The influence of intimate hygiene on vaginal microbiota and health

The word hygiene is derived from the Greek hygienos meaning “healthy” or “healthful.” It denotes a science dealing with the influence of the environment on human life and health. It aims to identify and eliminate negative factors and to promote the positive ones. The overall goal is to provide society with conditions fostering the full psychological and physical development of its members. From the modern perspective, hygiene is defined as actions and practices that limit the spread and transmission of pathogenic microorganisms, thereby reducing the prevalence of diseases [1]. Intimate hygiene is a part of personal hygiene and involves genital care in women and men.

The development of society is associated with increasing awareness of healthy behaviors, which influences consumer behaviors, driving the demand and supply. Products and practices related to intimate hygiene differ depending on users’ geolocation, age, socioeconomic status, culture, and beliefs. The main motivator of these actions is to prevent intimate infections. Worldwide, women use different genital care practices, for instance vaginal irrigation, vulva cleansing with a shower gel or aseptic liquid, and vaginal washing with lemon juice, salt water or vinegar [2]. However, probably the most common practice in developed countries is to use intimate hygiene washes.

All factors disrupting the physiological composition of the vaginal microbiota may lead to intimate infections connected with vaginal discharge, itching, and burning, which significantly impair the quality of life [3]. In pregnant women, this condition can cause preterm delivery, resulting in prematurity and its complications for babies [4]. Both conditions, apart from personal hardship, place a significant financial burden on the health system related to the treatment of infections and their complications. Therefore, with a view to benefiting patients, it is important to take all preventive measures against intimate infections in women.

Currently, there is a large selection of intimate hygiene products for women on the market. In the present review, we analyze if and how their use can influence the physiological vaginal microbiota, women’s intimate health, and infection prevention. In this review, we also summarize the role of selected active ingredients of intimate hygiene products in maintaining a proper balance of the vaginal microbiota.

The term microbiota encompasses bacteria, fungi, archeons, and viruses colonizing the human body, including commensal, symbiotic, and pathogenic organisms [5]. On the other hand, microbiome, often used interchangeably with microbiota, originally meant the collection of genomes of the above-mentioned organisms [6]. It was discovered with the use of modern molecular techniques, which replaced in vitro cultures and microscopic methods. One of the most frequently used molecular techniques, high-throughput DNA sequencing (HTS), makes use of two strategies: whole genome sequencing (entire microbiota) and the so-called metataxonomy, which consists of sequence analysis of bacterial 16S ribosomal RNA (16S rRNA) specific for a given bacterial species [7]. The application of these methods also enabled deciphering vaginal microbiota in women.

The first studies confirming the presence of bacteria of the genus Lactobacillus (lactic acid rods) in the vagina were conducted by Doderlein in 1892, and for many years these bacteria were called by the common name “Doderlein’s rods.” In order to systematize the bacteria in more detail, the CTS (Community State Types) system was introduced [8]. In this system, four of five groups belong to the genus Lactobacillus: CST I – L. crispatus, CST II – L. gasseri, CST III – L. iners, CST V – L. jensenii. Group IV includes anaerobic bacteria of genera such as Prevotella, Dialister, Atopobium, Gardnerella, Megasphaera, Peptoniphilus, Sneathia, Eggerthella, Aerococcus, Finegoldia, Mobiluncus, none of which clearly predominates. Numerous bacteria from CST VI have been associated with the risk of bacterial vaginosis (BV), which can increase the risk of sexually transmitted diseases, premature childbirth, and miscarriages in pregnant women [7]. Because of the differences in metabolic mechanisms, the domination of CST I Lactobacillus spp. is associated with vaginal state of health, while CST III with higher risk of dysbiosis [9].

In 2020, a new bacteria classification system named VALENCIA (VAginaL community state typE Nearest CentroId clAssifier) was created on the basis of taxonomic profiles collected from 1975 women in the United States [10]. The new system can be used independent of geographical origin and ethnicity of patients, which makes this classification more unequivocal and more standardized.

