Developmental programming or imprinting refers to the phenomenon whereby changes in the early development of an organism can exert long-lasting impacts that manifest during adulthood (1). Antenatal glucocorticoid use among pregnant women at risk of preterm delivery to assist the maturation of lungs of newborns is an example (2). Recent studies suggest the development of metabolic and cardiovascular abnormalities during adulthood in the offspring of mothers receiving glucocorticoids (3). This phenomenon has been replicated in primate models recently, whereby antenatal glucocorticoid exposure led to obesity in adult male baboons (4). The prevailing theory about the mechanism of developmental programming is epigenetic regulation by DNA methylation and histone modification induced by various factors (5).

The skeletal system is responsive to hormones and hormone-like substances. Prenatal and neonatal exposure of the skeletal system to hormonal stimuli might alter the trajectory of skeletal development. A single dose of estrogen at the first postnatal day could increase the bone mineral density (BMD) and cortical thickness of male mice at the prepubertal stage, but the inverse happened at the stage of peak bone mass acquisition (6). Prenatal and postnatal exposure of bisphenol A, a synthetic xenoestrogen, lowered the femoral bone stiffness of female rats but not in males (7). These studies highlight that early exposure of xenoestrogens can potentially impact bone development.

Isoflavones are phytoestrogens present abundantly in soy-based products (8) and are also an important source of dietary xenoestrogens to human infants. The average isoflavone content in soy infant formula ranges from 25 to 28 mg/100 g of powder, consisting of daidzein (DAI), genistein (GEN), and glycitein (9). Additionally, these isoflavones are also present in breast milk (10). Setchell et al. reported that circulating isoflavones of infants were 13,000–22,000 times higher than the estradiol level, and the exposure level was 6–11 times higher than the dose in adults with regular soy food intake (11). The American Academy of Pediatrics Committee on Nutrition only recommends soy formula to infants with inherited galactosemia, lactose intolerance, or from vegan families (12). Soy formulation is not recommended to infants with cow milk protein allergy because some of them also experience allergy to soy protein (12). A study in the United States showed that 11.6% of the infants ≤ 12 months consuming formula were being fed with soy-based products (13). The prevalent use of soy infant formula that may imply the reason to consume these products is based on preference rather than medical reasons. Other committees around the world take a stricter stand against the use of soy infant formula over concerns of the biological activities of phytoestrogens (14).

The skeletal effects of soy isoflavones take decades to develop, and the planning of epidemiological studies or interventional trials on this topic is difficult. Animal models of prenatal and neonatal programming enable us to have a glimpse of the potential effects of soy isoflavones on skeletal developments. Animals like rodents have a relatively short life cycle, so developmental changes can be examined within a shorter time. The purpose of this review is to examine the skeletal effects of early-life exposure to soy isoflavones in animal models of prenatal and neonatal programming.

In toxicity testing, the neonatal life stage refers to the period from birth to 3 weeks of age (15), but the definition is debatable because newborn rodents are very immature due to the short gestation period, and some researchers suggest they better resemble human preterm infants (16). A similar definition has been used for rats (17). Thus, this review will focus on rodent models supplemented with soy isoflavones within this period. We noted that most neonatal and prenatal studies are the collective work of one research group, so the reader should take note of potential publication bias.

Most studies examining neonatal exposure of soy isoflavones on the skeletal system used mice as the model organism. The soy isoflavones studied included DAI, GEN, or a combination of both. Since the binding affinity of different types of isoflavones on biological targets, like estrogen receptors (18), are different, this could contribute to the variation of the results among the studies. Notably, a study reported that skeletal effects of a combination of GEN and DAI were less prominent compared to individual isoflavones (19), suggesting that the less active isoflavone could attenuate the effects of the more potent isoflavone.

The soy isoflavones were administered subcutaneously during the first few postnatal days, and the skeletal outcomes were assessed when the mice reached maturity (4 months of age), with diethylstilbestrol (DES) as the positive control. The dose and type of isoflavone used in each study were different [GEN, 4 μg/day sc or 5 mg/kg body weight (bw)/day sc; DAI, 2 mg/kg bw/day sc; GEN + DAI, 7 mg/kg bw/day sc]; thus, the skeletal effects may not be comparable between studies. Other variations of the models exist. Two studies assessed the skeletal effects of prenatal exposure of folic acid (FA) and postnatal exposure of isoflavones on mice (20, 21); thus, the effects of isoflavones were confounded by prenatal FA treatment.

