Role of Vaginal Microbiota Dysbiosis in Gynecological Diseases and the Potential Interventions.

The role of microbiota in non-infectious gynecological diseases was long underestimated, until recently when mounting evidence showed its significance. Herein, we introduce the common non-infectious diseases that were caused by physical injury, fertility problems, and endocrine disorders, and discuss the treatment effects of restoring Lactobacillus -dominated vaginal microbiota by antibiotic and probiotic interventions ( Figure 2 ). Therapy effects of restoring Lactobacillus -dominated vaginal microbiota in non-infectious diseases. Lactobacillus -dominated vaginal microbiota cannot only benefit vaginal health by preventing vaginal infections and creating a non-inflammatory environment but also prevent pathogens in the vagina moving to the cervix and uterus. Besides, vaginal Lactobacillus can move to the upper reproductive tract, preventing pathogens infecting the cervix and uterus, and creating a non-inflammatory cervical and uterine environment. Induced abortion is a fairly common gynecological operation worldwide, and whether it is drug-induced or surgery-induced, it can cause a great damage to the female reproductive tract and predispose to serious complications such as incomplete abortion, heavy bleeding, infection, scarring of endometrium, adhesions of the uterine cavity and cervix, and endometriosis. Among these, upper genital tract infection is the most concerned and common clinical problem, which can further cause endometritis, salpingitis, and infertility ( Mary and Mahmood, 2010 ; Carlsson et al., 2018 ). Studies showed that postabortal infection was usually caused by pathogens from the lower genital tract, such as Chlamydia , N. gonorrhoeae , Mycoplasma , and BV-related bacteria, moving through the cervix to the uterus and even to fallopian tubes, causing infections and inflammation of the entire reproductive system ( Bjartling et al., 2010 ). In this regard, World Health Organization (WHO) recommended the use of antibiotic prophylaxis to prevent the abortion-induced infection, and a meta-analysis of randomized clinical studies showed that perioperative antibiotics (metronidazole, nitroimidazoles, etc.) could effectively reduce the risk of postabortal infection by 50% ( Sawaya et al., 1996 ; Carlsson et al., 2018 ). However, if the homeostasis of the uterus is not fully restored and is still conducive to microbial disorders, antibiotics cannot protect the women from being reinfected ( Low et al., 2012 ). Therefore, as probiotics have a great potential in restoring the normal vaginal microbiota and improving the uterine environment, perioperative use of antibiotics and probiotics can prevent the postabortal infection. Intrauterine adhesions is a condition in which the scar tissues build up inside the uterine cavity, which in many cases will cause adhesion of the opposing endometrium ( Chi et al., 2018 ). Though the adhesions can be removed through the transcervical resection, the postoperative recurrence is high ( Pabuccu et al., 2008 ). A clinical study showed that IUA patients had disturbed vaginal microbiota, characterized by obviously reduced probiotic Lactobacillus , and increased pathogenic Gardnerella and Prevotella , which may promote the uterine pathology and cause recurrence ( Liu et al., 2019 ). Therefore, antibiotics and/or probiotics can promote the recovery and reduce the recurrence of IUA by correcting the vaginal microbiota. Miscarriage, defined as a spontaneous pregnancy loss before the expected point, is the most prevalent pregnancy complication ( Smith et al., 2019 ). The etiology is multifactorial and usually secondary to other disorders, such as uterine malformation, infections, chromosomal abnormalities, and hormone deficiency (progestogen) ( Haas et al., 2019 ). In this regard, the preventive and curative measures are always aimed at addressing the primary causes ( Haas et al., 2019 ). Previous studies showed that pregnant women with BV had around two-fold higher risk of miscarriage than those without BV, which was thought to be caused by ascending of the BV-related bacteria from the vagina to the uterus, leading to endometritis, deciduitis, chorioamnionitis, and amniotic fluid infection ( Ralph et al., 1999 ; Goldenberg et al., 2000 ). Studies exploring the specific mechanisms of BV favoring miscarriage showed that BV-related bacteria could produce lytic enzymes (proteases, phospholipases, etc.) to cause lysis of the fetal membranes, and induce the formation of prostaglandin, which could promote