1. Introduction
Vulvovaginal infections are the most common gynaecological illnesses among women, with recurrences that are defined as more than three episodes per year, affecting nearly 8% of women globally [
1]. The three common vulvovaginal infections are bacterial vaginosis, vulvovaginal candidiasis and trichomoniasis. Changes in the vulvovaginal regions as affected by these infections often create a niche for the pathogenesis of other infections, leading to mixed infections and co-infections, which when left untreated, will not only affect the female reproductive health, but may also result in many foster infections/diseases and adverse pregnancy outcomes [
2]. Vulvovaginal candidiasis (VVC) is the most prevalent human candida infection resulting in inflammation of the vulva or vagina and affecting 75% of reproductive-age women. The majority of VVC is caused by
Candida albicans and
Candida glabrata, which are opportunistic fungi that invade the mucous membrane of the vagina, leading to exuberant mucosal inflammatory responses [
3]. This eventually leads to symptoms of vaginal itchiness, irritation, swelling, pain during sexual intercourse, soreness, discomfort, redness and vaginal discharge [
4].
VVC is also the most common form of fungal infection in pregnant mothers, which may cause systemic infections in neonate and has been associated with low birth weight and premature delivery [
5]. Pregnancy increases the frequency of vaginal
Candida colonization and it is thought to be the consequences of increased levels of circulating estrogens and deposition of glycogen and other substrates in the vagina during pregnancy [
6]. Azole-based antifungal drugs remain as the top choice to treat VVC including the pregnant population. However, this has caused various concerns attributed to the emergence of drug-resistant yeasts, while prolonged exposure to fluconazole, as in cases of recurrences, can shift the predominant vaginal yeast flora from
Candida albicans to more intrinsically azole-resistant species such as
Candida krusei or
Candida glabrata, typically in immunosuppressed women, and may be exacerbated in healthy women by use of over-the-counter products for self-treatment of VVC. Cross-resistance between OTC drugs (miconazole, clotrimazole, and tioconazole) and fluconazole has been observed in clinical isolates of
C. glabrata and
C. albicans [
7,
8].
Probiotics are live microorganisms that exert health benefits to the host upon consumption in sufficient amounts [
9]. Although probiotics have been used commonly for gut health [
10], increasing evidence over the years has illustrated the benefits of probiotics beyond that of gut maintenance [
11,
12]. Probiotics have been documented to maintain and modulate microbiota profiles in the gut and vagina, and they are able to inhibit pathogenic
Candida species. These have given rise to the concept of using probiotics for the treatment of VVC. We have previously reported the use of a multi-species lactobacilli probiotic (SynForU-HerCare) to reduce the recurrences of vaginal candidiasis (VC) in pregnant women. SynForU-HerCare is a probiotic product containing dairy-isolated lactobacilli, namely
Lactiplantibacillus plantarum (former
Lactobacillus plantarum) LP115,
Lactobacillus helveticus LA25 (previously identified as
L. acidophilus [
13,
14]),
Lacticaseibacillus rhamnosus (former
Lactobacillus rhamnosus) LRH10,
Lacticaseibacillus paracasei (former
Lactobacillus paracasei) LPC12,
Limosilactobacillus fermentum (former
Lactobacillus fermentum) LF26, and
Lactobacillus delbrueckii subsp.
Lactis LDL114. These strains of lactobacilli were developed primarily for women’s health with patents in Taiwan (2013) and China (2018), specifically for their ability to adhere to HeLa cells (a human cervical carcinoma cell line) and to produce hydrogen peroxide, which inhibits infectious urogenital bacteria in women such as
Salmonella, Escherichia coli,
C. albicans, and
Gardnerella vaginalis [
13,
14]. Our previous randomized, double-blind and placebo-controlled study involving 78 pregnant women with VC showed that the oral administration of SynForU-HerCare for 8 weeks reduced VVC symptoms and recurrences of VC, accompanied by improved emotional and social distress attributed to VC as compared to the placebo, indicating that probiotics could be a potential strategy for the maintenance of vaginal health during pregnancy [
8].
This study is a continuation of our previous work, where we continue to evaluate the effects of SynForU-HerCare against the abundance of Candida and Lactobacillus in the high- and low-vaginal and cervicovaginal regions of pregnant women with VC, accompanied by assessments for inflammatory responses in these regions. We hypothesize that the recurrences of VC in pregnant women are attributed to a disruption of vaginal lactobacilli, leading to overgrowth of vaginal Candida, while probiotics were able to restore such a disruption.
4. Discussion
While VVC is one of the most common vulvovaginal infections in women, a higher prevalence is detected in pregnant women with increased risks attributed to recurrent episodes of infections. Symptomatic recurrences among pregnant women are also highest during the second and third trimester during pregnancy, mainly attributed to decreased cell-mediated immunity, increased estrogen levels and increased vaginal mucosal glycogen production that facilitates adherence of yeast to vaginal mucosal epithelial cells during these phases of pregnancy [
19]. Evidence has suggested that VVC during pregnancy is associated with an increased risk of premature rupture of membranes and poor pregnancy outcome, while eradication of VVC in pregnancy via the use of clotrimazole reduces the risk of preterm birth. Although VVC-induced chorioamnionitis is rare, several cases have been reported of intraamniotic infection caused by
C. albicans and
C. glabrata, leading to preterm rupture of membranes or preterm labour, where such a progression could lead to foetal fatality [
20]. While treatment using clotrimazole has rare and few side effects, a small number of patients (<10%) have reported vulvar or vaginal burning sensation, rash, hives, blisters, burning, itching, peeling, redness, swelling, pain, or other signs of skin irritation. It is thus hoped that a natural dietary intervention such as that of using probiotics may alleviate these side effects of treatment, albeit rare, and prevent recurrences among pregnant women that ultimately may reduce pregnancy and birth complications.
