Nádia Cristina Polpeta PT, MsC1 ;
Paulo César Giraldo, MD, PhD1;
Cássia Raquel Teatin Juliato, MD, PhD1;
Fernando Guimarães, PhD1
Marcela Grigol Bardin PT, MsC1
Joziani Beghini, MD, PhD1
Filiation:
1 Department of Gynecology and Obstetrics - University of Campinas, Campinas, São
Abstract
Objective: to examine variation in L-lactic acid (L-LAC) concentrations and measurements of pH and vaginal temperatures in women with and without vulvovaginitis (VV) undergoing pelvic floor muscle training (PFMT).
Design: Clinical Trial
Location: Campinas, Brazil
Sample: 80 women.- 40 women without vulvovaginal infection (control group) and 40 women with VV – 22 with vulvovaginal candidiasis (VVC)- and 18 with bacterial vaginosis (BV).
Methods: Vaginal L-Lac concentrations were obtained before and after PFMT and determined by enzyme-linked immunosorbent assay (ELISA). Vaginal pH was measured by pH strip (MERCK® KGaA, DARMSTADT, Germany) and vaginal temperature was recorded with a digital thermometer (Walgreens pharmacist recommended Ltda). Repeated measures ANOVA, Tukey’s Test, a paired t-test, Fisher's Exact Test and the chi-square test were the tests used for stastical analysis. Main methods for outcome measurement: Levels of L-lactic acid, pH, vaginal temperature were observed before and after PFMT.
Results: L-Lac concentrations and pH increased immediately after PFMT in total of the 80 women (L-Lac p=0.0001 and pH = 0.0006), in the control group (p˂0.0001 L- Lac and pH) and in VVC patients (L-Lac p˂0.0001 and pH p=0.0062), but not in BV patients. The vaginal temperature did not change significantly in any of the groups (p˃0,05).
Conclusions: PFMT increases L-Lac concentration and vaginal pH in women wtih VVC and without vulvovaginal infections.
Keywords: L-lactic acid, vulvovaginitis, vaginal candidiasis, bacterial vaginosis, pelvic floor, physical therapy.
Introduction
The balance of vaginal microbiota is responsible for maintaining local pH control at acidity levels ranging from 3.8 to 4.5, inhibiting anaerobic bacterial growth which produces proteolytic enzymes. These are key factors in protecting the vaginal microbiota against the development of vulvovaginitis, endocervicitis, including sexually transmitted diseases (1).
Lactic acid has a major role in vaginal acidification. The acid is present in its optical isomers levo (L) and dextro (D). Lactobacilli are the largest producers of lactic acid in the vaginal cavity, generating the D and L isoforms of lactic acid. Other bacteria
such as: Atopobium sp, Megasphaera sp and Leptotrichia sp, as well as vaginal
epithelial cells are also capable of producing low concentrations of L-lactic acid (L-Lac)
(2) (3). Until now, D-lactic acid has only been known as a vaginal acidifying agent (4).
In contrast, L-Lac participates in a number of immunologic activities. It activates the Th17 lymphocyte pathway, induces vaginal epithelial cells to release proinflammatory cytokines in the presence of synthetic viral RNA (5), and inhibits bacteria associated with bacterial vaginosis (BV)(6).
Vulvovaginal candidiasis (VVC) and BV are two types of vulvovaginitis that have a high prevalence worldwide (7). Both vulvovaginal infections are related to a misbalance of vaginal homeostasis and may have significant consequences for a woman, including pain, itching, foul odor and even marital maladjustments (8). The usually low vaginal concentrations of L-Lac in BV patients (9), may explain why BV is more commonly associated with other infections such as Chlamydia, Neisseria gonorrhoeae Trichomonas and HPV (Human Papillomavirus) and is especially related to a higher probability of acquiring HIV (Human Immunodeficiency Virus) (10, 11).
Recent discoveries have suggested that microbicidal activity of vaginal acidity and competition of lactobacilli inhibiting the proliferation of other bacteria have broader implications, involving innate immune response of the vaginal epithelium, where lactic acid could participate in maintaining homeostasis of this complex
ecosystem (5). Since L-Lac maintains the balance of the vaginal environment, it would
be of interest to find a way to stimulate lactic acid production.
