Association between a soy-based infant diet and the onset of puberty: A systematic review and meta-analysis.

The objective of this systematic review was to evaluate the association between a soy-based infant diet and the onset of puberty. We included studies in which children were fed a soy-based diet, and we compared them with those who were not. The primary outcomes were the onset of puberty in girls (thelarche, pubarche, and menarche age), boys (pubarche, voice change, testicular and penis enlargement age), and both (risk of delayed and precocious puberty [PP]). Search strategies were performed in PubMed, Embase, LILACS, and CENTRAL databases. Two reviewers selected eligible studies, assessed the risk of bias, and extracted data from the included studies. The odds ratio (OR) and mean difference (MD) were calculated with a 95% confidence interval (CI) as a measure of the association between soy consumption and outcomes. We used a random-effects model to pool results across studies and the Grading of Recommendations Assessment, Development, and Evaluation to evaluate the certainty of evidence. We included eight studies in which 598 children consumed a soy-based diet but 2957 did not. The primary outcomes that could be plotted in the meta-analysis were the risk of PP and age at menarche. There was no statistical difference between groups for PP (OR: 0.51, 95% CI: 0.09 to 2.94, 3 studies, 206 participants, low certainty of evidence). No between-group difference was observed in menarche age (MD 0.14 years, 95% CI -0.16 to 0.45, 3 studies, 605 children, low certainty of evidence). One study presented this outcome in terms of median and interquartile range, and although the onset of menarche was marginally increased in girls who received a soy-based diet, the reported age was within the normal age range for menarche. We did not find any association between a soy-based infant diet and the onset of puberty in boys or girls. Trial Registration: PROSPERO registration: CRD42018088902.

Introduction

As a consequence of the interaction of genetic, endocrine, and environmental factors in the last four decades, the onset of puberty in girls has occurred earlier [1]. Certain components in the diet and environment are affecting the endocrine system. This has consequently led to the early development of secondary sexual characteristics in both girls and boys.

Efforts to implement healthy eating habits that are associated with the possible benefits of soy when consumed early in life have resulted in an increase in the consumption of soy products [2,3]. Additionally, soy foods have been the base of supplements, infant formulas, flour, milk, juices, soy sauce, tofu, and they are included in many industrialized foods that are consumed predominantly in childhood. Moreover, these foods are important sources of polyphenols, also called isoflavones, that can function as estrogen agonists/antagonists or selective estrogen receptor (ER) modulators.

These ERs can interact with a wide variety of compounds. Despite their low binding affinities, isoflavones exhibit estrogenic activities. Therefore, these compounds are referred to as phytoestrogens and can interfere with the metabolism of steroids [4]. This binding effect has a higher affinity for ER beta than for ER alpha. Sometimes, they are classified as natural selective estrogen receptor modulators (SERMs)—mixed estrogen agonists/antagonists [5]. Isoflavones, and in particular genistein, have the potential to exert physiological effects. They affect the signal transduction pathways and inhibit the activity of many enzymes (e.g., protein kinase tyrosine, kinase activated by mitogens, and DNA topoisomerase). They also regulate the cellular factors that control cell growth and differentiation [6,7]. Isoflavones are often classified as endocrine disruptors, that is, chemicals capable of altering the function of the endocrine system and potentially resulting in adverse health effects [8]. Because of their hormonal activity, it is concerning that these phytoestrogens might promote the onset of early puberty.

Experimental animal studies have shown that xenobiotics, such as polychlorinated biphenyls (PCBs), phytoestrogens, fungicides, and pesticides may affect sexual differentiation [9]. This interference has a high probability of affecting both reproductive physiology and behavior at several stages of life [10]. In female rats, early exposure (late embryonic and/or early postnatal) to low doses of PCBs [10] or soy significantly affected mating behavior [11].

Genistein, the main phytoestrogen in soy, has a wide range of biological activities. It binds to ER alpha and ER beta, although it also has an antiestrogenic action [12]. At low concentrations, genistein acts as an estrogen and has an inhibitory effect on lipogenesis. There are also sexual differences in the effect of genistein on adipose deposition and insulin resistance, an effect that involves ER beta [13]. At higher concentrations, genistein promotes lipogenesis through the PPAR gamma pathway and the ER-independent pathway [12]. In female mice, post-weaning dietary genistein consumption advanced puberty by decreasing the age of the vaginal opening, increased the length of the estrus stage, and accelerated mammary gland development [14].

A few studies have evaluated the effects of isoflavones on sexual development in humans. The literature reports controversial results regarding their influence on the onset of puberty. According to Cheng et al., girls with a high intake of isoflavones (423.4–19,178 μg/day) in the pre-pubertal period had breast development of Tanner stage 2. This was 0.7 years later than girls whose diet was in the lowest isoflavone tertile intake [15]. Conversely, when exclusively observing male adolescents, Segovia-Siapco et al. concluded that moderate/high consumption (3–20 mg and >20 mg/day) of isoflavones lead to an earlier puberty [16]. In another study, only with adolescent girls, the same authors found no association between isoflavone intake and the age of menarche onset [1]. Strom et al. found no adverse effects on the reproductive health of adults who consumed soy milk during childhood [17]. The American Academy of Pediatrics does not recommend restricting the consumption of soy-based formulas in childhood. They claim that there is a low affinity of soy phytoestrogens to ERs and a low estrogenic potency in bioassays [18]. Thus, any possible effect of the abundance of phytoestrogens in an infant’s diet would be balanced by the low affinity of these compounds to ERs.

Considering this conflicting evidence in the literature, this study aimed to evaluate the association between a soy-based infant diet and the onset of puberty in girls and boys.

Materials and methods

A systematic review was conducted and reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) Statement [19]. The protocol was registered in the International Prospective Register of Systematic Reviews (CRD42018088902).

Eligibility criteria

We included observational (cohort and cross-sectional studies) and randomized controlled trials (RCT) that met the PECO structure described below.

Participants (P)

The participants were children, adolescents, or adults of both sexes from all ethnic backgrounds where it was possible to assess the onset of puberty, presence/absence of precocious puberty, or delayed puberty.

Exposure (E)

The exposure group was comprised of individuals who consumed a soy-based diet. We considered a soy-based infant diet to be the consumption of soy-based products (e.g., soy-based formula or milk), higher food intake of isoflavones, or the use of soy protein-based supplements. Given that the mean intake of isoflavones varies from country to country and there is no definition of either a mean or high intake of this phytoestrogen in children, we did not use a cut-off point to separate participants with a high intake from those with a low intake. However, the studies were included if the authors used any tool to classify children as high or low consumers. When the mean daily intake of soy and its’ derivatives was not available, we included patients who were known to have a higher consumption of soy than the general population, such as vegetarians and Asian populations. The assessment of soy consumption was determined through dietary records, food frequency questionnaires, or the participant’s use of soy supplements.

Comparison (C)

We considered participants who did not consume a soy-based infant diet as a comparison group.

Outcomes (O)

Primary outcomes were related to the onset of puberty in girls and boys. In girls, this included the onset of breast development (thelarche age), first appearance of pubic hear (pubarche age), and first menstrual cycle (menarche age). In boys, this included the age at onset of testicular growth, age at onset of pubarche, age at onset of voice change, and age at penis enlargement. In both girls and boys, the primary outcomes were the risk of precocious puberty (development of secondary sexual characteristics before the age of 8 years in girls and 9 years in boys) and the risk of delayed puberty (absence of testicular enlargement in boys by 14 years or lack of breast development in girls by 13 years). The secondary outcomes were other indicators of puberty such as height at the last follow-up visit (measurement at the beginning of puberty, during puberty, or as adult height), adverse events, and for boys, age at first ejaculation.

Exclusion criteria

We excluded studies without a comparison group and studies in which phytoestrogens were not derived from soy. As our objective was to compare the onset of puberty in children who consumed soy with children who did not, we excluded case-control studies wherein participants with early or precocious puberty were compared with those who had soy consumption.

Identification of the studies

Electronic databases

General research strategies were applied to the main electronic health databases: Embase (Elsevier, 1980–2020), Medline (by PubMed), LILACS (by Virtual Health Library), Controlled Clinical Trials of Cochrane Collaboration (CENTRAL), Trip database, SCOPUS, and Web of Science. Databases were searched on December 17, 2017 and updated on April 02, 2020. The Medical Subject Headings that were used included: “Soy Foods,” “Soy Milk,” “Soybeans,” “Soybeans Protein,” “Soybean oil,” “Puberty,” “Puberty, precocious,” “Sexual Maturation,” “Menstruation Disturbances.” The search strategies for the primary databases are included in the Supporting Information (S1 File). We also searched for unpublished studies among dissertations and theses (ProQuest Dissertation & Theses Global, WorldCatDissertations, The Digital Library of Theses and Dissertations of the University of São Paulo, Catalog of Theses & Dissertations-CAPES), ClinicalTrials.gov website, and Brazilian Registry of Clinical Trials (ReBec).

