Women’s health and the microbiome

A targeted combination of the beneficial bacterium Lacticaseibacillus rhamnosus CA15, plant extracts of saffron and olive leaves, and vitamins B3 and B7

The combination of the probiotic bacterium Lacticaseibacillus rhamnosus CA15 (CA15), plant extracts of saffron and olive leaves, and vitamins B3 and B7 was selected on the basis of their scientifically demonstrated positive effects on restoring and maintaining the balance of the vaginal microbiota as well as on the management of symptoms associated with PMS or the menopause.

Clinical study

• 30-day
• randomised
• double-blind
• placebo-controlled
• in a cohort of 60 women with signs and symptoms of vaginal dysbiosis

In the study, oral consumption of the probiotic strain L. rhamnosus CA15 led to a significant improvement in clinical signs and symptoms not only 10 days after the start of treatment (T1) but also 30 days after the end of treatment (T2).

Introduction

The vaginal microbiota (VM) is a dynamic ecosystem consisting of bacteria, viruses, archaea, fungi and protozoa (1, 2). Under physiological conditions the VM plays a key role in women’s health as a defender against pathogenic microorganisms. It is generally reported that in healthy women of reproductive age a balanced VM is dominated by lactobacilli (3).

Through the production of metabolites (e.g. lactic acid) and antimicrobial substances such as bacteriocins and hydrogen peroxide, and through interaction with the host’s innate immune system, lactobacilli can protect the vaginal mucosa from potentially harmful microorganisms (4). Where the VM is not lactobacillus-dominant, a high risk of adverse health outcomes is reported. A reduction or loss of lactobacilli is associated in particular with vaginal infections such as bacterial vaginosis (BV), vulvovaginal candidiasis (VVC), mixed vaginitis, sexually transmitted infection (STI) and spontaneous preterm birth (1–3).

Standard treatment of BV involves the use of metronidazole (topical or oral) or clindamycin (topical) (4), while polyenes (e.g. nystatin) and azoles are usually given in the case of VVC (5).

Unfortunately, antibiotics and antimycotics are not always effective, because the microorganisms responsible for BV and VVC can resist by forming biofilms or acquiring resistance (6, 7). In addition, antibiotics that do not act selectively against pathogens can adversely affect endogenous lactobacilli and consequently potentially promote recurrence of BV and VVC (8). On the basis of this evidence, alternative strategies are needed to balance the VM and maintain a healthy vaginal ecosystem. Data from clinical studies and meta-analyses suggest that probiotics may play a positive role in the treatment of vaginal dysbiosis (9–11).

The present randomised, double-blind, placebo-controlled study was carried out to evaluate the efficacy of the potential probiotic strain L. rhamnosus CA15, administered orally, for the treatment of vaginal dysbiosis. Accordingly, the women’s clinical parameters and subjective vaginal symptoms were assessed.

Methods

The study enrolled 60 healthy women of reproductive age (18–45 years) with vaginal signs and symptoms of vaginal infections, confirmed by clinical and microbiological analyses.

Patients assigned to the active group took 1 oral capsule for 10 consecutive days. Each active capsule contained 10x10^9 CFU/g of the probiotic strain L. rhamnosus CA15. The placebo consisted of an identical capsule containing maize starch as an excipient. Examinations of each woman were scheduled at three time points: baseline (T0), 10 days after the start of treatment (T1) and 30 days after the end of treatment (T2).

In addition, quality of life was assessed using the WHO quality of life questionnaire at the start of treatment (T0) and 30 days after the end of treatment (T2).

Results

Diagnostic parameters

The results for clinical signs and symptoms recorded at sampling times T0, T1 and T2 are shown in Fig. 1 (panels A and B).

Compared with baseline (T0), a statistically significant reduction in the symptom-related score reported by the women themselves was seen in the active group 10 days after the start of treatment (T0 vs. T1). Essentially unchanged results were obtained 30 days after the end of treatment (T0 vs. T2).

On the other hand, the scores recorded in the placebo group at both sampling times T1 and T2 were much closer to the scores observed at T0 and were not statistically significant (Fig. 1B).

In addition, a significant reduction in the Nugent score was found in the active group at both sampling times T1 and T2. More interestingly, at sampling time T2 all participants assigned to the active group had a Nugent score between 0 and 3. The scores of this group always showed a negative correlation over time. The scores of the placebo group did not correlate, as they showed only a slight and non-significant fluctuation at T1.

