316 C. R. Cederroth et al. Phyto-oestrogens and male fertility major isoflavones, genistein and daidzein, are present in soy as b-d-glycosides, namely genistin and daizin, which are biologically inactive (Setchell, 1998). The conversion to their corresponding bioactive aglycones (genistein and daidzein) by bacterial b-glucosidases in the intestinal wall permits their absorption by the intestinal tract. Daidzein can be further metabolized to equol, and indeed this compound along with genistein and daidzein are the major isoflavones detected in the blood and urine of humans and animals (Setchell, 1998). In rodents, equol is the major circulating metabolite among isoflavones, representing up to 70–90% of all circulating isoflavones. While all rodents are equol producers, only 30% of humans are able to metabolize daidzein into equol (Atkinson et al., 2005). In addition to its oestrogenic activity, genistein has also been reported to act through other mechanisms, including acting as a tyrosine kinase inhibitor or an antioxidant (Akiyama et al., 1987; Vedavanam et al., 1999). Numerous studies have investigated the plasma concentrations of phyto-oestrogens and their metabolites in humans and animals consuming a diet with or without soy (Adlercreutz et al., 1993a,b; Coward et al., 1996; Morton et al., 1994). In humans consuming soy-free diets, plasma concentration of isoflavones are usually in the nanomolar range £40 nm see (Morton et al., 1994; van Erp-Baart et al., 2003). By contrast, acute ingestion of dietary soy leads to a rapid increase in the plasma concentration of isoflavones to the micromolar range (Adlercreutz et al., 1993a,b; Xu et al., 1994; King & Bursill, 1998; Watanabe et al., 1998). Certain Asian populations may consume up to 1 mg ⁄ kg of body weight per day, and infants fed soy-based formula ingest even higher amounts of isoflavones relative to their body mass: the mean daily consumption of total isoflavones ranges from 6 to 9 mg ⁄ kg in 4-month-old infants fed exclusively with soy-based infant formulas (SBIFs), resulting in plasma isoflavone concentrations (980 lg ⁄ L) much higher than that of infant fed cow’s milk formula or human breast milk (9.4 and 4.7 lg ⁄ L respectively) (Setchell et al., 1997, 1998). Soy isoflavones have received much attention as a result of their potential health benefits. Soy consumption in oriental countries correlates with low incidences of breast and invasive prostate cancers, improvements in metabolic parameters and amelioration of age-related diseases e.g. cardiovascular diseases, osteroporosis (Cederroth & Nef, 2009; Sacks et al., 2006; Setchell, 1998; Tham et al., 1998). However, exposure to high levels of phytooestrogens, either from a lifetime of exposure or during a critical period of development, could also have potentially detrimental effects on fertility and reproductive functions. In particular, some concerns have been raised with regard ª 2009 The Authors Journal compilation ª 2010 European Academy of Andrology • to the high isoflavone content of SBIFs and the possible adverse effects on infants (Irvine et al., 1995, 1998a,b; Setchell et al., 1997, 1998). Based on current estimates, the prevalence of the feeding of infants with SBIFs is high (25% in the USA), and these products represent the breast milk substitute of choice (Barrett, 2006). It has been reported that human infants fed exclusively on SBIFs have blood levels of isoflavones that are at least fivefold higher than adult humans who eat a soy-rich diet (Setchell et al., 1997; Irvine et al., 1998a,b). Experimental and clinical studies addressing the effects of soy and phyto-oestrogens on the reproductive system have produced conflicting and somewhat confusing results. The purpose of this review is to examine the evidence, or the lack of evidence, regarding the potential detrimental effects of soy and phyto-oestrogens on male reproductive function and fertility in animals and humans. Lack of standardization of animal and clinical studies Numerous studies have investigated the potential detrimental effects of soy and ⁄ or isoflavones on reproductive function (see Tables 1–5). Unfortunately, comparisons between different animal or clinical studies are hampered by the lack of standardization of soy nomenclature, the various formulations (soy proteins, pure isoflavones, etc), doses and routes of exposure (dietary, injection and gavage) and the differences in time (gestation, perinatal or adult) and duration of exposure. Comparisons are further hampered by major disparities in the subsequent analyses performed to evaluate the effects and elucidate the mechanisms by which phyto-oestrogens and soy potentially affect reproductive and endocrine functions. All of these variables make it difficult to compare and evaluate the absence or presence of putative beneficial ⁄ detrimental effects of soy and phyto-oestrogens on male fertility. In fact, numerous studies have described an absence of effects on serum testosterone levels, testis weight and sperm abundance. Most of them assessed organ weights as an indicator of endocrine disruption, without evaluating the reproductive efficiency per se through fertility tests, which represent the true physiological outcome of a disruptive effect of a given compound. Reproductive and hormonal effects of soy and phyto-oestrogens in men and primates There is a relative paucity of studies available designed to evaluate the effects of soy and phyto-oestrogens on fertility or reproductive hormones in human males (Table 1). Mitchell et al. evaluated the effects of phyto-oestrogen International Journal of Andrology 33 (2010), 304–316 305 306 Nagata et al., (2001) Nagata et al., (2000) T: No effect T: Decreased ND ND ND ND ND ND ND ND Soymilk Soy product Japanese Japanese 76.8 30 Soy food Caucasian For control groups, the number of individuals (n) is shown in parentheses. ND, not determined; G, gensitein; D, daidzein; T, testosterone. ND ND No effect No effect Decreased ND Soy extract Caucasian <22.3 Less and more than 2 months 8 weeks Life long 100 G: 1 D: 0.5 ND 15 2 months Tofu Caucasian 40 17 (17) 69 Chavarro et al., (2008) Mitchell et al., (2001) T, LH, FSH: No effect ND No effect No effect No effect Habito et al., (2000) T: No effect ND ND ND G: 201 D: 401 ND ND 21 (21) 4 weeks Diet 70 Sperm motility Sperm production Urine isoflavone levels (ng ⁄ lmol creatinine) Plasma isoflavone levels (lM) No. subjects Duration of exposure Isoflavone intake (mg ⁄ day) C. R. Cederroth et al. Ethnic group Human studies evaluating the effects of isoflavone intake on male reproduction Table 1 Human studies evaluating the effects of gestational and post-natal exposure to isoflavones on fertility and male hormones Seminal volume Blood hormone levels Reference Phyto-oestrogens and male fertility supplementation (500 mg of a