EndNote X9 citation management software was used to download the references and remove duplicate entries. The initial screening of abstracts and titles was performed using the free web application Rayyan QCRI [20].

Study selection

Three reviewers (FRKO, AFSG, and RBG) independently selected the titles and abstracts identified during the literature search. The studies selected for full-text review were subsequently assessed for adequacy of the proposed “PECO” structure. In case of disagreement, a final consensus was reached between the reviewers and the project coordinator (VSN-N).

Data extraction and management

For the studies selected for inclusion, the reviewers used a standardized extraction form. This ensured that all information contained in each study (number of patients, average age, study design, inclusion and exclusion criteria, exposure, unexposed, outcomes analyzed, follow-up time, and risk of bias) could be compared.

Assessment of risk of bias in the included studies

For each selected randomized study, the risk of bias was evaluated according to the criteria described in the Cochrane Collaboration tool (RoB 1). This tool encompasses seven domains: the process of randomization, concealing allocation, blinding of participants and researchers, blinding of outcome assessors, whether the losses were included in the final analysis, selective reporting of outcomes, and others [21].

For each selected observational study, the risk of bias was evaluated according to the criteria described by the Newcastle–Ottawa Scale (NOS) for cohort studies. This scale encompasses three domains: selection (four items), comparability (one item), and outcome (three items) [22].

Unit of analysis

The unit of analysis was the data published in the included studies.

Data analyses

Similar outcomes were plotted in the meta-analysis using Stata Statistical Software 16 (Stata Statistical Software: Release 16. College Station, TX, StataCorp LLC, USA). Continuous data were expressed as means and standard deviations. Differences between means (MD) with 95% confidence intervals (CIs) were used to estimate the exposure effect. For the risk of precocious puberty, we present a meta-analysis that included and excluded both-armed zero-event studies [23]. In the first scenario, we added 0.5 to each cell of the 2 × 2 table for the trials with zero events in both arms and the odds ratio (OR) was calculated with a 95% CI [23]. For the second method, we used the Peto one-step OR method [24]. We selected a random-effects model for the meta-analysis. The studies were evaluated separately according to their design (observational versus RCT).

When sufficient studies were available, we conducted sensitivity analyses to assess the robustness of our results. These analyses were performed by comparing studies according to the risk of bias, length of exposure, and assessment of pubertal development through self-report or physical examination.

Assessment of heterogeneity

Inconsistencies among the study results were ascertained by visually inspecting a forest plot and using the Higgins or I2 statistic, in which an I2 > 50% indicated a moderate probability of heterogeneity. When sufficient studies were available, the potential causes of heterogeneity among the studies could be evaluated by subgroup analysis. In case of considerable inconsistency (I2 > 50%) associated with variation in the direction of association and heterogeneity could not be explained, we did not perform a meta-analysis.

Quality of the evidence

The quality of evidence for estimating the effect of exposure on outcomes that could be plotted in the meta-analysis was generated in accordance with the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) Working Group [25]. GRADE is a structured process for rating the quality of evidence in systematic reviews or in guidelines for health care [25]. Randomized controlled trials begin as high-quality evidence; however, the confidence in the evidence may decrease if the studies have major limitations that may interfere with the estimates of the treatment effect [25]. These limitations include the risk of bias, inconsistency of results, indirectness of evidence, imprecision, and reporting bias [26]. Conversely, observational studies are initiated with a low certainty of evidence. However, the quality of evidence can increase when studies rigorously present one of the following criteria: the magnitude of the treatment effect is very large, there is evidence of a dose–response relationship, or all plausible biases would decrease the magnitude of the treatment effect [27].

When more than 10 studies were included in the meta-analysis, we used a funnel plot to investigate the presence of publication bias [24].

Results

After removing duplicates, the search strategies yielded 962 references, and we selected 16 studies for the final examination (Fig 1). Eight studies met our eligibility criteria and were therefore included in this review.

Fig 1

Flow diagram of the selection of studies.

A total of eight studies were excluded from the final examination for the following reasons: one was a literature review, one was a case report, two presented only laboratory outcomes, one was an animal study, one did not describe clinical outcomes related to puberty (only behavioral factors), and in two studies the time of follow-up was insufficient to evaluate the health outcomes [28-35].

Included studies

We included eight studies, seven observational studies (five were longitudinal studies and two were cross-sectional studies) [1,15-17,36-38], and one RCT [39]. The mean follow-up time was 9.3 years in the longitudinal studies. Regarding the included participants, six studies were conducted with a healthy population, and two studies did not mention if participants with chronic illness were excluded [1,16]. Regarding exposure, four studies used soy-based formula or milk [17,36-38], one used a soy protein supplement [39], and one assessed soy intake by dietary records (the highest and lowest dietary isoflavone tertile were considered exposure and unexposed, respectively) [15]. One study classified soy intake into four categories. We considered unexposed individuals to be those who ate soy foods less than once a week, and as exposed if they consumed soy products more than three times per day [1]. In the Segovia-Siapco et al. study, we regarded as exposure when the total isoflavone intake was higher than 20 mg/day and as unexposed when the total isoflavone intake was less than 3.0 mg/day. In the Sinai et al. study, the exposure group comprised children with an IgE-mediated cow’s milk allergy. They ingested soy-based formula for longer than 3 months, usually at the end of breastfeeding or in combination with breastfeeding. The unexposed group comprised infants considered “healthy” as no gastrointestinal symptoms associated with food were reported.

Regarding potential confounders, two studies did not mention pre-pubertal weight. However, six studies reported that at 7–9 years of age, there was no significant difference between the groups in BMI [15,17,34,36,37,39]. Four studies reported that exposed and unexposed children were similar with respect to birth weight, maternal pre-pregnancy BMI, prenatal smoking status, marital status, and maternal education. However, Cheng et al. reported that unexposed girls were younger at baseline, and they were more likely to be from a smoking household, consume less fruit fiber, and have a lower baseline energy intake. In the Segovia-Siapco et al. studies, there was a significant difference between the groups in terms of site of study (California vs Michigan), type of school (public vs private) and exposure participants were more likely to have parents with a graduate-level education [1,16].

Table 1 shows the main characteristics of the included studies, eligibility criteria, and Table 2 shows outcome results.

Table 1

Characteristics of included studies.

