Hum Reprod. 2006 Apr;21(4):896-904.
Infant feeding with soy formula milk: effects on puberty progression, reproductive function and testicular cell numbers in marmoset monkeys in adulthood.
Tan KA, Walker M, Morris K, Greig I, Mason JI, Sharpe RM.
MRC Human Reproductive Sciences Unit, Centre for Reproductive Biology and Division of Reproductive and Developmental Science, The Queen's Medical Research Institute, University of Edinburgh, Edinburgh, UK.
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BACKGROUND: This marmoset study addresses concerns about feeding human male infants with soy formula milk (SFM). METHODS: From age 4 to 5 days, seven male co-twin sets were fed standard formula milk (SMA) or SFM for 5-6 weeks; blood samples were subsequently collected at 10-week intervals. Testes from co-twins killed at 120-138 weeks were fixed for cell counts. RESULTS: SFM- and SMA-fed twins showed normal weight gain; puberty started and progressed normally, based on blood testosterone measurements. Body weight, organ weights (prostate, seminal vesicles, pituitary, thymus and spleen) and penis length were comparable in co-twins. All SMA- and 6/7 SFM-fed males were fertile. Unexpectedly, testis weight (P = 0.041), Sertoli (P = 0.025) and Leydig cell (P = 0.026) numbers per testis were consistently increased in SFM-fed co-twins; the increase in Leydig cell numbers was most marked in males with consistently low-normal testosterone levels. Seminiferous epithelium volume per tubule showed a less consistent, non-significant increase in SFM-fed males; raised germ cell numbers per testis, probably due to increased Sertoli cells, conceivably resulted in larger testes. Average lumen size, although greater in SFM-fed group, was inconsistent between co-twins and the difference was not significant. CONCLUSIONS: Infant feeding with SFM has no gross adverse reproductive effects in male marmosets, though it alters testis size and cell composition, and there is consistent, if indirect, evidence for possible 'compensated Leydig cell failure'. Similar and perhaps larger changes likely occur in adult men who were fed SFM as infants.

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In Western countries, many infants are fed with formula milk instead of being breastfed. Although feeding with formula milk was restricted initially to cow’s milk-based formulae, within the last half century it has become common in some parts of the world for infants to be fed with soy formula milk (SFM) (Polack et al., 1999). In most European countries, feeding with SFM is restricted to infants in whom intolerance to other formula milks has occurred, and as a result the prevalence of SFM feeding in these countries is <2% (UK Working Group on Phytoestrogens and Health, 2003). In contrast, in the USA, the prevalence of feeding with SFM at any stage after birth can be as high as 36% (Merritt and Jenks, 2004), and many of these infants are fed with SFM from soon after birth and throughout infancy (Polack et al., 1999). There has been an intense debate as to whether or not feeding with SFM poses a potential health risk to infants (Badger et al., 2002; Mendez et al., 2002; Chen and Rogan, 2004; Merritt and Jenks, 2004). As a consequence of this debate, there have been health recommendations in some countries that SFM should only be used when all other alternatives have been ruled out (Australian College of Paediatrics, 1998; Ministry of Health, Wellington, New Zealand, 1998; COMA UK, 2000). In contrast, in the USA, the American Academy of Paediatrics did not recommend any restriction on the use of SFM (American Academy of Paediatrics Committee on Nutrition, 1998).

The lack of agreement between health recommendations in USA and elsewhere is largely a reflection of uncertainty about the available data that have assessed whether or not SFM, or components of SFM such as the phytoestrogens genistein and daidzein, pose any health risk (Badger et al., 2002; Tuohy, 2003). Much of the evidence pointing to the potential adverse effects of SFM or phytoestrogens derives from studies in laboratory animals, mainly rodents, with some of these studies indicating that there may be impaired reproductive development and function (Irvine et al., 1998a; Badger et al., 2002; Chen and Rogan, 2004) or impaired development of the immune system after phytoestrogen exposure perinatally (Yellayi et al., 2002, 2003). Prompted by the latter findings, recent studies in human infants reared on SFM have revealed no evidence of impaired development of the immune system or of the ability to generate an appropriate antibody response to vaccination (Cordle et al., 2002; Ostrom et al., 2002). Similarly, one retrospective study of infants fed with SFM (Strom et al., 2001) concluded that there was no evidence for any detectable adverse effects on reproductive development. However, this study was conducted by telephone interview and did not involve any direct measurements of hormone levels or of reproductive function in the individuals concerned and has been criticized for a number of reasons (Goldman et al., 2001).

Apart from potential species differences in response to SFM or its components, there are two additional problems related to the safety evaluation of exposure to soy in infancy. The first, which relates only to the human studies, is that detailed assessment of reproductive development, in particular the development of cell types within the testis and their normal function, either requires invasive techniques or must await attainment of adulthood and the direct testing of fertility potential. Invasive approaches are precluded for ethical reasons, and so far no direct assessments of fertility potential or of other reproductive health parameters (e.g. prostatic disease) have been undertaken in adult men or women who have been reared as infants on SFM. The second issue, which relates to laboratory animal studies, is that some of these have either administered components of SFM such as genistein and/or have used a route of administration that is not directly relevant to human formula-fed infants, such as subcutaneous or intraperitoneal injection, or maternal dietary administration of the test material (Mendez et al., 2002; Chen and Rogan, 2004). On the other hand, some of these studies have highlighted that abnormalities resulting from phytoestrogen (genistein) exposure in rodents may not emerge until later in adulthood (Newbold et al., 2001; Jefferson et al., 2005), a period for which there is no relevant information from human studies.

