Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative treatment for hematological malignancies, but it is associated with substantial gut microbial dysbiosis linked to graft-versus-host disease, infections, and transplant-related mortality. The conditioning regimen represents the first major iatrogenic insult delivered within the transplant pathway, but its specific contribution to microbiota injury, independent of subsequent aplasia, antibiotic exposure, and mucositis, remains incompletely characterized. We conducted a prospective, single-center, observational longitudinal study at Fundeni Clinical Institute between August 2024 and February 2025, analyzing paired stool samples collected before conditioning (T0) and on the day of stem cell infusion (T1) from 47 adult allo-HSCT recipients. Microbial diversity was assessed using 16S rRNA gene sequencing (V4–V5 region), with the Shannon diversity index as the primary outcome. Statistical analyses were performed using SPSS, including Wilcoxon signed-rank tests for paired comparisons, Mann–Whitney U tests for between-group differences, and Kruskal–Wallis H tests. Shannon diversity decreased substantially between T0 and T1 (median 4.91 vs. 3.51; Wilcoxon P < 0.001; effect size r = 0.62), with 37 of 47 patients (78.7%) showing decline. This conditioning-induced injury was reproducible across three stratification dimensions: agent type (chemotherapy vs. TBI-containing; P = 0.67), intensity (RIC vs. MAC; P = 0.92), and number of cytotoxic agents (2 vs. 3; P = 0.54), with statistically significant within-subgroup decline observed in the four most represented regimens. Beta diversity analysis revealed a significant shift in community composition (PERMANOVA P < 0.001) without differential dispersion. Conditioning produces a substantial, biologically coherent microbiota injury before aplasia-associated factors come into play, but further studies on large cohorts are needed.