Author	Country	Study population	N°	Infant diet		Mean (SD) age at baseline		Mean (SD) BMI		Confounders
				Exposure	Unexposed	Exposure	Unexposed	Exposure	Unexposed
Adgent 2012	UK	White, healthy or with minor problems, girls from the Avon region of the UK, who were expected to deliver between April 1, 1991, and December 31, 1992	2,178	Formula or milk soy-based/Unknown	Early formula with no soy	Between 8 and 14.5 ys	Between 8 and 14.5 ys	Age 7–9 yearsEver > 85th Percentile: 26%Never > 85th Percentile: 74%	Age 7–9 yearsEver > 85th Percentile: 35%Never > 85th Percentile: 65%	Exposure girls were similar to unexposed with respect to birthweight, childhood BMI, as well as maternal pre-pregnancy BMI, prenatal smoking status, marital status, and prenatal vegetarian diet.
Cheng 2010	Germany	Healthy, white, children	79	Isoflavone intakea Girls: 1,199 μd/d Boys: 1,338 μd/d	Isoflavone intakea Girls: 13 μd/d Boys: 13μd/d	Girls: 6.8 ys ± 1.1 Boys: 9.0 ys ± 0.8	Girls: 7.4 ys ± 0.9 Boys: 8.8 ys ± 1.0	Age 7–9 years Girls: 15.9 ± 1.6 Boys: 16.4 ± 1.6	Age 7–9 years Girls: 16.3 ± 2.1 Boys: 16.8 ± 2.4.	Unexposed girls were younger at baseline, and they were more likely to be from a smoking household, consume less fruit fiber, and have a lower baseline energy intake. Boys in exposure group consumed less total fiber.
Duitama 2018	Colombia	Healthy boys and girls, between 7 and 9 years old, from public schools and attending community meal locations.	51	45 g of soy protein supplement dissolved in fruit juice, Monday to Saturday for 12 months	No intervention	Girls: 8.3 ys ±0.8Boys: 8.8 ys ±0.7	Girls: 8.2 ys ±0.9Boys: 8.4 ys ±0.8	Age 7–9 yearsGirls: 16.2 ± 1.2Boys: 16.6 ± 1.2	Age 7–9 yearsGirls: 16.4 ± 1.3Boys: 17.4 ± 1.5	-
Giampietro 2004	Italy	Exposure: children in prevention or treatment of cow’s milk allergy. Unexposed: healthy children	66	Exclusive feeding of soy-based formulas for at least 6 months of life (150 kcal / kg)	No feeding of soy-based	3.1 ys ±2	3.8 ys ±1.9	-	-	Height and weight, as BMI, were within the normal range compared with those of children of the same age, sex, and race.
Segovia-Siapco 2014	United State	Girls ages 12 to 18 years attending middle and high schools near two Seventh-day Adventist universities in in California and Michigan	131	Soy consumption >3×/day	Soy consumption < 1x/week	14.9 ys ±1.5	15.2 ys ±1.8	21.5 (3.8) b	22.3 (3.7) b	There was a significant difference between the groups in terms of site of study (California vs Michigan), type of school (public vs private) and exposure participants were more likely to have parents with a graduate-level education.
Segovia‑Siapco 2017	United State	Boys aged 12–18 years attending middle and high schools near two Seventh-day Adventist universities in in California and Michigan	150	Isoflavone intake > 20 mg/day	Isoflavone intake < 3 mg/day	14.9 ys ±1.7	15.0 ys±1.8	0.07±0.94 c	0.36±1.05 c	There was a significant difference between the groups in terms of site of study (California vs Michigan), type of school (public vs private) and exposure participants were more likely to have parents with a graduate-level education.
Sinai 2018	Israel	Newborns born during 2004–2006 were followed up through telephone for assessing the development of milk allergy during the first 6 months of life calls, and for milk formula intake until age 3 years.	89	Soy-based formula for more than 3 months	Not receiving soy formula.	8.92 ys (8.21, 9.42)d	8.99 ys (8.35, 9.42)d	0.67 ± 1.01c	0.53 ± 1.02c	There were no significant differences between groups with respect to age, birthweight, gender distribution, maternal characteristics, weight for gestational age, family history, or behavioral habits. Children who had early signs of puberty reported less weekly physical activity compared to those with no pubertal signs.
Strom 2001	United State	Mostly white healthy adults, who as children participated in a controlled observational study (cow’s milk versus soy milk)	811	Milk (formula) classified as soy based	Cow’s milk	Adults aged 20 to 34 ys	Adults aged 20 to 34 ys	Women: 22.8 ±3.3Men: 25.6 ±4.6	Women: 22.9 ±3.7Men: 24.8 ±3.6	-

UK: United Kingdom; N°: Children followed in each study,—no information provided

a mean

b median BMI (IQR)

c Mean BMI-for-age z scores (SD)

d Median and IQR.

Table 2

Outcome results.

Author year	Follow-up	N° of PP	♀ Age at onset of thelarche time (n/mean/SD)	♂ Age at onset of pubarche time (mean/SD)	♀ Age at menarche (mean/SD or median/IQR)	♂ Age at onset of testicular growth (n/mean/SD)	♂ Age at onset of Voice Change (n/mean/SD)	Age at first ejaculation n/mean/SD)	Height in the last visit
Adgent 2012	Age at menarche was assessed through questionnaires annually. between ages 8 and 14.5	-	-	-	E (54): 12.4 ys [IQR, 11.6–13.3].Une (2124): 12.8 ys [IQR, 12–13.6]	-	-	-	-
Cheng 2010	3 to 6 months until adulthood	-	E (40):10.7 ys ± 1Une (39):9.9 ys ± 1.2	-	E (40): 13.1 ys ± 1.2Une (39):12.6 ys ± 1.0	E (36):10.8 ys ± 0.9Une (36):11.2 ys ± 1.0	E (36):13.8 ys ± 1.0Une (36):13.5 ys ± 1.2	-	-
Duitama 2018	12 months	E (29):0Une (22): 0	-	-	-	-	-	-	Mean height (SD) in cm at baseline:E: 127 ± 1 Une: 124 ± 1Mean height (SD) in cm 12 months after exposure:E—boys: 130 cm ± 0.05/girls: 125 cm ± 0.07Une -boys: 124 cm ± 0.04/girls: 122 cm ± 0.06
Giampietro 2004	12 months	E (48):0Une (18): 0	-	-	-	-	-	-	-
Segovia-Siapco 2014	Cross-sectional	-	-	-	E (69):12.6 ys ± 1.3Une (62):12.5 ys ± 1.4	-	-	-	-
Segovia‑ Siapco 2017	Cross-sectional	-	-	♂ E (81): 12.5 ys ± 0.768♂ Une (69): 13 ys ± 0.87	-	-	-		-
Sinai 2018	7.8 and 10.5 years	E (29):1Une (60): 4	-	-	-	-	-	-	Mean height z scores at beginning of puberty (±9 years):E: - 0.17 ± 1.08/Une: - 0.16 ± 1.01
Strom 2001	1965–1978	-	E (128): 12.6 ys ± 1.4Une (267): 12.7 ys ± 1.3	♂ E (115): 13.9 ys ± 1.6♂ Une (286): 13.7 ys ± 1.7	E (128): 12.6 ys ± 1.4Une (267): 12.7 ys ± 1.3	-	E (111): 14.3 ys ± 1.7Une (262): 14.0 ys ± 1.5	E (108): 13.2 ys ± 1.2Une (274): 13 ys ± 1.4	Mean height (SD) in inches at adult height.MenE: 71.7 ± 2.5; Une: 71.4 ± 2.6WomenE: 65.3 ± 2.3; Une: 65.5 ± (2.5)

PP: Precocious puberty; E: Exposure; Une: Unexposed; BMI: Body mass index; ATO: Age at take-off; PHV: Peak height velocity; IQR: Interquartile range;—no information provided.

Bias risk assessment

For the RCT, due to lack of information, the risk of bias was unclear in the seven domains assessed. Table 3 shows the risk of bias in the observational studies. In all the studies, the exposed cohort met our eligibility criteria, and the unexposed cohort originated from the same community as that of the exposed participants. Regarding assessment of exposure, four studies used questionnaires [1,16,17,38], three used food records, and in the RCT, the supplement was provided and supervised by nutritionists [39]. Only one study demonstrated the outcomes of interest at the initiation of the study [15]. All the groups were comparable at baseline. Pubertal development was assessed by a physician in three studies [36,37,39], it was self-reported in four [1,16,17,38], and by nurses in one [15]. In three studies, the follow-up period was insufficient to assess delayed puberty [36,37,39]. Most studies reported loss of follow-up; thus, the data on outcomes for these participants could not be assessed.

Table 3

Newcastle–Ottawa Scale for observational studies.

Author (year)	Selection	Comparability	Outcome	TOTAL
Adgent (2012)	4/4	2/2	1/3	7/9
Cheng 2010	2/4	2/2	1/3	5/9
Giampietro (2004)	2/4	2/2	2/3	6/9
Segovia-Siapco (2014)	3/4	2/2	2/3	7/9
Segovia- Siapco (2017)	3/4	2/2	2/3	7/9
Sinai (2018)	3/4	2/2	2/3	7/9
Strom (2001)	3/4	2/2	2/3	7/9

Studies included in the meta-analysis

Risk of precocious puberty. Regarding the risk of precocious puberty, there was no significant difference between the children who were exposed or unexposed (OR: 0.51, 95% CI: 0.09–2.93, 3 studies, 206 participants, low certainty of evidence, Fig 2, Table 4). Excluding the studies with no events in both arms, no difference between groups remained (OR: 0.55, 95% CI: 0.08–3.72, 1 study, 89 participants, low certainty of evidence, supporting information, S1 Fig).

Fig 2

Meta-analysis of the frequency of precocious puberty: Soy-based diet versus non-soy-based diet.

Table 4

Summary of findings–quality of evidence according to GRADE approach of association between soy-based infant diet and the onset of puberty.

