Vaginal Samples Research Articles

Association between vaginal microbiome alteration and povidone iodine use during delivery

BackgroundThe vaginal microbiome is a dynamic community of microorganisms in the vagina. Its alteration may be influenced by multiple factors, including gestational status, menstrual cycle, sexual intercourse, hormone levels, hormonal contraceptives, and vaginal drug administration. Povidone iodine has been used before delivery to reduce infection that may be caused by the ascendance of pathogenic and opportunistic bacteria from the vagina to the uterus. This study aimed to elucidate the impact of povidone iodine use during delivery on the vaginal microbiome.MethodsThis study enrolled a total of 67 women from maternity services in three hospitals. During the delivery process, we have applied povidone iodine in three doses such as low dose, medium dose, and high dose based on the amount of povidone iodine administered, thus, we studied the three groups of women based on the doses applied. Vaginal swab samples were collected both before and immediately after delivery, and the microbial communities were characterized using 16 S rRNA sequencing. The identification of differentially abundant microbial taxa was performed using ZicoSeq software.ResultsBefore delivery, the vaginal microbiome was dominated by the genus Lactobacillus, with different percentage observed (86.06%, 85.24%, and 73.42% for the low, medium, and high dose groups, respectively). After delivery, the vaginal microbial community was restructured, with a significant decrease in the relative abundance of Lactobacillus in all three groups (68.06%, 50.08%, and 25.89%), and a significant increase in alpha diversity across all 3 groups (P < 0.01). Furthermore, as the dose of povidone iodine used during delivery increased, there was a corresponding decrease in the relative abundance of Lactobacillus (P < 0.01). Contrary, there was an increase in microbial diversity and the relative abundances of Pseudomonas (0.13%, 0.26%, and 13.04%, P < 0.01) and Ralstonia (0.01%, 0.02%, and 16.07%, P < 0.01) across the groups. Notably, some functional metabolic pathways related to sugar degradation were observed to have significant change with increasing use of povidone iodine.ConclusionPovidone iodine was associated with the vaginal microbiome alterations after parturition, and its significant change was associated to the dosage of povidone iodine administered. The escalation in iodine dosage was linked to a decrease in Lactobacilli abundance, and elevated prevalence of Pseudomonas and Ralstonia. There is a need for longitudinal studies to clearly understanding the effect of povidone iodine use on maternal and infant microbiome.

Changes in Uterine Metabolome Associated with Metritis Development and Cure in Lactating Holstein Cows.

The objective of this study was to identify alterations in the vaginal discharge (VD) metabolome and potential biomarkers to predict metritis development and a cure in dairy cows. This prospective cohort study was conducted on two dairies located in CA and TX. Vaginal discharge was evaluated and collected using the Metricheck® device. Cows were examined for metritis at 4, 7, and 9 days in milk (DIM). Cows with a fetid, watery, and reddish-brown uterine discharge were classified as having metritis and randomized to receive ceftiofur (n = 10) or remain untreated (n = 7). A cure was defined as the absence of a fetid, watery, reddish-brown uterine discharge at 14 d after enrollment. Vaginal discharge samples were collected from 86 cows within 6 h after parturition, at 4 and 7 DIM, at metritis diagnosis, and at 4 and 7 days after metritis diagnosis. Cows with metritis (MET; n = 17) were paired with counterparts without metritis (HTH) of a similar DIM and parity (n = 34). The uterine metabolome was evaluated using untargeted gas chromatography time-of-flight mass spectrometry (GC-TOF-MS). Metabolomic data were analyzed using the MetaboAnalyst 5.0. Data were log-transformed and auto-scaled for normalization. Univariate analyses, including the fold-change, were performed to identify the metabolites linked to metritis development and its cure and principal component analysis and partial least squares discriminant analysis were performed to explain metabolite variance between animals developing or not developing metritis and being cured or not being cured of metritis. Comparing HTH with MET cows at calving, 12 metabolites were upregulated, and one was downregulated. At four and seven DIM, 51 and 74 metabolites, respectively, were altered between MET and HTH cows. After metritis development, three and five metabolites were upregulated in cows that were cured and in cows that received treatment and were cured, respectively. In all scenarios, the metabolites lignoceric, malic, and maleic acids, ornithine, and hypotaurine, which are associated with arginine/aminoacyl-tRNA biosynthesis and taurine/purine metabolism, were upregulated in HTH cows. Metritis was associated with changes in the uterine metabolome. Cows not being cured of metritis had changes in the uterus metabolome independent of receiving ceftiofur or remaining untreated. Metabolome analysis may be an important tool to understand the vaginal discharge changes during postpartum and the dynamics of metritis development and cures and help to identify biomarkers to predict metritis being cured.

Comparison of Three Methods for Diagnosing Trichomoniasis in Female Patients with Sexual Activity Attended at a Hospital in Peru.