Physical therapy has grown over the past years, showing excellent results in the field of women’s health: urinary incontinence (12), chronic pelvic pain(13), sexual dysfunctions (14), vulvodynia(15). In other clinical areas, inspiratory muscle training
has proven to be efficient at reducing pulmonary infections (16) and aerobic exercises decrease the number of respiratory infections of the upper airways(17).
Little is known about the effect of physical therapy on the vaginal ecosystem and upon vulvovaginitis. It may be assumed that pelvic floor muscle training (PFMT) provided by physical therapy could improve homeostasis of the vaginal environment. PFMT is the most widely used resource in physical therapy (12, 14, 15). It is a practical intervention that has good results in uroginecology, stimulating lactic acid production. Pelvic floor training may be used particularly as an adjuvant treatment for vulvovaginal infections and in the prevention of their recurrences.
The aim of this study was to observe the variation in L-Lac concentrations, pH measurements, and vaginal temperatures in women with and without vulvovaginitis (VV) undergoing pelvic floor muscle training (PFMT).
Methods Subjects
A clinical trial was conducted with 120 sexually active women aged 18 to 45 years, with and without a history of vulvovaginal infection, seen at the Outpatient Facility of Genital Infections and outpatient Family Planning Facility of the Department of Obstetrics and Gynecology of the State University of Campinas School of Medicine (DTG/FCM/Unicamp) from August 2014 to July 2015.
Women using antibiotics, antifungals, corticosteroids, imunossupressants or vaginal creams in the last 2 weeks, those who had engaged in sexual activity in the last 48 hours or were having their menstrual period, pregnant women, patients with urinary and/or fecal incontinence, vaginal stenosis, pelvic organ prolapse, psychiatric disease, malignancies, chronic degenerative disease, muscular dystrophy and osteomuscular restriction were excluded from the study.
Also excluded were women who did not understand how to contract the pelvic floor muscles (PFM) (n=3), those diagnosed with cytolytic vaginosis (n= 10) and aerobic vaginitis (n= 2), VVC+BV (n= 4) or women with incomplete diagnosis (n=3), those without vulvovaginal infection and with a great amount of leukocytes in the vaginal smear (n=11), scanty vaginal flora (n=4), presence of spermatozoa (n=3) (Flow chart).
Eighty (80) women remained in the study: 40 women without vulvovaginal infection (control group) and 40 with VV. Of these patients with vulvovaginal infection,
22 had vulvovaginal candidiasis (VVC) and 18 had bacterial vaginosis (BV) (Flow chart).
The study was approved by the Research Ethics Committee at the University of Campinas in April, 2013 under nº 260.401 and in August 2014 addendum nº 756.429 was approved. All participants signed a written informed consent term.
Data Collection
After signing the consent term, all women responded the sociodemographic data form and were questioned about a history of vaginal discharge. Then these women were required to lie in a supine position for: vaginal sample collection;
evaluation of the PFM; perform PFMT. All data were collected by the main researcher
and identified by the number of research subject. Testing was performed with the researcher blinded to clinical data.
Vaginal sample collection
Vaginal samples were collected by swabbing the lateral wall with sterile cotton swabs. VVC was detected in symptomatic women by the presence of yeast or pseudohyfae in the vaginal discharge by microscopy, confirmed by inoculation of a Sabouraud CC dextrose agar wedge (Becton-Dickinson, Sparks, MD) and by optical microscopy analysis of Gram stain. BV was identified by the Nugent criteria ≥7 (18). Women from the control group had no complaints of vaginal discharge and vaginal content smear with no alterations (Lactobacillus predominat flora and no signs of inflammation) plus negative fungal culture.
Vaginal pH was assessed, before and immediateley after PFMT, with a strip for pH identification – PAP INDIC pH 4.0 – 7.0 (MERCK® KGaA, DARMSTADT, Germany). Variation included values from 4 to 7 with intervals of 0.3. The pH strip was inserted into the vaginal cavity, in contact with the vaginal wall and avoiding contact with the cervical mucus. After 1 minute, the strip was removed and pH was determined (19).
Vaginal temperature was measured, before and immediately after PFMT, with a digital thermometer (Walgreens pharmacist recommended Ltda) used with a disposable plastic sheath (Walgreens pharmacist recommended Ltda). A digital thermometer was introduced into the vaginal cavity which beeped after the temperature was measured in Fahrenheit (0F).
