Short stature, defined as height < -2 standard deviations below the mean for age, sex, and population, is a common complaint in pediatric endocrinology (1, 2). It is considered idiopathic in the absence of systemic, endocrine, nutritional, or genetic abnormalities. Despite technological advances and extensive research, the underlying cause of idiopathic short stature (ISS) remains largely unknown. Studies in mice have shown that germ-free (GF) mice exhibit stunted growth and reduced weight gain on a standard breeding diet (3). Antibiotic treatment in GF mice colonized with gut microbiota (GM) of specific pathogen-free mice resulted in decreased serum insulin-like-growth-factor (IGF)-1 levels and inhibited bone formation, while supplementation of the antibiotic-treated mice with short-chain fatty acids, products of microbial metabolism, restored IGF-1 levels and bone mass to the levels of non-antibiotic-treated mice (3). Recent research in malnourished mice has shown that a strain of Lactiplantibacillus plantarum (Lp^WJL) improved circulating levels and activity of IGF-1 and insulin by activating the NOD2 receptor in intestinal epithelial cells (4, 5). The role of nutrition and GM on linear growth in humans has mainly focused upon malnourished children in developing countries. Those studies found that a less diverse GM was associated with the severity of stunting (6), but such findings may be skewed due to differences in nutrition, sanitation, and healthcare accessibility. Indeed, genetic, environmental, and geographical factors are recognized as having an impact on the GM, prompting the consideration that members of the same family may have quite similar GM (7).

In the present study of the role of GM in the pathogenesis of ISS, we enrolled healthy pre-pubertal ISS children from an Israeli middle-class population and their normal-height siblings who served as controls. The study goals were to determine whether there is a difference in the GM composition between ISS children and their normal-height siblings and to investigate the relationship between GM and linear growth by transplanting feces from both groups into GF mice and monitoring their growth.

This case-control study was conducted at the Institute for Endocrinology and Diabetes at the Schneider Children’s Medical Center (SCMCI), Israel, from April 2018 through November 2020. It was approved by the Rabin Medical Center’s ethics review committee (study number 0238-17-RMC). All study participants’ parents signed a written informed consent form.

Patients: This study included 30 healthy pre-pubertal, short, and lean children (height ≤ 3^rd and body mass index (BMI) <10^th percentile for age and sex (2000 Centers of Disease Control [CDC] growth charts) from middle-class families referred to our clinic for growth assessment (one short child per family). In all ISS participants, a comprehensive medical work-up revealed no underlying systemic, hormonal, or skeletal pathologies as a cause of their ISS. Their general blood tests (complete blood count, renal and liver function tests, and a lipid profile performed at SCMCI’s central laboratory) fell within the normal range, and all were growth hormone (GH)‐sufficient with a peak stimulated GH levels ≥7.5 μg/L using the standard secretagogues of glucagon/clonidine/arginine.

The controls included their 30 healthy pre-pubertal siblings (one per participant) with normal percentiles of height (>25^th), weight (25^th-50^th), and body mass index (BMI: 25^th-75^th).

The eligibility criteria for the ISS children and their siblings were as follows: Inclusion criteria: Born appropriate-for-gestational-age; aged 3-10 years; prepubertal - boys: testicular volume ≤3 mL; girls: breast Tanner stage 1; GH‐treatment naïve. Exclusion criteria: genetic syndromes, chromosomal abnormalities, known chronic diseases, gastrointestinal diseases, malabsorption syndromes, metabolic disorders, malignancy, overweight/obesity, use of medications affecting growth/appetite, and antibiotic use within 3 months prior to enrollment.

In Israel, genetic screening that includes a panel for short stature is carried out in accordance with the Ministry of Health guidelines. Genetic screening is performed only in children with a suspected genetic syndrome associated with short stature or a familial extreme short stature defined as more than 3 standard deviations below the mean. Since none of our participants fulfilled these criteria, they did not undergo genetic analysis such as G-banding and mutation screening of ISS causal genes.

The results of this analysis of the GM of children with ISS and their normal-height siblings revealed that the phylum Euryarchaeota was present exclusively in the ISS group. Within the ISS group, the GM analysis identified two subgroups, with Methanobrevibacter, the predominant species in the ISS group’s archaeal gut community, found solely in subgroup 2. Post hoc analysis comparing the two ISS subgroups disclosed that children in subgroup 2 were shorter, and their height SDS exhibited a significant negative correlation with the abundance of Methanobrevibacter. This observation of diversity within the ISS group is innovative and implies a potential role of GM in understanding variations in the underlying causes of ISS.

ISS is a complex term that encompasses underlying mechanisms that are still unidentified. The GM and growth have recently been connected, with microbial diversity correlated with stunting severity (6). Our findings suggest that the composition of GM may play a role in explaining disparities in linear growth in some of the ISS children participating in this study, namely children with higher Methanobrevibacter levels (ISS-subgroup 2). However, in the ISS-subgroup with GM similar to that of their normal height siblings (ISS-subgroup 1) the underlying causes of ISS may be attributed to a wide range of other as-yet-unidentified variables.

