The Futility of Nugent Scoring as a Diagnostic Tool for Neovaginal Bacterial Dysbiosis in Transfeminine People

The Futility of Nugent Scoring as a Diagnostic Tool for Neovaginal Bacterial Dysbiosis in Transfeminine People | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The Futility of Nugent Scoring as a Diagnostic Tool for Neovaginal Bacterial Dysbiosis in Transfeminine People Jessica Prodger, Reeya Parmar, Bern Monari, Emery Potter, Jorge Rojas-Vargas, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7428168/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Feb, 2026 Read the published version in Communications Medicine → Version 1 posted You are reading this latest preprint version Abstract Background : Transfeminine people were assigned male at birth and experience a female or feminine gender identity. Many elect to undergo vaginoplasty, a surgical procedure that constructs a neovagina, typically using penile and scrotal tissue. Like cisgender females, transfeminine people experience gynecological symptoms, including pain, discharge, and malodor. In cisgender females, clinicians attribute these symptoms to bacterial dysbiosis and can be diagnosed by Nugent scoring of gram-stained vaginal smears. The Nugent score assesses the abundance of large gram-positive rod vs. small or curved gram-variable rod morphotypes, traditionally for the detection of Lactobacillus spp., Gardnerella vaginalis , and Mobiluncus spp. (curved rod), respectively. Although unvalidated for neovaginal samples, this method is frequently applied to diagnose dysbiosis in transfeminine people with vaginoplasty. Objective: This study assessed the Nugent score’s utility as a clinical tool for diagnosing neovaginal dysbiosis in transfeminine people who underwent penile inversion vaginoplasty. Study Design : As a part of the TransBiota study, n=39 transfeminine participants self-collected neovaginal smears. Smears were Gram stained and Nugent scored, and Nugent scores were correlated with existing data on neovaginal bacterial composition (16S rRNA gene sequencing), neovaginal cytokines (Luminex multiplex immunoassay), and self-reported symptoms. Results: More than 70% of smears fell in the 7-10 range that would indicate Bacterial Vaginosis in cisgender women. However, Nugent score failed to correlate with the abundance of Nugent-targeted bacteria. Bacteria with similar morphotypes, but not belonging to Lactobacillus , Gardnerella , or Mobiluncus , were highly abundant and prevalent in the neovagina. Nugent score also failed to predict local inflammation or clinical symptoms. Conclusion : The Nugent score is not an effective tool to identify neovaginal dysbiosis or indicators of health in transfeminine individuals. Clinicians need the development of accurate, evidence-based diagnostic tools for the neovagina. Biological sciences/Microbiology/Clinical microbiology Biological sciences/Microbiology/Microbial communities/Microbiome Biological sciences/Immunology/Cytokines Health sciences/Health care/Diagnosis Biological sciences/Biological techniques/Microscopy Bacterial Vaginosis Gynecology Microbiome Transgender Vaginoplasty Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Transfeminine individuals (TF) were assigned male at birth and experience a female/feminine gender identity. Numerous communities fall under this umbrella, including transgender women, non-binary, and other gender diverse individuals 1 , 2 . Many TF elect to undergo gender-affirming medical care, often through feminizing hormone therapy or surgery. Vaginoplasty is a widely performed gender-affirming surgery that creates a clitoris, vulva, and vaginal canal. The most prevalent technique is penile inversion vaginoplasty, involving orchiectomy, dissection of the space between the bladder and rectum, and lining the newly formed space with penile and scrotal tissue 3 . Although less common, sigmoid or peritoneal tissue may also be used to augment the vaginal canal 4 – 6 . Penile inversion vaginoplasty results in a vaginal canal that is visually and functionally similar to that of cisgender females (CF) but lined with soft-cornified skin. We use the term “vagina” to refer to the vaginal canal of those born with a vagina, and “neovagina” to refer to the surgically created vaginal canal of TF. Our aim in using two terms is to clinically distinguish between vaginal canals lined with epithelia of different origins. In 2021, an estimated 20% of TF in the US had undergone genital surgery, with an additional 67% desiring it 7 . Like CF, TF with vaginoplasty often experience genital symptoms, including itching, burning, discharge, and malodor; however, the source of these symptoms has not been explored. To address this gap, we performed a study of the neovaginal microenvironments of 47 TF living in Canada 8 , 9 . In this study, 56% of participants reported neovaginal symptoms within the past 30 days 10 . In reproductive-aged CF (rCF), similar symptoms are caused by bacterial vaginal dysbiosis termed “bacterial vaginosis” (BV), which occurs when beneficial Lactobacillus spp. are replaced by a polymicrobial microbiome, including Gardnerella vaginalis , Prevotella spp., Ca. Lachnocurva vaginae, and other anaerobic bacteria 11 – 13 . Lactobacillus spp. play a critical role in rCF vaginal health by producing antimicrobial compounds and lactic acid, which lowers pH and inhibits colonization by pathogens 14 , 15 . Even without symptoms, dysbiotic microbiomes in rCF are associated with increased susceptibility to sexually transmitted infections, including chlamydia, gonorrhea, HSV-2, and HIV-1, underscoring the importance of the vaginal microbiome in sexual health 14 – 23 . Clinicians usually diagnose BV in rCF using the Nugent score. The Nugent score is a Gram stain-based diagnostic measure that rates the relative abundance of gram-positive rods (indicative of beneficial Lactobacillus spp.) vs. small or curved gram-variable rods (morphotypes of BV-associated bacteria) 12 , 24 . Scores from 0–3 are considered optimal, 4–6 inconclusive or intermediate, and 7–10 indicative of BV. The Nugent score is often applied to diagnose TF experiencing genital symptoms; however, the neovaginal microbiota is distinct from the vaginal microbiota 25 – 27 , and results from TransBiota suggest bacteria associated with inflammation in the neovagina differ significantly from those in the rCF vagina 8 . Bacteria associated with neovaginal immune activation include Lawsonella , Howardella , Fusobacterium , and Parvimonas , while higher abundances of Ezakiella , Fastidiosipila , Murdochiella , and Peptoniphilus associated with reduced inflammation 8 . Core neovaginal bacteria are listed in Supplemental Table 1 . The substantial differences in microbiome composition and associated inflammation found in the rCF vagina and the neovagina raise questions about the utility of the Nugent score as a diagnostic tool in TF. Nevertheless, clinicians lack alternative diagnostics and the Nugent score continues to be frequently employed 28 . This study evaluated the Nugent score’s relevance as a diagnostic tool for neovaginal dysbiosis by assessing its accuracy in identifying bacteria targeted by the score, and its ability to predict markers of bacterial dysbiosis (cytokines and symptoms) in the neovagina. METHODS Participants TransBiota was a study investigating the genital microenvironments of trans and other gender diverse people receiving gender-affirming medical care 8 – 10 . Eligible TF participants were Canadian residents 18 years or older, who underwent vaginoplasty > 1 year prior to study entry. Research ethics board approval was obtained from Western University (REB #115503) and University of Maryland (IRB #HP-00096952), and all participants provided written informed consent. Participants were recruited online through social media and community groups, healthcare provider referral, and re-contact of consenting Trans PULSE Canada participants 29 . Participants were mailed a study kit containing instructions and self-collection materials. Demographic, behavior, and symptom data were collected through an online questionnaire. Participants (n = 47) returned three weekly neovaginal sample sets by mail using pre-paid envelopes. This analysis includes the first timepoint for each participant for which there is a corresponding 1) scorable gram-stained slide, 2) microbiome data, and 3) immune analyte data. All research activities were conducted in accordance with institutional and ethical regulations, and participant data were deidentified prior to analysis. Microbiota and Cytokine Analyses Participants self-collected neovaginal swabs at each timepoint for Nugent scoring, microbiota analysis, and cytokine quantification. Sample collection and analysis of neovaginal microbiota and cytokines have been described in detail elsewhere 8 . In brief, participants were instructed to insert each swab (Puritan HydraFlock) 5cm into the neovaginal canal, rotate 3 times, and place in collection media. Swabs for microbiota analysis were collected into 1ml of Qiagen C2.1 solution. DNA extractions were performed using the MagAttract PowerMicrobiome DNA/RNA kit (Qiagen), and 16S rRNA gene V3-V4 region amplicon sequencing (amplified via two step PCR) was conducted at University of Maryland Institute for Genomic Sciences 30 . Reads were processed using a QIIME-dependent script with DADA2 to generate amplicon-sequence variants (ASVs). Taxonomy was assigned with the RDP naïve-Bayes classifier trained on the SILVA v138.2 16S rRNA database and refined species-level calls with SpeciateIT v2.0.0. ASVs sharing identical taxonomy were collapsed. Swabs for cytokine analysis were collected into 500µl of a PBS-based stabilizing buffer, and IL-1a, IL-1b, IL-6, IL-8, MIG, MIP-1b, and RANTES concentrations were quantified on a Luminex MAGPIX system. Nugent Scoring Participants rolled collection swabs onto charged glass microscope slides (USA Scientific) and returned them to Western University in secured plastic cases. Neovaginal smears were heat-fixed and Gram stained using standard techniques. In brief, slides were stained with crystal violet (1 minute), iodine mordant (1 minute), decolorizing solution (until solution ran clear), and safranin (30 seconds) at room temperature. Slides were rinsed with running tap water between steps. Slides were observed under a 100x oil-immersion light microscope. For each smear, bacterial morphologies were individually scored in 10 representative fields of view (FOVs) following Nugent criteria 24 . The criteria assign a decreasing score from 0–4 for the abundance of Lactobacillus -like gram-positive large rods, an increasing score from 0–4 for the abundance of Gardnerella -like short gram-variable straight rods, and an increasing score from 0–2 for the abundance of Mobiluncus -like gram-variable curved rods. Scores for each morphotype were summed to yield a total score from 0–10 for each FOV. Final mean score across the 10 FOVs was calculated for each participant. In rCF, scores of 0–3 are optimal, 4–6 inconclusive/intermediate, and 7–10 indicative of BV 24 . Data Analysis Multiple unpaired Mann-Whitney U nonparametric tests were used to assess differences in Nugent scores between asymptomatic and symptomatic participants. Fisher’s exact test was used to assess differences in bacterial prevalence, and Mann-Whitney U test was used to assess differences in relative abundance between asymptomatic and symptomatic participants. Associations between Nugent score and cytokines (IL-1α, IL-1β, IL-6, IL-8, MIG, MIP-1β, and RANTES) were assessed by Spearman’s correlation. Relationship between neovaginal bacteria taxa and cytokines, as well as bacterial taxa and Nugent score was also evaluated using Spearman’s correlations. Only taxa with a neovaginal prevalence of > 25% were included in analyses. GraphPad Prism 8 and R Studio (version 4.3.2) were used to create graphs and perform statistical analyses. RESULTS Of the n = 47 TransBiota participants, 8 were excluded (17%) due to no scorable slides from destruction during heat-fixation from smearing on the incorrect side of the slide; damage to slides during shipping; or insufficient sample for Nugent scoring (defined at < 30 bacteria/FOV). Demographics on the remaining n = 39 participants included in this study are displayed in Table 1 . Median time on hormone therapy was 5.6 years and median time since vaginoplasty 2.8 years. Table 1 Participant demographics. Participants (n = 39) Age, years (median, range) 39 (26–67) Ethnoracial Identity (%) White 89.7% Latin American 2.6% Jewish 2.6% Mixed Ethnicity 5.1% Months on hormone therapy (median, range) 67 (35–265) Months since vaginoplasty (median, range) 33 months (12–229 months) Circumcised prior to vaginoplasty (%) 56.4% Symptoms, past 7 days (%) 23.1% Bleeding 5.1% Discharge 5.1% Itching/burning 2.6% Malodor 12.8% Pain 2.6% pH (median, range) 5.5 (4.5-8) Neovaginal smears contained nucleate and anucleate epithelial cells, with most bacteria clustered near these epithelial cells (Fig. 1 a, e). Four slides (10.3%) were visibly bloody with erythrocytes and leukocytes visible by microscopy (Fig. 1 b, e). Gram-variable rods and cocci dominated the majority of smears (Fig. 1 c, e ) . Although all smears contained gram-variable straight rods, only 48.7% of slides displayed large gram-positive rods ( Lactobacillus -like morphotype), and just three (7.7%) were dominated by large gram-positive rods (Fig. 1 d, e). The majority of neovaginal smears (71.8%) fell within the BV Nugent range (7–10; Fig. 2 a). The most common score was 7 (46.2% of participants) indicating abundant gram-variable rods and minimal Lactobacillus -like rods. Only two participants (5.1%) scored an optimal Nugent score range for rCF (0–3). Lactobacillus showed a moderate negative correlation with Nugent score (Fig. 2 b) while Gardnerella and Mobiluncus showed no correlation (Fig. 2 c, d). Nugent Score and traditional Nugent-targeted taxa are not associated with neovaginal symptoms Nine participants (23.1%) reported neovaginal symptoms (malodor, discharge, bleeding, pain/burning) within 7 days prior to sample collection. There was no difference in Nugent scores between symptomatic and asymptomatic participants (Fig. 3 a). Additionally, no relationship was observed between prevalence (Fig. 3 b) or proportional abundance (Fig. 3 c) of the traditional Nugent score bacteria ( Lactobacillus , Gardnerella , Mobiluncus ) and the presence or absence of neovaginal symptoms. Nugent Score is not associated with neovaginal cytokines Nugent scores were analyzed in relation to pro-inflammatory cytokine concentrations, but no significant correlations were observed (Fig. 4 a – g ). Representative images are shown of gram-stained smears from one participant with high cytokine levels (87.8pg/ml IL-1α, 65.4pg/ml IL-1β, 34.7pg/ml IL-6, 85.3pg/ml IL-8, 61.1pg/ml MIG, 21.7pg/ml MIP-1β, 33.1pg/ml RANTES) and one participant with low cytokine levels (83.9pg/ml IL-1α, 35.6pg/ml IL-1β, 6.3pg/ml IL-6, 57.1pg/ml IL-8, 11.8pg/ml MIG, 21.7pg/ml MIP-1β, undetectable RANTES). Both participants had a Nugent score of 7 with neovaginal smears dominated by gram-negative rods (Fig. 4 h – i ). Bacteria with Nugent-targeted morphotypes in the neovagina A comprehensive description of neovaginal bacterial communities of TransBiota participants has been previously published 8 . A summary of the most prevalent neovaginal bacteria (> 25% prevalence) are listed in Fig. 5 , separated by the morphotypes targeted by Nugent scoring. Traditional Nugent-targeted taxa ( Lactobacillus , Gardnerella , and Mobiluncus ) frequently appeared in the neovagina, but at low relative abundance (Fig. 5 ). Several other taxa detected in the neovagina had similar morphologies to Nugent-targeted genera. Lawsonella clevelandensis is a large gram-positive rod that can have similar appearance to Lactobacillus spp. on Gram staining 31 , 32 . Lawsonella clevelandensis (87.2% prevalence; 0.3% median abundance) outnumbered Lactobacillus (64.1%; 0.2%). Lawsonella abundance positively correlated with IL-1α, IL-1β, IL-8 and RANTES (p < 0.05), whereas Lactobacillus showed no correlation with cytokines, and correlated with lower Nugent scores (Fig. 5 ; p < 0.03). Three participants exhibited predominantly large rods; two had low Nugent scores of 2 (72.6% Lactobacillus relative abundance) and 3 (58.0% Lactobacillus relative abundance) and one scored 4 (16.5% Lactobacillus relative abundance), owing to mixed morphotypes. Gardnerella vaginalis , was detected in 33.3% of samples at a median relative abundance of 0.4%. Fannyhessea vaginae (formerly Atopobium vaginae ), although not a traditional Nugent bacteria, is also a BV-associated rod and may contribute to the diagnostic power of the Nugent score in rCF 33 . Fannyhessea was detected in 28.2% of samples at a median relative abundance of only 1.7%. Short straight rods in the neovagina were more likely to be Hoylesella (previously Prevotella; 97.4% prevalence, 9.7% relative abundance), Prevotella spp. (92.3%; 8.5%), or Porphyromonas spp. (97.4%; 8.3%). Prevotella was positively associated with increased cytokines (IL-1α; p < 0.01), while Gardnerella, Fannyhessea, Porphyromonas , and Hoylesella had no significant correlation. None had significant correlations with Nugent score. Mobiluncus , the gram-variable curved rod traditionally targeted by Nugent scoring, was detected in 76.9% of samples at 0.8% median relative abundance. Other abundant neovaginal curved rods included Varibaculum (92.3% prevalence, 2.6% abundance) and Campylobacter (89.7%; 1.7%). Abundances of Campylobacter were inversely correlated with neovaginal cytokines (IL-1β, IL-8; p < 0.05) while Mobiluncus and Varibaculum showed no significant correlation with cytokines. Both Mobiluncus (p < 0.02) and Campylobacter (p < 0.02) correlated with higher Nugent scores. Of note, in addition to F. vaginae , BV-associated Ca. Lachnocurva vaginae (formerly BVAB1; curved rod) and Sneathia amnii (curved Gram-negative rod) posses similar morphotypes to traditional Nugent bacteria and may contribute to the diagnostic power of the Nugent score in rCF 34 . However, both were absent from neovaginal samples. As noted in Fig. 1 E, many neovaginal smears contained high abundance of cocci, which are not considered by Nugent criteria. Based on V3-V4 16S rRNA gene sequences, 4/10 core neovaginal bacteria have morphotypes not considered during Nugent scoring ( Supplemental Table 1 ). Peptoniphilus , Ezakiella and Anaerococcus are all gram-positive cocci. Of these, Ezakiella is associated with reduced neovaginal cytokines (IL-8, MIG, p < 0.05), while Anaerococcus is associated with increased cytokines (IL-6, MIG, MIP-1β; p < 0.05) (Fig. 5 ). DISCUSSION Principal findings This study provides strong evidence that the Nugent score is not suitable for clinical diagnosis of neovaginal dysbiosis in TF with penile inversion vaginoplasty. Most neovaginal bacteria belong to taxa not targeted by the Nugent score, and the score does not associate with predictors of genital dysbiosis, including neovaginal symptoms and cytokines. Nugent scoring in rCF relies on the eubiotic nature of Lactobacillus spp. predominance, and vaginal polymicrobialism as dysbiotic. However, Lactobacillus spp. predominance in the neovagina is very rare 8 , 27 , 28 , 35 , potentially due to differences in carbon sources available in the rCF vagina and TF neovagina. Vaginal epithelial cells in rCF are rich in glycogen and constantly shed into the vaginal lumen 15 , 36 , 37 . In contrast, neovaginal epithelium derived from penile skin lacks glycogen despite exposure to estrogen levels similar to rCF, and is instead soft-cornified with a lipid-rich extracellular matrix 15 , 36 – 39 . In rCF, vaginal Lactobacillus spp. metabolize glycogen products to produce lactic acid with anti-inflammatory properties, inhibiting colonization by non-lactobacilli, including pathogens 40 , 41 . In our cohort Lactobacillus rarely dominated and showed similar abundance in symptomatic and asymptomatic TF. Future research is warranted to determine if the neovaginal epithelium can support Lactobacillus predominance, and if this confers any benefit. Further, there is no evidence that neovaginal microbiomes rich in gram-variable rods are necessarily dysbiotic. Neovaginal abundances of traditional Nugent morphotypes Gardnerella and Mobiluncus were not different between symptomatic and asymptomatic TF and did not