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... Unexpectedly, our findings show that testicular weight and somatic cell numbers are significantly increased in animals that have been fed with SFM as infants. The latter changes did not result in any values in SFM-fed males that lay outside of the control range of values, and these differences would not have been detectable if the present study had not used a paired, co-twin design. However, there is some concern that these changes may reflect a degree of ‘compensated Leydig cell failure’, which merits further investigation.

It is likely that the present findings in marmosets are directly relevant to the concerns about the human health effects of infant feeding with SFM or with exposure to soy or its constituents in various other foods during infancy, although this assumes similar uptake and metabolism of the SFM components in infant marmosets and humans. Our choice of using the marmoset as a model for the human was based on the comparability of the phases of male reproductive development in these two species (Mann and Fraser, 1996; McKinnell et al., 2001), together with the ability to use a co-twin design, which minimized the use of animals. This also allowed a more sophisticated evaluation of potentially subtle effects by using non-identical male twins, which experience has shown are far more comparable for various reproductive parameters than are unrelated males of the same age (Sharpe et al., 2000, 2002, 2003). The fact that we were able to administer SFM to the marmosets in a manner analogous to that in formula-fed human infants (Sharpe et al., 2002) was also a key factor in our studies. Our infants were fed on demand at regular intervals during the day, though for technical reasons (manpower limitations and animal welfare concerns), we were unable to use 100% feeding with SFM and therefore only achieved intakes of SFM that amounted to 40–87% (Sharpe et al., 2002) of that reported in human infants at age 4 months who were fed solely with SFM (Setchell et al., 1997). Based on these comparisons, it is logical to presume that similar effects will be observed in human infants fed with SFM as those that are described presently in SFM-fed marmosets; it is also likely that any such effects may be of greater magnitude if the human infants have been fed solely with SFM. This being the case, the issue is then whether such effects might be considered adverse and/or have health implications, either beneficial or adverse.

In the present study, SFM feeding of infant marmosets, which we had previously shown to attenuate the neonatal testosterone rise (Sharpe et al., 2002), had no obvious or significant effect on the timing or progression of puberty, on fertility in adulthood or on penis development and length. These findings are largely consistent with earlier studies in marmosets, and in other non-human primates, in which total ablation of the neonatal testosterone rise, via administration of a GnRH antagonist, had either no effect (fertility) or relatively minor effects (slightly retarded puberty and penis growth) (Mann et al., 1993; Lunn et al., 1994, 1997; Mann and Fraser, 1996). Nevertheless, it must be kept in mind that the present study was based only on seven sets of twins and is therefore not powered to detect low prevalence effects. In this regard, we cannot exclude that infant feeding with SFM can impair fertility in some males. Similarly, although SFM feeding of marmosets also had no effect on the weights of the seminal vesicles or prostate, it would be prudent to evaluate prostatic size in older men who were reared as infants on SFM, in view of the well-described effects of perinatal estrogen exposure on prostate development (Harkonen and Makela, 2004), and the long-term effects of neonatal genistein exposure on uterine adenocarcinoma in rodents (Newbold et al., 2001). Detailed analysis of prostatic histology in SFM-fed co-twins from the present study may also be instructive in this regard.

The most significant, and unexpected, of the present findings were the significant increases in testicular weight and numbers of Sertoli cells and Leydig cells in the testes of marmosets fed with SFM as opposed to SMA in infancy. At face value, these increases are not obviously ‘adverse’, though the fact that infant exposure to SFM has induced permanent changes to the cell composition of the testis does raise the possibility of effects in other organ systems that might be adverse. There is also concern that the increase in Leydig cell numbers in SFM-fed co-twins in the presence of normal serum testosterone levels could be indicative of some degree of ‘compensated Leydig cell failure’ (de Kretser, 2004). The finding that two of the SFM-fed marmosets had testosterone levels in adult samples (90–120 days of age) that were consistently towards the lower end of the normal range supports such a view, especially as these two animals showed by far the largest increases (214% increase in co-twin 1 and 55% increase in co-twin 6) in Leydig cells per testis in comparison with their co-twin controls. The fact that Leydig cell number was increased to a lesser extent in other SFM-fed males would be consistent with a milder degree of compensated Leydig cell failure in these animals. One sign of compensated Leydig cell failure can be the presence of supranormal serum levels of LH (de Kretser, 2004), but unfortunately it was not possible to assess this in the present study as there is still no available serum LH assay for marmosets. In this regard, it may prove useful to monitor serum levels of LH and testosterone in groups of young adult men who have been reared as infants on SFM or on other formula milk to establish whether or not there is a normal LH : testosterone ratio.