In the prenatal and postnatal rat model (22, 23), soy isoflavones were administered through food, in contrast to subcutaneous for studies in mice. Soy isoflavones like DAI are known to be metabolized by gut microbiomes to equol and O-desmethylangolensin with different biological activities compared to the parent compounds (24). However, in all studies reviewed, the bioavailability of the isoflavones and their metabolites were not measured. Along with the doses that were not readily translatable between the mouse and rat studies, these factors prevent effective comparisons between different research groups.

Another study exposed the mouse pups with isoflavones, castrated them in the fourth month, and assessed their bone health status in the eighth month (25). The castration model is a classic bone loss model due to sex hormone deficiency (26), so the mentioned study examined the antiosteoporosis effects of early exposure to isoflavones.

The composition of the control diet is essential to ensure a fair comparison between the isoflavone-supplemented and control groups. Long-term supplementation of different diet regimens with and without soy isoflavones or protein can modify body composition and metabolic profile of the animals (27). Most studies indicated the use of soy-free diet. Modified AIN93G diet without isoflavones is the most common diet used (19, 23, 28), while other studies indicated the use of diet without DAI (23) or isoflavones (22), or amino acid-based diet without isoflavones (20, 21). The difference in the composition of these diets could cause variation in the results of the studies reviewed.

The design of the studies is shown in Table 1 for comparison.

Other studies reported a longer period of isoflavone exposure involving the prenatal and neonatal periods. We limit our discussion to studies with postnatal exposure within the limit of the neonatal period of life. Tousen et al. (23) reported on the skeletal effects of rat dams fed with DAI from gestation till postnatal day 13 and their offspring fed with control diet until weaning. The whole BMD of the male and female offspring was significantly lower at the prepubertal stage (day 35) compared to the control rats (23). Upon sexual maturation (day 77), the BMD of the male remained lower compared to that of the normal control, but the BMD of the female returned to control values (23). The serum osteocalcin level of the exposed rats was lower compared to that of the control rats at the prepubertal stage, but the level returned to control values upon sexual maturity (23). Another study reported a lower tibial BMD in female offspring of rat dams exposed to GEN or isoflavones during gestation and lactation period (postnatal 21 days) (22). These effects persisted when the rats reached adulthood, but it should be noted that the offspring were fed with the same diet as the dams' lifelong (22).

Overall, in contrast to neonatal exposure, prenatal plus postnatal exposure of isoflavones exerted negative effects to the bone. This observation might be related to the critical period of intrauterine exposure. It was reported that soy isoflavones could regulate the epigenetic process of embryonic stem cells (37). Whether these changes are beneficial or harmful to the development of the skeletal system remains a question.

Current findings from animal experiments are summarized in Figure 1.

Prepubertal exposure of soy isoflavones also influences skeletal health at adulthood. Zhang et al. (38) showed that female rats fed with soy protein isolates for 14 days from postnatal day 20, had higher bone formation markers (alkaline phosphatase, ALP, and osteocalcin), bone resorption markers (RatLap), and sclerostin level (a negative regulator of bone formation) compared to the control. Chen et al. (39) compared the rats fed with casein or soy protein isolate-based diet starting from the postnatal day 24 till postnatal day 55 and subsequently switched to standard diet or continue with soy protein isolates till 6 months old. Some of the rats were castrated at the age of 6 months and sacrificed 1–3 weeks later. They reported that the total BMC and BMD, and trabecular and cortical BMD of the soy protein isolate group were greater than those of the casein group. After castration, rats on short-term or long-term soy protein isolates had better bone health than those on the casein diet. Overall, prepubertal exposure of soy isoflavones exerts skeletal beneficial effects similar to those of neonatal exposure models. In contrast, prenatal and postnatal exposures of soy isoflavones exert negligible or negative effects on rats in adulthood. However, apart from the period of exposure, the dose and type of isoflavones supplemented should be considered in interpreting the results of these studies.