correlate with increased cytokines. While the relationship between neovaginal inflammation and sexual health outcomes has not been adequately explored, genital inflammation is strongly correlated with negative health outcomes in cisgender individuals 16 , 26 , 42 – 45 . In rCF, Mobiluncus and Gardnerella are positively associated with vaginal cytokines such as IL-1β and IL-8 42,46–48 . Instead in the neovagina, Campylobacter , a more abundant curved rod, showed negative correlations with cytokines. More abundant short gram-variable rods such as Hoylesella (predominantly H. timonensis and H. buccalis , both previously Prevotella ), Porphyromonas and Dialister lacked any association with inflammation, while Fenollaria correlated inversely with cytokines. Other bacteria that normally contribute to the diagnostic power of the Nugent score in rCF ( Fannyhessea , Sneathia amnii , Ca. L. vaginae) showed no correlation to cytokines or were absent from the neovagina. These data suggest that gram-variable rod abundance on neovaginal smears does not provide interpretive information on neovaginal health. Lawsonella abundance correlated strongly with inflammation, yet Nugent scoring classifies Lawsonella morphotypes under the “beneficial rod” category. Likewise, pro-inflammatory cocci such as Anaerococcus lay outside the Nugent framework. These findings are consistent with a study by Weyers et al. examining neovaginal cytology, reporting that, despite over 50% of participants being diagnosed with BV, no correlation was observed with neovaginal inflammation 49 . Additionally, Weyers et al. noted a significant presence of inflammatory cells in the neovagina. In our study, we frequently observed blood cells, suggesting participants may be experiencing epithelial erosion. Clinical implications Nugent scoring of neovaginal smears risks misdiagnosis and promotes futile antibiotic use. Nearly all neovaginal smears in this study fell outside the optimal Nugent range (0–3), indicating most would be classified as dysbiotic and indicative of BV. BV in rCF is commonly treated with metronidazole, which spares Lactobacilli but is bactericidal for BV-associated gram-negative anaerobes, including Gardnerella 50 , 51 . However, the gram-positive bacteria associated with inflammation in the neovagina, such as Lawsonella and Anaerococcus , are unlikely to be susceptible to metronidazole 50 , 51 . TF who receive a Nugent score in the BV range may feel distress or use products or home remedies aimed at treating BV and restoring Lactobacilli in rCF. TransBiota participants who used diverse solutions for douching (povidone-iodine, soapy water, vinegar) were more likely to have high abundances of inflammation-associated bacteria 8 . Many also reported using oral probiotics or probiotic suppositories designed for rCF when experiencing neovaginal symptoms, and referred to having BV in their questionnaires 10 . Additional research is urgently needed to better characterize the causative agents of neovaginal inflammation and symptoms, and to design effective diagnostic tools to identify them in a clinical setting. However, these findings apply chiefly to mature penile-skin lined neovagina, and microbial dynamics might differ in bowel-segmented or peritoneal graft neovaginas. Limitations This study’s mail-in methodology enabled broader participation, but resulted in unusable smears due to inadequate sampling or slide damage during shipping. This study also did not address sampling location within the neovaginal canal; while participants were instructed to collect swabs 5cm into the neovaginal canal, sampling depth may have varied between participants and microbiomes vary by distance from the introitus. Although participants were asked to refrain from inserting anything into their neovagina 24h prior to sampling, variable practices outside this window may have introduced variability 52 , 53 . Unmeasured behavioral or hormonal variables may also confound associations. Conclusions The Nugent score is an ineffective tool for predicting neovaginal dysbiosis in TF with penile inversion vaginoplasty. Bacteria traditionally targeted by the Nugent score are rare in the neovagina, while other taxa with similar morphotypes are abundant. Nugent scores did not correlate with inflammation or symptoms in the neovagina. Using the Nugent score on neovaginal smears may result in misdiagnosis, inappropriate antibiotic use, and misplaced efforts by TF and clinicians to “correct” neovaginal microbiomes, possibly disrupting an optimal microbiome. These findings highlight that vaginal dysbiosis differs fundamentally between rCF and TF and underscores the need to establish evidence-based neovaginal diagnostics. Declarations ACKNOWLEDGEMENTS The authors would like to thank Jason Hallarn and Greta Bauer for their contributions establishing TransBiota. Funding: This work was supported by the Canadian Institute of Health Research [PJT 180322] and the National Institutes of Health [R21 AI157912]. JLP is supported by the Canada Research Chairs Program [CRC-2020-00175]. AS is supported by a Canada Graduate Scholarship from the Canadian Institute of Health Research. This research was undertaken, in part, thanks to funding from the Canada Foundation for Innovation [CFI 42343]. The funders played no role in study design, data collection, analysis and interpretation of data, or the writing of this manuscript. There are no conflicts of interest or financial disclosures to declare. AUTHOR CONTRIBUTIONS Conceptualization: J.R., J.L.P., Y.K, E.P. Methodology: R.P., B.M., H.W., D.Z. Formal analysis: R.P. and J.R-V. Interpretation: R.P., J.R-V., B.M., J.R. and J.L.P. Writing: R.P. and J.L.P, Editing: R.P., B.M., E.P., J.R-V., H.W., D.Z., A.P., A.C.S., V.L., Y.K, J.L.P. and J.R. Funding acquisition: J.R. and J.L.P. DATA AVAILABILITY All source codes used to analyze the data and generate the figures presented are available in GitHub at github.com/prodgerlab/TransBiota/tree/main/Nugent_scoring_paper. The cytokine, microbiome, and Nugent scoring datasets used in this study are also accessible through this repository. References James, S. E., Herman, J., Keisling, M., Mottet, L. & Anafi, M. 2015 U.S. Transgender Survey (USTS): Version 1. ICPSR - Interuniversity Consortium for Political and Social Research https://doi.org/10.3886/ICPSR37229.V1 (2019). 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Health care access among transgender and nonbinary people in Canada, 2019: a cross-sectional survey. cmajo 9 , E1213–E1222 (2021). Holm, J. B. et al. Ultrahigh-Throughput Multiplexing and Sequencing of >500-Base-Pair Amplicon Regions on the Illumina HiSeq 2500 Platform. mSystems 4 , e00029-19 (2019). Bell, M. E. et al. Lawsonella clevelandensis gen. nov., sp. nov., a new member of the suborder Corynebacterineae isolated from human abscesses. Int J Syst Evol Microbiol 66 , 2929–2935 (2016). Morton, A. B., Boyle, E., Pettengill, M. A. & Gancher, E. The Brief Case: Strictly Anaerobic and Staining Acid Fast. J Clin Microbiol 61 , e0015022 (2023). Muzny, C. A. et al. Identification of Key Bacteria Involved in the Induction of Incident Bacterial Vaginosis: A Prospective Study. J Infect Dis 218 , 966–978 (2018). Srinivasan, S. et al. More than meets the eye: associations of vaginal bacteria with gram stain morphotypes using molecular phylogenetic analysis. PLoS One 8 , e78633 (2013). Weyers, S. et al. Microflora of the penile skin-lined neovagina of transsexual women. BMC Microbiol 9 , 102 (2009). Cruickshank, R. The conversion of the glycogen of the vagina into lactic acid. J. Pathol. 39 , 213–219 (1934). Dinh, M. H., Okocha, E. A., Koons, A., Veazey, R. S. & Hope, T. J. Expression of Structural Proteins in Human Female and Male Genital Epithelia and Implications for Sexually Transmitted Infections1. Biology of Reproduction 86 , (2012). Krakowsky, Y. et al. The Effect of Gender-Affirming Medical Care on the Vaginal and Neovaginal Microbiomes of Transgender and Gender-Diverse People. Front. Cell. Infect. Microbiol. 11 , 769950 (2022). Dekker, J. J. M. L., Hage, J. J., Karim, R. B. & Bloemena, E. Do histologic changes in the skin-lined neovagina of male-to-female transsexuals really occur? Ann Plast Surg 59 , 546–549 (2007). Hearps, A. C. et al. Vaginal lactic acid elicits an anti-inflammatory response from human cervicovaginal epithelial cells and inhibits production of pro-inflammatory mediators associated with HIV acquisition. Mucosal Immunol 10 , 1480–1490 (2017). Delgado-Diaz, D. J. et al. Lactic acid from vaginal microbiota enhances cervicovaginal epithelial barrier integrity by promoting tight junction protein expression. Microbiome 10 , 141 (2022). Mitchell, C. & Marrazzo, J. Bacterial vaginosis and the cervicovaginal immune response. Am J Reprod Immunol 71 , 555–563 (2014). Chen, X., Lu, Y., Chen, T. & Li, R. The Female Vaginal Microbiome in Health and Bacterial Vaginosis. Front. Cell. Infect. Microbiol. 