Certainty assessment							Summary of findings
N° of Participants (studies) Follow up	Risk of bias	Inconsistency	Indirectness	Imprecision	Publication bias	Overall certainty of evidence	Study event rates (%)		Odds ratio (95% CI)	Anticipated absolute effects
							Non-soy-based infant diet	Soy-based infant diet		Effect with non-soy-based infant diet	Effect size with soy-based infant diet
Menarche Age
605 (3 studies) Childhood to Adulthood	serious a	not serious	not serious	serious b	none c	⨁⨁◯◯ LOW d,e	-	-	-	The mean menarche age was 12.6 years	MD 0.14 years higher (95% CI from 0.16 lower to 0.45 higher)
Precocious Puberty
206 (3 studies) 1 to 2 years	serious a	not serious	not serious	serious b	none c	⨁⨁◯◯ LOWd,e	4/100 (4.0%)	1/106 (0.9%)	0.52 (0.09 to 2.94)	The risk of PP was 4 per 100	Risk difference was 3 fewer per 100 (95% CI from 7% less to 1% more)
Height (centimeters)
862 (2 studies) Childhood to Adulthood/ 1 year	serious a	not serious	not serious	serious b	none c	⨁⨁◯◯ LOW d,e	-	-	-	The mean height was 151 cm	MD 0.43 cm higher (95% CI from 1 cm lower to 1.9 cm higher)
Height (z score)
89 (1 study) 7.8 to 10.5 years	serious a	not serious	not serious	serious b	none c	⨁⨁◯◯ LOWd,e	-	-	-	The mean z score of height was 0.16	MD 0.01 score lower (95% CI from 0.46 lower to 0.48 higher)

GRADE: Grading of Recommendations Assessment, Development, and Evaluation; CI: Confidence interval; MD: Mean difference; PP: Precocious puberty; RCT: Randomized controlled trial

s

a. In most observational studies the representativeness of the exposed cohort was a selected group. Most studies reported loss of follow- up, and data on outcomes which prevented the assessment of the participants. Pubertal development was self-reported in four studies. In the RCT there is no information regarding randomization process, allocation concealment and attrition bias.

b. All meta-analyses presented a small sample size, and the optimal information size was not achieved, the effect size crossed the line of no effect, and confidence interval was wide.

c. Investigation of publication bias was not possible due to the small number of included studies (<10). However, as we performed a huge search strategy comprising published and unpublished studies, we did not consider rating down the quality of evidence in this domain.

d. Most of the patients included were from observational studies that did not meet any of the criteria that could increase the certainty of evidence (such as the large magnitude of the treatment effect, the dose-response relationship, the plausible biases decreasing the magnitude of the exposure effect).

e. Low certainty of evidence: Since our confidence in the effect estimate is limited, the true effect may be substantially different.

Age at menarche. Regarding age at menarche, the meta-analysis of three studies showed no difference between the groups for the onset of menarche (MD 0.14 years, 95% CI -0.16 to 0.45 years, 605 children, low certainty evidence, Fig 3, Table 4). One study presented data of age at menarche in median and interquartile range (IQR). Although this timing was marginally increased in girls who consumed a soy-based diet as an infant, the reported age was within the normal age range for menarche (12.4 years; IQR 11.6 to 13.25; 54 girls) [38].

Fig 3

Meta-analysis of mean age of menarche: Soy-based diet versus non-soy-based diet.

WMD: Unstandardized mean difference.

Height in the last follow-up. Four studies evaluated the height of patients at the last visit of the study [17,36,37,39]. Height was assessed at the beginning of pubertal signs in three studies and at an adult age in one study [17]. In the meta-analysis for the Duitama and Strom studies (height measurement at beginning of puberty and at adult age, respectively) no clinically significant difference between groups was found (MD 0.432 cm, 95% CI -1.03 to 1.9 cm, 2 studies, 862 individuals, low certainty evidence, Fig 4, Table 4). The subgroup analysis according to sex did not show an association between exposure and height at the last follow-up (Fig 4). However, in the Duitama et al. study, when comparisons were made between groups in SD and by gender, there was a statistically significant difference between exposed and unexposed girls (-0.11 vs -0.8, p = 0.016). We could not plot the data from the Giampietro et al. study and the Sinai et al. study in the meta-analysis (the height was presented only by figures and in SD, respectively). In the first study, the authors reported that the children’s height was within the normal range, and in the second study, there was no difference in SDs between the groups (MD 0.01 scores, 95% CI -0.46 to 0.48, 89 participants).

Fig 4

Meta-analysis of height in the final follow-up: Soy-based diet versus non-soy-based diet.

WMD: Unstandardized mean difference. In the Duitama study height was measured at the beginning of puberty, and in the Strom study height was measured as an adult.

Investigation of publication bias was not possible owing to the small number of included studies (<10) [40].

Studies included in the qualitative synthesis

Only the Strom et al. study [17] assessed the age at first ejaculation, and no significant differences were found between the groups. Testicular growth was analyzed only by Cheng et al. [15], and no association between exposure and this outcome was found. Segovia-Siapco et al. [16] and Strom et al. [17] evaluated pubarche in boys. Segovia-Siapco et al. [33] reported that the mean age of pubarche was 12.5 years in exposed and 13 in unexposed (MD -0.5 years, 95% CI -0.77 to -0.23). Strom et al. [17] reported that the mean age was 13.9 in the exposed group and 13.7 years in the unexposed group (MD 0.2, 95% CI -0.15 to 0.55).

Cheng et al. [15] and Strom et al. [17] evaluated the age of thelarche. In the first study, the mean age for this outcome was 10.7 years for the exposed and 9.9 for the unexposed, with a mean difference of 0.8 years (95% CI 0.31 to 1.29, 79 patients). In the second study, mean age was 12.3 years in both groups (MD -0.01; 95% CI -0.33 to 0.32; 395 patients). The heterogeneity between these two studies can be explained by the criteria used for thelarche. Cheng et al. considered it to be self-reported Tanner stage 2 for breast development, while Strom et al. considered it to be when the breast developed sufficiently to begin wearing a bra.

No study assessed the risk of delayed puberty, as well as the age at onset of pubarche in girls.

Discussion

In the current review, we evaluated the association between a soy-based infant diet and the onset of puberty in girls and boys. Eight studies were included, with a total of 598 children exposed to a soy-based diet, and 2957 were unexposed. The outcomes that were plotted in the meta-analyses were the risk of precocious puberty, age at menarche, and height at the last follow-up. We did not find any association between exposure and these outcomes. In one study, the age at thelarche was marginally increased in girls with a soy-based infant formula. However, the reported age was within the normal age range for menarche [38]. Segovia-Siapco et al. [16] also reported that a high total soy isoflavone intake was significantly associated with earlier median age pubarche in boys. Cheng et al. reported that girls whose diet was in the highest dietary isoflavone tertile experienced Tanner stage 2 for breast development later than did girls whose diet was in the lowest isoflavone tertile [15]. However, in both situations, the reported age was within the normal age range for pubarche in boys and thelarche in girls.

This is the first systematic review to evaluate the association between a soy-based infant diet and the onset of puberty in girls and boys. The controlled studies (exposed vs. unexposed) on this topic were included in this review. Two Korean case–control studies have also evaluated the association between soy and puberty onset. In the first study, Kim et al. compared serum isoflavone concentrations in 101 girls with idiopathic central precocious puberty with 91 age-matched controls [41]. Serum concentrations of daidzein, genistein, and total isoflavones were significantly higher in children with precocious puberty than in normal children. In the second study, equol, genistein, and daidzein were measured in the plasma of 150 girls with precocious puberty and compared with 90 control subjects. Diadzein and equol levels in the plasma of precocious puberty patients were respectively 1.37 and 1.3 times higher than those of the control group. However, there was no significant difference. Genistein was significantly 2.67 times higher in girls with precocious puberty than in normal girls [42]. Thus, in both studies, the authors concluded that high serum isoflavones may be associated with the risk of precocious puberty in Korea. The important point that distinguishes the two case–control studies from the studies included in our review is that we compared the risk of precocious puberty and the age at the onset of puberty in participants who were exposed to a soy-based infant diet. Conversely, in the two Korean studies, children with central precocious puberty were compared with normal children in terms of isoflavone serum concentrations. After a clinical observation, the first step to explore the association of causality between exposure and outcome is to perform a case-control study [43]. It is an efficient method of investigation when a disease occurrence is rare. However, there were important limitations inherent in the study design. For example, in a case–control study, as exposure is assessed after the outcome, it can be difficult to establish if it was preceded by exposure. As in cohort studies, exposure precedes the disease, if a case–control study shows evidence that a certain exposure is suspected, the next step should be to carry out a cohort study [43]. The cohort studies included in our review did not confirm this association in the Korean studies.

However, our systematic review has some limitations. The main limitation is related to the small number of studies that evaluated the risk of precocious puberty. Additionally, two of the three studies that assessed this outcome were studies with no events in either arm. These studies were naturally excluded from meta-analyses of OR and relative risk [24]. However, there was no consensus regarding whether these studies should be included. Xu et al. showed that excluding such studies may influence the magnitude and direction of the treatment effects [44]. Cheng et al. recommend including studies with no events in both arms in meta-analyses when treatment effects are unlikely, although they should be excluded when there is treatment effect [23]. In our review, we performed meta-analyses including and excluding these studies. In both analyses, there was no statistical difference between the groups in the risk of precocious puberty. Second, because of the small number of studies included in the meta-analyses, we could not perform sensitivity analyses. The third limitation was that, in most studies, the exact amount of soy consumption was not reported. Fourth, our results were predominantly from retrospective studies that did not meet any of the criteria that could increase the certainty of evidence. Additionally, for children who consumed soy-based milk, only Sinai et al. and Giampietro et al. reported that the exposure group consisted of patients in prevention or with an allergy to cow’s milk. Although most studies reported that participants were from healthy populations, many of the children could have had a condition that forced the parents to use soy milk instead of cow’s milk. We could not evaluate whether this condition could interfere with the association between soy and the onset of puberty. The fifth limitation was that in most studies included reports of puberty stage, progression of puberty, the expected mid-parental height and parental puberty were not assessed by a physician.