Hotchkiss et al. (40) compared the skeletal effects of GEN in rats supplemented continuously for 2 years or switched to the control diet after postnatal day 140. Lifelong exposure to high-dose GEN resulted in lower lumbar BMC and area, as well as higher pyridinoline (a bone resorption marker) compared to those of rats supplemented with lower doses. In female rats treated till postnatal day 140, pyridonoline was lower and ALP was higher with higher GEN dose, indicating that GEN induced higher bone formation rate in these rats. In rats treated with GEN till weaning and male rats at all life stages, no skeletal effects were observed. The data may indicate that androgenic regulation is essential for skeletal development in males and that phytoestrogenic effects of isoflavones may not be as important as in females.

Soy isoflavones affect skeletal development through direct and indirect action on bone cells. In this section, the actions of soy isoflavones on bone cells, through DNA methylation and thyroid hormones (TH), are discussed.

GEN exerts a positive effect on bone remodeling by reducing osteoclast and increasing osteoblast number. It lowers the osteoclast number in neonatal bone marrow cell culture through decreasing the survival of osteoclasts or attenuating their formation (41). It also increases the differentiation and migration of primary rat calvarial osteoblasts by estrogen receptor (ER) and nitric oxide synthase pathway (42). Mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) transduction systems are shown to involve in the pro-osteogenesis effects of GEN (42). Surprisingly, in a coculture of osteoblasts and monocytes, GEN increased the formation of osteoclast-like cells (42), probably through stimulating factors from osteoblasts. Another study suggested the upregulation of Ereg and Efcab2, as well as downregulation of Hrc, Gli, and Ifim5 in MC3T3-E1 preosteoblastic cells (43).

Similarly, DAI promoted apoptosis of osteoclast-like cells derived from porcine bone marrow cells by caspase-3 cleavage and upregulation of nuclear ER (44). DAI improved osteoblastic differentiation of OCT-1 cells via upregulation of bone morphogenetic protein-2 (BMP-2) and activation of the SMAD pathway (44). DAI also improved proliferation, differentiation of MG-63 osteoblasts primarily through ERα signaling and at least partly through MAPK kinase/extracellular regulated kinase (MEK/ERK) and the PI3K/protein kinase B (Akt) signaling pathway (45). Another study showed an increased expression of ERα, ERβ, and steroid receptor (SR) comprising the classic estrogen-responsive elements in MG-63 osteoblasts induced by DAI (46). It also reduced the expression of stimuli for osteoclastogenesis [RANKL and interleukin-6 (IL-6)] while increasing the expression of OPG in MG-63 osteoblasts (46).

Equol and O-desmethylangolensin, derived from DAI metabolism, could also inhibit the formation of osteoclast-like cells from bone marrow cells from male mice (47). Equol also promoted osteoblast differentiation from primary osteoblast precursors, probably through ER and the protein kinase C-α (PKC-α) pathway (48).

DNA methylation is an epigenetic process mediating gene expression, which plays an important role in skeletal development. It is regulated by DNA methyltransferase (Dnmt) (49). The differentiation of mesenchymal stem cells to osteoblastic or non-osteoblastic progenitors is linked to the methylation rate of critical genes encoding BMP-2 and ALP, which is responsive to the Wnt signaling pathway (50). Methylation of the gene encoding interferon regulatory factor eight facilitates osteoclastogenesis (51). Previous studies have demonstrated that the antiestrogenic effects of isoflavones could be partially attributed to its effects on the methylation of steroidogenic factor 1 (49, 52). Exposure of mouse embryonic tissues to GEN also modified the regulation level of genes (37). Since DNA methylation is the prevailing theory for neonatal imprinting effects, the effects of prenatal FA plus postnatal isoflavones on Dnmt in the bone for female mice have been explored. Postnatal isoflavone exposure decreased Dnmt3a expression in the adequate FA group but increased Dnmt3a expression in the supplemental FA group, indicating a significant interaction between FA and isoflavones (21). Gene expression analysis demonstrated that neuropeptide Y (NPY) level, a potent osteogenesis inhibitor, was suppressed in the adequate FA plus isoflavone group (21). Other epigenetic factors, like histone modifications, could also influence bone health, but the role of soy isoflavones in mediating bone health through this mechanism is less clear.