11 , (2021). Arnold, K. B. et al. Increased levels of inflammatory cytokines in the female reproductive tract are associated with altered expression of proteases, mucosal barrier proteins, and an influx of HIV-susceptible target cells. Mucosal Immunology 9 , 194–205 (2016). Prodger, J. L. et al. Chemokine Levels in the Penile Coronal Sulcus Correlate with HIV-1 Acquisition and Are Reduced by Male Circumcision in Rakai, Uganda. PLoS Pathog 12 , e1006025 (2016). Joseph, A. et al. Intrauterine colonization with Gardnerella vaginalis and Mobiluncus mulieris induces maternal inflammation but not preterm birth in a mouse model. Am J Reprod Immunol 90 , e13749 (2023). Campos, A. C. C., Murta, E. F. C., Michelin, M. A. & Reis, C. Evaluation of Cytokines in Endocervical Secretion and Vaginal pH from Women with Bacterial Vaginosis or Human Papillomavirus. ISRN Obstet Gynecol 2012 , 342075 (2012). Anahtar, M. N. et al. Cervicovaginal bacteria are a major modulator of host inflammatory responses in the female genital tract. Immunity 42 , 965–976 (2015). Weyers, S. et al. Cytology of the ‘penile’ neovagina in transsexual women. Cytopathology 21 , 111–115 (2010). Verwijs, M. C., Agaba, S. K., Darby, A. C. & van de Wijgert, J. H. H. M. Impact of oral metronidazole treatment on the vaginal microbiota and correlates of treatment failure. Am J Obstet Gynecol 222 , 157.e1-157.e13 (2020). Goldstein, E. J. C., Tyrrell, K. L. & Citron, D. M. Lactobacillus species: taxonomic complexity and controversial susceptibilities. Clin Infect Dis 60 Suppl 2 , S98-107 (2015). Muthusamy, S., Varghese, J., Raveendran, V., Ezilarasan, K. & Easow, J. Evaluation of interobserver reliability of Nugent score for diagnosis of bacterial vaginosis. Indian J Sex Transm Dis 39 , 120 (2018). Mohanty, S., Sood, S., Kapil, A. & Mittal, S. Interobserver variation in the interpretation of Nugent scoring method for diagnosis of bacterial vaginosis. Indian J Med Res 131 , 88–91 (2010). Additional Declarations There is NO Competing Interest. Supplementary Files NugentScoreData.xlsx Dataset 1 MicrobiomeData.xlsx Dataset 2 CytokineData.csv Dataset 3 SUPPLEMENTALMATERIAL.docx Cite Share Download PDF Status: Published Journal Publication published 03 Feb, 2026 Read the published version in Communications Medicine → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7428168","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":506537384,"identity":"fff6b2f9-1934-4f6e-917f-b228ac6d1b22","order_by":0,"name":"Jessica Prodger","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAr0lEQVRIiWNgGAWjYBACxgYgwcPAIEe6FmMSrQJqSWwgWjXz7N6HD978sknfcCP5AMOPGmIcNue4seHcvrTcDTfSEhh7jhGjZUYamzRvz+HcDbdzDJgZ2IjX8j/dAKzlH7FaeH4cSABrYWwjRsucY8yGcxuSDWfef5ZwsLePCC2Gs9sYH7z5YyfPd+bwwQc/vhGjZQbIKqh7DhChgYFBXgJE/iFK7SgYBaNgFIxUAACtizhy/HILFQAAAABJRU5ErkJggg==","orcid":"","institution":"Western University","correspondingAuthor":true,"prefix":"","firstName":"Jessica","middleName":"","lastName":"Prodger","suffix":""},{"id":506537385,"identity":"a26e977b-87a0-41cc-a6ad-7c8580b1d0ab","order_by":1,"name":"Reeya Parmar","email":"","orcid":"https://orcid.org/0009-0001-7374-2203","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Reeya","middleName":"","lastName":"Parmar","suffix":""},{"id":506537386,"identity":"1f435dd2-f3df-4739-8af7-9db48c27b988","order_by":2,"name":"Bern Monari","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Bern","middleName":"","lastName":"Monari","suffix":""},{"id":506537387,"identity":"ef91e2d5-fe3c-415f-b3bb-4af31157461b","order_by":3,"name":"Emery Potter","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Emery","middleName":"","lastName":"Potter","suffix":""},{"id":506537388,"identity":"410c80eb-f258-4292-8b20-3e56cbb99581","order_by":4,"name":"Jorge Rojas-Vargas","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jorge","middleName":"","lastName":"Rojas-Vargas","suffix":""},{"id":506537389,"identity":"745f075a-1b8e-4f8a-9d7d-3240ab144dfb","order_by":5,"name":"Hannah Wilcox","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Hannah","middleName":"","lastName":"Wilcox","suffix":""},{"id":506537390,"identity":"5c04b163-e569-4fa8-b64a-5ee993f0a45b","order_by":6,"name":"David Zuanazzi","email":"","orcid":"","institution":"University of Western Ontario","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Zuanazzi","suffix":""},{"id":506537391,"identity":"ac409f93-1cb0-430e-a9d1-2ce934839eb9","order_by":7,"name":"Annabel Poon","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Annabel","middleName":"","lastName":"Poon","suffix":""},{"id":506537392,"identity":"c03cd56c-9eb7-4d09-984f-4544a3b1f503","order_by":8,"name":"Ainslie Shouldice","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Ainslie","middleName":"","lastName":"Shouldice","suffix":""},{"id":506537393,"identity":"45574881-3d5b-4545-9ecd-8b977d6c8451","order_by":9,"name":"Vonetta Edwards","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Vonetta","middleName":"","lastName":"Edwards","suffix":""},{"id":506537394,"identity":"bb0864b3-7c65-4f38-b1f7-6b595c378294","order_by":10,"name":"Yonah Krakowsky","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yonah","middleName":"","lastName":"Krakowsky","suffix":""},{"id":506537395,"identity":"3c137009-ff23-4502-ba90-29d052f5b887","order_by":11,"name":"Jacques Ravel","email":"","orcid":"https://orcid.org/0000-0002-0851-2233","institution":"University of Maryland, Baltimore","correspondingAuthor":false,"prefix":"","firstName":"Jacques","middleName":"","lastName":"Ravel","suffix":""}],"badges":[],"createdAt":"2025-08-21 16:51:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7428168/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7428168/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s43856-026-01410-2","type":"published","date":"2026-02-03T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":90792649,"identity":"60a9e30d-f067-4e13-910c-08d624254e30","added_by":"auto","created_at":"2025-09-08 08:29:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":536777,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepresentative images and descriptive characteristics of neovaginal smears. \u003c/strong\u003eSelf-collected neovaginal smears (n=39) were heat-fixed, Gram-stained, and observed under a 100x oil-immersion light microscope. Ten fields of view (FOV) were scored per participant; representative FOVs showing \u003cstrong\u003e(a)\u003c/strong\u003e proximity of bacteria to epithelial cells, \u003cstrong\u003e(b)\u003c/strong\u003e abundance of red and white blood cells, \u003cstrong\u003e(c)\u003c/strong\u003edominance with Gram-variable rods and cocci, and \u003cstrong\u003e(d)\u003c/strong\u003e dominance with \u003cem\u003eLactobacillus\u003c/em\u003e-like morphotype. Descriptive characteristics of neovaginal smears are shown in \u003cstrong\u003e(e)\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7428168/v1/a15dd2a946bea92d2380aa4e.png"},{"id":90792648,"identity":"d2d53f68-19f7-464f-9a63-750516bc0645","added_by":"auto","created_at":"2025-09-08 08:29:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":53800,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNugent scores and correlation with traditional Nugent-targeted taxa across neovaginal smears. (a)\u003c/strong\u003e Distribution of Nugent scores from n=39 TF participants. Scores represent the median score per participant over 10 fields of view (FOVs) acquired at 100x oil-immersion magnification.\u003cstrong\u003e \u003c/strong\u003eLower scores from 0–3 are considered optimal, intermediate scores of 4–6 are inconclusive, and higher scores of 7–10 are considered indicative of BV. Spearman’s correlations are shown between the relative abundances of \u003cstrong\u003e(b)\u003c/strong\u003e \u003cem\u003eLactobacillus \u003c/em\u003e(p\u0026lt;0.03), \u003cstrong\u003e(c)\u003c/strong\u003e \u003cem\u003eGardnerella \u003c/em\u003e(p=0.67), \u003cstrong\u003e(d)\u003c/strong\u003e and \u003cem\u003eMobiluncus \u003c/em\u003e(p=0.85) within neovaginal smears and Nugent score.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7428168/v1/a683fdab75c8aff95627960a.png"},{"id":90792653,"identity":"bfb76090-1388-45d5-9da2-c8f0eff73bae","added_by":"auto","created_at":"2025-09-08 08:29:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":44520,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNo differences in Nugent score or traditional Nugent score bacteria between symptomatic and asymptomatic participants.\u003c/strong\u003e \u003cstrong\u003e(a) \u003c/strong\u003eMedian Nugent score did not vary between asymptomatic participants and participants who self-reported neovaginal malodor, discharge, bleeding, or pain/burning in the last 7 days (Mann-Whitney U, asymptomatic participants vs. each symptom, all p\u0026gt;0.1). Comparison of the \u003cstrong\u003e(b) \u003c/strong\u003eprevalence and \u003cstrong\u003e(c)\u003c/strong\u003erelative abundance of \u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003eGardnerella\u003c/em\u003e, and \u003cem\u003eMobiluncus\u003c/em\u003ebetween asymptomatic and symptomatic participants (prevalences compared by Fisher’s exact test, relative abundances by Mann Whitney U, all p\u0026gt;0.8). \u0026nbsp;Median and interquartile range are shown.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7428168/v1/5dace1157c5720f138f6d0e0.png"},{"id":90792662,"identity":"acd3c242-260c-44b9-805e-37e1934c308d","added_by":"auto","created_at":"2025-09-08 08:29:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":187929,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNeovaginal cytokines do not correlate with Nugent score\u003c/strong\u003e. Self-collected neovaginal smears from transfeminine participants (n=39) were heat-fixed, Gram stained, and observed under a 100x oil-immersion light microscope. Cytokine concentrations in neovaginal swabs were measured by multiplex immunoassay (Luminex). Spearman’s correlations were used to assess associations between\u003cstrong\u003e \u003c/strong\u003eNugent score and\u003cstrong\u003e (a)\u003c/strong\u003e IL-1α,\u003cstrong\u003e (b)\u003c/strong\u003eIL-1β, \u003cstrong\u003e(c)\u003c/strong\u003e IL-6, \u003cstrong\u003e(d)\u003c/strong\u003e IL-8, \u003cstrong\u003e(e)\u003c/strong\u003e MIG, \u003cstrong\u003e(f)\u003c/strong\u003e MIP-1β, and \u003cstrong\u003e(g)\u003c/strong\u003e RANTES. Representative images of gram-stained neovaginal smears from participants with low (\u003cstrong\u003eh\u003c/strong\u003e) and high (\u003cstrong\u003ei\u003c/strong\u003e) cytokine levels. Images have been cropped for visualization.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7428168/v1/6f476793eb998eed4ab9b171.png"},{"id":90792651,"identity":"8252f278-7c82-4c7a-8058-68f8054c04c0","added_by":"auto","created_at":"2025-09-08 08:29:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":217081,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNeovaginal bacterial prevalence, relative abundance, and association with cytokines and Nugent score. \u003c/strong\u003eTaxa typically scored by Nugent criteria (\u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003eGardnerella\u003c/em\u003e, \u003cem\u003eFannyhessea\u003c/em\u003e, and \u003cem\u003eMobiluncus\u003c/em\u003e) at the start of each morphotype category. Bacterial taxa with a prevalence of \u0026gt;25% are included. Median relative abundance is measured among prevalent participants and interquartile range are shown. Bacterial prevalence and relative abundance were measured from neovaginal swabs (n=39) using 16S rRNA gene sequencing. Cytokine concentrations were measured from swab eluent by multiplex immunoassay (Luminex). Spearman’s correlations were used to assess associations between\u003cstrong\u003e \u003c/strong\u003etaxa and\u003cstrong\u003e \u003c/strong\u003eIL-1α,\u003cstrong\u003e \u003c/strong\u003eIL-1β, IL-6, IL-8, MIG, MIP-1β, RANTES and Nugent score. Spearman’s correlations p values are shown in \u003cstrong\u003eSupplemental Table 3.