Trichomoniasis is a sexually transmitted infection that mainly affects women. The diagnosis is made by different methods that identify the presence of Trichomonas vaginalis; therefore, sensitivity, specificity, and performance are essential to guarantee an adequate diagnosis. Our study is aimed at comparing three methods for diagnosing trichomoniasis in patients treated at a hospital in Peru during the year 2018. We designed a cross-sectional study and enrolled women whose vaginal secretion samples were evaluated by direct examination, Papanicolaou staining, and culture in the Diamond medium. We evaluated 134 women with a mean age of 36.6 ± 12.1 years and a beginning of sexual activity of 18.5 ± 3.0 years. We found leukocyte infiltration, fetid odor, and strawberry cervix in 66.4%, 35.1%, and 18.7%. The prevalence of trichomoniasis by the culture method, direct examination, and Papanicolaou was 32.1, 21.6, and 11.2%. The direct examination's sensitivity, specificity, and diagnostic performance (AUC) were 65.1%, 98.9%, and 82.0%, while for the Pap smear, they were 32.6%, 98.9%, and 65.7%%. The culture of T. vaginalis is the gold standard for diagnosing trichomoniasis; however, direct examination is a fast, specific alternative with good diagnostic performance. The Pap test has low sensitivity and should not be used in settings where the prevalence and risk factors for trichomoniasis are high.

Prevalence and sociodemographic predictors of high-risk vaginal human papillomavirus infection: findings from a public cervical cancer screening registry

IntroductionHigh-risk human papillomavirus (HPV) screening is vital for early cervical cancer detection and treatment. With the introduction of the national cervical cancer screening programme and screening registry in Malaysia, there is a need to monitor population-based HPV screening uptake and high-risk HPV prevalence as part of cervical cancer surveillance.ObjectiveTo determine the prevalence and sociodemographic factors predicting high-risk HPV infection in Malaysia based on a public, community-based cervical cancer screening registry targeting women at risk of getting HPV infection.MethodsThe study used data from the Malaysian cervical cancer screening registry established by the Family Health Development Division from 2019 to 2021. The registry recorded sociodemographic data, HPV test details and results of eligible women who underwent HPV screening at public primary healthcare facilities. A vaginal sample (via self-sampling or assisted by a healthcare provider) was used for DNA extraction for HPV detection and genotyping. Registry data were extracted and analysed to determine prevalence estimates of high-risk HPV infection. Multifactorial logistic regression analysis was conducted to determine predictors of high-risk HPV infection. All analyses were performed using Stata version 14.ResultsThe programme screened a total of 36,738 women during the study period. Women who attended the screening programme were mainly from urban areas, aged 30–39 years, and of Malay ethnicity. The prevalence of high-risk HPV infection was 4.53% among women screened, with the yearly prevalence ranging from 4.27 to 4.80%. A higher prevalence was observed among urban settling women, those aged 30–49 years, those of Indian ethnicity, and those without children. The results from logistic regression showed that women from urban areas, lower age groups, of Indian or Chinese ethnicity, and who are self-employed were more likely to be infected with high-risk HPV.ConclusionTargeted and robust strategies to reach identified high-risk groups are needed in Malaysia. In addition, the registry has the potential to be expanded for an improved cervical cancer elimination plan.Trial registrationTrial registration number: NMRR ID-22-00187-DJU.

Composition, function, and timing: exploring the early-life gut microbiota in piglets for probiotic interventions

BackgroundThe establishment of a robust gut microbiota in piglets during their early developmental stage holds the potential for long-term advantageous effects. However, the optimal timeframe for introducing probiotics to achieve this outcome remains uncertain.ResultsIn the context of this investigation, we conducted a longitudinal assessment of the fecal microbiota of 63 piglets at three distinct pre-weaning time points. Simultaneously, we gathered vaginal and fecal samples from 23 sows. Employing 16S rRNA gene and metagenomic sequencing methodologies, we conducted a comprehensive analysis of the fluctuation patterns in microbial composition, functional capacity, interaction networks, and colonization resistance within the gut microbiota of piglets. As the piglets progressed in age, discernible modifications in intestinal microbial diversity, composition, and function were observed. A source-tracking analysis unveiled the pivotal role of fecal and vaginal microbiota derived from sows in populating the gut microbiota of neonatal piglets. By D21, the microbial interaction network displayed a more concise and efficient configuration, accompanied by enhanced colonization resistance relative to the other two time points. Moreover, we identified three strains of Ruminococcus sp. at D10 as potential candidates for improving piglets' weight gain during the weaning phase.ConclusionsThe findings of this study propose that D10 represents the most opportune juncture for the introduction of external probiotic interventions during the early stages of piglet development. This investigation augments our comprehension of the microbiota dynamics in early-life of piglets and offers valuable insights for guiding forthcoming probiotic interventions.

Prevalence and incidence of sexually transmitted infections among South African women initiating injectable and long-acting contraceptives.

