Children with idiopathic short stature and growth hormone deficiency exhibit similar changes in gut microbiota

Abstract

Children with idiopathic short stature (ISS) and growth hormone deficiency (GHD) exhibit imbalances in gut microbiota (GM), and the latter is related to endocrine hormones (such as insulin-like growth factor 1 (IGF-1)). The current study investigated the compositional and functional variations in GM between children with ISS and GHD, employing 16S rRNA sequencing technology. Sequencing results from 15 children with ISS and 18 children with GHD indicated no significant differences in GM alpha diversity or phylum-level diversity between the ISS and GHD groups. At the genus level, the abundance of Terrisporobacter was significantly greater in the ISS group compared to the GHD group, whereas the abundance of Acidovorax was reduced. The abundance of Prevotella stercorea and uncultured Sutterella sp. at the species level was significantly lower in the ISS group compared to the GHD group. The third level (L3) of the Kyoto Encyclopedia of Genes and Genomes (KEGG) database revealed functional variations in GM, with children in the ISS group having higher levels of intestinal bacteria Mobility Proteins and Background Chemotaxis. Despite these differences, the overall composition and function of GM between ISS and GHD children were not significantly different, indicating that the mechanisms by which GM influences the growth and development of children in both groups may be similar. This study was registered with the Medical Research Registration and Record System with the registration number MR-44-24-045472.

1. Introduction

Idiopathic short stature (ISS), growth hormone deficiency (GHD), constitutional delayed puberty (CDGP), familial short stature (FSS), and congenital hypothyroidism (CH) have been identified as the top five common causes of short stature in children [1]. The two most frequent causes of short stature are ISS and GHD; the incidence of ISS ranges from 40% to 80% [2, 3], while the incidence of GHD is approximately 38.6% [4]. The precise etiology of ISS is unknown, and its pathophysiology is somewhat complicated. Currently, the known causes of ISS may include [5]: (1) Insufficient or disordered secretion of the growth hormone (GH); (2) Reduced activity or structural abnormalities in GH; (3) Abnormal function or decreased activity of GH receptors; (4) The concentration of GH binding protein could be too high; (5) Abnormal synthesis or secretion of IGF-1. The term “GHD” primarily refers to a growth disorder condition brought on by changes in the normal molecular structure of growth hormone or inadequate or absent GH production and secretion by anterior pituitary cells. Recombinant human growth hormone (rhGH) treats both ISS and GHD because, from an etiological perspective, both conditions show failure in GH synthesis, secretion, or binding. However, the therapeutic efficacy of rhGH in children with GHD has been demonstrated to be superior at a lower dosage. On the other hand, children with ISS may develop resistance to GH, which leads to a slightly reduced therapeutic effect and a higher dosage [6, 7].

The GM functions as an endocrine organ, secreting various hormones that influence growth and development. The intestinal tract is home to many endocrine cells, such as K cells, I cells, L cells, and intestinal Chromaffin cells. These cells are capable of effectively regulating the levels of Adrenocorticotropic hormone (ACTH), ketone (CORT), and Adrenocorticotropic hormone Release factor (CRF), as well as influencing brain function and host behavior through the endocrine pathway and gut microbiota structure [8]. GM can modulate the levels of a variety of hormones secreted by the endocrine cells in the intestinal mucosa and submucosa, including cholecystin (CCK), motilin (MTL), Secretin, leptin (LP), Adrenocorticotropic hormone (ACTH), and CRF [9-11]. Furthermore, GM can impact the secretion of insulin, glucagon, leptin, and other hormones essential for controlling growth and development by acting on the liver, pancreas, fat, and other secretory organs [12, 13]. Many studies have shown that probiotic supplementation influences intestinal hormone secretion [14-16], which further supports the idea that alterations in GM can influence intestinal endocrine hormone release.