Conclusion

Implications for practice

We did not observe any association between a soy-based infant diet and the onset of puberty in boys or girls. The meta-analyses did not show any significant differences between groups in the risk of precocious puberty and menarche age. Individual studies have not shown significant clinical differences between groups regarding menarche age, age of testicular enlargement, pubarche, and voice change in boys. No study assessed the risk of delayed puberty as well as the age at onset of pubarche in girls.

Implications for research

Cohort studies with large sample sizes and appropriate methodology are required to evaluate whether the lack of an association between a soy-based infant diet and the onset of puberty remains when the results are compared considering the length of exposure, amount of soy consumption, and assessment of pubertal development through self-report or physical examination.

Figure of meta-analysis of the frequency of precocious puberty (excluding the studies with no events in both arms).

(DOCX)

Click here for additional data file.

Search strategy.

(DOCX)

Click here for additional data file.

PRISMA checklist.

(DOCX)

Click here for additional data file.

25 Sep 2020

PONE-D-20-20990

Effect of Soy-Based Diet in the Timing of Puberty: A Systematic Review and Meta-Analysis

PLOS ONE

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2) Please revise your introduction to ensure that all statements are supported by appropriate references.  Moreover, we note that reference 10 refers to a study conducted on animals, not on human participants; please revise the statement made in the introduction referring to this citation.

3)  In your Abstract, please consider including a statement regarding the overall quality  of evidence.

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- https://academic.oup.com/edrv/article/30/4/293/2355049

In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: I Don't Know

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors reviewed an extremely important topic that is highly debatable among pediatricians and pediatric endocrinologist. I have comments that are important to improve the reporting and end-user utilization:

- please add a reference to this statement is not "Efforts to implement healthier eating habits have resulted in an increase in the consumption of 47 soy-based products."

- the definition of population need to be more specific: were children with chronic illness excluded, did you included children from all ethnic background

- what did you consider an intake above the population mean for soy product?

- line 114: please correct the age for delayed puberty for girls is 13, boys 14

- do you mean final adult height? Or height assessed at the beginning of pubertal signs?

- how was the pubertal assessment done? was it assessed by a physician or self-reported

- in the statistical analysis section the authors need to elaborate on the method used to analyze the frequency of precocious puberty. Please see comment below regarding analysis of zero events rate.

- please describe GRADE evaluation in brief for the readers

-In the results please give details of the exposure in the included studies e.g dose, length of exposure, product type, the starting age of follow up

-Line 200-2001 Please clarify the sentence “ the exposed cohort was a selected group, and not as a representative of the average population in the community type of selected”

-In the meta-analysis, please do appropriate statistical correction for analyzing count data that include zero events. The current analysis model is not appropriate.

- Why the age of thelarche, pubarche, voice change were not meta-analyzed?

-For the height outcome, it is hard to interpret not knowing if height is assessed at similar pubertal stage or final adult height. However, in the figure the interpretation of data can be simplified for clinicians by changing the label of the figure to something like” taller with soy” vs. “taller with control”. It seems that the children consuming Soy are taller than controls, although the CI is touching zero. This needs to be heightened in the results & discussion.

From this data its suspicious that those children had gone through puberty earlier.

- please do sensitivity analysis to compare results of length of exposure to soy product, if puberty was assessed vs. self-reported, boys vs. girls, cohort year (because of the secular trend in pubertal achievement).

- table 1 in the supplementary material should be moved to the manuscript

- why heterogeneity was not explored?

-Please organize the authors in table 1 in alphabetical order, include the length of follow up, type of population (healthy, children with CMP allergy), amount of consumed soy products per day, ethnic group as separate column. Can you include possible effect modifers

-There are a list of outcomes planned in the methods section to be reported with no results reported. Can you please provide data on these outcomes.

-The results were not expected, can you please provide possible explanations. Also, can you please compare the intake reported in the included studies to the typically reported to cause precocious puberty

- your review has a unique opportunity to report on methods used to report puberty assessment. Please add a paragraph in the discussion to discuss the appropriateness of the used methods in the included studies and ways that could improve future research report.

-Please discuss gaps to be addressed in future research

- the conclusion statement need to be re-phrased to capture the evidence quality

Reviewer #2: I appreciate the effort of the authors in gathering collective evidence on the status of soy intake in relation to pubertal onset. The supplementary material indicates that there had been a thorough search of literature for articles that could potentially be included in the review. It seems that the authors also made sure to test the suitability of conducting a meta-analysis. However, I have the following concerns:

(1) The aim of the study, stated as “evaluate the EFFECT of exposure to a soy-based infant feeding or to a soy-rich diet during childhood on the timing of onset of puberty in girls and boys” was obviously not consistent with the choice of articles included in the review. One can only determine “effect” in the context of a well-designed clinical trial/experimental study, particularly a randomized controlled trial.

(2) Observational studies, where comparisons between the exposed group (ie, consumed soy/soy constituents) and an unexposed group (i.e., those that didn’t consume soy/soy constituents ), had been repeatedly called “controlled” studies. Considering that there were only 8 studies, and all but one (an RCT) are observational studies, this is misleading. A more appropriate term should be used to be more consistent with the types of articles that were included in the review.

(3) Although understandable, several outcome variables were considered for meta-analysis even if in some cases, only 1 or 2 studies was/were the source of “evidence” on which to base conclusions for a particular outcome. The heterogeneity tests all turned out non-significant possibly due to this (although already expected since the findings from the articles are not dissimilar). Very few studies are currently existing on this topic and this limits the ability to extract a well-informed or solid evidence on associations between soy intake and timing of puberty. Based on the existing evidence, however, can something conclusive be determined? If so, why is that so?

Here are additional comments/feedback and questions on the work:

1. Abstract:

lines 20-22 --Often, we measure the risk (of precocious puberty, in this case); why was "frequency" used and what does it mean? Did you mean mean this to be "count" (i.e., number of children who have precocious puberty)? THis is not clear.

2. Introduction:

lines 63-64 -- The study you cited is an animal study (on rats). We don't call female animals "women".

lines 63-64: The statement is not clear --What substances and what receptors?

line 73: Are you sure about the unit for this value (423.4 mg/day? The article says microgram/day (ug/day).

line 76: median intake is 10 milligrams/day (mg/day), as indicated in that study.

lines 84-86: "Effect" is the main aim of the review. However, the studies included in the review are mostly observational studies except for 1 RCT. Exposure mentioned here is "soy" but articles chosen included soy constituents (specifically the isoflavones). This aim needs rewriting.

3. Methods:

Criteria for eligibility section -- This part is a bit confusing because I'm expecting this to be focused on your criteria for selection of articles, but in some parts of this section, you are referring to individuals/study participants instead of the studies. Your focus should be on the articles and not study participants/individuals.

lines 103-104: What "population" serves as the reference then? Did you consider this as your "cut-off" for soy intake? Isn't it that exposure or non-exposure to soy in its different forms (soy foods, products, formula) and/or its constituents (soy isoflavones) regardless of amount eaten is your independent /exposure variable?

Lines 111-117: There are so many outcomes listed here and they appear to be different or not even related to each other. In your aim, you stated that you want to determine how soy exposure --> timing of puberty. I suggest that you stick to that and then state under this section what you consider to be measurements of timing of puberty before you list all that you have in the section (ie, early (such as in precocious puberty) or delayed onset of secondary sexual characteristics, in both males (pubarche, testicular growth, penile enlargement, etc.) and females (menarche, pubarche, thelarche) and other indicators of puberty (e.g., first ejaculation, voice change, growth spurt, etc.). This way, it would be clear to the reader that these "outcomes" you listed are all related to the timing of puberty. Also, why did you not consider separating the pubertal timing in boys and the pubertal timing in girls separately in your meta-analysis?

Lines 120-122: Did you mean studies without a "comparison group"? Please note that "uncontrolled studies" imply a clinical or experimental study design. Even if there is a comparison group, the exposed group cannot be considered to be a "controlled" comparison grp if the study design is not experimental.

Line 131: Please specify the databases you used for these types of studies (PhD/master's theses).