South Africa is among the countries with the highest prevalence of sexually transmitted infections (STIs), including Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG). In 2017, there were an estimated 6 million new CT, 4.5 million NG and 71 000 Treponema pallidum infections among South African men and women of reproductive age. We evaluated STI prevalence and incidence and associated risk factors in 162 women aged 18-33 years old, residing in eThekwini and Tshwane, South Africa who were part of the Evidence for Contraceptive Options and HIV Outcomes (ECHO) trial. Women were randomised to use depot medroxyprogesterone acetate (n = 53), copper intrauterine device (n = 51), or levonorgestrel (n = 58) implant. Lateral vaginal wall swab samples were collected prior to contraceptive initiation and at months one and three following contraceptive initiation for STI testing. There were no significant differences in STI incidence and prevalence across contraceptive groups. At baseline, 40% had active STIs (CT, NG, Trichomonas vaginalis (TV), Mycoplasma genitalium (MG) or herpes simplex virus-2 shedding across all age groups- 18-21 years (46%), 22-25 years (42%) and 26-33 years (29%). The incidence of STIs during follow-up was exceptionally high (107.9/100 women-years [wy]), with younger women (18-21 years) more likely to acquire CT (75.9/100 wy) compared to 26-33 year olds (17.4/100 wy; p = 0.049). TV incidence was higher in the 26-33 year old group (82.7/100 wy) compared to the 18-21 year olds (8.4/100 wy; p = 0.01). Although the study participants received extensive counselling on the importance of condom use, this study highlights the high prevalence and incidence of STIs in South African women, especially amongst young women, emphasising the need for better STI screening and management strategies.

PSXIV-9 Effects of Dietary Restriction and One-Carbon Metabolite Supplementation During Early Gestation on the Maternal Gut and Vaginal Microbiota in Cattle

Abstract Maternal diet quality and quantity have significant impacts on both maternal and fetal health and development. The composition and function of the maternal gut microbiome is also significantly influenced by diet. However, little is known about the impact of gestational nutrient restriction on the maternal microbiome during early gestation, which is a critical stage for many developmental processes. Here, we evaluated the effects of dietary restriction and one-carbon metabolite (OCM) supplementation on the ruminal and vaginal microbiota of beef heifers during early pregnancy. For this, 32 beef heifers (approximately 14-month-old) were used in a 2 × 2 factorial experiment with main factors including targeted gain (CON; 0.45 kg/d gain vs. RES; -0.23 kg/d gain), and OCM supplementation (OCM vs. noOCM) (n = 8/treatment). OCM heifers received ruminal-protected methionine (20 g/day) and choline (60 g/day) daily via a fine-ground corn carrier, with weekly intramuscular injections of folate (320 mg) and vitamin B12 (20 mg). The noOCM heifers received only corn carrier and saline injections. Heifers were individually fed and began treatment at breeding (d 0) and remained on treatment until d 63 of gestation. Ruminal fluid and vaginal swab samples were collected at d -2, d 35, and d 63 (at necropsy). Microbiota from samples were assessed using 16S rRNA gene (V3-V4) sequencing. Overall community structure of the ruminal microbiota was affected by gain (PERMANOVA: R2 = 0.04, P &amp;lt; 0.0001), OCM (R2 = 0.013, P = 0.03), time (R2 = 0.16, P &amp;lt; 0.0001) and by their interactions (R2 = 0.08, P = 0.004). The greatest difference in ruminal microbial community structure was between RES and CON heifers (R2 ≥ 0.16, P ≤ 0.003) on d 63. Overall microbial richness (total ASVs) was greater in RES (2,900 vs. 2,737; P = 0.005) but was unaffected by OCM (P = 0.25). Analysis via MaAsLin2 revealed that phyla Firmicutes (31% vs. 22%), Actinobacteriota (0.65% vs. 0.36%) and Chloroflexi were enriched (P &amp;lt; 0.05) in RES heifers. Twenty-five of the 34 differentially abundant genera in the rumen microbiota, including Christensellaceae R7 (5.0% vs 2.6%), Lachnobacterium (2.0% vs. 0.46%), and Butyrivibrio were more relatively abundant in RES; whereas 9 genera including Prevotella (16.5% vs. 31.1%), Ruminococcus (1.6% vs. 2.5%) and Saccharofermentans (0.4% vs. 0.5%) were reduced in RES (P &amp;lt; 0.05) on d 63. Overall vaginal microbiota structure was affected by gain (R2 = 0.02, P = 0.023) and time (R2 = 0.34, P &amp;lt; 0.0001) but not OCM (P &amp;gt;0.05). Community structure of RES was distinct from CON (R2 = 0.16, P = 0.032) on d 63, but microbial community richness and diversity were not influenced by OCM (P &amp;gt; 0.05). On d 63, RES heifers had increased Actinobacteriota (30.5% vs. 17.7%), but reduced Proteobacteriota (6.2% vs. 9.4%) and Fusobacteriota (0.02% vs. 0.94%; P &amp;lt; 0.05). In summary, restricted dietary intake during early gestation induced significant alterations in the ruminal microbiota and its impact also extended to the vaginal microbiota. The composition of these two microbial communities remained largely unaffected by OCM supplement.