According to the following two studies, short-chain fatty acid-producing bacteria were found to be significantly less common in both ISS and GHD children. Li et al. [3] found that ISS children had significantly lower relative abundances of the family Ruminococcaceae, general Faecalibacterium, and Eubacterium. However, there was a significant increase in Clostridium and generic Parabacteroides. Moreover, the total concentrations of intestinal short-chain fatty acids and butyrate in ISS children were substantially diminished, and the dysbiosis of gut microbiota together with aberrant metabolites demonstrated a correlation with serum insulin-like growth factor 1 (IGF-1) levels and height. Another study [17] found previously that the abundance of genus Prevotella in GHD children increased, but the abundance of Lachnospiracea incertae sedis, Clostridium XlVa, and Megamonas decreased. Further, the function of GM was also altered, and some GM was related to endocrine hormones. The current study hypothesized that the dysregulation of gut microbiota influenced energy metabolism and growth hormone secretion in children with growth hormone deficiency, resulting in growth restriction. However, Li et al.’s study [18] did not observe any changes in the abundance of those, as mentioned earlier, short-chain fatty acid-producing bacterial genera. Li et al. [18] compared the short stature (GHD and ISS) data with those of healthy children. They found that short-stature children exhibited a higher relative abundance of Prevotellace-NK3B31_group at the genetic level compared to healthy children when Rodentibacter, Rothia, and Pelomonas showed lower abundance. As a result, the current study randomly selected data from 18 children with GHD from the previous study [17] and paired them with 15 children with ISS for comparative analysis to determine any differences in GM composition and function between the two groups.

2. Materials and Methods

This study was registered with the Medical Research Registration and Record System with the registration number MR-44-24-045472. The Ethics Committee of Shenzhen Longgang District Maternity and Child Health Care Hospital approved the study (Approval number: LGFYKYXMLL-2024-14). Informed consent was obtained from the legal guardians of all participating children.

2.1. Patients

This study randomly selected data from 18 children with GHD from a previous study [17] and included 15 children with ISS who visited our hospital between January 2021 and January 2023. Both groups of children ranged in age from 6 to 14 years, with no statistically significant differences observed in gender, age, weight, or height (p > 0.05). However, the GH pack and IGF-1 values in the ISS group were observed to be significantly higher in comparison to those in the GHD group, with statistical significance (p < 0.05) (Table 1). Both groups of children participated in growth stimulation tests at the Longgang Maternity and Child Institute of Shantou University Medical College (Longgang District Maternity & Child Healthcare Hospital of Shenzhen City) and received diagnoses following the completion of relevant examinations. Exclusion criteria used were: 1) Children suffering from familial inherited short stature, constitutional delayed puberty, chromosomal diseases, skeletal developmental disorders, and metabolic diseases; 2) Children suffering from systemic diseases, such as congenital heart defect, chronic liver disease, asthma, long-term malnutrition, etc.; 3) Antibiotics or probiotic preparations were used within one month before testing. Informed consent from the guardians of both groups of children was obtained before collecting the fecal specimens.

Table 1 Comparison of general information between two groups of children ($\bar{x}\pm s$)

project	ISS group	GHD group	Statistical value (X2/F/Z)	p
gender (male/female)	9/6	12/6	X2 = 0.157	0.692
Age (Y)	8.00 ± 2.30	8.72 ± 1.97	F = 0.948	0.338
Weight (kg)	20.78 ± 4.69	23.99 ± 6.40	F = 2.601	0.117
Height (cm)	117.33 ± 10.98	123.43 ± 9.93	F = 2.806	0.104
GH peak (IU/L)	15.24 ± 9.65	3.54 ± 1.20	Z = 23.83	<0.0001
IGF-1 (μg/L)	284.84 ± 54.14	141.47 ± 44.02	Z = 22.10	<0.0001

2.2. Sampling, sequencing, and bioinformatics

Five grams of midcourse feces were promptly collected and frozen at –80°C. Whole DNA was extracted using the PowerSoil^® DNA Isolation Kit (MoBio, USA) and amplified with primers targeting the 16S rDNA V3–V4 regions for next-generation sequencing (Illumina, USA). The raw sequencing data underwent quality control, was assembled into tags using FLASH (v1.2.11), and clustered into operational taxonomic units (OTUs). The taxonomic annotation was implemented based on Greengene (v201305). The GM function was afterward annotated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Variation analysis was conducted between different groups or subgroups at the genus and phylum taxonomic levels.