daily supplement containing 40 mg of genistein, daidzein and glycitein) for 2 months among 14 young men. Semen quality was not influenced by isoflavone extracts, nor did serum concentrations of oestradiol, testosterone, FSH and LH differ from pre-supplementation values (Mitchell et al., 2001). By contrast, a cross-sectional analysis comparing dietary intake of soy food and isoflavones to semen quality parameters among 99 men found an inverse correlation between soy ⁄ isoflavone intake and sperm concentration (Chavarro et al., 2008). Neither sperm motility, sperm morphology nor ejaculate volume was affected. These last results should be taken with caution, in particular as a result of the fact that men enrolled for this study were selected from subfertile couples at an infertility clinic. Furthermore, the intakes of total and specific isoflavones (daidzein, genistein and glycitein) were not measured in blood serum, but instead estimated by summing the isoflavone contributions of dietary habits in a questionnaire. Two additional studies investigated the effects of dietary soy intervention on male reproductive hormones. Habito et al. (2000) performed a crossover study of 42 men who consumed either 150 g lean meat or 290 g tofu daily (approximately 70 mg of isoflavones) for four consecutive weeks. Soy intake did not influence oestradiol, testosterone and dihydrotestosterone levels, although SHBG levels were 9% higher (p = 0.01). Similarly, another study performed with 35 men consuming 400 mL of soymilk (approximately 48 mg isoflavones) daily for 8 weeks, did not find an effect on blood concentration of oestradiol, total or free testosterone, or SHBG (Nagata et al., 2001). By contrast, Nagata et al. (2000) have reported an inverse correlation between soy product intake and serum androgen and oestrogen concentration in men. This cross sectional analysis of soy product intake and reproductive hormones was performed with 69 Japanese men with an average isoflavone intake of 22 mg ⁄ day. Of note, the significance of the inverse correlation with the serum concentration of total and free testosterone was borderline (r = )0.25, p = 0.05 and r = )0.25, p = 0.06 respectively). Isoflavones may also alter the maturation or capacitation of human spermatozoa. It has been recently shown that low concentration of genistein (at a concentration of 1, 10 and 100 nm) caused an accelerated capacitation and acrosome loss in human spermatozoa in vitro (Fraser et al., 2006). If such response were to occur in vivo, this could result in a larger percentage of the sperm population lacking the ability to fertilize an ovum – previously acrosome-reacted spermatozoa have lost the plasma membrane over the anterior part of the sperm head and thus are unable to bind to the zona pellucida, a prerequisite for successful fertilization (de Lamirande et al., 1997). Journal compilation ª 2010 European Academy of Andrology • ª 2009 The Authors International Journal of Andrology 33 (2010), 304–316 C. R. Cederroth et al. Phyto-oestrogens and male fertility Table 2 Animal studies evaluating the adult effects of gestational and post-natal exposure to isoflavones on male reproduction Species and strain Period of exposure Serum isoflavone levels No. Litter animals size Sperm production Testis weight ND ND ND No effect No effect No effect ND Fielden et al., (2003) ND ND ND No effect Increased Kang et al., (2002) 4.0 ND ND ND No effect No effect No effect ND 10–20 ND 12 (12) ND No effect No effect ND ND ND ND ND No effect No effect ND Dose Oral gavage of genistein Mice mg ⁄ kg ⁄ day B6D2F1 E12 to P20 0.1–10 (C57BL ⁄ 6 · DBA ⁄ 2) Rats Sprague–Dawley E6 to P20 0.4 Rabbits New Zealand E0 to P29 Dietary supplementation with aglycone genistein Mice mg ⁄ kg ⁄ day NIH ⁄ S E0 to P21 8 C57BL ⁄ 6 mice Seminal vesicle weight Increased Blood hormone levels ND ND Reference Cardoso & Bao, (2008) Ryokkynen et al., (2006) Wisniewski et al., (2005) E0 to P21 5; 300 (ppm) ND 7 (10) ND No effect No effect No effect T: No effect Mink Wild type E0 to P21 mg ⁄ kg ⁄ day 8 ND ND ND No effect +11% Rats Sprague–Dawley ppm E15 to P10 20–1000 ND 5 (5) ND ND No effect ND E0 to P21 5 ND 12 (12) ND No effect No effect No effect T: )52% 300% ND 12 (12) ND No effect No effect No effect T: )40% 18% ND 12 (12) ND No effect No effect ND ND Cardoso & Bao, (2008) lM 5–100 ND 9 (11) ND ND +9% )60% Henry & Witt, (2006) Long Evans Rabbits New Zealand E0 to P29 Drinking solution of resveratrol Rats Sprague–Dawley E0 to P22 ND ND T: No effect Ryokkynen et al., (2005) ND Masutomi et al., (2003) Wisniewski et al., (2003) For control groups, the number of individuals (n) is shown in parentheses. ND, not determined; ppm, parts per million; E, embryonic day; P, post-natal day; T, testosterone. Fraser et al. also demonstrated that, when genistein was tested in combination with other endocrine disruptors such as non-ylphenyl and 8-pre-nylnaringenin, their adverse effects on sperm were more pronounced. This highlights the importance of testing different mixtures of EDCs, as humans and animals are likely to be exposed to more than one xenobiotic. Although these studies have investigated the reproductive and endocrine effects of adult exposure to soy food and isoflavones, one major issue remaining is whether exposure during infancy causes detrimental male reproductive effects in adulthood. So far, in the USA, millions of infants have been fed with soy formulas over decades without detrimental effects (Badger et al., 2009). One of the rare studies that has assessed the adult reproductive ª 2009 The Authors Journal compilation ª 2010 European Academy of Andrology • effects of soy formula consumption during infancy in humans reports that no significant differences in onset of puberty or in reproductive functions were found in comparison with those fed with milk formulas (Strom et al., 2001). However, the true relevance of this study has been the subject of controversy, as result of the lack of direct measurements of hormone levels and reproductive functions (Goldman et al., 2001; Tan et al., 2006). Using the marmoset monkey as an animal model, the group of Richard Sharpe investigated the effects of soybased formula on testicular development and function (Sharpe et al., 2002; Tan et al., 2006). Seven co-twin sets of male marmoset infants were fed during the first 6 weeks of age either with a standard or a soy-based formula. Like human male infants, marmosets exhibit a International Journal of Andrology 33 (2010), 304–316 307 Phyto-oestrogens and male fertility C. R. Cederroth et al. Table 3 Animal studies evaluating the adult effects of post-natal exposure to isoflavones on male reproduction. Species and strain Period of exposure Dose Serum isoflavone levels Subcutaneous injections of single isoflavones Mice (mg ⁄ kg ⁄ day) ICR P1–P5 0.1 genistein