The research team, led by pediatric endocrinologists at Fuzhou Children’s Hospital of Fujian Medical University, randomly assigned female C57BL/6 mice to receive either a multi-strain probiotic suspension containing Bifidobacterium longum, Lactobacillus bulgaricus, and Streptococcus thermophilus, or a distilled water control, from postnatal day 21 to 27. After gavage-related losses, the final sample sizes were nine probiotic-treated mice and six controls. Fecal samples were collected during the three days preceding vaginal opening, the standard marker of pubertal onset in mice, for 16S rRNA gene sequencing, PICRUSt2 functional prediction, and targeted short-chain fatty acid quantification.

Sequencing of the V3–V4 region of the bacterial 16S rRNA gene, processed through a contemporary DADA2/ASV pipeline against the SILVA 138.1 database, identified 2,946 amplicon sequence variants from 15 fecal samples. The probiotic group exhibited significantly lower alpha diversity across four indices—Observed species, Chao1, ACE, and Shannon—all with P values below 0.05. This reduction in within-sample richness suggests that probiotic administration promoted the dominance of specific taxa at the expense of overall community variety, a pattern consistent with the concept that introducing exogenous live microorganisms can transiently narrow the ecological space available to resident bacteria.

Beta-diversity analysis using PERMANOVA revealed a significant difference in overall community structure between the two groups, with Bray-Curtis R² = 0.193 (P = 0.013) and Jaccard R² = 0.158 (P = 0.008). Importantly, multivariate dispersion testing showed no significant difference in within-group variability, supporting the interpretation that the PERMANOVA result reflects genuine compositional shifts rather than differences in dispersion. The finding that probiotic supplementation altered the entire community architecture, not merely individual taxa, highlights the improved sensitivity of ASV-based approaches for detecting community-level changes that might be missed by older operational taxonomic unit methods.

At the genus level, differential abundance analysis using ANCOM-BC2 identified no taxon that remained significant after Benjamini-Hochberg correction for multiple comparisons. At a nominally significant level, Prevotellaceae UCG-001 and an unclassified Bacteroidales genus were depleted in the probiotic group, while Dubosiella, Parabacteroides, and an unclassified Lachnospiraceae genus were enriched. The most biologically notable trend involved Akkermansia, a mucin-degrading commensal widely recognized for its beneficial effects on intestinal barrier integrity and metabolic health, which showed a strong shift toward enrichment in the probiotic group (9.39% in controls versus 45.85% in probiotic-treated mice; ANCOM-BC2 log2FC = +2.15, P = 0.179, FDR = 0.395). Although the Wilcoxon rank-sum test yielded a nominally significant P value of 0.016 for this genus, the signal did not persist after FDR correction, and the high inter-individual variability in Akkermansia abundance warrants caution when interpreting this trend.

PICRUSt2 functional prediction identified 63 MetaCyc pathways at nominal significance between the two groups, but none survived Benjamini-Hochberg correction, with a minimum FDR of 0.106. The authors emphasize that PICRUSt2 provides predicted rather than measured functional capacity, and these inferences require validation through shotgun metagenomics or metatranscriptomics. The absence of corrected significance in predicted metabolic function suggests that the compositional changes, while real at the community level, may not have been large enough or consistent enough across individuals to produce detectable shifts in predicted pathway abundance.

Targeted metabolomics quantified seven short-chain fatty acids in fecal samples collected during the three days preceding vaginal opening. At a nominally significant level, the probiotic group exhibited reduced caproic acid concentration (P = 0.039) and elevated acetic acid levels (P = 0.039), neither surviving FDR correction (FDR = 0.121). Valeric acid showed a trend toward reduction in the probiotic group (P = 0.052). The direction of these changes is biologically plausible: acetic acid is a primary fermentation product of both Bifidobacterium and Akkermansia species, both of which showed trends toward enrichment in the probiotic group. Spearman’s rank correlation analysis, after correction for 42 comparisons, identified two associations remaining significant at the exploratory FDR < 0.1 threshold: a negative correlation between Prevotellaceae UCG-001 and acetic acid (rho = −0.693, FDR = 0.066), and a negative correlation between Akkermansia and valeric acid (rho = −0.670, FDR = 0.066). The authors stress that FDR < 0.1 is a permissive exploratory threshold rather than a conventional significance level, and that correlation does not establish causation.

Despite the substantial microbial remodeling, probiotic supplementation did not detectably influence pubertal timing or reproductive organ development under the conditions studied. Body weight at weaning and at vaginal opening, age at vaginal opening, uterine weight, and ovarian weight were all comparable between groups. Combined with the team’s previous findings in the same cohort, where probiotics obviated the puberty-accelerating effect of the soy isoflavone daidzein, this suggests that probiotic-induced gut microbial remodeling alone may not be sufficient to alter pubertal timing in the absence of exogenous endocrine-active compounds. The authors caution, however, that the small sample size, the absence of circulating hormone measurements, and the single time point of assessment mean a modest effect on pubertal development cannot be excluded.

Several limitations temper the strength of these conclusions. The small sample size (n = 6–9 per group) limited statistical power, and no genus-level differences survived FDR correction. Animals were group-housed with cage mates belonging to the same treatment group, with only two cages per group, meaning treatment and cage are substantially confounded and individual mice cannot be considered fully independent experimental units. The possibility of pseudoreplication cannot be excluded, and the authors explicitly state that no causal inference regarding the treatment effect on microbial community structure can be drawn from the PERMANOVA results. Probiotic viability after preparation was not independently verified, and qPCR validation of key taxa was not conducted due to limited sample availability.

The study provides a baseline characterization of probiotic-induced microbial remodeling at the pubertal window using current analytical standards, including an ASV-based DADA2 pipeline, the SILVA 138.1 reference database, and compositionally aware differential abundance methods. The authors frame their findings as exploratory and hypothesis-generating, emphasizing that the community-level restructuring they observed—along with the trend toward Akkermansia enrichment and the exploratory shifts in short-chain fatty acid metabolism—warrants confirmation in larger, adequately powered studies with longer intervention periods, multiple sampling time points, and direct measurement of circulating gonadotropins and estradiol. The work contributes foundational data for understanding gut microbiota–puberty interactions and clarifies the specific microbial and metabolic changes elicited by early-life probiotic intervention in the absence of exogenous endocrine disruptors.

Subject of Research: Gut Microbiota

Article Title: Early post-weaning probiotic supplementation alters gut microbial community structure and predicted metabolic function in female juvenile mice

Article References: Yuan, X., Shangguan, H., Zhang, Y., & Chen, R. (2026). Early post-weaning probiotic supplementation alters gut microbial community structure and predicted metabolic function in female juvenile mice. Frontiers in Endocrinology, 17, Article 1970147. https://doi.org/10.3389/fendo.2026.1970147

Image Credits: AI Generated

DOI: 10.3389/fendo.2026.1970147

Keywords: Early, post-weaning, probiotic, supplementation, alters, microbial, community, structure, predicted, metabolic, function, female