Line 134: Give a few examples of your search terms (you cannot expect readers to go to your supplementary file just for this)

Line 150: Did you mean randomized controlled trial?

4. Results:

In general, this section needs to be rewritten in order to be consistent with the other needed revisions (e.g., revised aim, etc.)

Line 211: Onset of menarche? timing of menarche

Line 219: Growth Spurt?

5. Discussion:

line 258: "controlled studies" would not be in the context of an experimental design. Please use a more appropriate term.

6. Conclusion: Considering that most of the evidence is from observational studies, there is a need to revise your statement here ("negatively alter" implies effect which is consistent with your current aim, but not appropriate based on where you got your evidence from, ie. observational studies).

7. Tables and Figures:

Figure 1:

--"Trabalhos excluidos" need to be translated to English;

--For the box "Full papers evaluated for eligibility), n=16. However, tracked articles (42) minus 29 under Trabalhos excluidos equals 13. Please correct this.

Table 1:

--This table would be more informational if the description of the studies were more succinct and if the study findings/conclusions were added. For example, "Outcome evaluation age" is redundant since the information is already under the "Patients" column (Why do you use the term patients instead of "Study Population"?The latter is more appropriate considering the study design of the articles). The exclusion criteria is not helpful--why not add information about variables/confounders that were controlled for by the study?

Table 2:

--This table is not helpful to my understanding of the quality of evidence. Please make it more stand-alone -- what do the symbols under the column "Certainty of the Evidence" mean? Also, there is no clear explanation in the text about how you came up with the quality of evidence. You only referred the reader to the table when discussing your results.

I hope this review will be helpful to the authors.

**********

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Reviewer #1: Yes: Reem Al Khalifah

Reviewer #2: Yes: Gina Segovia-Siapco

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8 Dec 2020

December 07, 2020

PLOS ONE

Editorial Board

Dear Editor:

Thank you very much for your email dated 09-Sep-2020 with reviews comments on our manuscript “The association between soy-based infant diet and the onset of puberty: A systematic review and meta-analysis”

The reviewers’ comments were very helpful. We thank you for the opportunity to address the comments in a revised version of the manuscript, and therefore we have revised the manuscript in accordance with the feedback.

On behalf of my co-authors, I am submitting the manuscript with the requested revision.

We hope that we have adequately addressed all the comments, and that this version is now acceptable for publication in the prestigious Plos One

Below are our responses to the specific issues raised in the comments:

Journal Requirements:

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOSONE style templates can be found at

R.: We have reviewed and made corrections

2. Please address the following points:

1) We note that publication bias has not been assessed. Please provide an assessment using both graphs (funnel plots) and statistical methods

R. We have not assessed publication bias either using graphs or statistical methods due to the number of studies (<10). We have emphasized this in the manuscript, line 243-244 and 324-325

2) Please revise your introduction to ensure that all statements are supported by appropriate references. Moreover, we note that reference 10 refers to a study conducted on animals, not on human participants; please revise the statement made in the introduction referring to this citation.

R. We have revised and corrected all introduction. “Experimental animal studies have showed that xenobiotics as polychlorinated biphenyls (PCBs), phytoestrogens, fungicides, pesticides may affect sexual differentiation [9]. This interference has a high probability of affecting both reproductive physiology and behavior at several stages of life [10]. In female rats, an early exposure (late embryonic and / or early postnatal) to low doses of PCBs [10] or soy significantly affected mating behavior”

3) In your Abstract, please consider including a statement regarding the overall quality of evidence.

R. We have included; line 39-40

4) Please consider reporting the full results of your quality assessment in the main text, and not in the Supplementary file.

R. We have reported the full results of quality assessment in the main text (Table 2 and 3)

3. We noticed you have some minor occurrence of overlapping text with the following previous publication(s), whichneeds to be addressed:

- https://academic.oup.com/edrv/article/30/4/293/2355049

In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text out side the methods section. Further consideration is dependent on these concerns being addressed.

R. We have revised all introduction in order to avoid this occurrence

4. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-textcitations to match accordingly. Please see our Supporting Information guidelines for more

R. We have included

5. We note that this manuscript is a systematic review or meta-analysis; our author guidelines therefore require that youuse PRISMA guidance to help improve reporting quality of this type of study. Please upload copies of the completed PRISMA checklist as Supporting Information with a file name “PRISMA checklist”.

[Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions

R. We have completed (S3_File)

1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

R. We have revised and updated the introduction, discussion and conclusion

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: I Don't Know

R. We have revised all methodology, especially the meta-analysis of precocious puberty, line 210-219

3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fullyavailable without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDFfile). The data should be provided as part of the manuscript or its supporting information, or deposited to a publicrepository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer 1

Thank you very much for your comments and suggestions that absolutely improved the quality of our review. So, we have updated the manuscript based on your comments and all your suggestions were included in the revised manuscript, as you can see below:

The authors reviewed an extremely important topic that is highly debatable among pediatricians and pediatric endocrinologist. I have comments that are important to improve the reporting and end-user utilization:

- please add a reference to this statement is not "Efforts to implement healthier eating habits have resulted in an increase in the consumption of 47 soy-based products."

R. We have updated this phrase and include the reference, line 86-87

- the definition of population need to be more specific: were children with chronic illness excluded, did you included children from all ethnic background

R. We have specified, line 140-143

- what did you consider an intake above the population mean for soy product?

R. We have rewritten this phrase, line 144-149

- line 114: please correct the age for delayed puberty for girls is 13, boys 14

R. We have corrected, line 158-159

- do you mean final adult height? Or height assessed at the beginning of pubertal signs?

R. We have changed this expression to height in the last follow-up, line 160

- how was the pubertal assessment done? was it assessed by a physician or self-reported

R. We have included this information, line 287-288

- in the statistical analysis section the authors need to elaborate on the method used to analyze the frequency ofprecocious puberty. Please see comment below regarding analysis of zero events rate.

R. We have updated the data analysis, line 210-217

- please describe GRADE evaluation in brief for the readers

R. We have included this information, line 232-242

- In the results please give details of the exposure in the included studies e.g dose, length of exposure, product type, the starting age of follow

R. We have included this information, line 258-267

-Line 200-2001 Please clarify the sentence “ the exposed cohort was a selected group, and not as a representative of the average population in the community type of selected”

R. We have updated all risk of bias assessment and this phrase was eliminated, line 281-290

-In the meta-analysis, please do appropriate statistical correction for analyzing count data that include zero events. The current analysis model is not appropriate.

R. We have updated the data analysis, line 210-217

- Why the age of thelarche, pubarche, voice change were not meta-analyzed?

R. They were not meta-analyzed because only one study evaluated this outcome, or two evaluated, but with heterogeneity between two results. We have included this information in the methodology, line 226 “If enough studies were available, the potential causes of heterogeneity among studies could be evaluated by subgroup analysis. In case of considerable inconsistency (I2 > 50%) associated to variation in the direction of association, and heterogeneity could not be explained, we did not perform a meta-analysis”

- For the height outcome, it is hard to interpret not knowing if height is assessed at similar pubertal stage or final adult height. However, in the figure the interpretation of data can be simplified for clinicians by changing the label of the figure to something like” taller with soy” vs. “taller with control”. It seems that the children consuming Soy are taller than controls, although the CI is touching zero. This needs to be heightened in the results & discussion. From this data its suspicious that those children had gone through puberty earlier.

R. Thank you for important information. First, I have double checked the results plotted in the meta-analyses, and we realized, that in one of the studies only the data for girls was plotted. So we corrected this mistake, and in the current graph we can see a non-difference between groups. Fig 4.

- please do sensitivity analysis to compare results of length of exposure to soy product, if puberty was assessed vs. self-reported, boys vs. girls, cohort year (because of the secular trend in pubertal achievement).

R. Unfortunately, we could not be performed sensitivity analyses due to small number of studies include, and have explained this in the main manuscript, line 219-222, and in the limitations paragraph.

- table 1 in the supplementary material should be moved to the manuscript - why heterogeneity was not explored? -Please organize the authors in table 1 in alphabetical order, include the length of follow up, type of population (healthy,children with CMP allergy), amount of consumed soy products per day, ethnic group as separate column. Can you include possible effect modifers

R. We have updated the table 1 and included all your suggestions, and we moved for it all information that was in the supplementary data. We have explored heterogeneity in the item “Studies included in the qualitative synthesis”, line 327

-There are a list of outcomes planned in the methods section to be reported with no results reported. Can you please provide data on these outcomes.