Irrespective of the aetiology of the increase in Leydig cell numbers in SFM-fed co-twin marmosets, this finding is consistent with our earlier observation in marmosets that complete suppression of the neonatal testosterone rise in infancy, via administration of a GnRH antagonist, results in increased Leydig cell number/volume per testis in adulthood (Sharpe et al., 2000). The mechanisms underlying such changes and the somewhat smaller (but consistent and significant) increase in Sertoli cell numbers in SFM-fed co-twin males in the present study are unclear, but elevation of gonadotrophin levels (both LH and FSH) could provide a logical explanation. These observations add to the growing evidence that events or exposures in perinatal life can have important consequences for testicular structure and/or function in adulthood, especially when effects on reproductive hormones are involved (Sharpe et al., 2000, 2003; Skakkebaek et al., 2001; Sharpe and Irvine, 2004).

The increase in testicular weight in SFM-fed marmosets in the present study could be a consequence of the general increase in cell numbers per testis or an increase in fluid content of the testis; the latter possibility arises because of the well-established effects of estrogens on fluid resorption from the excurrent duct system of the testis (Hess et al., 2001), and exposure to SFM during infancy would have resulted in supranormal estrogen exposure via the component phytoestrogens (Setchell et al., 1997, 1998; Irvine et al., 1998a,b). The possibility of any major such effects was excluded in the present studies based on analysis of seminiferous tubule lumen volume per tubule, which was shown to be similar in SFM-fed and SMA-fed co-twins. Although indirect evidence of disruption of the excurrent duct system was observed in one SFM-fed marmoset (number 7) and was shown to result in increased lumen volume per testis and the occurrence of SCO tubules, the absence of any such findings in the other six SFM-fed marmosets suggests that the findings in this particular animal most likely have another cause that is unrelated to the method of feeding in infancy.

It is therefore more likely that the increase in testicular weight in SFM-fed marmosets is a result of an increase in cell numbers per testis. The finding of higher numbers of Leydig and Sertoli cells per testis in SFM-fed animals is therefore consistent with this interpretation, as is the finding of a less consistent increase in the volume of seminiferous epithelium per tubule in the same animals, as the latter reflects mainly germ cell volume, which is the main determinant of testis size in an adult male. Because we determined seminiferous epithelial volume per tubule in the present study, it was not strictly legitimate to convert this to a volume per testis by multiplying by testis weight. However, if such a correction was applied, it revealed an average 26% increase in seminiferous epithelial volume per testis in SFM-fed compared with SMA-fed males, suggesting that increase in germ cell volume per testis is the most likely explanation for the increase in testis weight in SFM-fed versus SMA-fed males. It is likely that this increase is in turn a consequence of the increase in Sertoli cell numbers in SFM-fed males, as Sertoli cell number is the ultimate determinant of germ cell volume per testis and testis size in adult males (Sharpe, 1994).

In experimental studies in various animals, most have reported no effects of perinatal phytoestrogen exposure on adult testis weight (Badger et al., 2001; Delclos et al., 2001; Nagao et al., 2001; Fritz et al., 2003; Masutomi et al., 2003; Chen and Rogan, 2004; Jung et al., 2004), although one study in mink reported a significant increase in testis weight (Ryokkynen et al., 2005). Though some studies have reported significant adverse effects on reproductive function in male rodents after perinatal phytoestrogen exposure (Delclos et al., 2001; Wisniewski et al., 2003), such observations have generally only been made after the administration of very high amounts of compounds, and it is well established in laboratory animals that perinatal exposure to high levels of estrogens can induce such abnormalities (Atanassova et al., 2000; Fritz et al., 2003). It will be important in human studies to test whether similar changes in testis size to those reported presently in the marmoset can be detected in adult men who were fed with SFM as infants. If such studies are to be undertaken, they will have to use large numbers of subjects, because adult testicular size is extremely variable in normal men and will have to use an accurate means of assessing testicular size such as ultrasound (Behre et al., 1989; Carlsen et al., 2000). It will not be possible in such men to evaluate whether or not there is any increase in Sertoli or Leydig cell numbers, as found in the present studies in SFM-fed marmosets, as this would require removal or biopsy of the testis.

In conclusion, the present studies show that infant feeding with SFM in primates does not have dramatically adverse reproductive consequences in the male in adulthood, such as have been reported in some rodent studies. The main caveat to this conclusion is the possibility that there is consistent but variable degrees of compensated Leydig cell failure in the SFM-fed males, although this is based largely on indirect evidence. This can probably be evaluated in human subjects by determination of the LH : testosterone ratio in adult men who were fed as infants with SFM and compared with appropriate controls. Even though the present findings are generally reassuring, they do not rule out the possibility that infant feeding with SFM might induce adverse effects in a small proportion of exposed males, as our study was based only on small numbers of animals. In this regard, our findings reaffirm that any intervention in perinatal life that involves altered hormone exposure of the infant is likely to have adult consequences, as is demonstrated in the present studies. As such consequences may not always be favourable or benign, it seems prudent to recommend that any hormonal exposures of the infant male, especially during the period of the neonatal testosterone rise (0–6 months of age), should be avoided whenever possible.

Categories: 2006, Soy, Sexual development, Testes, Nutrition and diet