Most research evaluating the effects of the reproductive tract microbiota on reproductive performance has been done in humans, thus far. In bovids, reproductive microbiota research is not as advanced, with preliminary conclusions, not supported by contamination checks or repeatability. Our studies concluded that endogenous reproductive hormones, days of gestation, and pregnancy status does not change the overall vaginal microbiota composition. Although, the overall composition did not change there were species level differences. These differences could have implications in reproductive performance and fertility in heifers. Heifers that undergo nutrient restriction have similar vaginal microbiota to adequately fed heifers with no species differences. The most impactful finding is that exogenous supplementation of melatonin was associated with changes in the vaginal microbiota in Brangus heifers during late gestation. The implications of this finding are not yet clear, but to date, this is the first hormone, in bovids, determined to change the composition of the vaginal microbiota.

A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine. / The human microbiome is the collection of microorganisms in the body that exist in a mutualistic relationship with the host. Recent studies indicate that perturbations in the microbiome may be implicated in a number of diseases, including cancer. More specifically, changes in the gut and vaginal microbiomes may be associated with a variety of gynecologic cancers, including cervical cancer, uterine cancer, and ovarian cancer. Current research and gaps in knowledge regarding the association between the gut and vaginal microbiomes and the development, progression, and treatment of gynecologic cancers are reviewed here. In addition, the potential use of probiotics to manage symptoms of these gynecologic cancers is discussed. A better understanding of how the microbiome composition is altered at these sites and its interaction with the host may aid in prevention, optimization of current therapies, development of new therapeutic agents and/or dosing regimens, and possibly limit the side effects associated with cancer treatment.

The cpn60 gene is a DNA barcode for bacteria. Recently, the PCR primers that have been used extensively to amplify the cpn60 Universal Target (UT) region of bacteria were redesigned to improve their utility for fungal taxa. Additional novel primers were designed to amplify other regions of the cpn60 gene, specifically from fungal genomes. Design of the redesigned and novel primers was based on 61 nucleotide full-length cpn60 reference sequences available in 2012, including Ascomycota (51), Basidiomycota (5), Chytridiomycota (2), Glomeromycota (1), and Oomycota (2). The research described here investigated the utility of these primers for detecting and identifying fungal taxa and for profiling mixed communities of bacteria and fungi. The redesigned primers were used to discover cpn60 UT sequences for Ascomycota (1), Basidiomycota (2), and Chytridiomycota (1). The novel primers were used to discover new cpn60 sequence data for Ascomycota (3), Basidiomycota (1), and Zygomycota (1). To be adopted for use in studies of microbial communities that are predominantly bacterial, the redesigned cpn60 UT primers must perform at least as well as the original primers for bacterial profiling. Bacterial profiles, created using the original and redesigned primers and two DNA template samples created by pooling DNA extracts from vaginal swabs from individual women, were compared. These included comparisons of diversity indices, rarefaction curve analysis and Operational Taxonomic Unit abundances. Diversity indices and rarefaction curve analysis for bacterial profiles with original and redesigned primers were similar. OTU abundance estimates with the original and redesigned primers were compared at higher and lower taxonomic levels. The overall patterns produced were similar. For one template only, the phylum Bacteroidetes had a greater apparent abundance with the original primers than with the redesigned primers. The greater apparent abundance of Bacteroidetes taxa was balanced by a lesser apparent abundance of taxa that were not assigned to a phylum. These differences may reflect differences in the performance of the two primer sets. At lower taxonomic level, most OTU were represented with apparently equal abundances with redesigned and original primers in same template. Very few OTU were represented with different proportional abundances with redesigned and original primers. Different OTU having same reference cpn60 UT sequence as best hit were sometimes represented by different proportional abundance with same primer in same template that made the analysis difficult. On the whole, the redesigned cpn60 UT primers behaved at least as good as the original cpn60 UT primers. The overall results showed that the redesigned and novel primers used in this study had substantial utility for the identification of fungal samples and mixed microbial communities.