PSIII-9 Bacterial Community Diversity in Reproductive Tissues and Fetal Fluids of Pregnant Gilts

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 &amp;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 &amp;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 &amp;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).

Sequencing and culture-based characterization of the vaginal and uterine microbiota in beef cattle that became pregnant or remained open following artificial insemination.

In this study, we evaluated the vaginal and uterine microbiota between beef cattle that became pregnant via artificial insemination (AI) and those that remained open to identify microbial signatures associated with fertility. We also characterized the culturable fraction of these microbiota and screened certain vaginal and uterine bacterial isolates for antimicrobial resistance. For this, vaginal and uterine swabs were collected before AI from two cohorts of Angus-crossbred cattle: mature cows (vaginal and uterine: 27 open and 31 pregnant) and heifers (vaginal: 26 open and 33 pregnant). The microbiota of these samples were assessed using 16S rRNA gene sequencing and culturing. Twenty-nine vaginal and uterine bacterial isolates were screened for resistance against 41 antibiotics. Within the vaginal microbiota, 11 amplicon sequence variants (ASVs) were more relatively abundant in the open heifers compared with the pregnant heifers. No differentially abundant ASVs were detected in the vaginal samples from pregnant versus open cows. Pregnant cows had a distinct uterine microbiota community structure (P = 0.008) and interaction network structure compared with open cows. Twenty-eight differentially abundant uterine ASVs were observed between the pregnant and open cows. Community structure and diversity were different between the cow's vagina and uterus. A total of 733 bacterial isolates were recovered from vaginal (512) and uterine (221) swabs under aerobic (83 different species) and anaerobic (69 species) culturing. Among these isolates were pathogenic species and those mostly susceptible to tested antibiotics. Overall, our results indicate that fertility-associated taxonomic signatures are present in the bovine uterine and vaginal microbiota.IMPORTANCEEmerging evidence suggests that microbiome-targeted approaches may provide a novel opportunity to reduce the incidence of reproductive failures in cattle. To develop such microbiome-based strategies, one of the first logical steps is to identify reproductive microbiome features related to fertility and to isolate the fertility-associated microbial species for developing a future bacterial consortium that could be administered before breeding to enhance pregnancy outcomes. Here, we characterized the vaginal and uterine microbiota in beef cattle that became pregnant or remained open via artificial insemination and identified microbiota features associated with fertility. We compared similarities between vaginal and uterine microbiota and between heifers and cows. Using culturing, we provided new insights into the culturable fraction of the vaginal and uterine microbiota and their antimicrobial resistance. Overall, our findings will serve as an important basis for future research aimed at harnessing the vaginal and uterine microbiome for improved cattle fertility.

Early Colonization of the Intestinal Microbiome of Neonatal Piglets Is Influenced by the Maternal Microbiome.

The intestinal microbiome plays a crucial role in animal health and growth by interacting with the host, inhibiting pathogenic microbial colonization, and regulating immunity. This study investigated dynamic changes in the fecal microbial composition of piglets from birth through weaning and the relationship between the piglet fecal microbiome and sows. Feces, skin, neonatal oral cavity, and vaginal samples were collected from eight sows and sixty-three piglets, and 16S genome sequencing was performed. The results revealed that Firmicutes, Bacteroidetes, and Proteobacteria dominated the piglet microbiome in the early stages, and Firmicutes and Bacteroidetes were crucial for maintaining a balance in the intestinal microbiome during nursing. The abundance of Christensenellaceae_R-7_group, Succinivibrio, and Prevotella increased in weaned piglets fed solid feed. Analysis of the microbiome from sows to piglets indicated a shift in the microbiome colonizing piglet intestines, which became a significant constituent of the piglet intestinal microbiome. This study supports the theory that the neonatal intestinal microbiome is vertically transmitted from the mother. Further research is required to integrate factors related to sows, piglets, and their environments to gain a better understanding of the early establishment of the intestinal microbiome in piglets.

Human Papillomavirus (HPV) Infections Among Participants Undergoing Chlamydia trachomatis Testing in Reunion Island (RUN-SurV-HPV Study): Protocol for a Prevalence Study.