Composition of the bacterial biota by number of microorganisms

Overall, at baseline (T0) bacteria with potentially imbalanced pathogenic microorganisms and a reduced density of microbial cells predominated in all participants in both the active and placebo groups. In the active group, oral administration of strain CA15 for 10 days (T1) statistically significantly reduced the number of pathogens studied. Conversely, a significant increase in the number of lactobacilli of approximately 4 log units was found. Thirty days after the end of treatment (T2) the compositions of the vaginal microbiota were quite stable and similar to those observed at sampling time T1. In fact, when the data obtained at sampling times T1 and T2 were compared, no significant differences in the composition of the bacterial biota were found, which suggests that the balanced state reached at T1 was not disturbed. Different behaviour was found in the placebo group, in which no statistically significant differences in the composition of the bacterial biota were found. Throughout the study a high number of potentially pathogenic bacteria and a low cell density of lactobacilli were found.

Conclusion

Oral consumption of the strain L. rhamnosus CA15 led to a significant improvement in clinical signs and symptoms not only 10 days after the start of treatment (T1) but also 30 days after the end of treatment (T2). The strain CA15 was a safe and effective approach for restoring and maintaining a balanced vaginal microbiota.

In vitro studies

Introduction

Bacterial vaginosis affects 23–29% of women of reproductive age. It is estimated that more than 75% of women will experience at least one episode of vulvovaginal candidiasis during their lifetime and that recurrent episodes are very common (12).

In studying the effect of CA15 on vaginal dysbiosis and vaginal discomfort, CA15 was shown to have a broad-spectrum inhibition of the adhesion and proliferation of pathogens, including fungi (i.e. Candida spp.) and bacteria.

In addition, CA15 can inhibit biofilm formation by Candida spp. through displacement and competition for nutrients.

To ensure that CA15 reaches the vaginal tract, this strain was tested in vitro and shown to survive passage through the GI tract and to adhere to intestinal muscle cell lines and to vaginal (VK2/E6E7) and intestinal (Caco-2) cell lines (13).

Other benefits of CA15 for women’s health include its anti-inflammatory and antioxidant activities. CA15 reduces the pro-inflammatory cytokine IL-6 and increases the adaptive immunity cytokine IL-2.

1) Antagonistic activity against pathogens

Antagonistic activity against pathogens whose presence is often associated with various dysbioses of the urogenital tract was assessed using the agar spot test. The strain L. rhamnosus CA15 showed an overall broad spectrum of antagonistic activity, with the highest antimicrobial activity demonstrated against Candida albicans ATCC10231, Candida krusei ATCC 14243, Gardnerella vaginalis ATCC 14018 and Streptococcus agalactiae DSM 2134. On the basis of CFS treatments, the antagonistic activity was in all cases attributed to the production of organic acids.

2) Anti-adhesive activity against biofilms of various Candida species

Anti-adhesive activity of the strain L. rhamnosus CA15 was demonstrated in pre-coating and co-incubation assays. The percentage of anti-adhesion ranged from 87% to 50.1% and from 48.4% to 20.1% in the pre-coating and co-incubation assays. Similarly, in the pre-coating assay, anti-adhesive activity was demonstrated against C. glabrata (87%) and C. albicans (84.6%), followed by C. parapsilosis (75.7%), C. tropicalis (71.6%) and C. krusei (50.1%). In the co-incubation assay the highest anti-adhesive activity was demonstrated against C. tropicalis, with a 48.4% reduction in biofilm formation.

3) Survival under gastrointestinal tract conditions in vitro

Tolerance to lysozyme, acidic conditions and bile salts, as well as the ability to survive during simulated gastrointestinal (GI) transit, were evaluated in vitro. Overall, the strain L. rhamnosus CA15 demonstrated the ability to survive under the stress conditions tested, with a survival rate (SR) higher than 80%. Similarly, the strain L. rhamnosus CA15 was able to survive in acidic conditions (pH 2.0 and 3.0) after both 2 and 4 hours of incubation. As regards tolerance to lysozyme, the strain CA15 was classified as lysozyme-resistant (14), since it showed an SR of 95% and 93% after 30 and 120 minutes of incubation. Tolerance of both 0.5% and 1% ox bile salts, with an SR higher than 80%, was recorded after 2 and 4 h of incubation respectively. An SR higher than 90% was observed after treatment with simulated gastric juice and simulated intestinal fluid. Starting from an initial viable cell count of 9.13 log CFU/ml, the strain L. rhamnosus CA15 showed 9.10 log CFU/ml of viable cells after treatment with simulated gastric juice and 8.74 log CFU/ml after treatment with simulated intestinal fluid.

4) In vitro antioxidant and anti-inflammatory activities and the ability to adhere to Caco-2 and human vaginal epithelial cells

In LPS-treated Caco-2 cell lines, after treatment with the culture filtrate of strain CA15, a significant reduction in IL-6 levels was found, which suggests the strain’s ability to reduce both oxidative stress and inflammation (Fig. 2A). Conversely, treating LPS-treated Caco-2 cell lines with the culture filtrate of strain CA15 was able to induce an increase in IL-2 levels (Fig. 2B).