The vaginal microbial community is critical to a woman’s health and the health of her family. Bacterial vaginosis (BV) is a polymicrobial syndrome characterized by a shift of the vaginal microbiota from a Lactobacillus dominated community to a dense biofilm containing a complex mixture of organisms. Although BV is an important risk factor for poor reproductive health outcomes, the etiology of BV is poorly understood. Gardnerella vaginalis is a hallmark species of BV. Phylogenetic analysis of cpn60 universal target sequences from metagenomic studies of the vaginal microbiome and from G. vaginalis isolates resolved four subgroups within the species. This subdivision, supported by whole genome similarity comparisons, demonstrated that these subgroups might represent different species. Among a group of African women, only G. vaginalis subgroup B was significantly more abundant in women with BV relative to women with Nugent scores not consistent with BV. To characterize the subgroups further, several phenotypic and molecular factors of G. vaginalis subgroups were assessed. Proteomic profiles of isolates within each subgroup formed unambiguous clusters. Sialidase gene sequences were detected in all subgroups, however enzymatic activity was detected only in subgroup B. Two isolates of subgroup B isolates (N153 and N101) were incapable of growth in 7% CO2. Given the well-known relationship between an anaerobic microbiota and BV, anaerobic isolates of G. vaginalis are potentially important players in the vaginal microbial community. To determine genome content differences that could account for the phenotypic difference, whole genome sequences of four G. vaginalis subgroup B isolates representing facultative and anaerobic phenotypes were determined. Comparison of genomes led to the identification of genes predicted to encode proteins involved in cell wall biogenesis and protection from oxidative damage that might account for the observed phenotypes. The cpn60 universal target based methodology that improved resolution of the vaginal microbiota including G. vaginalis was applied in a prospective study of the vaginal microbiome of women with preterm premature rupture of membranes (PPROM). The objectives were to characterize the vaginal microbiota of women following PPROM, and to determine if microbiome composition at the time of rupture predicts latency duration and perinatal outcomes. Only 13/70 samples collected from 36 women were dominated by Lactobacillus spp., the expected profile for healthy women, while Megasphaera type 1 and Prevotella spp. were detected in all samples. Microbiome profiles at the time of membrane rupture did not cluster by gestational age at PPROM, or latency duration. Microbial profiles were unstable over the latency period, with dramatic shifts in composition between weekly samples, and an overall decrease in Lactobacillus abundance. Mollicutes were detected by PCR in 81% (29/36) of women, and these women had significantly lower gestational age at delivery and correspondingly lower birth weight infants than Mollicutes negative women. Taken together, the results presented in this thesis demonstrate the value of high resolution profiling of the vaginal microbiome using cpn60 UT sequences. The resolution of subgroups within G. vaginalis has potentially significant implications for women's health diagnostics, requiring a shift away from considering G. vaginalis as a single entity. The PPROM study provides foundational information that may lead to the identification of informative sequence patterns, providing clinicians with better tools for expectant management following PPROM.