R. We have updated this infomration and justified that No study assessed the risk of delayed puberty, as well as the age of onset of pubarche in girls, line 340

-The results were not expected, can you please provide possible explanations. Also, can you please compare the intake reported in the included studies to the typically reported to cause precocious puberty

R. We have provided this in the discussion section, line 357-379

- your review has a unique opportunity to report on methods used to report puberty assessment. Please add a paragraph in the discussion to discuss the appropriateness of the used methods in the included studies and ways that could improve future research report.

R. We have included this information, line 404-407

-Please discuss gaps to be addressed in future research

R. We have included this information, line 390-393

- the conclusion statement need to be re-phrased to capture the evidence quality

R. We have updated this information, line 395

Reviewer 2

Thank you very much for your comments and suggestions that absolutely improved the quality of our review. So, we have updated the manuscript based on your comments and all your suggestions were included in the revised manuscript, as you can see below:

I appreciate the effort of the authors in gathering collective evidence on the status of soy intake in relation to pubertal onset. The supplementary material indicates that there had been a thorough search of literature for articles that could potentially be included in the review. It seems that the authors also made sure to test the suitability of conducting a meta-analysis. However, I have the following concerns:

(1) The aim of the study, stated as “evaluate the EFFECT of exposure to a soy-based infant feeding or to a soy-rich diet during childhood on the timing of onset of puberty in girls and boys” was obviously not consistent with the choice of articles included in the review. One can only determine “effect” in the context of a well-designed clinical trial/experimental study, particularly a randomized controlled trial.

R. Thank you for important observation. I have updated all manuscript according to your suggestion. I have changed the term effect for association, and control for unexposed.

(2) Observational studies, where comparisons between the exposed group (ie, consumed soy/soy constituents) and an unexposed group (i.e., those that didn’t consume soy/soy constituents), had been repeatedly called “controlled” studies. Considering that there were only 8 studies, and all but one (an RCT) are observational studies, this is misleading. A more appropriate term should be used to be more consistent with the types of articles that were included in the review.

R. Thank you for important observation. I have updated all manuscript according to your suggestion. I have changed the term effect for association, and control for unexposed.

(3) Although understandable, several outcome variables were considered for meta-analysis even if in some cases, only 1or 2 studies was/were the source of “evidence” on which to base conclusions for a particular outcome. The heterogeneity tests all turned out non-significant possibly due to this (although already expected since the findings from the articles are not dissimilar). Very few studies are currently existing on this topic and this limits the ability to extract a well-informed or solid evidence on associations between soy intake and timing of puberty. Based on the existing evidence, however, can something conclusive be determined? If so, why is that so?

R. Thank you for important comments. I have updated all manuscript in order to change the idea of effect for association. Additionally, we have emphasized the quality of evidence according GRADE approach. The no association does not mean no effect, due to that, we think that the current version our conclusion is more apropriate.

Here are additional comments/feedback and questions on the work:

1. Abstract: lines 20-22 --Often, we measure the risk (of precocious puberty, in this case); why was "frequency" used and what does itmean? Did you mean mean this to be "count" (i.e., number of children who have precocious puberty)? This is not clear.

R. In all manuscript we have changed for risk of precocious puberty

2. Introduction: lines 63-64 -- The study you cited is an animal study (on rats). We don't call female animals "women". lines 63-64: The statement is not clear --What substances and what receptors? line 73: Are you sure about the unit for this value (423.4 mg/day? The article says microgram/day (ug/day). line 76: median intake is 10 milligrams/day (mg/day), as indicated in that study. lines 84-86: "Effect" is the main aim of the review. However, the studies included in the review are mostly observational studies except for 1 RCT. Exposure mentioned here is "soy" but articles chosen included soy constituents (specifically the isoflavones). This aim needs rewriting.

R. We have corrected all these smistake, sorry for this, line 92, 103-106, 116-121. In line 144 we have included: The exposure group comprised individuals exposed to a soy-based infant diet. We considered as a soy-based infant diet a soy-based infant feeding (e.g., formula or milk soy-based), higher food intake of isoflavones, or the use of soy protein-based supplements

3. Methods: Criteria for eligibility section -- This part is a bit confusing because I'm expecting this to be focused on your criteria for selection of articles, but in some parts of this section, you are referring to individuals/study participants instead of the studies. Your focus should be on the articles and not study participants/individuals. lines 103-104: What "population" serves as the reference then? Did you consider this as your "cut-off" for soy intake? Isn'tit that exposure or non-exposure to soy in its different forms (soy foods, products, formula) and/or its constituents (soy isoflavones) regardless of amount eaten is your independent /exposure variable? Lines 111-117: There are so many outcomes listed here and they appear to be different or not even related to each other. In your aim, you stated that you want to determine how soy exposure --> timing of puberty. I suggest that you stick to that and then state under this section what you consider to be measurements of timing of puberty before you list all that you have in the section (ie, early (such as in precocious puberty) or delayed onset of secondary sexual characteristics, in both males (pubarche, testicular growth, penile enlargement, etc.) and females (menarche, pubarche, thelarche) and other indicators of puberty (e.g., first ejaculation, voice change, growth spurt, etc.). This way, it would be clear to the reader that these "outcomes" you listed are all related to the timing of puberty. Also, why did you not consider separating the pubertal timing in boys and the pubertal timing in girls separately in your meta-analysis? Lines 120-122: Did you mean studies without a "comparison group"? Please note that "uncontrolled studies" imply a clinical or experimental study design. Even if there is a comparison group, the exposed group cannot be considered to be a "controlled" comparison grp if the study design is not experimental. Line 131: Please specify the databases you used for these types of studies (PhD/master's theses). Line 134: Give a few examples of your search terms (you cannot expect readers to go to your supplementary file just forthis) Line 150: Did you mean randomized controlled trial?

R. We have updated the methods section, and we included all your suggestions, line 131-181. Thank you so much for all them. I have specified the two outcomes not evaluate, because the studies included did not report them, line 340.

4. Results: In general, this section needs to be rewritten in order to be consistent with the other needed revisions (e.g., revised aim,etc.) Line 211: Onset of menarche? timing of menarche Line 219: Growth Spurt?

R. We have updated the methods section, and we included all your suggestions line 292-341, and in the method section (outcomes)we gave the definition for menarche age.

5. Discussion: line 258: "controlled studies" would not be in the context of an experimental design. Please use a more appropriate term.

R. We have updated this term for observational studies

6. Conclusion: Considering that most of the evidence is from observational studies, there is a need to revise your statement here ("negatively alter" implies effect which is consistent with your current aim, but not appropriate based on where you got your evidence from, ie. observational studies).

R. We have updated the conclusion, and changed the term effect for association

7. Tables and Figures: Figure 1: --"Trabalhos excluidos" need to be translated to English. R. Ok; --For the box "Full papers evaluated for eligibility), n=16. However, tracked articles (42) minus 29 under Trabalhos excluidos equals 13. R. OK, we have correcte.

Please correct this. Table 1: --This table would be more informational if the description of the studies were more succinct and if the studyfindings/conclusions were added. For example, "Outcome evaluation age" is redundant since the information is alreadyunder the "Patients" column (Why do you use the term patients instead of "Study Population"?The latter is more appropriate considering the study design of the articles). The exclusion criteria is not helpful--why not add information about variables/confounders that were controlled for by the study?

R. We have updated table 1 according to your suggestions.

Table 2: --This table is not helpful to my understanding of the quality of evidence. Please make it more stand-alone -- what do the symbols under the column "Certainty of the Evidence" mean? Also, there is no clear explanation in the text about how you came up with the quality of evidence. You only referred the reader to the table when discussing your results. I hope this review will be helpful to the authors.

R. R. We have included information about GRADE in lines 232-242, and we have updated the table 2 in order to be more precise.

14 Jan 2021

PONE-D-20-20990R1

The association between soy-based infant diet and the onset of puberty: A systematic review and meta-analysis

PLOS ONE

Dear Dr. Nunes,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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PLOS ONE

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Reviewer #1: All comments have been addressed

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Reviewer #1: the authors made significant improvement to the manuscript and addressed most of the comments. There are few comments need to be addressed:

1- the hight outcome need to be revisited. I notice that there are 2 studies that have reported height at measurement at beginning of puberty and one as a final adult height. 1- Table 1 should specify if the data is in Cm or SD. 2- present the effect estimate of the reported height at measurement at beginning of puberty and one as a final adult height as separate and as combined. 3- convert the effect estimate to absolute data either SD or Cm rather than leaving it as SMD to improve understanding of the results.

2- all meta-analysis figures did not include label for the direction of the effect estimate.

3- across the manuscript the authors mention that they could or couldn't present meta-analysis figure, but the correct wording is to say that meta analysis could or couldn't be performed. The figure is just a away of the results presentation but meta-analysis is a statistical technique.