2.3. Statistical analysis

The ade4 package (v1.7-22) in R software (v3.3.3) was employed for principal components analysis (PCA). Variation analysis among groups or subgroups was conducted using the Wilcoxon sum-rank test at the taxonomic levels of phylum, genus, species, and GM function. Statistical processing was performed using SPSS 25.0. Measurement data from the two groups of children were analyzed, with gender assessed through the Chi-squared test, while other variables were evaluated using One-Way ANOVA.

3. Results

3.1. Comparison of diversity of GM composition

The analysis results indicated that: 1) The α diversity of GM did not differ significantly between the two groups (p = 0.13) (Fig. 1A); 2) Principal Component Analysis (PCA) conducted on the samples from both groups of children revealed nearly overlapping samples in the ISS and GHD groups, suggesting minimal differences in GM between the two groups (Fig. 1B).

Fig. 1  (A) Alpha diversity analysis shows no significant difference in gut microbiota (GM) between the ISS group and the GHD group; (B) Principal component analysis (PCA) reveals an overlap between samples from the two groups, indicating similar gut microbiota composition; (C) Similarity clustering of samples based on Bray-Curtis distance does not show clear aggregation trends, suggesting insignificant β-diversity differences between groups; (D) A slight difference in Bray-Curtis distance between the ISS group and the GHD group suggests high inter-group similarity among children samples.

This study compared the differences between two groups of children’s samples in Bray-Curtis distance similarity clustering. The clustering of each sample was observed to be relatively scattered, with no discernible trend in the clustering of samples between groups. This observation suggested that the differences across groups in the sample were not significant for beta diversity. The difference in GM between the ISS group and the GHD group was relatively insignificant (Fig. 1C). Furthermore, a minor discrepancy in Bray-Curtis distance has been observed between the ISS and the GHD groups, suggesting a greater inter-group sample similarity among both groups of children with GM (p = 0.45) (Fig. 1D).

3.2. Comparison of GM composition

The present study employed the Wilcoxon method to assess the differences in species and functions between the two groups, subsequently applying a screening criterion of p < 0.05 at the phylum, genus, and species classification levels. Furthermore, at the phylum level, the composition and relative abundance of the top five phyla between the two groups of children were not substantially different (p > 0.05) (Fig. 2). Moreover, at the genus level, the composition and relative abundance of the top 15 Genus (the dominant bacteria) between the two groups of children was also not significantly different (p > 0.05) (Fig. 3). The composition and relative abundance of Terrisporobacter and Acidovorax (non-dominant bacteria) exhibited a significant difference (p < 0.05) between the two groups of children (Table 2). Moreover, the composition and relative abundance of Prevotella stercorea (T) and uncultured Sutterella sp. (bacterial species) between the two groups of children demonstrated a significant difference (p < 0.05) (Table 3).

Fig. 2  The phyla between the ISS and GHD groups

At the phylum level, the composition and relative abundance of the top five major bacterial phyla in the gut microbiota of children from both groups were compared, with results showing no significant differences.

Fig. 3  The genera between the ISS and GHD groups

At the genus level, the composition and relative abundance of the top 15 major genera in the gut microbiota of children from both groups are presented, with results indicating no significant differences.

