Vulvar lichen sclerosus (VLS) is a chronic inflammatory skin disease of the vulva that may lead to vulvar scarring and sexual dysfunction. VLS affects premenarchal children and postmenopausal women, with an incidence rate ranging from 1 in 70 to 1 in 1,000 (Fruchter et al., 2017). However, the actual incidence may be underestimated. The pathogenesis of VLS remains uncertain. Autoimmune factors, genetic background, hormonal factors, and local factors have been associated with the pathogenesis of VLS. With the development of microbiome sequencing technology, it has been demonstrated that changes in the composition of the human microbiota may contribute to the development of chronic inflammatory diseases. Symbiotic bacteria can beneficially regulate host immunity, reducing the risk of infection-induced autoimmune disease and/or inflammation, while maladaptive dysbiosis of the microbiome may lead to disruption of immune homeostasis and disease. It has been reported that colonization and alteration of the skin microbiota are associated with a variety of skin diseases, such as dermatitis, rosacea, and psoriasis (Holmes et al., 2013; Nakatsuji et al., 2017; Thio et al., 2018; Wang et al., 2020). Currently, microbiome profiling has been used to explore the microbial composition of the vulvar skin and the role of the microbiome in vulvar health and disease (JayaramA et al., 2014; Pagan et al., 2021; Murina et al., 2020). However, few studies have focused on the correlation between vulvar skin microbiota and VLS. The aim of our study was to evaluate the compositional characteristics of the vulvar skin microbiota between VLS patients and asymptomatic women using 16S rRNA sequencing.

Skin microecology is a skin microbiota composed of millions of bacteria, fungi, and viruses (Byrd et al., 2018). Like the microbes in the gut, skin microbes play a crucial role in the regulation of the immune system. The composition of the microbial community depended on the physiological state of the skin, and the relative abundance of bacteria changed with different humidity and sebaceous microenvironments. The vulva microbiota is diverse, and the normal vulva microbiota includes the characteristics of the vagina, urethra, and colon (Brown et al., 2007; Pagan et al., 2021). Previous studies on the vulvar skin microbiota, from cultivation to culture-independent molecular techniques, have shown that Lactobacillus and Corynebacterium spp., as well as Staphylococcus aureus and Prevotella, were the main bacterial components of healthy vulvar skin. (Elsner and Maibach, 1990a; Costello et al., 2009; Bruning et al., 2020). Wolf et al. (2017) transplanted vulvar skin of VLS patients onto the thigh and returned to normal skin, while normal skin was transplanted onto the vulvar area of VLS patients, and the skin turned into VLS. This suggested that the local microenvironment could be associated with the pathogenesis of VLS. Therefore, the natural microbiome of the vulvar skin may play a significant role in immunizing and maintaining vulva health, as well as preventing invasive organisms from gaining a foothold.

Lactobacillus is the permanent probiotic of the vaginal mucosa, which constitutes a majority of the vaginal flora. As the portal of the female reproductive tract, the flora composition of the vulva is influenced by the flora of the vagina. Therefore, lactobacilli dominate the vulva flora of healthy women (Brown et al., 2007; Costello et al., 2009; Murina et al., 2020). Researchers have used 16S rRNA sequencing to study the vulvar flora in premenopausal nonpregnant women. Samples were taken from 10 Japanese women before and on the second day of menstruation. Lactobacillus was dominant in all 7 subjects before menstruation and during the menstruation period (Shiraishi et al., 2011). In our study, we also found that the relative abundance of Lactobacillus in the asymptomatic control group was 54.17%, while the proportion of other bacterial groups was small. However, in the study of Chattopadhyay et al. (2021), sequencing and analysis of the vulva skin flora of 5 VLS child patients and 3 healthy children found that at the genus level, Porphyromonas spp., Prevotella, Corynebacterium and Peptoniphilus were dominant in the two groups, which may be due to the difference in vulva flora between women and children.

