Reproductive Tract Microbial Transitions from Late Gestation to Early Postpartum Using 16S rRNA Metagenetic Profiling in First-Pregnancy Heifers

Abstract

Studies in recent years indicate that reproductive tract microbial communities are crucial for shaping mammals’ health and reproductive outcomes. Following parturition, uterine bacterial contamination often occurs due to the open cervix, which may lead to postpartum uterine inflammatory diseases, especially in primiparous individuals. However, investigations into spatio-temporal microbial transitions in the reproductive tract of primigravid females remain limited. Our objective was to describe and compare the microbial community compositions in the vagina at late gestation and in the vagina and uterus at early postpartum in first-pregnancy heifers. Three swab samples were collected from 33 first-pregnancy Holstein Friesian heifers: one vaginal sample at gestation day 258 ± 4, and vaginal and uterine samples at postpartum day 7 ± 2. Each sample underwent 16S rRNA V4 region metagenetic analysis via Illumina MiSeq, with bioinformatics following Mothur MiSeq SOP. The reproductive tract bacterial communities were assigned to 1255 genus-level OTUs across 30 phyla. Dominant phyla, accounting for approximately 90% of the communities, included Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, and Fusobacteria. However, the results revealed distinct shifts in microbial composition between the prepartum vagina (Vag-pre), postpartum vagina (Vag-post), and postpartum uterus (Utr-post). The Vag-pre and Utr-post microbial profiles were the most distinct. The Utr-post group had lower relative abundances of Proteobacteria but higher abundances of Bacteroidetes, Fusobacteria, and Tenericutes compared to Vag-pre, while Vag-post displayed intermediate values for these phyla, suggesting a transitional profile. Additionally, the Utr-post group exhibited lower bacterial richness and diversity compared to both Vag-pre and Vag-post. The unsupervised probabilistic Dirichlet Multinomial Mixtures model identified two distinct community types: most Vag-pre samples clustered into one type and Utr-post samples into another, while Vag-post samples were distributed evenly between the two. LEfSe analysis revealed distinct microbial profiles at the genus level. Overall, specific microbial markers were associated with anatomical and temporal transitions, revealing a dynamic microbial landscape during the first pregnancy and parturition. These differences highlight the complexity of these ecosystems and open new avenues for research in reproductive biology and microbial ecology.

Keywords

• 16S rRNA next-generation sequencing
• uterus
• vagina
• pregnancy
• primigravida
• cow