In contrast to Li et al.’s recent study (22), which reported variations in GM composition and metabolite levels between children with ISS and the general population in eastern China, our analysis revealed no difference in microbial richness between children with ISS and their siblings. However, we did observe 3 notable variations, including the presence of Methanobrevibacter, which not only distinguished the short children from their normal-height siblings but also delineated 2 distinct subclasses within the ISS group. We also noted increased predicted levels of metabolic enzymes and decreased levels of specific AAs in those children. The differences between our findings and those of Li et al. could be attributed to differences in study design. While the former authors compared children with ISS to normal-height children from the general population, we conducted a comparison of each ISS child to his/her normal-height sibling, thereby isolating differences related to short stature rather than genetics, home environment, dietary habits, or demographics.

The only clinical difference between the 2 subgroups of ISS was the significantly shorter stature of the subgroup displaying elevated levels of Methanobrevibacter. Serum levels of Alkaline Phosphatase, a potential marker of bone elongation and osteoblast differentiation, were also significantly low in ISS Subgroup 2. This finding suggests that Methanobrevibacter, possibly through specific metabolites, may have an adverse impact on chondrocytes and osteoblasts, consequently impeding bone elongation. Nonetheless, in our study cohort, the causal relationship between Methanobrevibacter and ISS remains unidentified. Food consumption and eating behavior were also similar in the two subgroups. These findings are in opposition to those of previous studies that had suggested that the presence of Methanobrevibacter is influenced by dietary habits as evidenced by elevated levels of Methanobrevibacter in the fecal samples of malnourished and anorexic patients (23). Previous research has demonstrated that the diverse roles of certain Methanobrevibacter species are often associated with complex sugar degradation. Additionally, several studies demonstrated that Methanobrevibacter can increase short-chain fatty acid (SCFA) production, either directly or through association with other bacterial strains in gut (24). Contrarily, our metabolomics analysis failed to identify any significant differences between the ISS children and their siblings. It is plausible that Methanobrevibacter might behave differently in healthy, well-nourished individuals that possess stable microbiota. Although our analysis did not identify significant differences between the groups, these assumptions are based on existing literature. In this study we focused on a panel of 66 bacterially associated metabolites. We found no evidence of altered complex sugar degradation, nor did PiCRUSt predictive analysis reveal any differentially abundant metabolites/pathways associated with Methanobrevibacter. More research would be needed to more deeply understand the role of this taxon in ISS.

Our bioinformatics analysis suggested a possible increase in genes encoding enzymes involved in the de novo biosynthesis of pyrimidines and purines, as well as flavin, thiamine, and other similar compounds in the ISS group ( Figure 1D ). Pyrimidines and purines are essential building blocks of DNA and RNA, and also play important roles in various metabolic and signaling processes. Flavin mononucleotide and flavin adenine dinucleotide are crucial active groups in most flavoproteins/flavo-coenzymes, which are involved in numerous vital physiological functions (25). However, it remains unclear what clinical significance an increase in fecal levels of these compounds would have in children with ISS.

Furthermore, when compared to their siblings, the ISS children had lower levels of the AAs leucine, isoleucine, glutamic acid, and threonine in their feces. Colonic bacteria use these AAs to produce a complex mixture of metabolic end-products, including SCFAs, which affects metabolism and growth by changing hormone levels, such as leptin, ghrelin, glucagon-like peptide 1, and peptide YY, as well as by increasing IGF-1 in serum, liver, and bone (3). Leucine, in particular, is a strong inducer of the mammalian target of rapamycin complex 1 (mTORC1), a protein complex that functions as a nutrient sensor as well as a regulator of protein synthesis (26). Furthermore, leucine is required for IGFBP-1 de-phosphorylation: without it, IGFBP-1 has significantly increased IGF-1 affinity, stabilizing the IGF-1-IGFBP-1-ALS complex and inhibiting IGF-1-stimulated cell growth (27). Although we did not find lower levels of SCFA in our ISS group, an association of lower levels of these AAs with slower linear growth cannot be ruled out.

Aside from the differences in GM between ISS children and their normal-height siblings, our study findings revealed that the ISS group itself is diverse, with varying levels of Methanobrevibacter. These findings highlight the potential role of the GM in explaining variations in linear growth among healthy children and could serve as a starting point for personalized therapies targeting the GM to improve growth in children with ISS.

The data presented in the study are deposited in the EBI repository, accession numbers: PRJEB72222 and ERP157006.

The studies involving humans were approved by Rabin Medical Center’s ethics review committee (study number 0238-17-RMC). The studies were conducted in accordance with the local legislation and institutional requirements. Consent to participate in this study was obtained from the participants legal guardians/next of kin. The animal study was approved by Bar Ilan University Animal Studies Committee. The study was conducted in accordance with the local legislation and institutional requirements.

LL: Conceptualization, Data curation, Formal analysis, Supervision, Validation, Writing – original draft, Writing – review & editing. AE: Writing – review & editing, Investigation, Methodology. LM: Methodology, Writing – review & editing, Data curation. MY-G: Writing – review & editing, Formal analysis. MB: Writing – review & editing, Methodology, Data curation. BS: Methodology, Writing – review & editing, Data curation. MN: Writing – review & editing, Formal analysis, Funding acquisition. MP: Funding acquisition, Writing – review & editing. ST: Writing – review & editing, Data curation, Formal analysis. OK: Formal analysis, Writing – review & editing, Supervision. GG: Formal analysis, Supervision, Writing – review & editing, Conceptualization, Data curation, Investigation, Validation, Writing – original draft.