Table 2 Significant differences in Genus between the ISS and GHD groups

Significantly different bacteria	GHD group		ISS group		p value	FDR
	mean value (%)	SD (%)	mean value (%)	SD (%)
Terrisporobacter	0.008	0.012	0.102	0.330	0.028	0.739
Acidovorax	0.001	0.002	0.000	0.000	0.009	0.739

Table 3 Significant differences in bacterial Species between the ISS and GHD groups

Significantly different bacteria	GHD group		ISS group		p value	FDR
	mean value (%)	SD (%)	mean value (%)	SD (%)
Prevotella_stercorea_(T)	1.046	2.862	0.066	0.029	0.020	0.904
uncultured_Sutterella_sp.	0.140	0.329	0.131	0.500	0.040	0.904

3.3. Difference between the functional groups of GMs

Functional differences between the two groups were evaluated using 16S rRNA sequencing data from the KEGG database following microbiota hypothesis and annotation. The differential analysis was conducted at the KEGG database’s levels 2 (L2) and 3. The L3 level is a functional subdivision of the L2 level, and no differential functions were found at the KEGG L2 level (Fig. 4). The two differential functions at the KEGG L3 level were Bacterial_motility_proteins and Bacterial_chemotaxis (p < 0.05) (Table 4 and Fig. 5).

Fig. 4  Microbiota functions of L2 level in the ISS and GHD groups

Functional differential analysis at KEGG database L2 level shows no significant functional differences between the two groups.

Table 4 Microbiota functions of L2 level in the ISS and GHD groups

Significant difference function	GHD group		ISS group		p value	FDR
	mean value (%)	SD (%)	mean value (%)	SD (%)
Bacterial_motility_proteins	0.339	0.307	0.357	0.116	0.046	1.000
Bacterial_chemotaxis	0.187	0.083	0.203	0.039	0.049	1.000

Fig. 5  Microbiota functions of L3 level in the ISS and GHD groups

At KEGG database L3 level, it was found that two functional categories—Bacterial motility proteins and Bacterial chemotaxis—had higher enrichment levels in the ISS group.

4. Discussion

GM functions as an endocrine organ and influences material metabolism and nutrition absorption. Through controlling hormone release and microbial metabolites, it can also have an impact on growth and development [19]. Children with ISS and GHD demonstrate impaired secretion and application of GH [5-7] and present with GM disorders [3, 17], which may negatively influence the secretion and synthesis of GH and IGF-1 via microbial metabolites. A previous study found that ISS patients show issues related to hormone secretion and are also affected by genetic, nutritional, and environmental factors. The interaction of these factors can result in diminished efficacy and potential GH resistance in children with ISS [20].

The findings presented by Li et al. [18] do not align with the findings of this investigation. According to this study, there were no appreciable variations in the phylum of bacteria between the ISS and GHD groups, and the diversity of GM was comparable. Only two significantly different genera were found, Terrisporobacter and Acidovorax, and two different bacterial species were identified: Prevotella_ Stercorea_ (T), uncultured_ Sutterella_ Sp. However, these genera (species) were not the most abundant bacteria. Terrisporobacter is predominantly associated with carbohydrate metabolism and can enhance the formation of short-chain fatty acids, which is beneficial to the survival and adaption of foxes [21, 22]. Acidovorax is related to the survival adaptation of different plants [23, 24]. This study found that the abundance of Prevotella_ Stercorea_ (T) in children with GHD was significantly higher than in the ISS group, consistent with our previous study [17]. Prevotella has been shown to effectively lower blood sugar and insulin levels, influencing nutrient absorption and facilitating weight loss [25]. Sutterella may play a significant role in the modulation of glucose metabolism; however, the functions of Prevotella_Stercorea_(T) and uncultured_Sutterella_sp. remain unclear. Further research is necessary to ascertain the potential relationship between the previously mentioned strains and GH regulation. A similar study has previously reported a decrease in the abundance of butyric acid-producing bacteria such as Lachnospiraceae incertae sedis as well as Clostridium XlVa in children with GHD, and the latter was associated with nutrient absorption, energy metabolism, and hormone secretion [17]. Li et al. [3] also reported that the abundance of butyric acid-producing bacteria, such as family Ruminococcaceae and general Faecalibacterium, decreased substantially in children with ISS, which could adversely affect the synthesis of SCFAs but was positively related to IGF-1. The study revealed that the abundance of butyric acid-producing bacteria, including Faecalibacterium, Lachnospiracea incertae sedis, Clostridium XlVa, and Ruminococcus, was identical in the gut microbiomes of both groups of children. This finding suggests a significant restriction in the synthesis of short-chain fatty acids in both groups, adversely impacting their growth and development. However, the abundance of Klebsiella and Alistipes in the intestines of two groups of children in this study showed an upward trend, consistent with our previous reports [17] but inconsistent with earlier reports [3]. The above bacterial genera can affect the production of neurotransmitters, which in turn can affect both growth and development through the gut-brain axis [26-28].