On the other hand, soy isoflavones exert their effects on the skeletal system indirectly through interference with TH homeostasis (53). Congenital and juvenile hypothyroidism can delay skeletal development, while thyrotoxicosis can accelerate skeletal aging and premature closure of growth plate. These phenomena have been replicated in genetically modified rodent models with altered thyroid hormone signaling (54). On the other hand, the skeletal effects of over hypo/hyperthyroidism and thyroid-stimulating hormone (TSH) are well-known (55, 56). Epidemiological studies showed that soy isoflavone consumption could influence TH levels. Intakes of soy isoflavones or protein are associated with high TSH, especially among women (57), which could be a result of the thyroid suppressive effect of isoflavones. In contrast, urinary isoflavone levels are found to be associated with higher serum thyroxine (T4) in women but not in men (58), in which the authors attribute the higher T4 to the suppressive effects of isoflavones on deiodination of T4 to T3. Mechanistically, soy isoflavones suppress the level of TH by inhibiting thyroid peroxidase (TPO) activity competitively (59) and inhibit the binding of TH to thyroid transport protein [transthyretin (TTR)] in the blood (60, 61). Soy isoflavones, especially GEN, are also deiodinase (Dio) inhibitors, specifically type 1 (Dio1) (62). However, Dio1 is not expressed in the bone (63). In silico analysis showed that isoflavones could bind to thyroid receptors, but this effect has not been validated in vitro (64). Isoflavones were reported to enhance transcription mediated by triiodothyronine-liganded thyroid receptor (64). Soy isoflavones also inhibit aromatase and 17-β-hydroxysteroid dehydrogenase (17-β-HSD) (65), which prevent the conversion of androgens to estrogens, thereby lowering thyroxine-binding globulin (TBG) and decreasing T4 level (66). However, TTR, instead of TBG, is the main carrier of thyroid hormones in rodent blood (67).

Soy isoflavones may also affect skeletal health through other indirect mechanisms. A multigeneration supplementation study showed that soy protein concentrates and soy isoflavones slowed down the weight gain and reduced the insulin level of the rats (27). The reduction in mechanical loading exerted by body weight and the bone anabolic insulin signaling may be harmful to the bone. Soy protein isolates also decreased the expression of caveolin-1, a membrane protein that anchors other proteins to the membrane, accompanied by activation of BMP-2/SMAD signaling in bone and osteoblasts (38). In another study, long-term soy protein isolate supplementation caused concurrent reductions in caveolin-1, p 53, p 21, and senescence-associated beta-galactosidase in the bone of rats and osteoblasts cultured with the serum of treated rats (39), thus, preventing senescence of osteoblasts. These are translated to a beneficial effect on the bone.

Overall, soy isoflavones modulate a complex network of factors influencing bone health. The net effects seem to be beneficial, considering the phenotypic response of the skeletal system on soy isoflavone supplementation. The mechanisms of action of soy isoflavones and soy protein isolates are summarized in Figure 2.

Early-life exposure to isoflavones, especially during the neonatal period, enhances BMD, skeletal microstructure, and strength in adulthood. Similarly, prepubertal or life-long supplementation suggested beneficial skeletal effects, which may reduce the skeletal negative effects of castration at adulthood. On the other hand, the prenatal plus postnatal exposure models reveal the negative effects of isoflavones. Further, basic research on the influence of isoflavones on the epigenetic process in embryos would explain the skeletal phenotypes observed. Mechanistically, the skeletal protection may be due to their direct actions on bone cells favoring osteogenesis and DNA methylation. The effects of isoflavones on TH homeostasis could bring more harm than benefits to the skeleton at this life stage. Therefore, soy infant formulation should not be initiated without proper evaluation of the risk and benefits. The evidence presented in this review should also be interpreted together with evidence from other organ systems, especially sex hormone-sensitive organs to assess the impacts of early-life exposure of isoflavones on developmental health holistically.

K-YC and K-LP conceived the review. K-YC performed the literature search and drafted the manuscript. K-LP validated the search results and reviewed the manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.