In the prenatal period, in female newborns, colonization of the vagina, just like the skin or the digestive tract, crucially depends on swallowing the amniotic fluid. Bacterial populations occurring in the placenta and amniotic fluid are similar, with the dominating type Proteobacteria and genera Enterobacter, Escherichia, and Shigella. Under normal physiological conditions, species composition of bacteria inhabiting the mother’s vagina matches that of the amniotic fluid, which suggests that bacteria can migrate through the cervix.

In female newborns born by natural delivery, the contact with the mother’s vaginal microbiota is decisive for the composition of their vaginal microbiota; therefore, for the first 2–3 weeks of a baby’s life, its vaginal microbiota are determined by maternal bacteria: Lactobacillus, Bacteroides, Bifidobacterium, Enterococcus, Blautia, Veillonella, Streptococcus, Planococcus, Escherichia, Staphylococcus, and Prevotella. The contribution of bacteria of maternal origin declines with age; however, microorganisms acquired during parturition were shown to be able to survive until the 6th to 24th months of life.

Bacteria of the genus Lactobacillus (L. crispatus, L. gasseri, L. iners, L. jensenii) constitute 60% to 95% of all vaginal microorganisms in European and US women. However, some differences related to geographic region and race have been observed; in Caucasian and Asian women, Lactobacillus spp. represents 80.2–89.7% of the vaginal microbiota, whereas in women of African and Hispanic descent, the respective values are 59.6–61.9% [11]. The lower percentage of Lactobacillus spp. implies a higher contribution of group IV bacteria, according to the CST system. It is also a cause of differences in vaginal pH in women of different ancestry: in ethnic groups characterized by a lower abundance of Lactobacillus spp. in the microbiome, vaginal pH is higher. Some studies revealed that the domination of Bifidobacterium can also occur in a healthy vagina. Vaginal Bifidobacterium produces lactic acid (the highest level of B. longum) and moderate amounts of hydrogen peroxide [12].

The vaginal microbiota also includes its mycobiome, which is strongly interlinked with vaginal bacteria. Fungi can be transmitted vertically from the maternal genital tract during labor. Microbiological studies of vaginal smears demonstrated the presence of fungi in 20–60% of cases. Candida albicans is the most abundant species but other species of Candida genus, as well as Saccharomyces, filamentous fungi from the genera Aspergillus, Alternaria, and Cladosporium are also present [13]. Factors influencing the mycobiome include the increased concentration of estrogens which stimulate vaginal mucosa colonization by single–cell (yeast-like) forms of Candida spp. Lactates and short-chain fatty acids produced by Lactobacillus spp. increase the abundance of Candida spp. in the vagina, preventing, however, the fungi from transitioning from unicellular to filamentous stage. Importantly, it is thought that this fungus occurring in the unicellular form is a commensal organism, while the development of its filamentous form (mycelium) leads to Candida spp. pathogenicity [14]. According to other studies, Candida spp. growing in the presence of lactates showed immunomodulating actions by increasing anti-inflammatory cytokine production (e.g. IL-10) and by reducing pro-inflammatory cytokine synthesis (such as IL-17); in an in vitro model, it exhibited a greater virulence than Candida spp. cultured in the presence of glucose. This observation calls for the need to analyze in more detail the influence of the presence of Lactobacillus spp. on the development of Candida spp. infections, taking into account the complexity of bacteria-fungi interactions. It is also important in the context of the development of vaginal candidiasis in postmenopausal women and during the use of hormonal contraceptives.