ND Rats Sprague – Dawley Wistar P1–P5 P2–P18 Oral gavage of genistein Sprague – P1–P5 Dawley Wistar P1–P75 No. animals Litter size Sperm production Testis weight Seminal vesicle weight Blood hormone levels 5 (5) ND ND No effect ND ND Adachi et al., (2004) Awoniyi et al., (1997) Atanassova et al., (1999) Nagao et al., (2001) Fisher et al., (1999) 0.1 coumestrol 4 gesnitein ND 8 (8) ND No effect No effect ND ND ND No effect ND )8% (P18) ND No effects on LH, FSH, T FSH: )35% 12.5–100 ND 23 (31) No effect No effect No effect T: No effect 4 ND ND No effect ND ND Increased ND ND Reference For control groups, the number of individuals (n) is shown in parentheses. ND, not determined; P, post-natal day; T, testosterone. similar period of neonatal testicular activity (Lunn et al., 1994). Feeding with soy-based formula initially attenuated the neonatal testosterone rise (Sharpe et al., 2002), but no adverse reproductive consequences were observed in adulthood. More precisely, no significant effects were reported on the timing of puberty, on fertility, on penis development and length, and on the weight of the prostate and seminal vesicles. Surprisingly, testicular weight and Sertoli and Leydig cell number were significantly increased in marmosets fed with soy-based formula as infants (Tan et al., 2006). Although these aforementioned studies did not measure isoflavone levels, several reports indicate that primates, including humans, are able to produce equol (Adlercreutz et al., 1986; Musey et al., 1995; Clarkson et al., 2001; Blair et al., 2003). However, the relevance of this particular isoflavone in affecting mammalian reproductive health remains to be investigated. Even though soy-based formula has been consumed by millions of infants over the past decades without apparent detrimental effects, caution should prevail. Infancy is a very sensitive period for endocrine disruption, and exposure to significant levels of phyto-oestrogens may ultimately lead to adult onset diseases. More generally, as a result of the scarcity of human data available, evidence linking soy ⁄ isoflavone consumption with adverse effects on semen quality and the reproductive hormonal profile is lacking. In this regard, paediatric and health organizations usually consider that there is no conclusive evidence from animal, adult human or infant populations that isoflavones or soy-based formula may adversely affect human 308 development, reproduction, or endocrine function (COT Report, 2003; National Toxicology Program, 2006; Bhatia & Greer, 2008). Although no long-term detrimental effects to growth or sexual maturation were identified, it should be noted that recently a review panel from the Center for the Evaluation of Risks to Human Reproduction (CERHR, established by the National Toxicology Program, NTP and the National Institute of Environmental Health Sciences, NIEHS) failed to issue a conclusive recommendation on the reproductive and developmental toxicity of soy protein-based formula because of the limited utility of available humans studies (National Toxicology Program, 2006). It is thus very important that this issue of developmental or reproductive toxicity of soy infant formula is examined further, ideally in randomized trials. Reproductive and hormonal effects of soy and phyto-oestrogens in rodents The initial recognition and identification of phyto-oestrogens as bioactive compounds were made in the 1940s when it was found that formononetin, an isoflavone present in red clover (Trifolium pratense L.), caused a devastating infertility syndrome in sheep grazing in clover pasture (Bennetts et al., 1946). In another study, high levels of phyto-oestrogens were found in the leaves of stunted desert annuals in a dry year, leading ultimately to impaired reproduction when ingested by the California quail (Lophortyx californicus). In wet years, these quails bred normally and phyto-oestrogens were largely absent in these herbs (Leopold et al., 1976). Journal compilation ª 2010 European Academy of Andrology • ª 2009 The Authors International Journal of Andrology 33 (2010), 304–316 Species and strain Period of exposure • International Journal of Andrology 33 (2010), 304–316 Subcutaneous injections of genistein Mice NMRI M10 for 7 days Oral gavage of genistein Mice ICR mice P1 to P56 ICR mice P21 to P56 ICR mice M6 for 5 weeks Dietary supplementation with genistein Rats Sprague–Dawley P21 to P35 NIH ⁄ S Wistar Adult W7 for 52 weeks Dose Serum isoflavone levels No. animals Litter size Sperm production Testis weight Seminal vesicle weight Blood hormone levels mg ⁄ kg ⁄ day 2.5 ND 5 (5) ND ND ND ND T: )80% LH: )40% Strauss et al., (1998) mg ⁄ kg ⁄ day 2.5 2.5–5.0 2.5 ND ND ND 10 (10) 10 (10) 10 (10) ND ND ND No effect No effect No effect No effect No effect No effect ND ND ND ND ND ND Jung et al., (2004) Lee et al., (2004a) Lee et al., (2004b) ppm 250 1000 8 5 1785 9640 ND BLD 8 (8) 8 (8) 10 (10) 30 ND ND ND ND ND ND ND ND No No No No effect effect effect effect ND ND ND ND T: No effect T: No effect T: +25% ND Fritz et al., (2003) 177 1108 30 30 ND ND ND ND No effect No effect ND ND ND ND G: 927 D: 142 G: 241 D: 191 G: 51 D: 736 10 (10) ND ND No effect No effect T: No effect 10 (10) ND ND )41% )73% T: )90% 10 (10) ND ND No effect )73% T: )85% 50 500 Dietary supplementation with isoflavones aglycone Mice mg ⁄ day (Gen:Daid) ApoE-null W6 to W16 120 (10:1) 120 (2:1) Rats Wistar-Unilever Monkeys Cynomolgus macaques W8 for 12 months Adult ppm 200a ND ND ND No effect No effect ND ND 2000a ND ND ND No effect No effect ND ND 94a 799a 30 (30) ND No effect No effect ND T: No effect 1188a 1458a 30 (30) ND No effect No effect ND T: No effect Ryokkynen et al., (2006) McClain et al., (2007) Cline et al., (2004) Faqi et al., (2004) Perry et al., (2007) 309 Phyto-oestrogens and male fertility 120 (1:10) Reference C. R. Cederroth et al. ª 2009 The Authors Journal compilation ª 2010 European Academy of Andrology Table 4 Animal studies evaluating the effects of adult chronic exposure to isoflavones on male reproduction 310 Sharpe et al., (2002) Tan et al., (2006) )55% T: No effect (from W40) ND No effect No effect +14% For control groups, the number of individuals (n) is shown in parentheses. ND, Not determined; P, post-natal day; W, week; M, month; BLD, below the limit of detection. a Total isoflavone content. Serum genistein values are expressed in nM. ND ND ND No effect ND ND mg ⁄ L (aglycone) 25.5a 25.5a Soy formula milk dietary Monkeys Marmoset P4 to P45 Marmoset P4 to P45 15 (15) 7 (7) 57 (57) 2224a ng ⁄ ml C. R. Cederroth et al. Dietary supplementation with dietary soy Rats ppm isoflavone Sprague–Dawley Adult 600a Species and strain Table 4 (Continued) Period of exposure Dose Serum isoflavone levels No. animals ND Litter size ND Sperm production Testis weight No effect ND Seminal vesicle