The functional analysis of GM in this study indicated no differential function was observed at the KEGG L2 level. Two differential functions were identified at the KEGG L3 level, showing enrichment in ISS children. Mobility proteins and their role in chemotaxis. No significant variations have been observed in the composition and function of the gut microbiota between the two groups of children. The quantity of butyric acid-producing bacteria in the intestines of children in both groups was significantly decreased. The significant increase in neurotransmitter-producing bacteria in both groups may result in reduced synthesis of SCFAs and enhanced synthesis of neurotransmitters, thereby influencing IGF-1 levels and hypothalamic function [17, 29].

5. Conclusion and Limitations

This study identified comparable GM changes in GHD and ISS while also indicating a reduction in the prevalence of short-chain fatty acid-producing bacterial genera, aligning with our previous study [17] and the findings of Li et al. [3]. GM is an endocrine organ that can influence GH secretion and IGF-1 synthesis by generating metabolites like SCFAs and neurotransmitters (Graphical Abstract). The hypothalamus-pituitary IGF-1 growth axis can then control growth and development. The previously stated findings may indicate that GM influences GH secretion similarly in children with GHD and ISS, but GHD children have issues with GH consumption.

Graphical Abstract

The small sample size and single-center study may have contributed to the lack of significant changes in the composition and function of GM between the two groups of children in this investigation, even though children with ISS and GHD were shown to have GM disorders. Furthermore, no metabolomic analysis of GM was carried out in this investigation. To thoroughly examine any potential variations in the GM’s composition, function, and metabolites between the two child groups, the following phase will seek to increase the sample size, conduct multicenter research, and perform macro gene sequencing.

6. Ethics Statement

The Ethics Committee of Shenzhen Longgang District Maternity and Child Health Care Hospital approved the study (study approval number LGFYYXLL-024). The legal guardians of the enrolled children provided written informed consent for their children’s inclusion in the study.

7. Data Availability Statement

The data set generated in this study can be accessed from the NCBI Sequence Archive (SRA) database (Accession Number: PRJNA1007950).

8. Conflict of Interest

All authors declare that there were no conflicts of interest in this study.

9. Author Contributions

Jing Han and Dongming Meng managed the project. Dongming Meng, Congfu Huang, and Jing Han were responsible for registering the clinical information of the enrolled children. Dongming Meng and Lingjuan Meng collected stool samples according to standard protocols. Congfu Huang was responsible for DNA extraction and analyzing biological information. Jing Han was responsible for tabulation and statistical analyses. Congfu Huang and Dongming Meng were responsible for interpreting the data and writing the manuscript. All authors have reviewed the final version of the paper.

10. Funding

This research was supported by the Longgang District Science and Technology Innovation Bureau (LGWJ2022-48), the Research Initiation Fund of Longgang District Maternity and Child Healthcare Hospital of Shenzhen City (Y2024011).

11. Acknowledgments

We sincerely thank all the enrolled children and their guardians for their trust and cooperation, which enabled us to complete this study successfully. We are also very grateful to all the medical staff members of Longgang Maternity and Child Institute of Shantou University Medical College (Longgang District Maternity & Child Healthcare Hospital of Shenzhen City) for their enormous efforts in this study. The authors would like to thank all the reviewers who helped in the review and MJEditor (www.mjeditor.com) for their linguistic assistance while preparing this manuscript.

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