The average normal vaginal pH is ca. 3.5, which promotes the maintenance of normobiosis [15]. It is an accumulated result of lactate production by the vaginal epithelium and its colonization by bacteria of the genus Lactobacillus [16]. As mentioned above, their domination in the vaginal niche is characteristic of healthy women [17]. Bacteria produce both D- and L-isoforms of lactate while the epithelium only produces the L-lactate. The average proportion of D-lactate in the healthy vagina reaches 55%, which shows the dominant role of Lactobacillus spp. in the production of lactic acid [16]. Lactobacilli are Gram-positive, catalase-negative, non-spore-forming bacteria capable of lactic acid production [9,17,18]. They are microaerophilic organisms carrying out fermentative metabolism. They inhabit environments rich in carbohydrates, amino acids, and derivatives of nucleotides. In the vagina, they generate energy through anaerobic fermentation of glucose, derived from glycogen released during exfoliation of the vaginal epithelium. They transform glucose to pyruvate, subsequently metabolized by NADPH-dependent lactate dehydrogenase to lactate, which accumulates and acidifies the vaginal environment [19]. In addition, lactobacilli compete with other bacteria present on the surface of the epithelium and produce bacteriocins and hydrogen peroxide possessing bacteriostatic and bactericidal properties, thereby contributing to the maintenance of vaginal normobiosis [20].

The second significant source of lactates in the vagina derives from non-keratinized, stratified, squamous epithelium. This epithelium is composed of three layers: the basal layer with columnar and stem cells responsible for monthly renewal of the intermediate layer containing cells with basophilic cytoplasm, and the apical layer built of sloughing cells with basophilic or acidophilic cytoplasm. During intense metabolism with a limited oxygen supply, cells generate energy through anaerobic glucose fermentation, leading to lactic acid production that accumulates in the extracellular environment [21]. Lactate production depends on glucose availability. The epithelial intermediate layer cells synthesize and store glycogen, while the glucose release from it is contingent upon hormonal status. There is a positive correlation between glycogenolysis and estrogens: that is, glucose release and lactate production increase with the rise of estrogen concentration, which contributes to the maintenance of low vaginal pH. Glycogen synthesis rate is also stimulated by estrogens [22]. Thus the vaginal pH varies during menstrual cycle, in pregnancy, and with age [23, 24, 25]. Its higher values are observed in the prepubertal period (pH=6.5–7.5) [26], during menstruation (day 2 pH=6.6, day 4 pH=5.3) [27], in pregnancy (pH> 4.5 in 45.5% of the tested women) [28], and after menopause (pH gradually increases with postmenopausal age [29]). The lowest values in mature women are determined on about the 14th to the 16th day of the menstrual cycle, during ovulation. These fluctuations are directly connected with estrogen concentration.

Acidic vaginal pH enables maintaining normobiosis and limits the development of pathogens. Many studies indicate that pH > 4.5 is connected with dysbiosis and increases the risk of bacterial infections, while pH < 4.5 precludes bacterial vaginosis [30]. Therefore, a greater incidence of bacterial vaginosis, sexually transmitted diseases, and vulvar and vaginal candidiasis can be observed in physiological conditions characterized by increased vaginal pH, such as menstruation, pregnancy, and the postmenopausal period [9]. Studies revealed that sexual intercourse of a healthy woman during menstruation with a man infected with N. gonorrhoeae increased the risk of transmission of the microorganism and infection to the woman [24].

Apart from endogenous factors (such as age, menstrual cycle, pregnancy), exogenous factors also influence vaginal pH and its microbiome. These factors are mostly related to sexual and hygienic behaviors. It is thought that a large number of sexual partners and not using condoms increases the risk of dysbiosis and development of bacterial vaginosis. Likewise, hygienic habits leading to increased vaginal pH, such as the use of basic soaps for intimate hygiene or vaginal irrigation can lead to dysbiosis and can raise the risk of pelvic inflammatory disease, endometriosis, and sexually transmitted infections [21].

Products dedicated to women’s intimate hygiene usually contain three basic types of components: washing substances, active substances, and preservatives [31].