the uterine muscle contraction, decrease the cervical resistance, and induce the release of metalloproteinases (MMPs) to degrade the chorioamniotic membranes ( Isik et al., 2016 ). Besides, the level of inflammatory cytokines IL-6 and IL-8 was also elevated in the amniotic fluid of pregnant women with BV ( Keelan et al., 1997 ). A randomized controlled trial showed that pregnant women treated with clindamycin for BV were five times less likely to miscarry than those given placebo ( Ugwumadu et al., 2003 ). This suggests that early screening and treatment of BV can prevent the infection-induced miscarriage. In addition, a clinical study suggested that hydrogen peroxide producing Lactobacillus in the vagina could not only benefit the vaginal health by improving the microbial and inflammatory status, but also create a favorable uterine environment for implantation and placentation ( Eckert et al., 2003 ). Thus, antibiotics and probiotics could prevent and treat infection-induced miscarriage by restoring the vaginal microbiota and improving the intrauterine environment. Plenty of evidence showed that BV could also increase the risk of preterm and infertility, mainly via facilitating STI and eliciting the intrauterine inflammation ( Peelen et al., 2019 ; Hong et al., 2020a ). Recent studies showed that Lactobacillus -dominated vaginal microbiota was negatively associated with preterm and infertility, and could prevent women from adverse fertility outcome by modulating vaginal microbiota and inflammatory cytokines IL-4 and IL-10 ( Vitali et al., 2012 ; Hong et al., 2020a ). Therefore, antibiotics and probiotics can prevent the preterm and infertility by improving the vaginal and uterine microbiota and eubiosis. Polycystic ovarian syndrome is one of the most prevalent endocrine disorders in reproductive women, which usually manifests as menstrual disorder, hirsutism, and infertility ( Hong et al., 2020b ). Though current treatments are various, such as oral contraceptives to inhibit the maturation of ovarian follicles as a long-term PCOS management, and ovulation induction for PCOS patients with fertility requirement, they cannot cure it basically and lifestyle modification (e.g., loss weight) is still the first-line and mainstream treatment ( Jin and Xie, 2018 ). A case–control study of 39 PCOS patients and 40 healthy people showed that the vaginal microbiota of PCOS patients was significantly different from that of healthy people, characterized by increased diversity and increased relative abundance of Mycoplasma and Prevotella , and decreased relative abundance of L. crispatus ( Hong et al., 2020b ). This suggests that vaginal microbiota dysbiosis may participate or contribute to the PCOS pathology, and therapies targeted at improving the vaginal microbiota are promising. A systemic review involving 855 PCOS patients investigated the effects of probiotics ( Lactobacillus ) in treating PCOS, and the results showed that probiotic supplementation in PCOS women significantly improved their hormonal index by reducing free androgen index (FAI) and increasing sex hormone binding globulin (SHBG), and their inflammatory index by increasing plasma nitric oxide (NO) and reducing blood malondialdehyde (MDA) ( Shamasbi et al., 2020 ). Besides, the authors also observed that patients given probiotics had increased total glutathione (GSH) and total antioxidant capacity (TAC) levels, and reduced testosterone, dehydroepiandrosterone sulfate (DHEAS, hormonal index), high sensitive C reactive protein (hsCRP, inflammatory index), and hirsutism score compared to those given placebo. As antibiotics and probiotics are aimed at restoring the normal vaginal microbiota and improving the environment of the reproductive system and beyond, they can be used to treat the PCOS symptoms and promote the recovery by improving the hormonal and inflammatory levels. Recently, microbiota disturbance has also been implicated in the uterine fibroid (benign tumors in the uterus) and menstrual disorders (e.g., menorrhagia and dysmenorrhea), characterized by increased relative abundance of vaginal L. iners , and increased relative abundance of uterine Gardnerella , Prevotella , Sneathia , and Veillonella , respectively ( Chen et al., 2017 ; Pelzer et al., 2018 ). Thus, antibiotics and/or probiotics are also promising in treating uterine fibroid and menstrual disorders.

TC and ZL provided ideas of this review and designed its framework. YH conducted the research and wrote the manuscript. All authors edited the manuscript. All authors read and approved the final manuscript.