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7428168/v1/fbc0cfc3c879455ce23b713c.png"},{"id":104379429,"identity":"c2ce53de-bdd2-443b-91a3-7fefbe666b1a","added_by":"auto","created_at":"2026-03-11 07:12:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2146605,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7428168/v1/726a2309-ff88-432b-83eb-ee36ea999de8.pdf"},{"id":90792647,"identity":"5d12fe67-7171-472d-88df-5d4eeec2899c","added_by":"auto","created_at":"2025-09-08 08:29:56","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":9811,"visible":true,"origin":"","legend":"\u003cp\u003eDataset 1\u003c/p\u003e","description":"","filename":"NugentScoreData.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7428168/v1/15d598b904d3b31ff03cc839.xlsx"},{"id":90793098,"identity":"b4bfd14b-3614-42b6-8d1a-23988191c6ad","added_by":"auto","created_at":"2025-09-08 08:37:57","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":49963,"visible":true,"origin":"","legend":"\u003cp\u003eDataset 2\u003c/p\u003e","description":"","filename":"MicrobiomeData.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7428168/v1/c0be6490f9804780af1c5456.xlsx"},{"id":90793099,"identity":"57e30ec8-db31-40ab-bbf2-4546b2c32359","added_by":"auto","created_at":"2025-09-08 08:37:57","extension":"csv","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":2118,"visible":true,"origin":"","legend":"\u003cp\u003eDataset 3\u003c/p\u003e","description":"","filename":"CytokineData.csv","url":"https://assets-eu.researchsquare.com/files/rs-7428168/v1/b571df3e3fcabb04b1eaee52.csv"},{"id":90793103,"identity":"31027615-5adf-400d-b0ff-733e38acb62b","added_by":"auto","created_at":"2025-09-08 08:37:57","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":30532,"visible":true,"origin":"","legend":"","description":"","filename":"SUPPLEMENTALMATERIAL.docx","url":"https://assets-eu.researchsquare.com/files/rs-7428168/v1/301f130db2500072661003b6.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"The Futility of Nugent Scoring as a Diagnostic Tool for Neovaginal Bacterial Dysbiosis in Transfeminine People","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eTransfeminine individuals (TF) were assigned male at birth and experience a female/feminine gender identity. Numerous communities fall under this umbrella, including transgender women, non-binary, and other gender diverse individuals\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Many TF elect to undergo gender-affirming medical care, often through feminizing hormone therapy or surgery. Vaginoplasty is a widely performed gender-affirming surgery that creates a clitoris, vulva, and vaginal canal. The most prevalent technique is penile inversion vaginoplasty, involving orchiectomy, dissection of the space between the bladder and rectum, and lining the newly formed space with penile and scrotal tissue\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Although less common, sigmoid or peritoneal tissue may also be used to augment the vaginal canal\u003csup\u003e\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Penile inversion vaginoplasty results in a vaginal canal that is visually and functionally similar to that of cisgender females (CF) but lined with soft-cornified skin. We use the term \u0026ldquo;vagina\u0026rdquo; to refer to the vaginal canal of those born with a vagina, and \u0026ldquo;neovagina\u0026rdquo; to refer to the surgically created vaginal canal of TF. Our aim in using two terms is to clinically distinguish between vaginal canals lined with epithelia of different origins.\u003c/p\u003e\u003cp\u003eIn 2021, an estimated 20% of TF in the US had undergone genital surgery, with an additional 67% desiring it\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Like CF, TF with vaginoplasty often experience genital symptoms, including itching, burning, discharge, and malodor; however, the source of these symptoms has not been explored. To address this gap, we performed a study of the neovaginal microenvironments of 47 TF living in Canada\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. In this study, 56% of participants reported neovaginal symptoms within the past 30 days\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. In reproductive-aged CF (rCF), similar symptoms are caused by bacterial vaginal dysbiosis termed \u0026ldquo;bacterial vaginosis\u0026rdquo; (BV), which occurs when beneficial \u003cem\u003eLactobacillus\u003c/em\u003e spp. are replaced by a polymicrobial microbiome, including \u003cem\u003eGardnerella vaginalis\u003c/em\u003e, \u003cem\u003ePrevotella\u003c/em\u003e spp., \u003cem\u003eCa.\u003c/em\u003e Lachnocurva vaginae, and other anaerobic bacteria\u003csup\u003e\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eLactobacillus\u003c/em\u003e spp. play a critical role in rCF vaginal health by producing antimicrobial compounds and lactic acid, which lowers pH and inhibits colonization by pathogens\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Even without symptoms, dysbiotic microbiomes in rCF are associated with increased susceptibility to sexually transmitted infections, including chlamydia, gonorrhea, HSV-2, and HIV-1, underscoring the importance of the vaginal microbiome in sexual health\u003csup\u003e\u003cspan additionalcitationids=\"CR15 CR16 CR17 CR18 CR19 CR20 CR21 CR22\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eClinicians usually diagnose BV in rCF using the Nugent score. The Nugent score is a Gram stain-based diagnostic measure that rates the relative abundance of gram-positive rods (indicative of beneficial \u003cem\u003eLactobacillus\u003c/em\u003e spp.) vs. small or curved gram-variable rods (morphotypes of BV-associated bacteria)\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Scores from 0\u0026ndash;3 are considered optimal, 4\u0026ndash;6 inconclusive or intermediate, and 7\u0026ndash;10 indicative of BV. The Nugent score is often applied to diagnose TF experiencing genital symptoms; however, the neovaginal microbiota is distinct from the vaginal microbiota\u003csup\u003e\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, and results from TransBiota suggest bacteria associated with inflammation in the neovagina differ significantly from those in the rCF vagina\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Bacteria associated with neovaginal immune activation include \u003cem\u003eLawsonella\u003c/em\u003e, \u003cem\u003eHowardella\u003c/em\u003e, \u003cem\u003eFusobacterium\u003c/em\u003e, and \u003cem\u003eParvimonas\u003c/em\u003e, while higher abundances of \u003cem\u003eEzakiella\u003c/em\u003e, \u003cem\u003eFastidiosipila\u003c/em\u003e, \u003cem\u003eMurdochiella\u003c/em\u003e, and \u003cem\u003ePeptoniphilus\u003c/em\u003e associated with reduced inflammation\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Core neovaginal bacteria are listed in \u003cb\u003eSupplemental Table\u0026nbsp;1\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eThe substantial differences in microbiome composition and associated inflammation found in the rCF vagina and the neovagina raise questions about the utility of the Nugent score as a diagnostic tool in TF. Nevertheless, clinicians lack alternative diagnostics and the Nugent score continues to be frequently employed\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. This study evaluated the Nugent score\u0026rsquo;s relevance as a diagnostic tool for neovaginal dysbiosis by assessing its accuracy in identifying bacteria targeted by the score, and its ability to predict markers of bacterial dysbiosis (cytokines and symptoms) in the neovagina.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eParticipants\u003c/h2\u003e\u003cp\u003eTransBiota was a study investigating the genital microenvironments of trans and other gender diverse people receiving gender-affirming medical care\u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Eligible TF participants were Canadian residents 18 years or older, who underwent vaginoplasty\u0026thinsp;\u0026gt;\u0026thinsp;1 year prior to study entry. Research ethics board approval was obtained from Western University (REB #115503) and University of Maryland (IRB #HP-00096952), and all participants provided written informed consent. Participants were recruited online through social media and community groups, healthcare provider referral, and re-contact of consenting Trans PULSE Canada participants\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Participants were mailed a study kit containing instructions and self-collection materials. Demographic, behavior, and symptom data were collected through an online questionnaire. Participants (n\u0026thinsp;=\u0026thinsp;47) returned three weekly neovaginal sample sets by mail using pre-paid envelopes. This analysis includes the first timepoint for each participant for which there is a corresponding 1) scorable gram-stained slide, 2) microbiome data, and 3) immune analyte data.\u003c/p\u003e\u003cp\u003e All research activities were conducted in accordance with institutional and ethical regulations, and participant data were deidentified prior to analysis.