Infection by human papillomavirus (HPV) induces various cancers, mainly cervical cancer but also anal and pharyngeal cancers. Reunion Island is a French department in the Indian Ocean. Due to the history of its settlement, the island's population has a wide variety of origins, and the population's sociodemographic characteristics differ from those of the French mainland. These characteristics make the island's population an original French population in its own right, particularly in terms of health. Cervical cancer incidence and mortality in Reunion Island are 10.4 per 100,000 and 4.4 per 100,000, respectively, both of which are much higher than those in mainland France. There is also a disparity in the prevalence of different HPV types, with types 33 and 52 being overrepresented and type 18 being underrepresented. However, vaccination and screening coverage in Reunion Island is low. It is important to understand the burden of infection and its risk factors in members of the young Reunionese population at risk of HPV infection to promote and evaluate the implementation of future vaccination and screening programs on a larger scale. The RUN-SurV-HPV study will have the following four objectives: (1) to describe the prevalence of HPV genotypes in a population at risk of sexually transmitted infections in Reunion Island; (2) to describe the prevalence of HPV genotypes by anatomical site, gender, and sexuality; (3) to describe the correlates and risk factors for HPV detection; and (4) to examine HPV genotypes between different anatomical sites. Cross-sectional analyses of 1200 routine vaginal, anal, pharyngeal, and urinary samples collected between October 2020 to December 2022 from female and male patients aged 16 to 30 years undergoing Chlamydia trachomatis testing at a sexually transmitted infection (STI) testing center at Reunion Island will be performed. The population included 333 women who all had vaginal and pharyngeal swabs, with 80 of them also having had an anal swab. There are 167 men who have sex with men who have had anal and pharyngeal swabs, and 120 men who have sex with women who have had a urine swab only. Results will be correlated with sociodemographic and clinical data collected routinely during the consultations. HPV detection and genotyping will be performed using the Anyplex II HPV28 detection assay (Seegene). The first polymerase chain reactions should begin in November 2023. The first results should be submitted for publication in 2024. The study will determine HPV prevalence by age, sex, male sexual preference, human immunodeficiency virus status, and STI co-infection. Collecting data from young patients not usually routinely screened for HPV infection will be a simple and reproducible way of better understanding local specificities, encouraging vaccination campaigns in the short-term, and evaluating their effectiveness in the future. DERR1-10.2196/47379.

4D DIA-PRM proteomic study identifying modulated pathways and biomarkers associated with pelvic organ prolapse

Pelvic organ prolapse (POP) is a highly disabling condition that negatively affects the quality of life of millions of women worldwide. However, the underlying mechanisms associated with the development and progression of the disease remain poorly understood. Here, an untargeted four-dimensional data-independent acquisition (4D DIA)-based proteomics approach was applied to vaginal wall tissue samples from POP (n = 19) and control (n = 8) patients to identify potential diagnostic biomarker(s) for POP and examine the molecular mechanisms underlying the disease. Of the 5713 tissue proteins that were detected, 1957 proteins were significantly changed in POP patients. Further bioinformatics analysis revealed that multiple biological processes including protein digestion & absorption, retrograde endocannabinoid signaling, tyrosine metabolism, and nucleotide metabolism were significantly enriched and associated with the pathogenesis of POP. Interestingly, 16 of these differentially expressed proteins associated with four pathways were also identified by targeted parallel reaction monitoring (PRM) proteomics analysis on the same 27 tissue samples. Changes in 94 % (15/16) of these proteins were consistent with the 4D DIA data. Furthermore, most proteins displayed good diagnostic accuracy with high area under the curve (AUC) values (AUC＞0.8). Specifically, five proteins including ELN, COL6A2, ENTPD1, AOC3, and COX7A2 distinguished between POP and control patients with very high accuracy (AUC ≥ 0.95) in both 4D DIA and PRM analyses, and may therefore be potential diagnostic biomarkers for POP. In summary, the present study not only provided several potential biomarker(s) for effective POP diagnosis, but extended our knowledge of the key regulatory pathways associated with the disease.

44 Identification of Vaginal Gene Expression Differences in Gilts Achieving Early or Late Puberty

Abstract A known predictor of reproductive success in swine breeding herds is age at puberty. Early puberty is associated with improved long-term reproductive performance. Additionally, it has been established that vaginal gene expression in gilts changes throughout reproductive development. The hypothesis was that vaginal gene expression was correlated with estrus expression and therefore likelihood of reproductive success. Pre-pubertal gilts (n = 29) were followed from 70 d of age until first estrus or 215 d of age. Vaginal epithelia were collected using a swabbing technique at five key timepoints during reproductive development [70 (on farm arrival), 100 (mid-folliculogenesis), 130, 160 d of age, and first estrus or end of trial]. Boar exposure began on 160 d of age with 15 minutes of fenceline contact at 0700 h daily with trained technicians observing for signs of standing estrus (i.e., erect ears, swollen, pink vulva, allowing back pressure). By 215 d of age, 19 females were classified as expressing estrus early (160 to 181 d), 4 were average (181 to 202), and 6 were late or as anestrus (202 to 215 d). A subset of 5 gilts that expressed estrus early and 5 gilts that expressed estrus late or as anestrus were used for further investigation of vaginal gene expression. Total RNA was isolated from vaginal samples at 3 timepoints (100, 130, and 160 d of age). Samples were sent to a commercial genomic sequencing laboratory for RNA sequencing. Each gene was scaled individually so that all genes were on the same scale for comparison, and the Welch two sample t-test was applied to determine statistical differences. A total of 53 genes were differentially expressed between the early estrus and late estrus or anestrus groups. Data were further screened to identify genes that were expressed in at least 3 gilts in either the early estrus or late estrus group at a given time point; this subset of 26 genes were selected for direct comparison at each time point. At 100 d of age, late estrus gilts had increased expression (P = 0.05) of a gene commonly found in females with polycystic ovary syndrome. At 130 d of age, genes associated with the innate immune response had greater relative expression (P &amp;lt; 0.05) in gilts expressing early estrus. At 160 d of age, genes associated with fat cell deposition had greater relative expression (P &amp;lt; 0.05) in the early estrus expression gilts. Distinct differences in transcripts during reproductive development may be putative biomarkers for detection of early estrus in pre-pubertal females.