4- the definition of high soy intake need to be included in the methods section, then in the results you report what sort of intake was there int he groups, and include the actual intake in table 1.

5- the authors need to describe how the reported findings in the risk of bias section from studies had influenced the risk of bias scores for each one. for example, did studies using self-reported pubertal data were given high or low risk of bias score? this paragraph need to be restructured as specific ROB domains, then overall.

6-the opening paragraph of the discussion need to be focused on the study findings and not to reiterate what was mentioned already in the introduction.

7- the GRADE table, please change to the table view " GRADE profile (V2)) to give details on judgments leading to low quality evidence since its not described in the text.

8- the outcome data in the risk of the outcome with no soy bean, how did you come up with the numbers? are these using the risk in the general population or in the reported comparison group from included studies?

please include foot note to explain this.

for example the mean timing of menarche should not be 0, rather it should be 12,7 years

9- in table 1, please highlight the country of the study in a different column rather than combining it with the population, also age & BMI need to be separated in to columns . All ages need to be standardized either years or months. add a column for total number of children followed in each study.

10- the results are a bit confusing with regard to PP, in table 2 it says 40 out of 1000 are expected to have PP in the unexposed group vs 8 out of 1000 are expected to have PP in the exposed group. although not statistically significant effect, but it is suggesting more PP with lack of exposure, that raises suspicion about the results. Siani 2018, was the only study to report PP while the others had 0 events, the children in his study had cows milk allergy. It is important to understand more information about those children such as: what was the other milk types they took, for how long. was the exposure to soy milk limited to the 1st year of life ?

the discussion section for such observation is important and need to highlight all these details. The current discussion is not focused on this element. please consider if leaving this study out of the analysis would be the most appropriate action because those children are considered different than typically other healthy children in terms of type of nutritional intake because of the allergy.

11- in the limitation section please include that future studies need to report physician assessed puberty stage, and the progression of puberty in those children, in addition to : expected mid parental height, parental puberty.

12- need to consider english editing

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28 Feb 2021

Dear reviewer

Again, I want to like to thank you for spending time and efforts to improve our review. Your comments have improved it a lot, and we have learned many methodological aspects by your revision. Again, thank you very much.

1- The height outcome needs to be revisited. I notice that there are 2 studies that have reported height at measurement at beginning of puberty and one as a final adult height.

R. Thank you for the important detail, we have emphasized the height outcome in the manuscript, line 370 to 380, and in the table 1.

2- Table 1 should specify if the data is in Cm or SD.

R. We have specified

3- present the effect estimate of the reported height at measurement at beginning of puberty and one as a final adult height as separate and as combined.

R. We have specified this in the table 3, actually only Duitama presented height at baseline and as effect estimate, the other two only presented the estimate effect

4- convert the effect estimate to absolute data either SD or Cm rather than leaving it as SMD to improve understanding of the results.

R. As Sinai et al presented data only in SD, we have excluded it from the meta-analysis. We performed meta-analysis only with Duitama e Strom, and in Strom we converted inches in centimeters, and we could perform the meta-analysis in MD (figure 4)

5- all meta-analysis figures did not include label for the direction of the effect estimate.

R. Sorry for this, we have included the direction of the effect estimate in the meta-analyses

6- across the manuscript the authors mention that they could or could not present meta-analysis figure, but the correct wording is to say that meta-analysis could or could not be performed. The figure is just an away of the results presentation, but meta-analysis is a statistical technique.

R. Sorry for this, we have corrected this

7- the definition of high soy intake needs to be included in the methods section, then in the results you report what sort of intake was there int he groups and include the actual intake in table 1.

R. Again, thank you for important observation. Unfortunately, during the protocol of this SR we could not define a high soy intake as eligibility criteria. This is because there is not an exact definition of normal soy intake in children. Additionally, our objective was to include studies who participants intake more soy than most population, as people from Asia. We have included the paragraph below in the manuscript (line 122 to 131).

Given that the mean intake of isoflavones varies from country to country and there is no definition of either a mean or high intake of this phytoestrogen in children, we did not use a cut-off point to separate participants with a high intake from those with a low intake. However, the studies were included if the authors used any tool to classify children as high or low consumers. When the mean daily intake of soy and its’ derivatives was not available, we included patients who were known to have a higher consumption of soy than the general population, such as vegetarians and Asian populations. The assessment of soy consumption was determined through dietary records, food frequency questionnaires, or the participant’s use of soy supplements.

8- the authors need to describe how the reported findings in the risk of bias section from studies had influenced the risk of bias scores for each one. for example, did studies using self-reported pubertal data were given high or low risk of bias score? this paragraph need to be restructured as specific ROB domains, then overall.

R. Actually we could apply Rob 1 only in Duitama, because the other studies were observational. However, the table 4 (Explanations) we have emphasized that this point was important to rate down the quality of evidence in the risk of bias.

9-the opening paragraph of the discussion need to be focused on the study findings and not to reiterate what was mentioned already in the introduction.

R. We have removed what was mentioned already in the introduction.

10- the GRADE table, please change to the table view " GRADE profile (V2)) to give details on judgments leading to low quality evidence since it’s not described in the text.

R. Thank you very much for this suggestion, we have change for Grade profile V2 and we have given details on judgments leading to low quality evidence

11- the outcome data in the risk of the outcome with no soybean, how did you come up with the numbers? are these using the risk in the general population or in the reported comparison group from included studies?

please include foot note to explain this.

for example the mean timing of menarche should not be 0, rather it should be 12,7 years

R. We have reviewed the table of GRADE with the data from the meta-analyses in order to become it clear than the first one.

12- in table 1, please highlight the country of the study in a different column rather than combining it with the population, also age & BMI need to be separated into columns. All ages need to be standardized either years or months. add a column for total number of children followed in each study.

R. We have highlighted in a different column all these data

13- the results are a bit confusing with regard to PP, in table 2 it says 40 out of 1000 are expected to have PP in the unexposed group vs 8 out of 1000 are expected to have PP in the exposed group. although not statistically significant effect, but it is suggesting more PP with lack of exposure, that raises suspicion about the results. Siani 2018, was the only study to report PP while the others had 0 events, the children in his study had cow’s milk allergy. It is important to understand more information about those children such as: what was the other milk types they took, for how long. was the exposure to soy milk limited to the 1st year of life?

the discussion section for such observation is important and need to highlight all these details. The current discussion is not focused on this element. please consider if leaving this study out of the analysis would be the most appropriate action because those children are considered different than typically other healthy children in terms of type of nutritional intake because of the allergy.

R. We have updated these data in the table of GRADE. I have included all details of Sinai 2018 in the lines 251 to 254. We have included this point in the discussion section (line 447 to 452). In the meta-analysis of height Sinai was excluded due to present data only in SD. We have not excluded it from the review but emphasized this as a limitation (line 447 to 452)

14- in the limitation section please include that future studies need to report physician assessed puberty stage, and the progression of puberty in those children, in addition to: expected mid parental height, parental puberty.

R. Okay, we have included

15- need to consider English editing

R. Okay, we used Editage for English editing

Submitted filename: 2 response revision.docx

Click here for additional data file.

31 Mar 2021

PONE-D-20-20990R2

Association between a soy-based infant diet and the onset of puberty: A systematic review and meta-analysis

PLOS ONE

Dear Dr. Nunes,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by May 15 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Ivan D. Florez, MD, MSc, PhD

Academic Editor

PLOS ONE

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Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewer #1: All comments have been addressed

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Reviewer #1: Yes

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Reviewer #1: Yes

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Reviewer #1: thank you for addressing the comments

please verify that all data in table 1 are using the same metrics; for example there is still some data in months while others in years.

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Reviewer #1: No

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20 Apr 2021

April 20, 2021

Dear Reviewer

We have revised the manuscript according to your comment about table 1. We have reviewed table 1 and table 2, and we used the same metrics (years) in all anthropometric data, as well as follow up time in the table 4.

Again, thank you so much for spending time reviewing and consequently improving our manuscript.

Your Sincerely,

Vania dos Santos Nunes Nogueira

Department of Internal Medicine – Botucatu Medical School – Sao Paulo State University/UNESP, Sao Paulo, Brazil. Tel: (55 14) 3880 11 71E-mail: vania.nunes-nogueira@unesp.br

Submitted filename: 3 response revision to reviewer 1.docx

Click here for additional data file.

23 Apr 2021

Association between a soy-based infant diet and the onset of puberty: A systematic review and meta-analysis

PONE-D-20-20990R3

Dear Dr. Nunes,

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Reviewers' comments:

10 May 2021

PONE-D-20-20990R3

Association between a soy-based infant diet and the onset of puberty: A systematic review and meta-analysis

Dear Dr. Nunes-Nogueira:

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