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMicrobiota and Cytokine Analyses\u003c/h3\u003e\n\u003cp\u003eParticipants self-collected neovaginal swabs at each timepoint for Nugent scoring, microbiota analysis, and cytokine quantification. Sample collection and analysis of neovaginal microbiota and cytokines have been described in detail elsewhere\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. In brief, participants were instructed to insert each swab (Puritan HydraFlock) 5cm into the neovaginal canal, rotate 3 times, and place in collection media. Swabs for microbiota analysis were collected into 1ml of Qiagen C2.1 solution. DNA extractions were performed using the MagAttract PowerMicrobiome DNA/RNA kit (Qiagen), and 16S rRNA gene V3-V4 region amplicon sequencing (amplified via two step PCR) was conducted at University of Maryland Institute for Genomic Sciences\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Reads were processed using a QIIME-dependent script with DADA2 to generate amplicon-sequence variants (ASVs). Taxonomy was assigned with the RDP na\u0026iuml;ve-Bayes classifier trained on the SILVA v138.2 16S rRNA database and refined species-level calls with SpeciateIT v2.0.0. ASVs sharing identical taxonomy were collapsed. Swabs for cytokine analysis were collected into 500\u0026micro;l of a PBS-based stabilizing buffer, and IL-1a, IL-1b, IL-6, IL-8, MIG, MIP-1b, and RANTES concentrations were quantified on a Luminex MAGPIX system.\u003c/p\u003e\n\u003ch3\u003eNugent Scoring\u003c/h3\u003e\n\u003cp\u003eParticipants rolled collection swabs onto charged glass microscope slides (USA Scientific) and returned them to Western University in secured plastic cases. Neovaginal smears were heat-fixed and Gram stained using standard techniques. In brief, slides were stained with crystal violet (1 minute), iodine mordant (1 minute), decolorizing solution (until solution ran clear), and safranin (30 seconds) at room temperature. Slides were rinsed with running tap water between steps. Slides were observed under a 100x oil-immersion light microscope. For each smear, bacterial morphologies were individually scored in 10 representative fields of view (FOVs) following Nugent criteria\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. The criteria assign a decreasing score from 0\u0026ndash;4 for the abundance of \u003cem\u003eLactobacillus\u003c/em\u003e-like gram-positive large rods, an increasing score from 0\u0026ndash;4 for the abundance of \u003cem\u003eGardnerella\u003c/em\u003e-like short gram-variable straight rods, and an increasing score from 0\u0026ndash;2 for the abundance of \u003cem\u003eMobiluncus\u003c/em\u003e-like gram-variable curved rods. Scores for each morphotype were summed to yield a total score from 0\u0026ndash;10 for each FOV. Final mean score across the 10 FOVs was calculated for each participant. In rCF, scores of 0\u0026ndash;3 are optimal, 4\u0026ndash;6 inconclusive/intermediate, and 7\u0026ndash;10 indicative of BV\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003eData Analysis\u003c/h2\u003e\u003cp\u003eMultiple unpaired Mann-Whitney U nonparametric tests were used to assess differences in Nugent scores between asymptomatic and symptomatic participants. Fisher\u0026rsquo;s exact test was used to assess differences in bacterial prevalence, and Mann-Whitney U test was used to assess differences in relative abundance between asymptomatic and symptomatic participants. Associations between Nugent score and cytokines (IL-1α, IL-1β, IL-6, IL-8, MIG, MIP-1β, and RANTES) were assessed by Spearman\u0026rsquo;s correlation. Relationship between neovaginal bacteria taxa and cytokines, as well as bacterial taxa and Nugent score was also evaluated using Spearman\u0026rsquo;s correlations. Only taxa with a neovaginal prevalence of \u0026gt;\u0026thinsp;25% were included in analyses. GraphPad Prism 8 and R Studio (version 4.3.2) were used to create graphs and perform statistical analyses.\u003c/p\u003e\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003eOf the n\u0026thinsp;=\u0026thinsp;47 TransBiota participants, 8 were excluded (17%) due to no scorable slides from destruction during heat-fixation from smearing on the incorrect side of the slide; damage to slides during shipping; or insufficient sample for Nugent scoring (defined at \u0026lt;\u0026thinsp;30 bacteria/FOV). Demographics on the remaining n\u0026thinsp;=\u0026thinsp;39 participants included in this study are displayed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Median time on hormone therapy was 5.6 years and median time since vaginoplasty 2.8 years.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eParticipant demographics.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eParticipants (n\u0026thinsp;=\u0026thinsp;39)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAge, years (median, range)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e39 (26\u0026ndash;67)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEthnoracial Identity (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWhite\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e89.7%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLatin American\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.6%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eJewish\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.6%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMixed Ethnicity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.1%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMonths on hormone therapy (median, range)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e67 (35\u0026ndash;265)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMonths since vaginoplasty (median, range)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e33 months (12\u0026ndash;229 months)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCircumcised prior to vaginoplasty (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e56.4%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSymptoms, past 7 days (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e23.1%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBleeding\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.1%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDischarge\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.1%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eItching/burning\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.6%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMalodor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e12.8%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePain\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.6%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003epH (median, range)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.5 (4.5-8)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eNeovaginal smears contained nucleate and anucleate epithelial cells, with most bacteria clustered near these epithelial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, e). Four slides (10.3%) were visibly bloody with erythrocytes and leukocytes visible by microscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, e). Gram-variable rods and cocci dominated the majority of smears (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, e\u003cb\u003e)\u003c/b\u003e. Although all smears contained gram-variable straight rods, only 48.7% of slides displayed large gram-positive rods (\u003cem\u003eLactobacillus\u003c/em\u003e-like morphotype), and just three (7.7%) were dominated by large gram-positive rods (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe majority of neovaginal smears (71.8%) fell within the BV Nugent range (7\u0026ndash;10; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The most common score was 7 (46.2% of participants) indicating abundant gram-variable rods and minimal \u003cem\u003eLactobacillus\u003c/em\u003e-like rods. Only two participants (5.1%) scored an optimal Nugent score range for rCF (0\u0026ndash;3). \u003cem\u003eLactobacillus\u003c/em\u003e showed a moderate negative correlation with Nugent score (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) while \u003cem\u003eGardnerella\u003c/em\u003e and \u003cem\u003eMobiluncus\u003c/em\u003e showed no correlation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, d).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eNugent Score and traditional Nugent-targeted taxa are not associated with neovaginal symptoms\u003c/h2\u003e\u003cp\u003eNine participants (23.1%) reported neovaginal symptoms (malodor, discharge, bleeding, pain/burning) within 7 days prior to sample collection. There was no difference in Nugent scores between symptomatic and asymptomatic participants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Additionally, no relationship was observed between prevalence (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb) or proportional abundance (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec) of the traditional Nugent score bacteria (\u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003eGardnerella\u003c/em\u003e, \u003cem\u003eMobiluncus\u003c/em\u003e) and the presence or absence of neovaginal symptoms.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eNugent Score is not associated with neovaginal cytokines\u003c/h3\u003e\n\u003cp\u003eNugent scores were analyzed in relation to pro-inflammatory cytokine concentrations, but no significant correlations were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea \u003cb\u003e\u0026ndash; g\u003c/b\u003e). Representative images are shown of gram-stained smears from one participant with high cytokine levels (87.8pg/ml IL-1α, 65.4pg/ml IL-1β, 34.7pg/ml IL-6, 85.3pg/ml IL-8, 61.1pg/ml MIG, 21.7pg/ml MIP-1β, 33.1pg/ml RANTES) and one participant with low cytokine levels (83.9pg/ml IL-1α, 35.6pg/ml IL-1β, 6.3pg/ml IL-6, 57.1pg/ml IL-8, 11.8pg/ml MIG, 21.7pg/ml MIP-1β, undetectable RANTES). Both participants had a Nugent score of 7 with neovaginal smears dominated by gram-negative rods (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh \u003cb\u003e\u0026ndash; i\u003c/b\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eBacteria with Nugent-targeted morphotypes in the neovagina\u003c/h3\u003e\n\u003cp\u003eA comprehensive description of neovaginal bacterial communities of TransBiota participants has been previously published\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. A summary of the most prevalent neovaginal bacteria (\u0026gt;\u0026thinsp;25% prevalence) are listed in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, separated by the morphotypes targeted by Nugent scoring.