The strain CA15 also showed anti-inflammatory activity in differentiated human macrophages treated with LPS. Similarly, compared with untreated cells, treatment with the CA15 culture filtrate determined downregulation of the COX-2 and IL-8 genes, while the IL-10 gene was upregulated. In addition, the tested strain showed binding ability to both Caco-2 cell lines and human vaginal epithelial cell lines (VK2/E6E7), with rates of 36.15% ± 0.32 and 34.71% ± 0.09 respectively.

Olive polyphenols and the menopause

It is known that vaginal pH changes considerably over a woman’s lifetime. The drivers of pH variation are oestrogen, glycogen and lactobacilli. In a woman’s active years, oestrogen levels begin to rise with ovarian function – the vaginal epithelium thickens and fills with glycogen, which through endogenous degradation provides a pH close to 5. If oestrogen levels are disturbed, the epithelium loses glycogen, which can upset the pH balance and lead to vaginal dysbiosis (15,16).

The menopause marks the end of a woman’s reproductive phase, driven by a decrease in the production of ovarian hormones such as oestrogen. It is often accompanied by a whole range of not only physical but also psychological symptoms that affect overall health and quality of life, such as hot flushes, sleep disturbances, fatigue, dry skin and reduced libido.

As we age, the balance between bone formation and bone turnover shifts negatively. This problem is accelerated even further in women precisely because of the menopause. The lowest incidence of osteoporosis in Europe is observed in the Mediterranean region, probably thanks to the usually high consumption of olive polyphenols (OP). The active and main component of OP is oleuropein with hydroxytyrosol and tyrosol. Oleuropein has an aromatic ring that is similar to oestrogen, and so it can bind to binding sites on oestrogen β-receptors.

Its effect in the menopause was studied in a 12-month intervention study with two parallel groups in 64 postmenopausal women. The placebo group received 1 g of Ca daily and the treatment group 250 mg of olive leaf extract and 1 g of calcium daily. The primary aim was to examine its effect on bone turnover in postmenopausal women with reduced bone mass. The secondary aim was to examine the effect on bone mineral density and serum lipid profiles in the same cohort.

Results

1) Bone turnover

A significant effect was observed in the primary endpoint, i.e. osteocalcin levels. A 32% increase in osteocalcin level was observed in the treated group after 12 months of olive leaf extract supplementation compared with placebo (Fig. 3). At the same time osteocalcin fell by 6% in comparison with placebo.

Likewise, the bone balance ratio (OC/CTX) fell significantly in the placebo group, while no significant change was observed in the active group. OC/CTX is a relevant indicator of the balance of bone turnover. A fall in it points to higher bone resorption activity.

The improved bone balance ratio is also reflected in DEXA measurement. Specifically, after 12 months, BMD in the cervical and lumbar spine decreased in the placebo group, while it remained stable in the active group.

2) Blood lipid profile

Serum lipid levels improved in the active group compared with baseline after 12 months (Fig. 4). A significant effect was observed for total cholesterol, LDL cholesterol and triglycerides. Total cholesterol fell by 11% in the active group and was significantly lower than in the placebo group. LDL cholesterol fell by 21% in the active group and was significantly lower than in the placebo group. In addition, in the active group triglyceride levels fell by 4%, whereas in the placebo group they rose by 15%.

Results

Clinical studies have shown that olive leaf extract can raise osteocalcin levels, ease joint pain and improve the blood lipid profile, and that it offers a natural, non-oestrogenic alternative for managing postmenopausal symptoms, thereby improving the quality of life of women after the menopause.

Saffron extract and PMS

Premenstrual syndrome (PMS) is one of the most common health problems in women, affecting 20–40% of women of reproductive age. It is a group of chronic, cyclical disorders related to menstruation, characterised by emotional, behavioural and physical symptoms in the second half (the luteal phase) of the menstrual cycle (18). There is a body of evidence pointing to changes in serotonergic transmission in the central nervous system in PMS. PMS is therefore thought to be partly linked to impaired serotonergic transmission. This possibility is supported by the positive therapeutic effect of serotonergic inhibitors in women suffering from PMS, although their use also brings many undesirable effects (19).

A serotonergic mechanism is thought to be involved in the antidepressant effect of saffron (Crocus sativus L.), so in 2007 a double-blind, placebo-controlled study was carried out to find out whether saffron can ease PMS symptoms. The study enrolled 50 women aged 20 – 45 with regular menstrual cycles and experience of PMS symptoms for at least 6 months. They were divided into two groups: an active group taking capsules containing 30 mg/day of saffron and a group taking placebo capsules, over two menstrual cycles (cycles 3 and 4). Cycles 1 and 2 were observational, to confirm PMS.

Results

The study found saffron to be effective in easing PMS symptoms. A significant difference in the effect of saffron was observed in cycles 3 and 4 in total daily premenstrual symptoms (Fig. 5) and on the Hamilton Depression Rating Scale.

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