SAT371 Gavaging Adult Female Rats with Rothia sp. Leads to Profound Disruption of the Estrous Cycles and Metabolic Consequences
Authors: Santos Roberta , Parodi Gonzalo , Leite Gabriela , Morales Walter , Weitsman Stacy R , Barlow Gillian , Rivera Ignacio , Sanchez Maritza , Flor Dilara , Choi Linette , Gonzales Sophia L , Faria Flavio , Villanueva-Millan Maria Jesus , Pimentel Mark , Mathur Ruchi 
Published: October 2023

Abstract
Disclosure: R. Santos: None. G. Parodi: None. G. Leite: None. W. Morales: None. S.R. Weitsman: None. G. Barlow: None. I. Rivera: None. M. Sanchez: None. D. Flor: None. L. Choi: None. S.L. Gonzales: None. F. Faria: None. M. Villanueva-Millan: None. M. Pimentel: None. R. Mathur: None.
Polycystic ovary syndrome (PCOS) is a major endocrine disorder affecting ∼12% of reproductive age women. It has diverse clinical manifestations, including hyperandrogenism, ovarian dysfunction and enlargement, infertility, insulin resistance, and others. The role of the gut microbiome in PCOS is poorly understood. We previously found higher testosterone (T) levels in PCOS stool samples vs. controls and identified bacterial candidates that correlated with testosterone levels. Here, we further investigate the role of one candidate microbe, Rothia sp., in the development of PCOS-like phenotypes. Methods: The presence of androgen biosynthesis associated genes in Rothia sp. was confirmed by RT-PCR. Rothia sp. was cultured in M9 minimal medium plus cholesterol. T production in the supernatant was measured by LC-MS/MS. Next, reproductive-age female rats were gavaged with 106 CFU/mL Rothia or with PBS (controls) daily for 2 weeks. H&E stained slides from daily vaginal lavages were used to assess estrous cycles. Serum T and progesterone (P) levels were measured by LC-MS/MS, and insulin levels by ELISA. Ovaries, uteri, and cardiac blood were harvested. Results: Genes encoding proteins associated with T biosynthesis (cytochrome P450, 3-beta hydroxysteroid dehydrogenase/isomerase family protein and NAD(P)H steroid dehydrogenase-like protein) were detected in Rothia sp. T production in Rothia sp. cultures was confirmed, with levels from 3.2±0.01 pg/mL after 48 hrs to 8.9±0.02 pg/mL after 6 days. T was not detected in negative controls (P&lt;0.001). In gavaged rats, analysis of vaginal slides (controls n=10; Rothia n=10) revealed a disruption of estrous cycles in Rothia rats, with 68% of days in luteal phase (metestrus & diestrus) vs. 46% in controls (P=0.001). The predominance of luteal phase days in Rothia rats was driven by longer time in diestrus (1.96 vs 1.67 days in controls, P=0.04), and shorter time in estrus (1.24 vs 1.91 days in controls, P=0.001). Peak P and T levels occurred in diestrus in both groups. Rothia rats spent more days in diestrus, and the cumulative exposure times to P (P=0.046) and T (P=0.036) in the diestrus phase were significantly higher in Rothia rats vs controls. Final serum insulin levels were higher in Rothia rats (1.8±0.5 ng/dL) vs controls (1.0±0.1 ng/dL; P=0.07). Rothia rats also had heavier ovaries vs controls (0.128±0.007g vs 0.097±0.01g; P=0.07), and higher uterus weights (0.48±0.04g vs. 0.36±0.04g; P=0.04). Conclusion: Here we show that a specific Rothia sp. can produce testosterone in vitro. Gavaging female rats with this microbe results in PCOS-like phenotypes, including higher insulin levels, enlarged ovaries, significant disruption of reproductive cycles and longer exposure to peak progesterone and testosterone. This longer exposure to peak progesterone and testosterone may result in reproductive and metabolic sequelae that warrant further investigation.
Presentation Date: Saturday, June 17, 2023