High-grade vaginal intraepithelial neoplasia and risk of progression to vaginal cancer. a multicentre study of the Italian Society of Colposcopy and Cervico-Vaginal Pathology (SICPCV)

OBJECTIVE: The aim of this study was to analyse the women with high grade vaginal intraepithelial neoplasia (HG-VaIN), in order to identify a subset of women at higher risk of progression to invasive vaginal cancer. MATERIALS AND METHODS: The medical records of all the women diagnosed with HG-VaIN, and subsequently treated, from January 1995 to December 2013 were analyzed in a multicentre retrospective case series. The rate of progression to invasive vaginal cancer and the potential risk factors were evaluated. RESULTS: 205 women with biopsy diagnosis of HG-VaIN were considered, with a mean follow up of 57 months (range 4-254 months). 12 cases of progression to vaginal squamocellular cancer were observed (5.8%), with a mean time interval from treatment to progression of 54.6 months (range 4-146 months). The rate of progression was significantly higher in women diagnosed with VaIN3 compared with VaIN2 (15.4% vs. 1.4%, p < 0.0001). Women with HG-VaIN and with previous hysterectomy showed a significantly higher rate of progression to invasive vaginal cancer compared to non-hysterectomised women (16.7% vs. 1.4%, p < 0.0001). A higher risk of progression for women with VaIN3 and for women with previous hysterectomy for cervical HPV-related disease was confirmed by multivariable logistic regression analysis. CONCLUSIONS: A higher rate of progression to vaginal cancer was reported in women diagnosed with VaIN3 on biopsy and in women with previous hysterectomy for HPV-related cervical disease. These patients should be considered at higher risk, thus a long lasting and accurate follow up is recommended

Species-level classification of the vaginal microbiome

Background The application of next-generation sequencing to the study of the vaginal microbiome is revealing the spectrum of microbial communities that inhabit the human vagina. High-resolution identification of bacterial taxa, minimally to the species level, is necessary to fully understand the association of the vaginal microbiome with bacterial vaginosis, sexually transmitted infections, pregnancy complications, menopause, and other physiological and infectious conditions. However, most current taxonomic assignment strategies based on metagenomic 16S rDNA sequence analysis provide at best a genus-level resolution. While surveys of 16S rRNA gene sequences are common in microbiome studies, few well-curated, body-site-specific reference databases of 16S rRNA gene sequences are available, and no such resource is available for vaginal microbiome studies. Results We constructed the Vaginal 16S rDNA Reference Database, a comprehensive and non-redundant database of 16S rDNA reference sequences for bacterial taxa likely to be associated with vaginal health, and we developed STIRRUPS, a new method that employs the USEARCH algorithm with a curated reference database for rapid species-level classification of 16S rDNA partial sequences. The method was applied to two datasets of V1-V3 16S rDNA reads: one generated from a mock community containing DNA from six bacterial strains associated with vaginal health, and a second generated from over 1,000 mid-vaginal samples collected as part of the Vaginal Human Microbiome Project at Virginia Commonwealth University. In both datasets, STIRRUPS, used in conjunction with the Vaginal 16S rDNA Reference Database, classified more than 95% of processed reads to a species-level taxon using a 97% global identity threshold for assignment. Conclusions This database and method provide accurate species-level classifications of metagenomic 16S rDNA sequence reads that will be useful for analysis and comparison of microbiome profiles from vaginal samples. STIRRUPS can be used to classify 16S rDNA sequence reads from other ecological niches if an appropriate reference database of 16S rDNA sequences is available

Longitudinal change in cervical length following vaginal or abdominal cervical cerclage: a randomized comparison