weight Blood hormone levels T: )50% LH: No effect Reference Weber et al., (2001) Phyto-oestrogens and male fertility To date, there are no experimental studies evaluating the effects of gestation-only exposure to soy ⁄ isoflavones on adult male reproduction. However, some reports have evaluated the long-term effects of exposures encompassing both foetal and post-natal life in rodents (Table 2). For instance, rats exposed to maternal dietary genistein (300 ppm) through gestation and lactation exhibited a decrease in the anogenital distance, testis size and serum testosterone levels (Wisniewski et al., 2003). By contrast, perinatal exposure to genistein (from 0.1 to 10 mg ⁄ kg ⁄ day by oral gavage), had no effect on testis and seminal weight, or on sperm abundance (Fielden et al., 2003). Similarly, oral gavage of genistein at 4 mg ⁄ kg ⁄ day during gestation and lactation had no consequences on testis and seminal vesicle weight or on sperm abundance in rats (Kang et al., 2002). Exposure to soy ⁄ isoflavone exclusively during post-natal life also yielded conflicting results (Table 3). Wistar rats exposed to genistein from post-natal day 2 (P2) to P18 only, through subcutaneous injections at 4 mg ⁄ kg ⁄ day, showed reduced FSH levels and testis weight (Atanassova et al., 1999). By contrast, oral gavage of genistein in Sprague–Dawley rats from P1 to P5, at doses ranging from 12.5 to 100 mg ⁄ kg ⁄ day, did not reveal any detrimental effects on male reproductive parameters such as litter size, sperm production, testosterone levels, testis and seminal vesicle weights (Nagao et al., 2001). Using the same window of exposure and experimental model, subcutaneous injections of coumestrol at 100 lg ⁄ kg ⁄ day had no effects on sperm abundance, testis weight, LH, FSH or testosterone levels (Awoniyi et al., 1997). Despite the known long-term effects on testicular function of foetal exposure to endocrine disruptors (Phillips & Tanphaichitr, 2008), only two studies have evaluated, by means of toxicogenomic analyses, the effects of in utero exposure to phyto-oestrogens on the foetal testis. Subcutaneous delivery of genistein at 100 mg ⁄ kg ⁄ day in rat dams from gestational day 11 (GD11) to GD20 shows only a modest effect on testicular gene transcription, with an alteration in expression for only five genes [‡2-fold; (Naciff et al., 2005)]. This includes a 3.0-fold reduction of Star transcripts and a 2.9-fold increase in transcripts coding for the progesterone receptor. Unfortunately, these variations have not been validated by qRT-PCR. Similarly, transcriptional changes were also absent in the testes of newborn pups whose mothers were fed with dietary soy during the gestation (Cederroth, Beny, Zimmermann, Schaad, Combepine, Doerge, Pralong, Vassalli, Nef, submitted). Some of the studies that have evaluated the effects of lifelong exposure to phyto-oestrogens also assessed for reproductive efficiency in vivo (Table 5). Atanassova et al., (1999) have shown that litter size is not affected in male Wistar rats exposed throughout life to dietary soy. Journal compilation ª 2010 European Academy of Andrology • ª 2009 The Authors International Journal of Andrology 33 (2010), 304–316 C. R. Cederroth et al. ª 2009 The Authors Journal compilation ª 2010 European Academy of Andrology Table 5 Animal studies evaluating the adult effects of lifelong exposure to isoflavones (from gestation until adulthood) on male reproduction Species and strain Period of exposure Oral gavage of genistein Rats Wistar Han E0 onward No. animals Litter size Sperm production Testis weight Seminal vesicle weight Blood hormone levels mg ⁄ kg ⁄ day 1 ND ‡15 )45% No effect No effect No effect Eustache et al., (2009) 10 ND ‡15 )40% Decreased No effect No effect No effects for LH, FSH, T FSH: no effect LH: no effect T: )40% ppm 200–1000 5 ND ND 6 (6) 8 (8) ND ND ND No effect No effect )14% ND No effect Casanova et al., (1999) Roberts et al., (2000) ND ND ND ND No effect No effect ND LH: )67% T: No effect ND ND 10 (21) ND ND )5% ND ND • Dose Serum genistein levels International Journal of Andrology 33 (2010), 304–316 Dietary supplementation with genistein Rats Sprague–Dawley E0 onward Sprague–Dawley E17 onward Sprague–Dawley E7 onward 5–1250 Dietary supplementation with dietary soy Mice ppm C57BL ⁄ 6CR Slc E0 onward 0.05%a Delclos et al., (2001) E0 onward E0 onward 600a ND 10a ND 20 (18) 22 (24) )21% ND )25% ND No effect +8% )30% +22% T: No effect ND Takashima-Sasaki et al., (2006) Cederroth et al., (2009) Ruhlen et al., (2008) E0 onward 15.5%a ND ND No effect ND )10% ND FSH: +12% Atanassova et al., (1999) For control groups, the number of individuals (n) is shown in parentheses. ND, Not determined; E, embryonic day; T, testosterone. a Total isoflavone content. Serum genistein values are expressed in lM. 311 Phyto-oestrogens and male fertility CD-1 CD-1 Rats Wistar Reference Phyto-oestrogens and male fertility C. R. Cederroth et al. By contrast, two studies have come to the conclusion that lifelong exposure to isoflavones may affect reproductive success. Wistar Han rats exposed by gavage to genistein at 1 or 10 mg ⁄ kg ⁄ day from conception through to adulthood displayed reductions in sperm production, motility and abnormal motion parameters (Eustache et al., 2009). When mated with untreated females, male fertility was reduced at both doses, as reflected by an increased rate of post-implantation losses and a decrease in litter size. Corroborating such findings with another experimental design, CD-1 male mice exposed throughout life to dietary soy, and by inference phyto-oestrogens ( 20 mg ⁄ kg ⁄ day), had decreased sperm abundance and seminal vesicle weight, and smaller litter sizes after mating (Cederroth et al., 2009). Curiously both studies are the exception, among some twenty others, in reporting significant negative effects of phyto-oestrogen exposure in males on reproductive success (Tables 2–5). Such discrepancies may reflect differences in experimental design, species and strains, formulations, doses, routes of exposure, time and duration of exposure and measurements to evaluate the testicular and reproductive effects. The negative effects described above may reflect additive detrimental effects as a result of life-long exposure to phyto-oestrogens, which encompasses all developmental periods (gestation, lactation and adulthood). This could explain, for example, the absence of negative reproductive effects in animals exposed post-natally to dietary soy and ⁄ or phytooestrogens (Table 3). In addition, potential multigenerational effects would require further consideration as a recent study from the National Toxicology Program reported a decrease in litter size at the F2 generation of parent rats exposed to 500 ppm of genistein during adulthood (National Toxicology Program, 2008). Hormonal