Estrus synchronization (ES) is a valuable technology that can help beef cow-calf producers to overcome infertility caused by prolonged anestrus. Protocols for ES that use progesterone (P4) supplementation are of particular value to cows with prolonged postpartum anestrus as P4 triggers them to begin cycling and allows them to have fertility similar to that of cycling cows. Supplementation of P4 intravaginally with the use of a controlled internal drug release device (CIDR) improves cycle induction when compared to oral administration of P4. Vaginitis has been reported as a side effect to CIDR use in cattle, which raises concerns about its downstream effects on fertility. More specifically, the effects of CIDR use on the vaginal environment requires exploration, as no studies have explored the changes in vaginal microbiome in response to CIDR based ES protocols. In cattle, the vaginal microbiome has not been widely explored. On the contrary, the human vaginal microbiome is a well-defined environment, rich in bacteria from the genus Lactobacillus, which are responsible for promoting an environment of acidic pH. The dominance of Lactobacillus in the human vagina, however, fluctuates according to steroid hormone concentrations, and disruptions in the vaginal environment will cause depletion of Lactobacillus species, increase in vaginal pH and decreased fertility. Based on this data in humans, our objectives were to describe incidence of vaginitis caused by the CIDR in beef cows, as well as the vaginal microbiome changes in response to CIDR based protocols, and explore their relation to fertility. We found high incidences of vaginitis caused by CIDR use, yet CIDR-induced vaginitis did not negatively affect pregnancy outcomes. However, at CIDR withdrawal, there was decreased bacterial diversity, increased vaginal pH, increased bacterial abundance, and increased vaginal inflammation when compared to what was observed prior to CIDR insertion. Furthermore, abundance of bacteria, vaginal inflammation, and bacterial diversity, but not vaginal pH, were restored to normal values by the day of timed artificial insemination. This important finding suggests that although the vaginal microbiome was disrupted by the use of CIDR, the vaginal microbiome is resilient and capable of restoring its natural conditions without intervention. Finally, cows that ultimately became pregnant were found to have had increased bacterial diversity and decreased vaginal pH prior to protocol initiation, suggesting that the vaginal microbiome may play a role in individual cow fertility. In conclusion, our results indicate that for beef cows a more diverse vaginal microbiome with decreased vaginal pH presents greater resilience of the microbiome towards disruptions caused by an ES protocol, which is translated in greater pregnancy success. / Doctor of Philosophy / According to the Food and Agriculture Organization, the world population is expected to grow by 51% by the year of 2100. The efficiency of food production must therefore be optimized, given the finite availability of farmable land. In beef production, cow fertility is of great importance, since it will ultimately determine the number of animals available for slaughter. The main reproductive issue that cow-calf producers face is that after calving, cows will undergo a period known as postpartum anestrus, in which cows fail to ovulate. Artificial insemination (AI) can help to optimize beef production efficiency, since it allows for the dispersal of semen from valuable bulls to facilitate the genetic enhancement of herds. The use of estrus synchronization (ES) protocols allows for induction and synchronization of ovulation, which allows AI to be performed at the same time for large groups of cows. Progesterone is often used in ES protocols and is the hormone responsible for inducing cyclicity in postpartum cows. Progesterone can be administered either orally or intravaginally via the use of a controlled internal drug release (CIDR). While the CIDR seems to be more effective at inducing cyclicity of cattle compared to oral progesterone administration, vaginal inflammation as response to the CIDR has been reported in cattle. Little is known about the downstream effects of this inflammation on the normal vaginal microbiota and fertility in cattle. In humans, the vaginal microbiome is predominated by a single genus of bacteria (Lactobacillus), that has an essential role in producing lactic acid, which results in the human vagina being remarkably acidic. In humans, depletion of this bacteria, a condition called bacterial vaginosis (BV), allows for other types of bacteria to grow, which results in an increased vaginal pH and decreased fertility. The bovine vaginal microbiome composition and pH in response to the hormones administered during ES protocols and its relation to fertility have not been widely explored. Our objectives were to document the incidence of vaginitis caused by the CIDR in beef cows and evaluate its effects on the vaginal microbiome changes and fertility. We found high incidences of vaginitis caused by the CIDR, yet no effects of CIDR-induced vaginitis were seen on pregnancy success to the protocol. However, decreased bacterial diversity, followed by increases in vaginal pH, abundance of bacteria and vaginal inflammation are all detected at CIDR withdrawal when compared to before CIDR insertion. Furthermore, abundance of bacteria, vaginal inflammation, and bacterial diversity, but not vaginal pH, were restored to normal values by the day of timed AI, indicating that although the vaginal microbiome was disrupted using CIDR, the vaginal microbiome can restore to natural conditions, and indicate resilience of the vaginal microbiome. Finally, cows that became pregnant to the protocol presented increased bacterial diversity and decreased vaginal pH prior to the protocol. In conclusion, our results indicate that for beef cows a more diverse vaginal microbiome with decreased vaginal pH presents greater resilience of the microbiome towards disruptions caused by an ES protocol, which is translated in greater pregnancy success.

<p dir="ltr">The vaginal microbiome and female sex hormones are both involved in the development and progression of gynecological pathologies. The individual mechanisms by which the vaginal microbiome leads to disease progression and how female sex hormones are known. However, the mechanisms by which the vaginal microbiome regulates female sex hormones, such as progesterone, are not well understood. This study seeks to understand how the vaginal microbiome regulates progesterone receptor (PGR) expression via secondary analysis of host and vaginal microbiome multi-omics data from the Partners PrEP cohort. This dataset consists of cervicovaginal samples of women enrolled in the Partners PrEP study. Partial Least Squares Regression (PLSR) models were created for each biological data type (microbial composition, metabolomics, metaproteomics) to assess how these factors regulate PGR expression. Significant factors were identified through variable importance of projection (VIP) and correlation analysis. Partial correlation analysis and follow-up PLSR models incorporating clinical and demographic variables were performed to assess the robustness of the vaginal microbiome-PGR associations. The PLSR models indicated lower PGR expression was associated with <i>G. vaginalis,</i> and higher PGR expression was associated with <i>Lactobacillus </i>species. Cytosine, guanine, and tyrosine were among metabolites significantly associated with higher PGR expression and experimentally determined to be produced by <i>Lactobacillus</i> species. Conversely, citrulline and succinate were associated with lower PGR expression and experimentally determined to be produced by <i>G. vaginalis</i>. The models indicated that bacterial metabolic pathways involved in glucose metabolism, such as glucagon signaling and starch and sugar metabolism, may regulate PGR expression. Demographic phenotypes were also considered from the dataset and did not significantly alter the association between the biological explanatory variables and PGR expression. The results indicate that guanine, cytosine, succinate, starch and sucrose metabolism, and glycolysis gluconeogenesis may be regulators of PGR abundance and function. The models suggest vaginal microbiome factors could play a role in gynecological conditions where progesterone signaling is suppressed. Future experimental work is needed to validate the results of these models and support their use as predictive tools to understand the role of the vaginal microbiome.</p>