BACKGROUND: Cervical cerclage has been shown to reduce the risk of recurrent spontaneous preterm birth in a high-risk patient population; however, the mechanism is not well understood. Transabdominal cerclage is superior to low and high vaginal cerclage in reducing early spontaneous preterm birth and fetal loss in women with previous failed vaginal cerclage. Cervical length measurements are commonly used to monitor high-risk women and may explain the mechanism of success. OBJECTIVE: This study aimed to evaluate the rate of change in longitudinal cervical length after randomized placement of low transvaginal, high transvaginal, or transabdominal cerclage in women with a previous failed vaginal cerclage. STUDY DESIGN: This was a planned analysis of longitudinal transvaginal ultrasound cervical length measurements from patients enrolled in the Vaginal Randomised Intervention of Cerclage trial, a randomized controlled trial comparing transabdominal cerclage or high transvaginal cerclage with low transvaginal cerclage. Cervical length measurements at specific gestational ages were compared over time and between groups, using generalized estimating equations fitted using the maximum-likelihood random-effects estimator. In addition, cervical length measurements were compared in women with transabdominal cerclage placed before and during pregnancy. The diagnostic accuracy of cervical length as a predictor of spontaneous preterm birth at <32 weeks of gestation was explored. RESULTS: This study included 78 women who underwent longitudinal cervical length assessment (70% of the analyzed cohort) with a history of failed cerclage, of whom 25 (32%) were randomized to low transvaginal cerclage, 26 (33%) to high transvaginal cerclage, and 27 (35%) to transabdominal cerclage. Abdominal cerclage was superior to low (P=.008) and high (P=.001) vaginal cerclage at maintaining cervical length over the surveillance period (14 to 26 weeks of gestation) (+0.08 mm/week, 95% confidence interval, -0.40 to 0.22; P=.580). On average, the cervical length was 1.8 mm longer by the end of the 12-week surveillance period in women with transabdominal cerclage (+1.8 mm; 95% confidence interval, -7.89 to 4.30; P=.564). High vaginal cerclage was no better than low cervical cerclage in the prevention of cervical shortening; the cervix shortened by 13.2 mm over 12 weeks in those with low vaginal cerclage (95% confidence interval, -21.7 to -4.7; P=.002) and by 20 mm over 12 weeks in those with high vaginal cerclage (95% confidence interval, -33.1 to -7.4; P=.002). Preconception transabdominal cerclage resulted in a longer cervix than those performed during pregnancy; this difference was significant after 22 weeks of gestation (48.5 mm vs 39.6 mm; P=.039). Overall, cervical length was an excellent predictor of spontaneous preterm birth at <32 weeks of gestation (receiver operating characteristic curve, 0.92; 95% confidence interval, 0.82-1.00). CONCLUSION: In women with a previous failed cervical cerclage, in the next pregnancy, the cervical length in women treated with vaginal cerclage funneled and shortened over time, whereas there was preservation of cervical length in women who receive transabdominal cerclage. Cervical length remained longer in transabdominal procedures performed before pregnancy than in transabdominal procedures performed during pregnancy. Overall, cervical length was an excellent predictor of spontaneous preterm birth in our cohort. Our findings may explain the mechanism of benefit for transabdominal cerclage, with its high placement better maintaining the structural integrity of the cervix at the level of the internal os

Correlates of the molecular vaginal microbiota composition of African women.

BACKGROUND: Sociodemographic, behavioral and clinical correlates of the vaginal microbiome (VMB) as characterized by molecular methods have not been adequately studied. VMB dominated by bacteria other than lactobacilli may cause inflammation, which may facilitate HIV acquisition and other adverse reproductive health outcomes. METHODS: We characterized the VMB of women in Kenya, Rwanda, South Africa and Tanzania (KRST) using a 16S rDNA phylogenetic microarray. Cytokines were quantified in cervicovaginal lavages. Potential sociodemographic, behavioral, and clinical correlates were also evaluated. RESULTS: Three hundred thirteen samples from 230 women were available for analysis. Five VMB clusters were identified: one cluster each dominated by Lactobacillus crispatus (KRST-I) and L. iners (KRST-II), and three clusters not dominated by a single species but containing multiple (facultative) anaerobes (KRST-III/IV/V). Women in clusters KRST-I and II had lower mean concentrations of interleukin (IL)-1α (p < 0.001) and Granulocyte Colony Stimulating Factor (G-CSF) (p = 0.01), but higher concentrations of interferon-γ-induced protein (IP-10) (p < 0.01) than women in clusters KRST-III/IV/V. A lower proportion of women in cluster KRST-I tested positive for bacterial sexually transmitted infections (STIs; ptrend = 0.07) and urinary tract infection (UTI; p = 0.06), and a higher proportion of women in clusters KRST-I and II had vaginal candidiasis (ptrend = 0.09), but these associations did not reach statistical significance. Women who reported unusual vaginal discharge were more likely to belong to clusters KRST-III/IV/V (p = 0.05). CONCLUSION: Vaginal dysbiosis in African women was significantly associated with vaginal inflammation; the associations with increased prevalence of STIs and UTI, and decreased prevalence of vaginal candidiasis, should be confirmed in larger studies