changes after exposure to soy-derived phyto-oestrogens LH levels and testis weight are both decreased in Sprague–Dawley rats exposed from embryonic day 17 (E17) onwards to a dietary supplement containing 5 ppm genistein (Roberts et al., 2000). In ApoE-null mice exposed during adulthood to both dietary genistein and daidzein, testosterone levels are lower and also correlate with a decrease in testis weight (Cline et al., 2004). By contrast, other studies have shown an absence of effect in testis weight despite a decrease in hormonal levels in various species of mammals with differing routes and time of exposure (Strauss et al., 1998; Sharpe et al., 2002; Wisniewski et al., 2003). Strikingly, some reports mention an increase of testis and seminal vesicles weights, but with no differences in sexual hormone levels (Fisher et al., 1999; Kang et al., 2002; Ryokkynen et al., 2005; Tan et al., 312 2006). Overall, these studies indicate that hormone levels and testis weight cannot serve as an indicator for disruption of reproductive functions. Interference with spermatogenesis upon lifelong exposure to phyto-oestrogens The potential mechanisms by which soy and isoflavones adverserly affect the male reproductive system remain poorly characterized. Potential mechanisms of spermatogenesis disruption by dietary phyto-oestrogens have been recently proposed (Cederroth et al., 2009). Life long exposure to dietary phyto-oestrogens decreased epididymal sperm abundance, which correlated with a reduction in the number of haploid cells in the testis of CD-1 mice, without altering testosterone levels in the serum. The authors have shown by quantitative real-time PCR that Sertoli cell maturation and function appears to be unaffected, and that markers that cover the various stages of early and mid-spermatogenesis remain unchanged (Cederroth et al., 2009). However, the spermatid specific marker Gapd-s (glyceraldehyde 3-phosphate dehydrogenase-s), which encodes for a protein that regulates glycolysis in the spermatozoa and is required for sperm motility and fertility (Miki et al., 2004), was downregulated in the testis of mice exposed to dietary phyto-oestrogens. This result indicates that late spermatogenesis is affected. Interestingly, androgen receptor regulated genes (i.e. Pem, Testin and tPA) were also downregulated, suggesting that androgen sensitivity is perturbed upon exposure to phytooestrogens. These data suggest that spermatogenesis is unaffected until the round-spermatid stage and that phyto-oestrogen exposure might interfere with the androgen receptor pathway and affect the late steps of spermatogenesis. A recent study investigated changes in the adult testis transcriptome of rats exposed to genistein from conception to adulthood (Eustache et al., 2009). In this study, the number of modified genes at a ‡2-fold threshold, was over 100 at 1 mg ⁄ kg ⁄ day but markedly lower at 10 mg ⁄ kg ⁄ day. Generally, different genes were modified in the two groups, although for the genes which were modified at both doses, the magnitude of the effects was more pronounced for the higher dose. Although this analysis was performed with RNAs extracted from whole testis – a very heterogenous population of cells – a functional classification of genes revealed a slight upregulation of genes implicated in the GnRH pathway. Potential reproductive effects of a low-dose mixture of EDCs including phyto-oestrogens The sources of exposure to EDCs are diverse and vary widely around the world. As our environment contains Journal compilation ª 2010 European Academy of Andrology • ª 2009 The Authors International Journal of Andrology 33 (2010), 304–316 C. R. Cederroth et al. Phyto-oestrogens and male fertility numerous pollutants, it is likely that individuals or populations are exposed to a mixture of endocrine disrupting compounds with potential additive or synergistic effects. However, the consequences of simultaneous exposure to phyto-oestrogens and other EDCs are only beginning to be investigated. Recently, it was reported that the relatively weak alterations in reproductive function of males exposed to low doses of genistein (1 mg ⁄ kg ⁄ day) were exacerbated when co-exposed with a low dose of the fungicide vinclozolin (1 mg ⁄ kg ⁄ day), an ubiquitous antiandrogenic food contaminant (Eustache et al., 2009). Indeed, the genistein ⁄ vinclozolin mixture induced greater alterations on the male reproductive tract and fertility endpoints when compared with the exposure to each compound in isolation, at the same dose. Interestingly, the effects were greater in the low dose mixture (1 mg ⁄ kg ⁄ day for both compounds) than in the high dose mixture (10 mg ⁄ kg ⁄ day genistein +30 mg ⁄ kg ⁄ day vinclozolin), and mimicked the effects of vinclozolin exposure alone at 30 mg ⁄ kg ⁄ day. Such results emphasize the need for further studies to assess the synergistic effects of natural and chemical EDC mixtures at low doses on male reproductive success. Conclusion Exposure to endocrine disruptors (e.g. BPA or dioxins) during critical periods of reproductive development increases the incidence of reproductive disorders. Given the popularity of soy-based formula, isoflavone supplements and soy-derived products, a better understanding of the influence of phyto-oestrogens on male development is needed. To date, there has been a lack of consistency in human and animal studies examining the effects of soy and phyto-oestrogens on reproductive parameters. These discrepancies certainly reflect the variety of experimental designs, the differences between the specific endpoints measured but also inadequate descriptions or insufficient sample size to permit confidence in the observed results. In humans for example, it would be important to investigate adult male reproductive and endocrine functions of healthy full-term infant fed soybased formula compared with breast-fed or cow milk formula-fed infants. These studies should investigate pubertal development and reproductive endpoints such as adult testicular function (testicular volume, spermiogram) and endocrine parameters (testosterone, DHT, oestradiol, LH, FSH, IGF1, INSL3, etc). The cohorts should be large enough to ensure statistical power to detect meaningful differences. Concerning animal studies, the choice of the animal model and nutritional differences in animal diets need to be considered carefully when designing experiment. It would be relevant to assess dose response ª 2009 The Authors Journal compilation ª 2010 European Academy of Andrology • relationships, mutigenerational studies and evaluation of both reproductive and post-natal endpoints. Finally, most studies are designed to investigate the effects of a single endocrine disrupting chemical. Although straightforward in term of scientific design, this approach fails to appreciate the