Surfactants (surface active agents, SAA), responsible for washing properties, have an amphiphilic structure and reduce surface tension of the liquid. After exceeding the critical micelle concentration (CMC), they form micelles which facilitate contaminants removal [32]. Surfactants contained in intimate hygiene products for women can be divided into: strong anionic SAA (sodium lauryl sulfate), mild anionic SAA (sodium lauryl glucose carboxylate, sodium lauroyl sarcosinate, disodium lauryl sulfosuccinate, sodium cocoyl glutamate), amphoteric surfactants (cocamidopropyl betaine, capryl/capramidopropyl betaine, sodium cocoamphoacetate, disodium cocoamphodiacetate), non-ionic surfactants (alkyl polyglucosides: lauryl glucoside, dodecyl glucoside, coconut glucoside; oxyethylene sorbitan esters: polysorbate 80, polysorbate 20, PEG-40), and natural surfactants – saponins [31]. These compounds are capable of penetrating the skin and mucous membranes, causing dissolution and leaching out of lipids, which result in irritations (pruritus, erythema, burning) [33]. Irritation results in itching and vaginal discharge, and the persistence of these symptoms prompts women to wash more frequently to obtain temporary relief. This is a vicious circle mechanism similar to the “itch-scratch cycle”, in which scratching is replaced by washing. This mechanism only promotes greater destabilization of the vaginal environment and increases discomfort [34]. Vaginal epithelial cells secrete a number of pro-inflammatory substances in response to a change of the dominating of bacteria in its niche (dominance of Lactobacillus spp. is a protective factor, reducing the secretion of pro-inflammatory molecules), including matrix metalloproteinase-8, stress-inducible 70 kDa heat shock protein (Hsp-70), neutrophil gelatinase-associated lipocalin, secretory leukocyte protease inhibitor, mannose-binding lectin, beta defensins, and cathelicidins. Damage of the epithelial cells may result in the release of these substances and the promotion of inflammation. Additionally, high concentrations of Hsp-70 are associated with inhibition of autophagy in vaginal epithelial cells and greater risk of developing infection [35]. Few studies show a positive effect of sodium lauryl sulfate in preventing sexually transmitted viral infections. It was shown that this substance inhibits the infectious potential of various strains of HSV-1, HSV-2, and HIV-1 [36].

Anionic surfactants are characterized by a stronger action than amphoteric and non-ionic ones, therefore, products which contain these ingredients have a greater irritating potential. Saponins are natural surface-active agents originating from plants, such as common soapwort. Their main advantages include slightly acidic pH value and anti-inflammatory, antibacterial, and antifungal properties [37]. Their strong antifungal effect resulting from production of reactive oxygen species that damage the C. albicans cell membrane was also reported. Since only limited, negligible toxic effects of saponin use were observed, intimate hygiene products based on these compounds may be considered important in the treatment of vaginal candidiasis [38]. In contrast to their synthetic counterparts, natural saponins have only minor irritative potential, even though their potential to induce hemolysis should be taken into consideration during their use, particularly in patients with anemia. A study was conducted to evaluate the effect of commercially available vaginal irrigation products based on vinegar, iodine, and baking soda on vaginal Lactobacillus, Escherichia coli, and the immune response of the epithelium. At the same time, the relationship between the degree of epithelium damage and the presence of Lactobacillus spp. was examined. It has been shown that the use of these products is associated with the induction of epithelial cell death, inhibition of the E. coli multiplication, and that the presence of any strain of Lactobacillus spp. has a protective effect on epithelial cells. The results of this study clearly indicate the negative effect of the tested substances on vaginal health, causing damage to the epithelium and reduction of the anti-inflammatory effect of Lactobacillus spp. [39]. Long-term use of such products may lead to frequent or chronic vaginitis and disruption of the cervicovaginal microflora. This condition in unfavorable for patients not only due to bothersome symptoms and reduced quality of life, but may also promote carcinogenesis through reduced immunity and inhibition of HPV virus proliferation [40].

Active substances are added to intimate hygiene washes in order to provide them with special properties. Among them, lactic acid is one of the most commonly used. A 4-week study was designed to evaluate the effect of daily use of an intimate wash supplemented with lactic acid (pH 4.2) on microflora of the vulvar skin in healthy women. The study showed a good tolerance of this wash without significant changes in pH or signs of irritation in intimate parts; no alterations in the vaginal and vulvar microbiota were also seen [41]. In another study a neutral effect of lactic acid on vaginal pH was presented; however, an increased incidence of anaerobic vaginal microbiota development was observed when used during menstruation. This circumstance may have a negative impact on vaginal microbiota well-being, thus increasing the risk of infections [42]. Other active substances are used to reduce the irritating action of washing agents or to alleviate irritations already progressing. This group comprises allantoin (increases neutrophil count, accelerates skin cell regenerative processes, stimulates cell proliferation) [43], panthenol (moisturizes, speeds up epidermis healing, alleviates irritations) [44], or bisabolol, a component of chamomile (has anti-inflammatory action and alleviates skin irritation) [45].