279 Maternal Programming of the Piglet Microbiome Through Supplementation with a Yeast Feed Additive

Abstract The objective of this study was to investigate effects of live yeast supplementation of sow diets during late gestation and lactation on sow reproductive performance and piglet microbiome development. Three dietary treatments were imposed on mixed-parity sows (n = 92) from three contemporary farrowing groups during late gestation and lactation. Dietary treatments were based upon the inclusion level (%) of Actisaf Sc47 in corn/soybean meal-based diets: Control (C, 0%), Low (L, 0.1%), and High (H, 0.5%). Sows were fed their assigned gestation diet (1.8 kg ∙animal-1∙day-1/) from days 85 to 110 of gestation. On day 111 of gestation, sows were fed their assigned lactation diet (3.6 kg/head/day) until parturition. Sows were allowed ad libitum access to their assigned lactation diet beginning day one postpartum until piglets were weaned. Fecal and vaginal swabs were collected from all sows on day 85 of gestation (T1-S) and the day before parturition (T2-S). Colostrum and milk samples were collected from 30 focal sows (10/treatment group), and fecal swabs were collected from 6 piglets per focal sow (n = 180) at 24 to 36 hours after birth (T1-P) and weaning (T2-P, ~19 days old). Extracted DNA from swab samples was sequenced on the MiSeq platform targeting the V4 variable region of the 16S rRNA bacterial gene, and sequence data were analyzed using DADA2 plus various packages within the R statistical software. Generalized linear mixed models were built for sow performance data using the R lme4 package, with treatment as a fixed effect, and farrowing group, parity, and sire as random effects. Piglet performance models also included sex as a fixed effect and maternal origin instead of sire as a random effect. No significant differences were observed among dietary treatment groups for gestation or lactation length, lactation feed intake, sow body weights, sow backfat depth, litter size at birth or weaning, pre-weaning piglet mortalities, or piglet growth performance. Although alpha and beta diversity did not differ among maternal dietary treatment groups in sow colostrum, milk, fecal, or vaginal samples (T1-S or T2-S), several taxa were significantly enriched or less abundant in sow samples based upon dietary yeast inclusion levels. Diversity of piglet fecal microbiomes was not different across maternal dietary treatment groups, but community composition was different at both time-points (T1-P: PERMANOVA, R2 = 0.13, P=0.02; T2-P: PERMANOVA, R2 = 0.01, P=0.048). These results indicate that yeast supplementation in sow diets in late gestation and lactation may influence development and composition of piglet gut microbiomes in early life.

188 Characterization of the Vagino-Uterine Microbiota in Pregnant and Non-Pregnant Beef Cattle Using 16S Rrna Gene Sequencing and Culturing