chemical soup that is more typical of the human or animal environment. Thus further investigation is needed to evaluate the consequences of simultaneous exposure to phyto-oestrogens and other EDCs on fertility and testicular function. Acknowledgements S.N. is supported by the Foundation Gertrude von Meissner and The Sir Jules Thorn Charitable Overseas Trust Reg., Schaan. C.R.C. is supported by the Ernst & Lucie Schmidheiny Foundation. Serge Nef declares is a founder of Amazentis S.A. and a member of its scientific advisory board. J.A. and F.E. have been supported in part by the French Program on Endocrine Disruption (PNRPE; contract MEDD CV 05147). References Adachi, T., Ono, Y., Koh, K. B., Takashima, K., Tainaka, H., Matsuno, Y., Nakagawa, S., Todaka, E., Sakurai, K., Fukata, H., Iguchi, T., Komiyama, M. & Mori, C. (2004) Long-term alteration of gene expression without morphological change in testis after neonatal exposure to genistein in mice: toxicogenomic analysis using cDNA microarray. Food Chemical Toxicology 42, 445–452. Adlercreutz, H., Musey, P. I., Fotsis, T., Bannwart, C., Wahala, K., Makela, T., Brunow, G. & Hase, T. (1986) Identification of lignans and phytoestrogens in urine of chimpanzees. Clinica Chimica Acta 158, 147–154. Adlercreutz, H., Fotsis, T., Lampe, J., Wahala, K., Makela, T., Brunow, G. & Hase, T. (1993a) Quantitative determination of lignans and isoflavonoids in plasma of omnivorous and vegetarian women by isotope dilution gas chromatography-mass spectrometry. Scandinavian Journal of Clinical and Laboratory Investigation. Supplement 215, 5–18. Adlercreutz, H., Markkanen, H. & Watanabe, S. (1993b) Plasma concentrations of phyto-oestrogens in japanese men. Lancet 342, 1209– 1210. Akiyama, T., Ishida, J., Nakagawa, S., Ogawara, H., Watanabe, S., Itoh, N., Shibuya, M. & Fukami, Y. (1987) Genistein, a specific inhibitor of tyrosine-specific protein kinases. Journal of Biological Chemistry 262, 5592–5595. Atanassova, N., McKinnell, C., Walker, M., Turner, K. J., Fisher, J. S., Morley, M., Millar, M. R., Groome, N. P. & Sharpe, R. M. (1999) Permanent effects of neonatal estrogen exposure in rats on reproductive hormone levels, sertoli cell number, and the efficiency of spermatogenesis in adulthood. Endocrinology 140, 5364–5373. Atkinson, C., Frankenfeld, C. L. & Lampe, J. W. (2005) Gut bacterial metabolism of the soy isoflavone daidzein: exploring the relevance to human health. Experimental Biology and Medicine (Maywood) 230, 155–170. Awoniyi, C. A., Roberts, D., Chandrashekar, V., Veeramachaneni, D. N., Hurst, B. S., Tucker, K. E. & Schlaff, W. D. (1997) Neonatal International Journal of Andrology 33 (2010), 304–316 313 Phyto-oestrogens and male fertility C. R. Cederroth et al. exposure to coumestrol, a phytoestrogen, does not alter spermatogenic potential in rats. Endocrine 7, 337–341. Badger, T. M., Gilchrist, J. M., Pivik, R. T., Andres, A., Shankar, K., Chen, J. R. & Ronis, M. J. (2009) The health implications of soy infant formula. American Journal of Clinical Nutrition 89, 1668S– 1672S. Barrett, J. R. (2006) The science of soy: what do we really know? Environmental Health Perspectives 114, A352–A358. Bennetts, H. W., Underwood, E. J. & Shier, F. L. (1946) A specific breeding problem of sheep on subterranean clover pastures in western Australia. Australian Veterinary Journal 22, 2–12. Bhatia, J. & Greer, F. (2008) Use of soy protein-based formulas in infant feeding. Pediatrics 121, 1062–1068. Blair, R. M., Appt, S. E., Franke, A. A. & Clarkson, T. B. (2003) Treatment with antibiotics reduces plasma equol concentration in cynomolgus monkeys (Macaca fascicularis). Journal of Nutrition 133, 2262–2267. Cardoso, J. R. & Báo, S. N. (2008) Morphology of Reproductive Organs, Semen Quality and Sexual Behaviour of the Male Rabbit Exposed to a Soy-containing Diet and Soy-derived Isoflavones during Gestation and Lactation. Reproduction in Domestic Animals doi: 10.1111 ⁄ j.1439-0531.2008.01121.x Casanova, M., You, L., Gaido, K. W., Archibeque-Engle, S., Janszen, D. B. & Heck, H. A. (1999) Developmental effects of dietary phytoestrogens in Sprague-Dawley rats and interactions of genistein and daidzein with rat estrogen receptors alpha and beta in vitro. Toxicological Sciences 51, 236–244. Cederroth, C. R. & Nef, S. (2009) Soy, phytoestrogens and metabolism: a review. Molecular and Cellular Endocrinology 304, 30–42. Cederroth, C. R., Beny, J. L., Zimmermann, C., Schaad, O., Combepine, C., Doerge, D. R., Pralong, F., Vassalli, J. D. & Nef, S. (2009) Potential detrimental effects of a phytoestrogen-rich diet on male fertility in mice. Submitted. Chavarro, J. E., Toth, T. L., Sadio, S. M. & Hauser, R. (2008) Soy food and isoflavone intake in relation to semen quality parameters among men from an infertility clinic. Human Reproduction 23, 2584–2590. Clarkson, T. B., Anthony, M. S. & Morgan, T. M. (2001) Inhibition of postmenopausal atherosclerosis progression: a comparison of the effects of conjugated equine estrogens and soy phytoestrogens. Journal of Clinical Endocrinology and Metabolism 86, 41–47. Cline, J. M., Franke, A. A., Register, T. C., Golden, D. L. & Adams, M. R. (2004) Effects of dietary isoflavone aglycones on the reproductive tract of male and female mice. Toxicologic Pathology 32, 91–99. COT Report (2003) Phytoestrogens and Health. Consumer Products and the Environment, Food Standards Agency, Department of Health, Committee on Toxicity of Chemicals in Food, United Kingdom, 1–444. Coward, L., Kirk, M., Albin, N. & Barnes, S. (1996) Analysis of plasma isoflavones by reversed-phase hplc-multiple reaction ion monitoring-mass spectrometry. Clinica Chimica Acta 247, 121–142. Delclos, K. B., Bucci, T. J., Lomax, L. G., Latendresse, J. R., Warbritton, A., Weis, C. C. & Newbold, R. R. (2001) Effects of dietary genistein exposure during development on male and female CD (Sprague-Dawley) rats. Reproductive Toxicology 15, 647–663. van Erp-Baart, M. A., Brants, H. A., Kiely, M., Mulligan, A., Turrini, A., Sermoneta, C., Kilkkinen, A. & Valsta, L. M. (2003) Isoflavone intake in four different european countries: the venus approach. British Journal of Nutrition 89(Suppl. 