There was limited evidence on the skin flora of VLS patients. Watchorn et al. found a significant increase in the relative abundance of Prevotella compared to the normal control group in a study of male sclerosing lichens (20 vs. 4%) (Watchorn et al., 2021). Chattopahyay et al. (2021) found that the relative abundance of Prevotella in the vulvar flora of VLS children was significantly higher than that of healthy children. However, we found that there was no significant difference in the relative abundance of Prevotella between the VLS group and the control group (17.69 vs. 8.96%, p = 0.3630), which indicated that the vulvar skin microbiota of VLS patients might vary with age and postmenopausal status.

In addition to Prevotella, the abundance of other anaerobes in the vulva skin flora of VLS patients was also higher than that of healthy women. Guet-Revillet et al. (2014) found that strict anaerobes, such as Prevotella, Peptonophilus, and Porphyromonas gingivalis, were present in 87% of chronic suppurative bacteria, suggesting that these bacteria may be involved in chronic inflammation. In the same way, these anaerobic bacteria may also be involved in the formation of chronic skin inflammation and the development of VLS through similar immune regulation. Our study found that Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria accounted for more than 97% of the total sequence in the vulvar skin swab samples in both groups. Compared to the control group, the number of Proteobacteria in the VLS group was significantly increased (19.22 vs. 2.66% p = 0.000997). Previous studies have shown that Corynebacterium predominate on wet skin, while the predominant colonization mixture is Proteobacteria and Flavobacteria on dry skin (Grice et al., 2009). Our result is consistent with the dry vulvar skin condition in patients with VLS.

Other potential factors influencing the vulvar skin microbiota include menstrual cycle, body mass index, pH, temperature, dryness, ethnicity, dietary habits, antibiotic use, and other factors, which play a crucial role in maintaining and stabilizing the microbiota. Vongsa et al. (2019) found that Lactobacillus spp. were more prevalent on the vulva of women with normal BMI, whereas Corynebacterium spp. and Anaerococcus spp. were more prevalent on the vulva of women with a high BMI. Therefore, it should be noted that our results may be influenced by BMI, as there were significant differences in BMI between the VLS patients and asymptomatic controls. It has been speculated that the vulva pH value may be somewhere between the skin value (estimated at pH 4.7) and the vagina value (average pH 3.5), which ranges from 3.8 to 4.2 in the menstrual cycle (Chen et al., 2017). A range of factors may affect the vulva pH value, including endogenous factors (e.g., humidity, sweat and vaginal discharge, menstrual cycle, urine and faecal pollution, anatomical folds, genetics, and age) and exogenous factors (such as soap, detergent, cosmetics, lubricants and spermicide, with tight clothes or sanitary napkin shade, shaving, and leather products). Long-term dryness of vulvar skin can significantly reduce its pH (Elsner and Maibach, 1990b), and pH can subsequently influence the growth of various microorganisms.

Therefore, our study has the following shortcomings. Due to the limitations of 16S rRNA, this study was not able to assess whether vulvar skin fungi and viruses changed in VLS patients. Second, questions have been raised regarding whether skin biopsy samples of the deep epidermis, dermis and glands might provide additional information on the skin microbiota (Grice et al., 2009). Therefore, swab sampling may have limitations in the research results. Finally, due to the limitations of the algorithm, our evaluation could not reach the species level, and metagenomics is required for further analysis in future work.

This study compared the compositional characteristics of the vulvar skin microbiota in patients with VLS and asymptomatic menopausal women. Although there was no significant difference in the α diversity, the β diversity differed significantly between the two groups. The predominant bacteria on the vulvar skin of asymptomatic women were Lactobacillus spp., while the proportion of Prevotella spp. was most prevalent in the VLS group. The relative abundance of Finegoldia spp., Ralstonia spp., Peptoniphilus spp., Anaerococcus spp., Campylobacter spp., Providencia spp. Kelbsiella spp., Ezakiella spp., and Escherichia-Shigella spp. was significantly increased in the VLS group. Metabolomics and transcriptomics are required to explain the relationship more completely between VLS and the vulvar skin microflora, which may provide a theoretical basis for clarifying the pathogenesis of VLS and new concepts for its treatment.