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTraditional Nugent-targeted taxa (\u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003eGardnerella\u003c/em\u003e, and \u003cem\u003eMobiluncus\u003c/em\u003e) frequently appeared in the neovagina, but at low relative abundance (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Several other taxa detected in the neovagina had similar morphologies to Nugent-targeted genera. \u003cem\u003eLawsonella clevelandensis\u003c/em\u003e is a large gram-positive rod that can have similar appearance to \u003cem\u003eLactobacillus\u003c/em\u003e spp. on Gram staining\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eLawsonella clevelandensis\u003c/em\u003e (87.2% prevalence; 0.3% median abundance) outnumbered \u003cem\u003eLactobacillus\u003c/em\u003e (64.1%; 0.2%). \u003cem\u003eLawsonella\u003c/em\u003e abundance positively correlated with IL-1α, IL-1β, IL-8 and RANTES (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), whereas \u003cem\u003eLactobacillus\u003c/em\u003e showed no correlation with cytokines, and correlated with lower Nugent scores (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e; p\u0026thinsp;\u0026lt;\u0026thinsp;0.03). Three participants exhibited predominantly large rods; two had low Nugent scores of 2 (72.6% \u003cem\u003eLactobacillus\u003c/em\u003e relative abundance) and 3 (58.0% \u003cem\u003eLactobacillus\u003c/em\u003e relative abundance) and one scored 4 (16.5% \u003cem\u003eLactobacillus\u003c/em\u003e relative abundance), owing to mixed morphotypes.\u003c/p\u003e\u003cp\u003e\u003cem\u003eGardnerella vaginalis\u003c/em\u003e, was detected in 33.3% of samples at a median relative abundance of 0.4%. \u003cem\u003eFannyhessea vaginae\u003c/em\u003e (formerly \u003cem\u003eAtopobium vaginae\u003c/em\u003e), although not a traditional Nugent bacteria, is also a BV-associated rod and may contribute to the diagnostic power of the Nugent score in rCF\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eFannyhessea\u003c/em\u003e was detected in 28.2% of samples at a median relative abundance of only 1.7%. Short straight rods in the neovagina were more likely to be \u003cem\u003eHoylesella\u003c/em\u003e (previously Prevotella; 97.4% prevalence, 9.7% relative abundance), \u003cem\u003ePrevotella\u003c/em\u003e spp. (92.3%; 8.5%), or \u003cem\u003ePorphyromonas\u003c/em\u003e spp. (97.4%; 8.3%). \u003cem\u003ePrevotella\u003c/em\u003e was positively associated with increased cytokines (IL-1α; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while \u003cem\u003eGardnerella, Fannyhessea, Porphyromonas\u003c/em\u003e, and \u003cem\u003eHoylesella\u003c/em\u003e had no significant correlation. None had significant correlations with Nugent score.\u003c/p\u003e\u003cp\u003e\u003cem\u003eMobiluncus\u003c/em\u003e, the gram-variable curved rod traditionally targeted by Nugent scoring, was detected in 76.9% of samples at 0.8% median relative abundance. Other abundant neovaginal curved rods included \u003cem\u003eVaribaculum\u003c/em\u003e (92.3% prevalence, 2.6% abundance) and \u003cem\u003eCampylobacter\u003c/em\u003e (89.7%; 1.7%). Abundances of \u003cem\u003eCampylobacter\u003c/em\u003e were inversely correlated with neovaginal cytokines (IL-1β, IL-8; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) while \u003cem\u003eMobiluncus\u003c/em\u003e and \u003cem\u003eVaribaculum\u003c/em\u003e showed no significant correlation with cytokines. Both \u003cem\u003eMobiluncus\u003c/em\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.02) and \u003cem\u003eCampylobacter\u003c/em\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.02) correlated with higher Nugent scores.\u003c/p\u003e\u003cp\u003eOf note, in addition to \u003cem\u003eF. vaginae\u003c/em\u003e, BV-associated \u003cem\u003eCa.\u003c/em\u003e Lachnocurva vaginae (formerly BVAB1; curved rod) and \u003cem\u003eSneathia amnii\u003c/em\u003e (curved Gram-negative rod) posses similar morphotypes to traditional Nugent bacteria and may contribute to the diagnostic power of the Nugent score in rCF\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. However, both were absent from neovaginal samples.\u003c/p\u003e\u003cp\u003eAs noted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, many neovaginal smears contained high abundance of cocci, which are not considered by Nugent criteria. Based on V3-V4 16S rRNA gene sequences, 4/10 core neovaginal bacteria have morphotypes not considered during Nugent scoring (\u003cb\u003eSupplemental Table\u0026nbsp;1\u003c/b\u003e). \u003cem\u003ePeptoniphilus\u003c/em\u003e, \u003cem\u003eEzakiella\u003c/em\u003e and \u003cem\u003eAnaerococcus\u003c/em\u003e are all gram-positive cocci. Of these, \u003cem\u003eEzakiella\u003c/em\u003e is associated with reduced neovaginal cytokines (IL-8, MIG, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while \u003cem\u003eAnaerococcus\u003c/em\u003e is associated with increased cytokines (IL-6, MIG, MIP-1β; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003ePrincipal findings\u003c/h2\u003e\u003cp\u003eThis study provides strong evidence that the Nugent score is not suitable for clinical diagnosis of neovaginal dysbiosis in TF with penile inversion vaginoplasty. Most neovaginal bacteria belong to taxa not targeted by the Nugent score, and the score does not associate with predictors of genital dysbiosis, including neovaginal symptoms and cytokines.\u003c/p\u003e\u003cp\u003eNugent scoring in rCF relies on the eubiotic nature of \u003cem\u003eLactobacillus\u003c/em\u003e spp. predominance, and vaginal polymicrobialism as dysbiotic. However, \u003cem\u003eLactobacillus\u003c/em\u003e spp. predominance in the neovagina is very rare\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, potentially due to differences in carbon sources available in the rCF vagina and TF neovagina. Vaginal epithelial cells in rCF are rich in glycogen and constantly shed into the vaginal lumen\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. In contrast, neovaginal epithelium derived from penile skin lacks glycogen despite exposure to estrogen levels similar to rCF, and is instead soft-cornified with a lipid-rich extracellular matrix\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan additionalcitationids=\"CR37 CR38\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. In rCF, vaginal \u003cem\u003eLactobacillus\u003c/em\u003e spp. metabolize glycogen products to produce lactic acid with anti-inflammatory properties, inhibiting colonization by non-lactobacilli, including pathogens\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. In our cohort \u003cem\u003eLactobacillus\u003c/em\u003e rarely dominated and showed similar abundance in symptomatic and asymptomatic TF. Future research is warranted to determine if the neovaginal epithelium can support \u003cem\u003eLactobacillus\u003c/em\u003e predominance, and if this confers any benefit.\u003c/p\u003e\u003cp\u003eFurther, there is no evidence that neovaginal microbiomes rich in gram-variable rods are necessarily dysbiotic. Neovaginal abundances of traditional Nugent morphotypes \u003cem\u003eGardnerella\u003c/em\u003e and \u003cem\u003eMobiluncus\u003c/em\u003e were not different between symptomatic and asymptomatic TF and did not correlate with increased cytokines. While the relationship between neovaginal inflammation and sexual health outcomes has not been adequately explored, genital inflammation is strongly correlated with negative health outcomes in cisgender individuals\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan additionalcitationids=\"CR43 CR44\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. In rCF, \u003cem\u003eMobiluncus\u003c/em\u003e and \u003cem\u003eGardnerella\u003c/em\u003e are positively associated with vaginal cytokines such as IL-1β and IL-8\u003csup\u003e42,46\u0026ndash;48\u003c/sup\u003e. Instead in the neovagina, \u003cem\u003eCampylobacter\u003c/em\u003e, a more abundant curved rod, showed negative correlations with cytokines. More abundant short gram-variable rods such as \u003cem\u003eHoylesella\u003c/em\u003e (predominantly \u003cem\u003eH. timonensis\u003c/em\u003e and \u003cem\u003eH. buccalis\u003c/em\u003e, both previously \u003cem\u003ePrevotella\u003c/em\u003e), \u003cem\u003ePorphyromonas\u003c/em\u003e and \u003cem\u003eDialister\u003c/em\u003e lacked any association with inflammation, while \u003cem\u003eFenollaria\u003c/em\u003e correlated inversely with cytokines. Other bacteria that normally contribute to the diagnostic power of the Nugent score in rCF (\u003cem\u003eFannyhessea\u003c/em\u003e, \u003cem\u003eSneathia amnii\u003c/em\u003e, \u003cem\u003eCa.\u003c/em\u003e L. vaginae) showed no correlation to cytokines or were absent from the neovagina. These data suggest that gram-variable rod abundance on neovaginal smears does not provide interpretive information on neovaginal health.\u003c/p\u003e\u003cp\u003e\u003cem\u003eLawsonella\u003c/em\u003e abundance correlated strongly with inflammation, yet Nugent scoring classifies \u003cem\u003eLawsonella\u003c/em\u003e morphotypes under the \u0026ldquo;beneficial rod\u0026rdquo; category. Likewise, pro-inflammatory cocci such as \u003cem\u003eAnaerococcus\u003c/em\u003e lay outside the Nugent framework. These findings are consistent with a study by Weyers et al. examining neovaginal cytology, reporting that, despite over 50% of participants being diagnosed with BV, no correlation was observed with neovaginal inflammation\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Additionally, Weyers et al. noted a significant presence of inflammatory cells in the neovagina. In our study, we frequently observed blood cells, suggesting participants may be experiencing epithelial erosion.