PSIII-9 Bacterial Community Diversity in Reproductive Tissues and Fetal Fluids of Pregnant Gilts
Authors: Hickman-Brown Kyle J , Smith Molly S , McAnally Brooke E , Cain Joe , Seo Heewon , Bazer Fuller W , Johnson Greg , Palanisamy Vignesh , Dass Sapna Chitlapilly , Wiegert Jeffrey , Poole Rebecca K 
Published: November 2023

Abstract
The fertility status of women has previously been linked to shifts in bacterial diversity within the reproductive tract. The objective of this study was to analyze the diversity of bacterial populations within reproductive tissues (i.e., vagina, cervix, endometrium, and chorion) and fetal fluids (i.e., allantoic and amniotic) of pregnant gilts. Crossbred gilts free of physical, health or reproductive-related issues were euthanized and hysterectomized during the preimplantation (D11, n = 7), implantation (D15, n = 5), mid-gestation (D60, n = 6), and late gestation (D90, n = 6) stages of pregnancy. Sterile swabs were rotated 8 times to collect samples from the mucosal surface of individual tissues and immediately placed in microcentrifuge tube for storage (-80°C) until sequencing. A one cm incision was made to expose the endometrium and cervix, whereas for vaginal sampling, sterile swabs were inserted 6 inches past the vulva and then rotated. For collection of samples of chorion, an incision was made in the endometrium, exposing the chorioallantois to allow sampling of the chorion adjacent to the endometrium, while the allantoic epithelial surface of the chorioallantois was sampled directly. For allantoic and amniotic fluids, 5 mL samples were collected and placed immediately in sterile 10 mL conical tubes and stored (-80°C) for sequencing targeting the V4 hypervariable region of the 16S rRNA gene. Quality of all samples was determined by FastQC before being processed for taxon analysis using the qiime2 pipeline. Alpha diversity (within sample metric) was analyzed using the Kruskal-Wallis test, whereas PERMANOVA was performed for beta diversity analysis (between sample metric). Shannon’s index, an alpha diversity metric, indicated contrasts in diversity by day (P = 0.02). A pairwise comparison indicated that D15 had greater alpha diversity (Shannon’s index) compared with D60 and D90 (4.89 ± 0.20 vs. 3.96 ± 0.19 and 4.10 ± 0.18, respectively; P &lt; 0.05). Differences in alpha diversity by tissue and fluid type were detected with a pairwise comparison indicating that amniotic fluid had the lowest alpha diversity compared with the cervix (3.44 ± 0.34 vs. 4.62 ± 0.22, respectively; P = 0.04). For beta diversity, PERMANOVA and PCoA plots for unweighted UniFrac demonstrated significant clustering by day (P &lt; 0.01), with clusters being D11 and D15 vs. D60 and D90. Additionally, PERMANOVA and PCoA plots for unweighted UniFrac indicated significant clustering by tissue and fluid type (P &lt; 0.01), with distinct clustering from the external portion of the reproductive tract (i.e., vagina) to the internal portion (i.e., amniotic and allantoic fluids). Based on these results, bacterial diversity within the reproductive tract shifts changes depending on the stage of pregnancy. Moreover, bacterial diversity decreases as you move from the external surface of the reproductive tubes towards the fetus (i.e., least in the amniotic fluid).