Abstract Emerging evidence suggests that urogenital microbiome-targeted approaches may provide a novel opportunity to reduce the incidence of reproductive failures in cattle. The objectives of this study were to: 1) characterize the vaginal and uterine microbiota between beef cattle that became pregnant via artificial insemination (AI) and those that did not; 2) identify differentially abundant taxa between pregnant and non-pregnant cattle; and 3) characterize the culturable fraction of the vaginal and uterine microbiota using extensive culturing. Vaginal and uterine swab samples were collected from two cohorts of Angus-crossbred cattle: mature cows (n =100) and heifers (n = 72, vaginal swab only) at the time of AI. At 35 days post-AI, pregnancy diagnoses were made via ultrasound. A subset of vaginal and uterine swabs from cows (27 non-pregnant and 31 pregnant) and heifers (26 non-pregnant and 33 pregnant) were selected and processed for microbiota assessment using 16S rRNA gene (V3-V4) sequencing. For culturing, a subset of cryopreserved vaginal and uterine swabs (128) were plated onto three different agar types and cultured, and isolates were identified by near-full length 16S rRNA gene sequencing. Sequencing results revealed 11 taxa that were more abundant in the vaginas of heifers that failed to become pregnant via AI than heifers that did become pregnant. While there was no significant difference in vaginal microbiota community structure (P = 0.21) between pregnant and non-pregnant cows, non-pregnant cows tended to have greater microbial richness and Shannon diversity (P ≤ 0.08) compared with pregnant cows. No differentially abundant taxa were detected in the vaginas of pregnant versus non-pregnant cows. Pregnant cows had a distinct uterine microbiota community structure (R2 = 0.032, P = 0.008) but similar alpha diversity (P &amp;gt; 0.1) compared with non-pregnant cows. Twenty-eight differentially abundant taxa were observed in the uterine microbiota between pregnant and non-pregnant groups, with 11 of them more abundant in pregnant cows. Methanobrevibacter ruminantium (archaea) and Fusobacterium necrophorum were among these 11 positive pregnancy-associated taxa. Although community structure was significantly different between the vagina and uterus, 277 “core taxa” (present in ≥ 80% of all samples) were shared between the two communities. A total of 733 bacterial isolates were recovered from vaginal (512) and uterine (221) swabs under aerobic (n = 363, 83 different species) and anaerobic (n = 370, 69 species) culturing. Potential pathogenic isolates within the Trueperella, Mannheimia and Histophilus genera were most exclusively identified from vaginal swabs. Only two Lactobacillus isolates were recovered. Overall, our results indicate that pregnancy-associated taxonomic signatures are present in the bovine uterine and vaginal microbiota. Among 39 pregnancy status-associated taxa, 41% were unclassified at the genus level, suggesting that shotgun metagenomics and culturomics are needed to identify and characterize the pregnancy-associated taxa in cattle.

Longitudinal analysis of vaginal microbiota during IVF fresh embryo transfer and in early pregnancy.

Non-Lactobacillus-dominated vaginal microbiota has been associated with poor gynecologic health and complications during pregnancy. Lactobacilli and especially Lactobacillus crispatus associate with good reproductive health and dominate the microbiota during healthy pregnancy. We examined whether the composition of vaginal microbiota at the time of fresh embryo transfer (ET) has an impact on the success of in vitro fertilization (IVF) and whether vaginal microbiota changes from IVF-ET to early pregnancy within individuals. Vaginal swab samples were collected from subfertile women at the time of IVF-ET (n = 76) and at the eighth gestational week (n = 21) from those who achieved clinical pregnancy. The microbiota composition was analyzed using 16S rRNA gene amplicon sequencing. L. crispatus was more abundant among the 30 women who achieved clinical pregnancy (46.9% vs. 19.1%, q = 0.039) and the 26 women who had live birth (43.3% vs. 23.1%, q = 0.32) compared to those who did not. Lactobacilli, mainly L. crispatus (76.2%), dominated all early pregnancy samples. Microbiota remained the same, i.e., Lactobacillus-dominated type in 52% (11/21), shifted from one Lactobacillus-dominated type to another in 24% (5/21), or shifted from mixed community to Lactobacillus-dominated type in 24% (5/21) women, but never from Lactobacillus dominance to non-lactobacilli dominance. Our results emphasize the role of L. crispatus in the success of IVF-ET and in early pregnancy. During pregnancy, the microbiota shifted toward L. crispatus dominance even if it was undetectable before pregnancy, indicating that most women hold a reservoir of this beneficial Lactobacillus in their reproductive tract.IMPORTANCEInfertility is a global public health issue which leads many couples to seek fertility treatments, of which in vitro fertilization (IVF) is considered to be the most effective. Still, only about one-third of the women achieve live birth after the first IVF embryo transfer (IVF-ET). Factors affecting embryo implantation are poorly known, but the female reproductive tract microbiota may play a key role. Our study confirms the beneficial role of vaginal lactobacilli, especially Lactobacillus crispatus, in the probability of achieving clinical pregnancy and live birth following IVF-ET. Our findings regarding the intra-individual shift of vaginal microbiota between non-pregnancy and pregnancy states are novel and provide new information about the dynamics of microbiota in the early steps of human reproduction. These findings may help clinicians in their attempts to optimize the conditions for ET by microbiota screening or modulation and timing the ET when the microbiota is the most favorable.

A citizen-science-enabled catalogue of the vaginal microbiome and associated factors

Understanding the composition and function of the vaginal microbiome is crucial for reproductive and overall health. Here we established the Isala citizen-science project to analyse the vaginal microbiomes of 3,345 women in Belgium (18–98 years) through self-sampling, 16S amplicon sequencing and extensive questionnaires. The overall vaginal microbiome composition was strongly tied to age, childbirth and menstrual cycle phase. Lactobacillus species dominated 78% of the vaginal samples. Specific bacterial taxa also showed to co-occur in modules based on network correlation analysis. Notably, the module containing Lactobacillus crispatus, Lactobacillus jensenii and Limosilactobacillus taxa was positively linked to oestrogen levels and contraceptive use and negatively linked to childbirth and breastfeeding. Other modules, named after abundant taxa (Gardnerella, Prevotella and Bacteroides), correlated with multiple partners, menopause, menstrual hygiene and contraceptive use. With this resource-rich vaginal microbiome map and associated health, life-course, lifestyle and dietary factors, we provide unique data and insights for follow-up clinical and mechanistic research.