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eClinical implications\u003c/h2\u003e\u003cp\u003eNugent scoring of neovaginal smears risks misdiagnosis and promotes futile antibiotic use. Nearly all neovaginal smears in this study fell outside the optimal Nugent range (0\u0026ndash;3), indicating most would be classified as dysbiotic and indicative of BV. BV in rCF is commonly treated with metronidazole, which spares \u003cem\u003eLactobacilli\u003c/em\u003e but is bactericidal for BV-associated gram-negative anaerobes, including \u003cem\u003eGardnerella\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. However, the gram-positive bacteria associated with inflammation in the neovagina, such as \u003cem\u003eLawsonella\u003c/em\u003e and \u003cem\u003eAnaerococcus\u003c/em\u003e, are unlikely to be susceptible to metronidazole\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. TF who receive a Nugent score in the BV range may feel distress or use products or home remedies aimed at treating BV and restoring \u003cem\u003eLactobacilli\u003c/em\u003e in rCF. TransBiota participants who used diverse solutions for douching (povidone-iodine, soapy water, vinegar) were more likely to have high abundances of inflammation-associated bacteria \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Many also reported using oral probiotics or probiotic suppositories designed for rCF when experiencing neovaginal symptoms, and referred to having BV in their questionnaires \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Additional research is urgently needed to better characterize the causative agents of neovaginal inflammation and symptoms, and to design effective diagnostic tools to identify them in a clinical setting. However, these findings apply chiefly to mature penile-skin lined neovagina, and microbial dynamics might differ in bowel-segmented or peritoneal graft neovaginas.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eLimitations\u003c/h2\u003e\u003cp\u003e This study\u0026rsquo;s mail-in methodology enabled broader participation, but resulted in unusable smears due to inadequate sampling or slide damage during shipping. This study also did not address sampling location within the neovaginal canal; while participants were instructed to collect swabs 5cm into the neovaginal canal, sampling depth may have varied between participants and microbiomes vary by distance from the introitus. Although participants were asked to refrain from inserting anything into their neovagina 24h prior to sampling, variable practices outside this window may have introduced variability\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Unmeasured behavioral or hormonal variables may also confound associations.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe Nugent score is an ineffective tool for predicting neovaginal dysbiosis in TF with penile inversion vaginoplasty. Bacteria traditionally targeted by the Nugent score are rare in the neovagina, while other taxa with similar morphotypes are abundant. Nugent scores did not correlate with inflammation or symptoms in the neovagina. Using the Nugent score on neovaginal smears may result in misdiagnosis, inappropriate antibiotic use, and misplaced efforts by TF and clinicians to \u0026ldquo;correct\u0026rdquo; neovaginal microbiomes, possibly disrupting an optimal microbiome. These findings highlight that vaginal dysbiosis differs fundamentally between rCF and TF and underscores the need to establish evidence-based neovaginal diagnostics.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Jason Hallarn and Greta Bauer for their contributions establishing TransBiota.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFunding: This work was supported by the Canadian Institute of Health Research [PJT 180322] and the National Institutes of Health [R21 AI157912]. JLP is supported by the Canada Research Chairs Program [CRC-2020-00175]. AS is supported by a Canada Graduate Scholarship from the Canadian Institute of Health Research. This research was undertaken, in part, thanks to funding from the Canada Foundation for Innovation [CFI 42343]. The funders played no role in study design, data collection, analysis and interpretation of data, or the writing of this manuscript.\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThere are no conflicts of interest or financial disclosures to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: J.R., J.L.P., Y.K, E.P. Methodology: R.P., B.M., H.W., D.Z. Formal analysis: R.P. and J.R-V. Interpretation: R.P., J.R-V., B.M., J.R. and J.L.P. Writing: R.P. and J.L.P, Editing: R.P., B.M., E.P., J.R-V., H.W., D.Z., A.P., A.C.S., V.L., Y.K, J.L.P. and J.R. Funding acquisition: J.R. and J.L.P.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll source codes used to analyze the data and generate the figures presented are available in GitHub at github.com/prodgerlab/TransBiota/tree/main/Nugent_scoring_paper. The cytokine, microbiome, and Nugent scoring datasets used in this study are also accessible through this repository.\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eJames, S. E., Herman, J., Keisling, M., Mottet, L. \u0026amp; Anafi, M. 2015 U.S. Transgender Survey (USTS): Version 1. 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Lactobacillus species: taxonomic complexity and controversial susceptibilities. \u003cem\u003eClin Infect Dis\u003c/em\u003e \u003cstrong\u003e60 Suppl 2\u003c/strong\u003e, S98-107 (2015).\u003c/li\u003e\n \u003cli\u003eMuthusamy, S., Varghese, J., Raveendran, V., Ezilarasan, K. \u0026amp; Easow, J. Evaluation of interobserver reliability of Nugent score for diagnosis of bacterial vaginosis. \u003cem\u003eIndian J Sex Transm Dis\u003c/em\u003e \u003cstrong\u003e39\u003c/strong\u003e, 120 (2018).\u003c/li\u003e\n \u003cli\u003eMohanty, S., Sood, S., Kapil, A. \u0026amp; Mittal, S. Interobserver variation in the interpretation of Nugent scoring method for diagnosis of bacterial vaginosis. \u003cem\u003eIndian J Med Res\u003c/em\u003e \u003cstrong\u003e131\u003c/strong\u003e, 88\u0026ndash;91 (2010).\u003cstrong\u003e\u003cbr\u003e\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Bacterial Vaginosis, Gynecology, Microbiome, Transgender, Vaginoplasty","lastPublishedDoi":"10.21203/rs.3.rs-7428168/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7428168/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: Transfeminine people were assigned male at birth and experience a female or feminine gender identity. Many elect to undergo vaginoplasty, a surgical procedure that constructs a neovagina, typically using penile and scrotal tissue. Like cisgender females, transfeminine people experience gynecological symptoms, including pain, discharge, and malodor. In cisgender females, clinicians attribute these symptoms to bacterial dysbiosis and can be diagnosed by Nugent scoring of gram-stained vaginal smears. The Nugent score assesses the abundance of large gram-positive rod vs. small or curved gram-variable rod morphotypes, traditionally for the detection of \u003cem\u003eLactobacillus\u003c/em\u003espp., \u003cem\u003eGardnerella vaginalis\u003c/em\u003e, and \u003cem\u003eMobiluncus\u003c/em\u003e spp. (curved rod), respectively. Although unvalidated for neovaginal samples, this method is frequently applied to diagnose dysbiosis in transfeminine people with vaginoplasty.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eObjective: \u003c/strong\u003eThis study assessed the Nugent score’s utility as a clinical tool for diagnosing neovaginal dysbiosis in transfeminine people who underwent penile inversion vaginoplasty.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStudy Design\u003c/strong\u003e: As a part of the TransBiota study, n=39 transfeminine participants self-collected neovaginal smears. Smears were Gram stained and Nugent scored, and Nugent scores were correlated with existing data on neovaginal bacterial composition (16S rRNA gene sequencing), neovaginal cytokines (Luminex multiplex immunoassay), and self-reported symptoms.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eMore than 70% of smears fell\u003cstrong\u003e \u003c/strong\u003ein the 7-10 range that would indicate Bacterial Vaginosis in cisgender women. However, Nugent score failed to correlate with the abundance of Nugent-targeted bacteria. Bacteria with similar morphotypes, but not belonging to \u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003eGardnerella\u003c/em\u003e, or \u003cem\u003eMobiluncus\u003c/em\u003e, were highly abundant and prevalent in the neovagina. Nugent score also failed to predict local inflammation or clinical symptoms.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e: The Nugent score is not an effective tool to identify neovaginal dysbiosis or indicators of health in transfeminine individuals. Clinicians need the development of accurate, evidence-based diagnostic tools for the neovagina.\u003c/p\u003e","manuscriptTitle":"The Futility of Nugent Scoring as a Diagnostic Tool for Neovaginal Bacterial Dysbiosis in Transfeminine People","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-08 08:29:52","doi":"10.21203/rs.3.rs-7428168/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-medicine","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsmed","sideBox":"Learn more about [Communications Medicine](http://www.nature.com/commsmed)","snPcode":"43856","submissionUrl":"https://mts-commsmed.nature.com/cgi-bin/main.plex","title":"Communications Medicine","twitterHandle":"@commsmedicine","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6bca43cf-2303-4e04-b4f5-a954afc83736","owner":[],"postedDate":"September 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":53805203,"name":"Biological sciences/Microbiology/Clinical microbiology"},{"id":53805204,"name":"Biological sciences/Microbiology/Microbial communities/Microbiome"},{"id":53805205,"name":"Biological sciences/Immunology/Cytokines"},{"id":53805206,"name":"Health sciences/Health care/Diagnosis"},{"id":53805207,"name":"Biological sciences/Biological techniques/Microscopy"}],"tags":[],"updatedAt":"2026-03-11T07:12:35+00:00","versionOfRecord":{"articleIdentity":"rs-7428168","link":"https://doi.org/10.1038/s43856-026-01410-2","journal":{"identity":"communications-medicine","isVorOnly":false,"title":"Communications Medicine"},"publishedOn":"2026-02-03 05:00:00","publishedOnDateReadable":"February 3rd, 2026"},"versionCreatedAt":"2025-09-08 08:29:52","video":"","vorDoi":"10.1038/s43856-026-01410-2","vorDoiUrl":"https://doi.org/10.1038/s43856-026-01410-2","workflowStages":[]},"version":"v1","identity":"rs-7428168","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7428168","identity":"rs-7428168","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}