The vagina is a stretchable, muscular duct connecting the uterus and external genitalia, and is responsible for the physiological functions of female sexual intercourse, menstrual discharge, and delivery of the fetus ( Farage and Maibach, 2006 ). Its mucosal system, consisted of a stratified squamous epithelium and cervicovaginal fluid (CVF), is vital in maintaining vaginal health by immune response, antimicrobial products (e.g., B-defensin), finely balanced microbial communities, etc. ( Torcia, 2019 ). Among these, the vaginal microbiota is the most changeable and vulnerable one in response to internal and external stimulus ( Gajer et al., 2012 ). Recently, the detailed composition and relative abundance of vaginal microbiota has been determined by high-throughput 16s rRNA sequencing, characterizing five microbial community state types (CST) in asymptomatic women ( Ravel et al., 2011 ). Four of them (CST-I, II, III, V) were dominated by Lactobacillus species, while CST-IV was heterogenous and polymicrobial, characterized by lower level of Lactobacillus and higher level of anaerobic bacteria including Gardnerella , Atopobium , Mobiluncus , Prevotella , Streptococcus , Mycoplasma , and Ureaplasma ( Ravel et al., 2011 ). At present, over 140 Lactobacillus species have been identified, but the only species that normally dominate the vaginal microbiota are L. crispatus , Lactobacillus gasseri , Lactobacillus jensenii , and L. iners ( Smith and Ravel, 2017 ). They are considered as keystones of vaginal health, as they can produce lactic acid, hydrogen peroxide, and bacteriocins, maintaining acidic environment and preventing pathogen growth ( Gupta et al., 2019 ); adhere to epithelium, repelling other bacteria adhesion ( Torcia, 2019 ); and regulate immune and inflammatory response, enhancing the resistance of vagina to diseases ( Aldunate et al., 2015 ). Thus, Lactobacillus dominance is generally considered as a hallmark of healthy vagina ( Ma et al., 2012 ). It is generally known that vaginal microbiota disturbance is highly related to various gynecological diseases, especially BV, which is characterized by the alteration of vaginal microbiome from Lactobacillus dominance to anaerobic and facultative bacteria ( Gardnerella , Atopobium , Prevotella , Megasphaera , Leptotrichia , Sneathia , etc.) dominance ( Ling et al., 2010 ; Srinivasan et al., 2012 ; Nasioudis et al., 2017 ). BV has been shown to be associated with various other reproductive tract disorders, including infertility, preterm, cervical cancer, and HIV acquisition ( Leitich and Kiss, 2007 ; van Oostrum et al., 2013 ; Torcia, 2019 ). It is also reported that many sextually transmitted infections (STI), such as infections of N. gonorrhoeae and C. trachomatis , are facilitated by vaginal microbiota dysbiosis and more prevalent in BV-positive women ( Lewis et al., 2017 ). In addition, as the microbiota research progresses intensively, a growing number of studies have linked vaginal microbiota dysbiosis to various gynecological non-infectious diseases, among which Liu et al. found that compared with healthy people, IUA patients had lower percentage of Lactobacillus , and higher percentage of Gardnerella and Prevotella ( Liu et al., 2019 ); Hong et al. found that patients with PCOS had lower Lactobacillus and higher Mycoplasma and Prevotella than controls ( Hong et al., 2020b ); and Chen et al. found that Lactobacillus were less abundant, while L. iners were more abundant in patients with uterine fibroid than individuals without ( Chen et al., 2017 ). Therefore, as the balanced vaginal microbiota plays a significant role in female health, interventions aimed at restoring the healthy microbiota composition can be a good and reasonable therapy for gynecological diseases.

Inspired by the similarity of the intestinal and vaginal microbiota, and the success of the fecal microbiota transplantation (FMT), VMT has also been proposed for the treatment of vaginal dysbacteriosis, which involves transplanting the entire vaginal microbiota of a healthy donor into the vagina of the patient to restore the overall diversity, stability, and normal composition of the microbiota ( DeLong et al., 2019 ). The procedures of VMT are shown in Figure 3 . The research of our group in 2017 showed that transplantation of healthy rat vaginal microbiota into the vagina of BV model rats restored the morphology of uterine tissue and reduced the serum inflammatory factors such as IL-6, IL-8, and TNF-α, showing obvious recovery effects on vaginal infections caused by dysregulation of vaginal microbiota. In 2019, the clinical study conducted by Lev-Sagie et al. (2019) further showed that VMT had a great effect on long-term recovery from recurrent, antibiotic unresponsive, and refractory BV. In this study, four of the five BV patients treated with VMT recovered effectively after 5–21 months of VMT treatment, showing significant improvements in symptoms, negative Amsel criteria, and Lactobacillus -dominated vaginal fluid under the microscopy, with a cure rate up to 80% and no observed adverse effect. Besides, the authors also found that patients with long-term resolution of BV had a dramatic change in microbial composition in the first month after VMT, which was dominated by increase of Lactobacillus and decrease of Bifidobacterium (closely related to Gardnerella ), accompanied with reduced Fannyhessea and Prevotella . In 2021, our group verified the feasibility of VMT in animal models and explored the specific mechanisms ( Chen et al., 2021 ). The results showed that vaginal secretions from healthy rats can be used to treat vaginal microbiota imbalance and prevent the recurrence in rats, which specifically manifested as the decrease of inflammatory cells, pro-inflammatory cytokines, and apoptotic factors in the uterine wall and the restoration of the diversity of vaginal