1), S25–S30. Eustache, F., Mondon, F., Canivenc-Lavier, M. C., Lesaffre, C., Fulla, Y., Berges, R., Cravedi, J. P., Vaiman, D. & Auger, J. (2009) Chronic 314 dietary exposure to a low-dose mixture of genistein and vinclozolin modifies the reproductive axis, testis transcriptome and fertility. Environmental Health Perspectives 117, 1272–1279. Faqi, A. S., Johnson, W. D., Morrissey, R. L. & McCormick, D. L. (2004) Reproductive toxicity assessment of chronic dietary exposure to soy isoflavones in male rats. Reproductive Toxicology, 18, 605–611. Fielden, M. R., Samy, S. M., Chou, K. C. & Zacharewski, T. R. (2003) Effect of human dietary exposure levels of genistein during gestation and lactation on long-term reproductive development and sperm quality in mice. Food and Chemical Toxicology 41, 447–454. Fisher, J. S., Turner, K. J., Brown, D. & Sharpe, R. M. (1999) Effect of neonatal exposure to estrogenic compounds on development of the excurrent ducts of the rat testis through puberty to adulthood. Environmental Health Perspectives 107, 397–405. Fraser, L. R., Beyret, E., Milligan, S. R. & Adeoya-Osiguwa, S. A. (2006) Effects of estrogenic xenobiotics on human and mouse spermatozoa. Human Reproduction 21, 1184–1193. Fritz, W. A., Cotroneo, M. S., Wang, J., Eltoum, I. E. & Lamartiniere, C. A. (2003) Dietary diethylstilbestrol but not genistein adversely affects rat testicular development. Journal of Nutrition 133, 2287–2293. Goldman, L. R., Newbold, R. & Swan, S. H. (2001) Exposure to soybased formula in infancy. JAMA 286, 2402–2403. Habito, R. C., Montalto, J., Leslie, E. & Ball, M. J. (2000) Effects of replacing meat with soyabean in the diet on sex hormone concentrations in healthy adult males. British Journal of Nutrition 84, 557–563. Henry, L. A. & Witt, D. M. (2002) Resveratrol: phytoestrogen effects on reproductive physiology and behavior in female rats. Hormones and Behavior 41, 220–228. Irvine, C., Fitzpatrick, M., Robertson, I. & Woodhams, D. (1995) The potential adverse effects of soybean phytoestrogens in infant feeding. New Zealand Medical Journal 108, 208–209. Irvine, C. H., Fitzpatrick, M. G. & Alexander, S. L. (1998a) Phytoestrogens in soy-based infant foods: concentrations, daily intake, and possible biological effects. Proceedings of the Society for Experimental Biology and Medicine 217, 247–253. Irvine, C. H., Shand, N., Fitzpatrick, M. G. & Alexander, S. L. (1998b) Daily intake and urinary excretion of genistein and daidzein by infants fed soy- or dairy-based infant formulas. American Journal of Clinical Nutrition 68, 1462S–1465S. Jung, E. Y., Lee, B. J., Yun, Y. W., Kang, J. K., Baek, I. J., Jurg, M. Y., Lee, Y. B., Sohn, H. S., Lee, J. Y., Kim, K. S., Yu, W. J., Do, J. C., Kim, Y. C. & Nam, S. Y. (2004) Effects of exposure to genistein and estradiol on reproductive development in immature male mice weaned from dams adapted to a soy-based commercial diet. Journal of Veterinary Medical Science 66, 1347–1354. Kang, K. S., Che, J. H. & Lee, Y. S. (2002) Lack of adverse effects in the f1 offspring maternally exposed to genistein at human intake dose level. Food and Chemical Toxicology 40, 43–51. King, R. A. & Bursill, D. B. (1998) Plasma and urinary kinetics of the isoflavones daidzein and genistein after a single soy meal in humans. American Journal of Clinical Nutrition 67, 867–872. Kuiper, G. G., Carlsson, B., Grandien, K., Enmark, E., Haggblad, J., Nilsson, S. & Gustafsson, J. A. (1997) Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors alpha and beta. Endocrinology 138, 863–870. Kuiper, G. G., Lemmen, J. G., Carlsson, B., Corton, J. C., Safe, S. H., van der Saag, P. T., van der Burg, B. & Gustafsson, J. A. (1998) Journal compilation ª 2010 European Academy of Andrology • ª 2009 The Authors International Journal of Andrology 33 (2010), 304–316 C. R. Cederroth et al. Phyto-oestrogens and male fertility Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta. Endocrinology 139, 4252–4263. de Lamirande, E., Leclerc, P. & Gagnon, C. (1997) Capacitation as a regulatory event that primes spermatozoa for the acrosome reaction and fertilization. Molecular Human Reproduction 3, 175–194. Lee, B. J., Jung, E. Y., Yun, Y. W., Kang, J. K., Baek, I. J., Yon, J. M., Lee, Y. B., Sohn, H. S., Lee, J. Y., Kim, K. S. & Nam, S. Y. (2004a) Effects of exposure to genistein during pubertal development on the reproductive system of male mice. Journal of Reproduction and Development 50, 399–409. Lee, B. J., Kang, J. K., Jung, E. Y., Yun, Y. W., Baek, I. J., Yon, J. M., Lee, Y. B., Sohn, H. S., Lee, J. Y., Kim, K. S. & Nam, S. Y. (2004b) Exposure to genistein does not adversely affect the reproductive system in adult male mice adapted to a soy-based commercial diet. Journal of Veterinary Science 5(3), 227–234. Leopold, A. S., Erwin, M., Oh, J. & Browning, B. (1976) Phytoestrogens: adverse effects on reproduction in california quail. Science 191, 98–100. Lunn, S. F., Recio, R., Morris, K. & Fraser, H. M. (1994) Blockade of the neonatal rise in testosterone by a gonadotrophin-releasing hormone antagonist: effects on timing of puberty and sexual behaviour in the male marmoset monkey. Journal of Endocrinology 141, 439– 447. Masutomi, N., Hibutani, M., Takagi, H., Uneyama, C., Takahashi, N. & Hirose, M. (2003) Impact of dietary exposure to methoxychlor, genistein, or diisononyl phthalate during the perinatal period on the development of the rat endocrine ⁄ reproductive systems in later life. Toxicology 192, 149–170. McClain, R. M., Wolz, E., Davidovich, A., Edwards, J. & Bausch, J. (2007) Reproductive safety studies with genistein in rats. Food and Chemical Toxicology 45, 1319–1332. Michael McClain, R., Wolz, E., Davidovich, A., Pfannkuch, F., Edwards, J. A. & Bausch, J. (2006) Acute, subchronic and chronic safety studies with genistein in rats. Food and Chemical Toxicology 44, 56–80. Miki, K., Qu, W., Goulding, E. H., Willis, W. D., Bunch, D. O., Strader, L. F., Perreault, S. D., Eddy, E. M. & O’Brien, D. A. (2004) Glyceraldehyde 3-phosphate dehydrogenase-s, a sperm-specific glycolytic enzyme, is required for sperm motility and male fertility. Proceedings of the National Academy of Sciences of the United States of America 101, 16501–16506. Mitchell, J. H., Cawood, E., Kinniburgh, D., Provan, A., Collins, A. R. & Irvine, D. S. (2001) Effect of a phytoestrogen food supplement on reproductive health in normal males. Clinical Science (London) 100, 613–618. Morton, M. S., Wilcox, G., Wahlqvist, M. L. & Griffiths, K. (1994) Determination of lignans and isoflavonoids in human female plasma following dietary supplementation. Journal of Endocrinology 142, 251–259. Musey, P. I., Adlercreutz, H., Gould, K. G., Collins, D. C., Fotsis, T., Bannwart, C., Makela, T., Wahala, K., Brunow, G. & Hase, T. (1995) Effect of diet on lignans and isoflavonoid phytoestrogens in chimpanzees. Life Sciences 57, 655–664. Naciff, J. M., Hess, K. A., Overmann, G. J., Torontali, S. M., Carr, G. J., Tiesman, J. P., Foertsch, L. M., Richardson, B. D., Martinez, J. E. & Daston, G. P. (2005) Gene expression changes induced in the testis by transplacental exposure to high and low doses of 17{alpha}-ethynyl estradiol, genistein, or bisphenol A. Toxicological Sciences 86, 396–416. ª 2009 The Authors Journal compilation ª 2010 European Academy of Andrology • Nagao, T., Yoshimura, S., Saito, Y., Nakagomi, M., Usumi, K. & Ono, H. (2001) Reproductive effects in male and female rats of neonatal exposure to genistein. Reproductive Toxicology 15, 