Orientador: Camila Marconi / Resumo: A microbiota vaginal normal é composta predominantemente por Lactobacillus spp. que conferem proteção contra infecções por patógenos, por meio da produção de ácido lático, peróxido de hidrogênio e bacteriocinas. Diferentemente, a vaginose bacteriana (VB) é caracterizada pela substituição da microbiota de Lactobacillus spp. por bactérias anaeróbias em sua maioria. A VB é a alteração de microbiota vaginal mais comum em mulheres de idade reprodutiva, acometendo aproximadamente 30% dessa população. Além disso, a VB é fator de risco para aquisição de infecções sexualmente transmissíveis (IST). Diversas características da população já foram associadas à VB, como idade, etnia, comportamentos sexual e de higiene. Entretanto, a real composição da microbiota vaginal só foi possível em 2011 com estudo utilizando o sequenciamento de nova geração do gene bacteriano RNA ribossômico 16S. Foi demonstrado que o microbioma vaginal pode ser classificado em cinco tipos de comunidades bacterianas (community-state types, CST). Quatro dessas CSTs tem predomínio de Lactobacillus: L. crispatus (CSTI), L. gasseri (CST II), L. iners (CST III) e L. jensenii (CST V), enquanto que a CST IV apresenta grande diversidade bacteriana e engloba a maioria dos casos de VB. Apesar de quatro CSTs apresentarem predomínio de Lactobacillus, o papel protetor da CST III, dominada por L. iners, contra aquisição de IST tem se demonstrado menor que os demais. Embora os estudos de microbioma tenham possibilitado conhecer me... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The normal vaginal microbiota is predominantly composed of Lactobacillus spp. which confer protection against pathogen infections through the production of lactic acid, hydrogen peroxide and bacteriocins. Differently, bacterial vaginosis (BV) is characterized by the replacement of the microbiota of Lactobacillus spp. by anaerobic bacteria for the most part. BV is the most common vaginal microbiota alteration in women of reproductive age, affecting approximately 30% of this population. In addition, BV is a risk factor for the acquisition of sexually transmitted infections (STIs). Several characteristics of the population have already been associated with BV, such as age, ethnicity, sexual and hygiene behaviors. However, the actual composition of the vaginal microbiota was only possible in 2011 with study using the new generation sequencing of the bacterial 16S ribosomal RNA gene. It has been shown that the vaginal microbiome can be classified into five types of community-state types (CST). Four of these CSTs have a predominance of Lactobacillus: L. crispatus (CSTI), L. gasseri (CST II), L. iners (CST III) and L. jensenii (CST V), while CST IV shows great bacterial diversity and involve most cases of BV. Although four CSTs have a predominance of Lactobacillus, the protective role of CST III, dominated by L. iners, against IST acquisition has been shown to be lower than the others are. Although microbiome studies have made it possible to know better the relationship between bact... (Complete abstract click electronic access below) / Mestre

Increasing evidence implicates the presence of bacteria in intrauterine tissues as an important risk factor for spontaneous preterm labor. Epigenetic alterations of innate immunity genes may increase the mother’s sensitivity to subclinical levels of bacteria. This study examined the presence of TET2, TLR-2, and TLR-9 in intrauterine tissue, and evaluated whether epigenetic alterations of these genes, as well as IL-8, changed their expression in human decidual tissue and a macrophage cell culture. Immunohistochemicalstaining was used to detect the presence of these proteins in intrauterine tissue. Gene expression changes were evaluated in stimulated monocytes and macrophages. Fluorescence immunohistochemistry was used to track translocation of TET2 in stimulated monocytes and macrophages. Secreted IL-8 concentration was detected with ELISA. Decidual expression of TET2, TLR-2, and TLR-9 increased in the order TNL < TL < sPTL < iPTL. This study found that TET2, TLR-2, TLR-9, and IL-8 are regulated by epigenetic mechanisms. This study was the first to report activation of TET2 involves its translocation from the cytosol to the nucleus in macrophages.