microbiota. Schematic diagram of VMT operation. As for donors, first, donors are recruited, preferably from the recipients’ first and second relatives. Second, collect blood/CVF/urine samples of donors and conduct a series of screening by questionnaire, PCR, blood, and urine analysis machines, cultures, microscopy, and NGS. Third, collect CVF sample from qualified healthy donor and process it. It is then transplanted into the recipient’s vagina. As for recipients, first, recipients undergo the same screening process for diagnosis and basic health assessment. Second, recipients are given intravaginal antibiotics to prepare for transplantation. Third, transplant the prepared CVF solution from healthy donor into the recipient’s vagina. Fourth, follow-up studies are carried out on recipients to assess the treatment outcome and adverse effects. VMT, vaginal microbiota transplantation; CVF, cervicovaginal fluid; PCR, polymerase chain reaction; NGS, next-generation sequencing. Preliminary studies of VMT have demonstrated the feasibility of VMT to treat BV, showing favorable therapeutic effects. Compared to other treatments for BV, VMT can completely restore the vaginal microbiota to a healthy state, thus showing better curative effects than conventional antibiotics and probiotics, while addressing the drug resistance, recurrence, and side effects associated with antibiotic treatment. Considering that besides BV, vaginal microbiota dysbiosis is also comprehensively involved in the progression of other gynecological diseases, and improving vaginal microbiota by antibiotics and probiotics shows good therapy effects, restoration of vaginal microbiota by VMT may also have favorable therapeutic effects in the treatment of various gynecological infectious and non-infectious diseases. However, the clinical implementation of VMT still faces many problems, such as insufficient VMT clinical trial (only one research with five subjects), lacking standard protocol, transmission of unidentifiable and antimicrobial-resistance pathogens, unintended pregnancy, immune rejection, and unclear long-term effects. Therefore, the improvement of the VMT requires multi-disciplinary cooperation. Relevant personnel should formulate VMT screening guidelines as soon as possible, continue to explore the application potential of VMT in the treatment of BV and other gynecological diseases, develop a safe and effective new treatment regimen, and develop safety evaluation criteria. We have reasons to believe that safe, standard, and efficient VMT will bring new hope to patients with gynecological diseases and have a good prospect of application.

The vagina is an important and complex ecosystem, dominated by Lactobacillus , but also containing a small number of fungi and parasites, and the balanced microbial communities are vital for female health ( Bradford and Ravel, 2017 ; Gupta et al., 2019 ). However, the microbial balance can be disrupted and leads to various infectious diseases, characterized by overgrowth of anaerobic bacteria (agent of bacterial vaginitis and atrophic vaginitis, BV and AV) and Candida albicans (agent of vulvovaginal candidiasis, VVC), and infections of Trichomonas vaginalis (agent of trichomonal vaginitis), Neisseria gonorrhoeae (agent of gonorrhea), Mycoplasma genitalium (agent of cervicitis), Chlamydia trachomatis (agent of pelvic inflammatory disease, PID), and various viruses including human papillomavirus (HPV, agent of cervical cancer), herpes simplex virus-2 (HSV-2, agent of genital ulcers), and human immunodeficiency virus (HIV, agent of acquired immunodeficiency syndrome, AIDS) ( Gupta et al., 2019 ). In addition, some non-infectious diseases, e.g., induced abortions (with BV microbiome, etc.), intrauterine adhesions (IUA, with reduced Lactobacillus and increased Gardnerella , Prevotella , etc.), miscarriage (with BV microbiome), preterm (with BV microbiome), infertility (with BV microbiome), polycystic ovarian syndrome (PCOS, with reduced Lactobacillus crispatus , and increased mycoplasma and Prevotella ), uterine fibroid (with increased Lactobacillus iners ), and menstrual disorders (with increased uterine Gardnerella , Prevotella , Sneathia , and Veillonella ), also show associations with microbial dysbiosis ( Hay, 2004 ; Chen et al., 2017 ; Pelzer et al., 2018 ; Liu et al., 2019 ; Hong et al., 2020a , b ), posing a serious threat to women’s reproductive health. At present, the most conventional treatment strategy for microbial disorders is antibiotics (metronidazole, clotrimazole, azithromycin, etc.), which has a good therapy effect while accompanied with various adverse effects and recurrence ( Cudmore et al., 2004 ; Bradshaw et al., 2006 ; Xie et al., 2017 ; Khosropour et al., 2018 ; Paez-Canro et al., 2019 ). Recently, probiotics based on Lactobacillus have shown promise in treating not only infectious diseases (e.g., BV, fungal infection, and urinary tract infections) but also non-infectious diseases (e.g., preterm, infertility, and PCOS) ( Hanson et al., 2016 ; Lopez-Moreno and Aguilera, 2020 ). However, the treatment outcome of probiotics is usually mixed, which may be due to the fact that these diseases are usually caused by multiple microbes rather than one. Excitingly in 2019, Ahinoam et al. conducted a clinical study of vaginal microbiota transplantation (VMT) in five patients with recurrent BV, finding that four of them achieved long-term remission and established a long-term vaginal microbiota dominated by Lactobacillus ( Lev-Sagie et al., 2019 ). Therefore, intervention on vaginal microbiota is significant and promising in treating gynecological diseases. Thus, in this review, we elaborated the role of microbiota dysbiosis in various gynecological infectious and non-infectious diseases, and the current and potential interventions.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.