Evaluation of an Isothermal Amplification HPV Assay on Self-Collected Vaginal Samples as Compared to Clinician-Collected Cervical Samples.

This study aimed to evaluate the concordance of HPV results between the SentisTM HPV assay (Sentis) (BGI Group, Shenzhen, China), an isothermal amplification-based HPV assay, on self-collected and clinician-collected samples and the agreement of Sentis on self-collected samples with the BD OnclarityTM HPV assay (Onclarity) (Becton, Dickinson, and Company, Franklin Lakes, New Jersey, USA), a PCR-based HPV assay, on clinician-collected samples. This was a prospective study of 104 women attending the colposcopy clinic for abnormal smears. After informed consent, participants self-collected vaginal samples before having clinician-collected cervical samples. Self-collected samples underwent HPV testing with Sentis (Self-Sentis HPV) and clinician-collected samples were tested with Sentis (Clinician-Sentis HPV) and Onclarity (Clinician-Onclarity), which was used as a reference standard. The concordance was assessed using Cohen's kappa. The prevalence of HPV and the acceptability of self-sampling were also evaluated. The concordance rate between Self-Sentis HPV and Clinician-Sentis HPV was 89.8% with a kappa of 0.769. The concordance rate between Self-Sentis HPV and Clinician-Onclarity was 84.4% with a kappa of 0.643. The prevalence of HPV was 26.0% on Clinician-Onclarity, 29.3% on Clinician-Sentis HPV, and 35.6% on Self-Sentis HPV. Overall, 65% of participants would undergo self-sampling again. This was attributed to mainly not feeling embarrassed (68%) and being convenient (58%). Our study showed a substantial agreement between Self-Sentis HPV with Clinician-Sentis HPV and Clinician-Onclarity. Self-sampling was also shown to be a generally well-accepted method of screening.

Microbiome alterations in women with pelvic organ prolapse and after anatomical restorative interventions

Pelvic organ prolapse (POP) is a benign gynecological disease in which the pelvic organ descends into the vagina and causes voiding, and defecatory dysfunction, mainly occurs in older women. This study aimed to investigate the vaginal microbiome of POP and associated changes after anatomical restorative pessary or reconstructive pelvic operation. We analyzed the vaginal microbiome using 16S ribosomal RNA gene sequencing and compared the results among patient groups with POP, pessary, and postoperation. We also measured 10 inflammation-related cytokines in vaginal swab samples using multiplex immunoassay. In pelvic organ prolapse, vaginal community status type IV was the most prevalent, which showed a low abundance of Lactobacillus with increased diversity and abundance of anaerobic species. The alpha diversity of species richness was highest in the POP group. The beta diversity distance differed significantly between the three groups (p = 0.001). While human intestinal taxa-associated bacteria were reduced after pessary or operation, vaginitis-associated bacterial composition was altered but vaginal microbiome homeostasis was not improved. IFN-γ, IL-10, IL-12p70, IL-1β, IL-4 and TNF-α levels increased in the pessary group. Therefore, in addition to anatomical restorative treatment, supplementary treatment focusing on the recovery of the vaginal microbiome may be needed to maintain the health of gynecological organs in old age.

Prevalence and Genotypic Diversity of Human Papillomavirus Infection Among Male and Female Sex Workers in Thailand.

Human papillomavirus (HPV) is associated with cancer. Female sex workers (FSWs) are known to be at risk for HPV, but little is known about male sex workers (MSWs). We examined HPV prevalence and associated risk factors in both populations. During 2022, HPV testing using vaginal or penile samples, HIV testing, and interviews were performed among 100 MSWs and 100 FSWs in Chiang Mai, Thailand. The prevalence of all HPV types was 63.5% (66% in MSW, 61% in FSW), HPV-16 prevalence was 14%, HPV-52 was 13%, and HPV-18 was 4%. There was no difference between MSW and FSW for these subtypes. The prevalence of HPV-16 or HPV-18 was 17%, and for HPV-16, HPV-18, or HPV-52, it was 26%. HIV-positive participants had a higher prevalence of all HPV types (94% vs. 60%, P = 0.004), HPV-16 or HPV-18 (39% vs. 15%, P = 0.018), and HPV-16, HPV-18, or HPV-52 (50% vs. 23%, P = 0.017). We demonstrated an equally high HPV prevalence across the sexes. Further studies are needed to determine if this indicates an equal risk for cancer. Increased HPV awareness, screening, and vaccination should be considered, regardless of gender.