Effects of low dose estrogen therapy on the vaginal microbiomes of women with atrophic vaginitis

www.nature.com/scientificreports OPEN received: 18 September 2015 accepted: 22 March 2016 Published: 22 April 2016 Effects of low dose estrogen therapy on the vaginal microbiomes of women with atrophic vaginitis Jian Shen1,2,6, Ning Song1,2, Christopher J. Williams3,5, Celeste J. Brown4,5, Zheng Yan7, Chen Xu1,2 & Larry J. Forney4,5 Atrophic vaginitis (AV) is common in postmenopausal women, but its causes are not well understood. The symptoms, which include vaginal itching, burning, dryness, irritation, and dyspareunia, can usually be alleviated by low doses of estrogen given orally or locally. Regrettably, the composition of vaginal bacterial communities in women with AV have not been fully characterized and little is known as to how these communities change over time in response to hormonal therapy. In the present intervention study we determined the response of vaginal bacterial communities in postmenopausal women with AV to low-dose estrogen therapy. The changes in community composition in response to hormonal therapy were rapid and typified by significant increases in the relative abundance of Lactobacillus spp. that were mirrored by a decreased relative abundance of Gardnerella. These changes were paralleled by a significant four-fold increase in serum estradiol levels and decreased vaginal pH, as well as nearly a twofold increase in the Vaginal Maturation Index (VMI). The results suggest that after menopause a vaginal microbiota dominated by species of Lactobacillus may have a beneficial role in the maintenance of health and these findings that could lead to new strategies to protect postmenopausal women from AV. Atrophic vaginitis (AV) is a common affliction that develops in 25% to 50% of postmenopausal women1. Its symptoms include vaginal itching, burning, dryness, irritation, and dyspareunia, which degrade a woman’s quality of life and are unlikely to diminish without treatment2. Studies have shown that AV is associated with estrogen deficiencies during menopause that cause reduced vaginal secretions, vulvovaginal atrophy, and decreased glycogen production by vaginal epithelial cells. It is accompanied by decreased numbers of lactobacilli and lactic acid production, causing an increased vaginal pH that possibly renders the vagina more susceptible to infections1,2. Although the occurrence of vulvovaginal atrophy and increased vaginal pH are nearly universal in menopause, most elderly women do not present with genital complaints, which suggests there are unknown factors that distinguish AV patients and asymptomatic women. So far the preferred treatment of AV symptoms is low-dose estrogen therapy, which minimizes the risk of endometrial and breast cancers that are responsive to estrogen3–5. However, the effects of this therapy on the vaginal microbiome are not well understood. The vaginal microbiome and host immunity are two components of a mutualistic relationship that play a pivotal role in maintaining health and minimizing risk to adverse urogenital problems such as AV. In reproductive age women the vaginal microbiome is often dominated by various species of Lactobacillus that occur in high numbers. This is widely thought to be a normal and healthy state. In contrast, communities in which lactobacilli are supplanted by any of various strictly anaerobic bacteria are often accompanied by symptoms of bacterial vaginosis (BV) that places these women at higher risk to disease as well as obstetric and gynecological complications6. Although considerable efforts have been made to better understand these communities in reproductive-age women, far less is known about the composition and function of these communities in women after menopause. 1 Department of Anatomy, Histology and Embryology, Shanghai Jiao Tong University School of Medicine, Shanghai, China, 200025. 2Shanghai Key Laboratory of Reproductive Medicine, Shanghai, China, 200025. 3Department of Statistics, University of Idaho, Moscow, ID, 83844, USA. 4Department of Biological Sciences, University of Idaho, Moscow, ID, 83844, USA. 5Institute for Bioinformatics and Evolutionary Studies (IBEST), University of Idaho, Moscow, ID, 83844, USA. 6Department of Obstetrics and Gynecology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China. 7Reproductive Medicine Center, Shanghai Ninth Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China. Correspondence and requests for materials should be addressed to C.X. (email: ) or L.J.F. (email: ) Scientific Reports | 6:24380 | DOI: 10.1038/srep24380 1 www.nature.com/scientificreports/ H group AV group Characteristic (n = 29) (n = 30) Age (year) 55.6 ±  2.6 55.8 ±  3.2 Time since menopause (month) 61.8 ±  26.5 64.6 ±  30.6 Body mass index 26.5 ±  0.8 26.5 ±  0.6 Systolic blood pressure (mmHg) 126.2 ±  6.6 126.0 ±  6.7 Diastolic pressure (mmHg) 72.6 ±  8.9 75.3 ±  8.4 Pulse rate (bpm) 75.4 ±  4.1 77.3 ±  4.2 Serum estradiol concentration (pmol/L) 44.8 ±  8.4 42.0 ±  7.7 Vaginal maturation index 29.2 ±  10.7 28.1 ±  11.5 Vaginal pH 6.6 ±  0.2 6.6 ±  0.2 Table 1. Baseline characteristics of subjects in H and AV groupsa. aNo significant differences between groups for any characteristic. Values are means ± standard error. In this study we compared the vaginal microbiota of healthy post-menopausal women (H group) to those of post-menopausal women with atrophic vaginitis (AV group). Women in the AV group were given low-dose estrogen therapy and followed over a four-week treatment period to observe the response of their vaginal microbiota to the treatment. These were compared to the vaginal microbiota of the untreated H group over the same period of time. Results Sixty of the 67 women originally enrolled completed this longitudinal study. Reasons for not continuing in the study included: loss to follow-up (N =  3 in H group), adverse events (nausea in one subject of the AV group), non-compliance (one subject of the AV group), and other reasons (N =  2, one from each group). The microbial community data from samples of one subject (subject 20) in the H group were not included in data analysis because the numbers of DNA sequence reads from vaginal samples were too low. Finally, 59 postmenopausal women with (N =  30) or without (N =  29) AV were enrolled in the present study. Overall, there were no major differences in the baseline characteristics of women in the two groups (Table 1). Subjects had a mean age of 56 years and the range of time since last menses was 21–118 months. The species composition and structure of vaginal bacterial communities were determined by classifying 16S rRNA gene sequences recovered from mid-vaginal swab samples collected by a physician at 0, 2 and 4 weeks. DNA sequencing using barcoded primers (Supplementary Table S1) produced a dataset that consisted of 982,704 high-quality 16S rRNA gene sequences from 177 samples. On average 5459 ±  2345 (SD) reads (...truncated)