Preservatives prevent the development of undesired microflora and ensure microbiological purity of the manufactured cosmetics. Their use and allowable concentrations are strictly defined in the Regulation (EC) No 1223/2009 of the European Parliament and of the Council of 30 November 2009 [46]. The most frequently used preservatives in intimate hygiene cosmetics include sodium benzoate and dehydroacetic acid [13]. Both compounds possess bacteriostatic and fungistatic properties. Sodium benzoate has a strong inhibitory effect on bacteria capable of butyric and acetic fermentation, yeasts, molds, and to a lesser extent, on lactic acid bacteria. There is no detailed data on the influence of dehydroacetic acid on different bacteria types. These substances are considered to be safe at strictly defined concentrations (sodium benzoate – 0.5%, dehydroacetic acid – 0.6%) [47]. Due to its bacteriostatic properties, they can have a potential negative impact on the vaginal microbiota; however, no studies have verified this hypothesis yet. Sodium benzoate seems to be a better choice since it shows less impact on bacteria engaged in lactic acid fermentation.

Intimate hygiene products can also contain antibacterial and antifungal substances, like tea tree oil, urea, or lavender essential oil. In vitro and in vivo studies have shown the effectiveness of a gel containing tea tree oil and cloves in the treatment of vaginal candidiasis [43]; however, more research is needed before these kinds of products can be introduced into clinical practice.

Bacterial vaginosis (BV) is a disease entity characterized by disturbances in physiological composition of the vaginal microbiome consisting in overgrowth of group VI bacteria according to the CST classification (see above). Customary treatment of BV and recurrence prevention relies on antibiotics, such as metronidazole or clindamycin administered orally or transvaginally [21]. However, due to an increasing resistance of bacteria to antibiotics, the recurrence rate is on the rise, reaching even 50% of cases within 6 months after the treatment has ended [48]. For this reason, studies are carried out to test alternative therapeutics able to reduce the risk of BV recurrence. These therapeutics include antiseptic drugs, probiotics, and preparations containing lactic acid, estradiol, and sucrose. Another group is composed of topical non-medicinal products, like cleansing gels or foams. They are considered to be early prevention hygienic products, as described above.

Antiseptics can be administered intravaginally and they include: polyhexamethylene biguanide (PHMB), dequalinium chloride, povidone iodine, silver ions, boric acid, chlorhexidine. The advantages of antiseptics emerge from their ability to effect biofilm destruction (PHMB, silver ions), poor resistance mechanisms to them in bacteria (except from reported chlorhexidine resistance of MRSA, Pseudomonas aeruginosa, Acinetobacter baumannii; plasmid encoded silver resistance of members of the Enterobacteriaceae), and broad spectrum action. Adverse effects of antiseptics include: thyroid dysfunction (povidone iodine should not be administered for longer than 7 days), vaginal candidiasis and discharge, itching/burning sensation (dequalinium chloride is contraindicated in epithelial ulcerations). Since these side effects are not as significant as those caused by antibiotics, the usage of antiseptics as first-line products in BV therapy should be practiced [49, 50].