Our present study shows that a probiotic consisting of a lactobacilli mixture in conjunction with clotrimazole prevented recurrences of
C. glabrata after 8 weeks in the lower vaginal region, although a similar effect was not observed for
C. albicans.
C. glabrata is the second most common
Candida species in humans and is a lesser pathogenic commensal of the vagina as compared to
C. albicans. This may be the reason that the inhibitory effect of lactobacilli was more prominent against
C. glabrata than
C. albicans. While clotrimazole was developed as a vaginal insert treatment that primarily targets
C. albicans, recent clinical isolates have identified increasing resistance of
C. glabrata against the drug [
21]. Our current data shows an alternative of using lactobacilli against
C. glabrata in the effort to possibly reduce dependency on clotrimazole.
Meanwhile, in the higher vaginal region, clotrimazole reduced the abundance of
C. albicans after 4 weeks, but such a reduction was not sustained over a prolonged period of time as seen in the placebo group, while a reduction was also observed at week 8 in the probiotic group. Patients on the placebo showed a consistent abundance of both
Candida species over time in the lower vaginal region but decreased abundance of
C. albicans after 4 weeks in the higher vaginal region, while it increased in both
C. albicans and
C. glabrata in the cervicovaginal region between week 4 and week 8. While
C. albicans and
C. glabrata are facultative anaerobes, they proliferate better under aerobic conditions [
22]. We postulate that clotrimazole was sufficient in preventing the overgrowth of these
Candida in the lower vaginal region, but insufficient to inhibit and prevent recurrences. The higher vaginal region would be more anaerobic than the lower vaginal regions.
C. albicans is prone to produce hyphae under anaerobicity which increases its survival, virulence and pathogenicity [
23]. While
C. glabrata is unable to produce hyphae; it is prone to attach to the hyphae of
C. albicans, leading to increased survival, virulence and pathogenicity as well. This may explain the unchanged abundance of
C. glabrata over time in higher vaginal regions despite treatment using clotrimazole in the placebo patients. The administration of probiotics reduced the abundance of both
C. albicans and
C. glabrata continuously over 8 weeks in the higher vaginal region, indicating not just growth inhibition against
Candida but also the prevention of recurrences typically in the higher vaginal regions, an area of stubborn virulence and pathogenicity.
The reduced abundance of
C. glabrata in the lower vaginal region upon administration of probiotics also occurred in a microenvironment with increased abundance of
L. crispatus, while the reduced abundance of
Candida in higher vaginal regions upon administration of probiotics had an increased abundance of
L. jensenii instead.
L. crispatus and
L. jensenii are the main lactobacilli species among a healthy vaginal microbiota, where a reduced abundance is often observed in vaginitis patients such as bacterial vaginosis and VVC [
24]. Predominant vaginal LABs are also grouped into community state types (CST) grouped as I, II, III, IV, V, where each is dominated by
L. crispatus,
L. gasseri,
L. iners, polymicrobial
Lactobacillus and bacterial vaginosis-associated bacteria, and
L. jensenii, respectively.
L. crispatus- and
L. jensenii-dominated vaginal microbiota form CST I and V, respectively, an indication of a healthy vaginal microenvironment. However, the influence of hormones particularly oestradiol during pregnancy can stimulate the transition of CST I (
L. crispatus-dominated) to other CSTs including those of transitional and diseases states [
25]. Based on these, our present study shows that the administration of probiotics shifted the vaginal microenvironment towards a healthier state, where lower vaginal regions were dominated by
L. crispatus (CST I) and higher vaginal regions dominated by
L. jensenii (CST V), amid the presence of vaginal candidiasis.
VVC is often accompanied by inflammation, primarily caused by the ability of
Candida, typically
C. albicans, to co-regulate with the expression of genes encoding for virulence factors such as secreted aspartyl proteases and candidalysin, causing tissue damages. While vaginal epithelial cells counter
Candida invasion via epithelial shedding, secretion of mucin and strong interepithelial cell connections, an increased fungal burden often overcomes the tolerance threshold, triggering an intense inflammatory response [
26]. In our present study, inflammation may have occurred in both low and high vaginal regions, predominantly observed by the increased concentrations of pro-inflammatory cytokine TNF-alpha. The administration of probiotics has shortened the period of inflammation in low vaginal regions, as observed from the increased pro-inflammatory cytokines IL-4 and IL-10 after 4 weeks in the probiotic group, while placebo did not show any changes over the same period but attributed to increased pro-inflammatory cytokine TNF-alpha over 8 weeks in the placebo group but only increased over 4 weeks in the probiotic group which decreased thereafter. This was also in line with the unchanged or increased abundance of
Candida in both low and high vaginal regions over time in the placebo patients, but reduced abundance of
Candida in patients consuming probiotics. Probiotics have been associated with inhibitory activities against a myriad of human pathogens [
27] and anti-inflammatory properties due to the production of inflammation-inhibitive metabolites [
28].