The truth about metagenomics: quantifying and counteracting bias in 16S rRNA studies

Background Characterizing microbial communities via next-generation sequencing is subject to a number of pitfalls involving sample processing. The observed community composition can be a severe distortion of the quantities of bacteria actually present in the microbiome, hampering analysis and threatening the validity of conclusions from metagenomic studies. We introduce an experimental protocol using mock communities for quantifying and characterizing bias introduced in the sample processing pipeline. We used 80 bacterial mock communities comprised of prescribed proportions of cells from seven vaginally-relevant bacterial strains to assess the bias introduced in the sample processing pipeline. We created two additional sets of 80 mock communities by mixing prescribed quantities of DNA and PCR product to quantify the relative contribution to bias of (1) DNA extraction, (2) PCR amplification, and (3) sequencing and taxonomic classification for particular choices of protocols for each step. We developed models to predict the “true” composition of environmental samples based on the observed proportions, and applied them to a set of clinical vaginal samples from a single subject during four visits. Results We observed that using different DNA extraction kits can produce dramatically different results but bias is introduced regardless of the choice of kit. We observed error rates from bias of over 85% in some samples, while technical variation was very low at less than 5% for most bacteria. The effects of DNA extraction and PCR amplification for our protocols were much larger than those due to sequencing and classification. The processing steps affected different bacteria in different ways, resulting in amplified and suppressed observed proportions of a community. When predictive models were applied to clinical samples from a subject, the predicted microbiome profiles were better reflections of the physiology and diagnosis of the subject at the visits than the observed community compositions. Conclusions Bias in 16S studies due to DNA extraction and PCR amplification will continue to require attention despite further advances in sequencing technology. Analysis of mock communities can help assess bias and facilitate the interpretation of results from environmental samples

Improving Appropriate Use of Antifungal Medications: The Role of an Over-the-Counter Vaginal pH Self-Test Device

Objectives: To determine whether patients can understand and use the vaginal pH device in the diagnosis of vaginitis. To compare whether vaginal pH readings determined by patients and healthcare providers are similar. To determine whether vaginalpHcan reduce inappropriate over-the-counter (OTC) antifungal medication use and improve the correct diagnosis of vaginitis. Methods: One hundred and fifty-one women indicated their belief about the cause of their vaginal infection, read the instructions of the vaginal pH device package insert, used the device and interpreted the findings. The patient interpretations were compared with results obtained by healthcare providers, blinded to patient findings. Results: Over 96% of patients stated that they could easily read the instructions, use the vaginal pH device and interpret the readings. They obtained the same readings as healthcare professionals (Kappa = 0.9). Restricting the use of OTC antifungal medications to those individuals with vaginitis symptoms and vaginal pH ≤ 4.5 significantly reduced inappropriate use by approximately 50%, Fisher's exact test,p-value = 0.018. Conversely, seeking healthcare provider assessment with vaginal pH > 4.5, leads to correct diagnosis of vaginitis. Conclusions: The vaginal pH device can be used as an OTC diagnostic tool by consumers when a vaginal infection is suspected. Vaginal pH readings would direct patients whether to purchase an antifungal medication or seek professional diagnosis from a healthcare provider. Understanding and use of this vaginal pH device could reduce inappropriate use of OTC antifungal medications by approximately 50% and improve the correct diagnosis of vaginitis