Probiotics, which can include bacteria such as Bifidobacterium spp. or Lactobacillus spp., are defined as live microorganisms which, if administered at appropriate doses, offer health benefits to the host. Preparations containing bacteria Lactobacillus spp. are available for intravaginal and oral use. The postulated mechanism of the beneficial effect of the oral probiotics is based on proximity of the rectum and the vagina, which can be easily crossed by bacteria that have survived the intestinal passage [21]. Especially oral L. gasseri, L. fermentum, L. plantarum were shown to produce the above-mentioned effect [5]. Many more studies have been dedicated to the action of intravaginal probiotics. Many of them were reported to possess beneficial therapeutic effects in BV, at least at the level similar to antibiotic therapy [51]. Other examples of probiotic strains of Lactobacillus spp. include L. reuteri RC-14 and L. rhamnosus GR-1, which could be administered as intravaginal capsules or fermented milk product. The required dosage is ≥ 10⁸ CFU for capsules and ≥ 5*10⁹ CFU for fermented milk products [21]. When used in combination with an antibiotic either concomitantly or after the therapy, they reduce BV recurrence rate, thereby showing a positive influence on the vaginal microbiome. Lactic acid can also be used in the form of vaginal gel. Studies comparing the therapeutic effect of this gel, metronidazole, and placebo demonstrated that efficacy of the gel with lactic acid in BV treatment was comparable to metronidazole [52].

Interestingly, it appears that the efficacy of probiotic therapy in women from different ethnic groups is not the same. Smaller health benefit of probiotics was observed in women of African descent; however, a greater risk of infections leading to BV and more frequent HIV infections, compared with women from other ethnic groups, might have contributed to this difference [53].

As mentioned above, natural estrogens have a beneficial effect on physiological bacterial flora. Studies examining the efficacy of BV treatment with a L. acidophilus in combination with 0.03 mg estradiol in comparison with antibiotic therapy showed an equivalent effect of both treatment methods, although recurrences were observed more often after probiotic+estradiol therapy than after the antibiotic. It can suggest a greater efficacy of antibiotic therapy during long-term follow-up [54]. Another disadvantage of estradiol-containing preparations is that they produce a positive effect on vagina colonization by C. albicans, which can lead to increased incidence of candidiasis.

Lactic acid bacteria are capable of utilizing sucrose as a substrate, which stimulates their growth in the form of biofilm. Two studies conducted to examine sucrose-containing gels demonstrated that their BV treatment efficacy was comparable with metronidazole [55, 56]. Unfortunately, there are no newer (published after 2018) studies on the use of this gel in BV patients; also, no recommendations as to BV prophylaxis with sucrose-containing gels in healthy women were found.

The market offers a wide range of products dedicated to intimate hygiene for women. Among them, there are preparations recommended for everyday use and for prevention/treatment of disturbances in the vaginal microbiome. From many perspectives, efforts to prevent pathological states are better than cure.

Literature fails to provide data on the indirect influence of ingredients of vaginal wash products on the vaginal microbiome. It is only postulated that they tend to cause irritations, which may impair the vaginal biocenosis. Considering the widespread and regular use of these products, studies examining their possible impact on vaginal microbiome composition appear to be of crucial significance.

Antiseptic drugs usage as a first-line therapy seems to be an attractive replacement for antibiotics. Studies on the vaginal wash products capable of preventing the vaginal microbiota disorders and supporting its stability revealed a beneficial effect of probiotics containing special bacterial strains of the genus Lactobacillus (e.g., L. gasseri, L. fermentum, L. plantarum, L. reuteri RC-14, and L. rhamnosus GR-1) for oral or intravaginal use, in the form of milk fermentation products or directly as lactic acid in intravaginal gel. Their efficacy was comparable to antibiotics tested in parallel. In addition, the combination of the above-mentioned probiotics with antibiotics reduced BV recurrence rate. Likewise, concomitant use of preparations containing L. acidophilus and estriol was also propitious and comparable to the antibiotic; however, BV recurrences were observed more often than with the antibiotic alone. Prophylaxis with sucrose-containing preparations is also worth mentioning as its efficacy was confirmed by a few published reports. The studies on the use of preparations with sucrose in BV prophylaxis should be complemented with determination of the risk of BV recurrences and general safety of their use, also in the context of the risk of candidiasis. Further investigations due to the possibility of therapeutic Bifidobacterium usage, as an alternative to Lactobacillus probiotics, is needed. It may lead to actualization of “healthy vagina” within the context of microbiota balance.