399–411. Nagata, C., Inaba, S., Kawakami, N., Kakizoe, T. & Shimizu, H. (2000) Inverse association of soy product intake with serum androgen and estrogen concentrations in japanese men. Nutrition and Cancer 36, 14–18. Nagata, C., Takatsuka, N., Shimizu, H., Hayashi, H., Akamatsu, T. & Murase, K. (2001) Effect of soymilk consumption on serum estrogen and androgen concentrations in japanese men. Cancer Epidemiology, Biomarkers and Prevention 10, 179–184. National Toxicology Program (2006) NTP-CERHR Expert Panel Report on the Reproductive and Developmental Toxicity of soy Formula. U.S. Department of Health and Human Services, Center for The Evaluation of Risks to Human Reproduction, Research Triangle Park, NC, 1–214. National Toxicology Program (2008) Multigenerational reproductive study of genistein (cas no. 446-72-0) in sprague-dawley rats (feed study). National Toxicology Program Technical Report Series TOX79, 1–98. Perry, D. L., Spedick, J. M., McCoy, T. P., Adams, M. R., Franke, A. A. & Cline, J. M. (2007) Dietary soy protein containing isoflavonoids does not adversely affect the reproductive tract of male cynomolgus macaques (Macaca fascicularis). Journal of Nutrition 137, 1390–1394. Phillips, K. P. & Tanphaichitr, N. (2008) Human exposure to endocrine disrupters and semen quality. Journal of Toxicology and Environmental Health. Part B, Critical Reviews 11, 188–220. Roberts, D., Veeramachaneni, D. N., Schlaff, W. D. & Awoniyi, C. A. (2000) Effects of chronic dietary exposure to genistein, a phytoestrogen, during various stages of development on reproductive hormones and spermatogenesis in rats. Endocrine 13, 281–286. Ruhlen, R. L., Howdeshell, K. L., Mao, J., Taylor, J. A., Bronson, F. H., Newbold, R. R., Welshons, W. V. & vom Saal, F. S. (2008) Low phytoestrogen levels in feed increase fetal serum estradiol resulting in the ‘‘fetal estrogenization syndrome’’ and obesity in CD-1 mice. Environmental Health Perspectives 116, 322–328. Ryokkynen, A., Nieminen, P., Mustonen, A. M., Pyykonen, T., Asikainen, J., Hanninen, S., Mononen, J. & Kukkonen, J. V. (2005) Phytoestrogens alter the reproductive organ development in the mink (mustela vison). Toxicology and Applied Pharmacology 202, 132–139. Ryökkynen, A., Kukkonen, J. V. & Nieminen, P. (2006) Effects of dietary genistein on mouse reproduction, postnatal development and weight-regulation. Animal Reproduction Science 93, 337–348. Sacks, F. M., Lichtenstein, A., Van Horn, L., Harris, W., Kris-Etherton, P. & Winston, M. (2006) Soy protein, isoflavones, and cardiovascular health: an american heart association science advisory for professionals from the nutrition committee. Circulation 113, 1034–1044. Setchell, K. D. (1998) Phytoestrogens: the biochemistry, physiology, and implications for human health of soy isoflavones. American Journal of Clinical Nutrition 68, 1333S–1346S. Setchell, K. D., Zimmer-Nechemias, L., Cai, J. & Heubi, J. E. (1997) Exposure of infants to phyto-oestrogens from soy-based infant formula. Lancet 350, 23–27. Setchell, K. D., Zimmer-Nechemias, L., Cai, J. & Heubi, J. E. (1998) Isoflavone content of infant formulas and the metabolic fate of these phytoestrogens in early life. American Journal of Clinical Nutrition 68, 1453S–1461S. International Journal of Andrology 33 (2010), 304–316 315 Phyto-oestrogens and male fertility C. R. Cederroth et al. Sharpe, R. M., Martin, B., Morris, K., Greig, I., McKinnell, C., McNeilly, A. S. & Walker, M. (2002) Infant feeding with soy formula milk: effects on the testis and on blood testosterone levels in marmoset monkeys during the period of neonatal testicular activity. Human Reproduction 17, 1692–1703. Skakkebaek, N. E., Rajpert-De Meyts, E. & Main, K. M. (2001) Testicular dysgenesis syndrome: an increasingly common developmental disorder with environmental aspects. Human Reproduction 16, 972– 978. Strauss, L., Makela, S., Joshi, S., Huhtaniemi, I. & Santti, R. (1998) Genistein exerts estrogen-like effects in male mouse reproductive tract. Molecular and Cellular Endocrinology 144, 83–93. Strom, B. L., Schinnar, R., Ziegler, E. E., Barnhart, K. T., Sammel, M. D., Macones, G. A., Stallings, V. A., Drulis, J. M., Nelson, S. E. & Hanson, S. A. (2001) Exposure to soy-based formula in infancy and endocrinological and reproductive outcomes in young adulthood. JAMA 286, 807–814. Takashima-Sasaki, K., Komiyama, M., Adachi, T., Sakurai, K., Kato, H., Iguchi, T. & Mori, C. (2006) Effect of exposure to high isoflavone-containing diets on prenatal and postnatal offspring mice. Bioscience, Biotechnology, and Biochemistry 70, 2874–2882. Tan, K. A., Walker, M., Morris, K., Greig, I., Mason, J. I. & Sharpe, R. M. (2006) Infant feeding with soy formula milk: effects on puberty progression, reproductive function and testicular cell numbers in marmoset monkeys in adulthood. Human Reproduction 21, 896–904. Tham, D. M., Gardner, C. D. & Haskell, W. L. (1998) Clinical review 97: potential health benefits of dietary phytoestrogens: a review of the clinical, epidemiological, and mechanistic evidence. Journal of Clinical Endocrinology and Metabolism 83, 2223–2235. 316 Toppari, J. (2002) Environmental endocrine disrupters and disorders of sexual differentiation. Seminars in Reproductive Medicine 20, 305– 312. Vedavanam, K., Srijayanta, S., O’Reilly, J., Raman, A. & Wiseman, H. (1999) Antioxidant action and potential antidiabetic properties of an isoflavonoid-containing soyabean phytochemical extract (spe). Phytotheraphy Research 13, 601–608. Watanabe, S., Yamaguchi, M., Sobue, T., Takahashi, T., Miura, T., Arai, Y., Mazur, W., Wahala, K. & Adlercreutz, H. (1998) Pharmacokinetics of soybean isoflavones in plasma, urine and feces of men after ingestion of 60 g baked soybean powder (kinako). Journal of Nutrition 128, 1710–1715. Weber, K. S., Setchell, K. D., Stocco, D. M. & Lephart, E. D. (2001) Dietary soy-phytoestrogens decrease testosterone levels and prostate weight without altering LH, prostate 5alpha-reductase or testicular steroidogenic acute regulatory peptide levels in adult male SpragueDawley rats. Journal of Endocrinology 170, 591–599. Wisniewski, A. B., Klein, S. L., Lakshmanan, Y. & Gearhart, J. P. (2003) Exposure to genistein during gestation and lactation demasculinizes the reproductive system in rats. Journal of Urology 169, 1582–1586. Wisniewski, A.B., Cernetich, A., Gearhart, J.P. & Klein, S.L. (2005) Perinatal exposure to genistein alters reproductive development and aggressive behavior in male mice. Physiology & Behavior 84, 327–334. Xu, X., Wang, H. J., Murphy, P. A., Cook, L. & Hendrich, S. (1994) Daidzein is a more bioavailable soymilk isoflavone than is genistein in adult women. Journal of Nutrition 124, 825–832. Journal compilation ª 2010 European Academy of Andrology • ª 2009 The Authors International Journal of Andrology 33 (2010), 304–316