Material was also collected from the vaginal wall to analyze L-Lac, before and after PFMT. A sterile swab was used to remove smear from the right vaginal wall in a uniform manner. Then, material was diluted in an eppendorf tube containing 1 ml of 2% PBS (phosphate buffer saline). Subsequently, it was centrifuged for 8 minutes at 12.000 rpm to separate the pellet from the supernatant. The supernatant was stored at -80°C.
Evaluation of PFM
To assess PFM, the PERFECT Scheme was used (20). The manual test was carried out with the woman in the dorsal lithotomy position on the examination table. The chest of the woman was elevated, legs abducted, knees flexed with feet in stirrups. A bidigital vaginal exam was performed. The hand of the examiner was pronated, with the middle and index fingers spread in a scissor-like manner. The test assessed PFM contractions, in the following parameters: endurance (E), resistance (R) and frequency (F). To score strength of PFM contraction, the Oxford Modified Scale was used 0-5 (20).
PFMT
The researcher was positioned next to the woman who remained in the supine position. Bidigital vaginal palpation was performed for proprioception – manual biofeedback. The patient, in supine position, was asked to perform 3 series of 10 rapid contractions, and 2 series of 10 contractions maintained for up to 10 seconds, 10- second intervals between sustained contractions and 1 minute between series. Then, without vaginal palpation, the patient performed 5 sustained contractions for up to 10 seconds and 6 rapid contractions, in 9 different positions. Six exercises were done in the supine position – lower limbs (LL) flexed with feet supported, LL in a “butterfly” position, LL in extension, pelvic antero-retroversion movement, “bridge”, lower limbs interlaced close to the body; and 2 exercises were done with the woman seated and her feet supported in stirrups, and 1 in the erect position (14). There was a total number of 140 contractions during 45 minutes.
Laboratory Analysis
Eppendorf tubes containing frozen supernatant at -80°C were analyzed in the Microbiology laboratory of the Department of Gynecology and Obstetrics of the
State University of Campinas to confirm vaginal L-Lac concentrations determined colorimetrically by the ELISA technique, using commercial kits (Bioassay Systems, Hayward, CA). Values were converted to mM de L-Lac, by reference to a standard curve generated in paralell for each assay. Lower sensitivity limits were 0.02 mM for L-Lac. All samples were analyzed with the researcher blinded to clinical findings.
Statistics
The variation in L-Lac levels, pH and temperature concentrations were obtained, using repeated measures ANOVA, followed by Turkey’s test. A paired t-test was used for a comparison between the time period before and after in the same group. P value p<0.05 was considered significant. SAS (Statistical Analysis System) Version 9.4 for Windows was the computer program used.
The sample power calculation was based on the variable L-Lac in women with and without VV. Number of cases in each group: 40 Control, 22 VVC, 18 BV and average before and after PFM: 0.81mM (Control), 1.04mM (CVV), 0.13 mM (BV) with standard deviation (SD=0.81), and alpha=0.05 - has a power of 90.7%. The general
procedure test power for one-way ANOVA, through the SAS program was the
computer program used.
Results
Analyzing the sociodemographic characteristics, there were no significant statistical differences between groups (Table 1). The majority of women used levonogestrel IUD, was overweight, had more than one pregnancy, had only one sex partner and half of these women led a sedentary lifestyle.
A complaint of previous vaginal discharge was present in all women investigated (VVC and BV). In 68.18% of VVC patients and 50% of BV patients, there were more than 3 episodes per year of vaginal discharge. The duration of discharge complaint in the VVC group was 1-3 years in 63.64% and more than 3 years in 36.37% of patients. In the BV group, 44.45% of women had a complaint lasting from 1 to 3 years and 55.56% had a complaint that lasted more than 3 years.
Assessment of the pelvic floor muscles using the PERFECT scheme,
showed that the BV group had a mean strength of 2.55 (±1.20), which was less than
p=0.044). There was no difference between groups in the endurance, resistance and frequency parameters.
Women were questioned about previous knowledge of pelvic floor muscle contraction. Only 40% had previous knowledge, although none had performed the exercises, and there were no differences between groups.
PFMT increased L-Lac concentration significantly (p=0.0001) and pH (p=0.0006) in the 80 women evaluated (Total). However, it did not influence vaginal temperature in the TOTAL (p=0.81) (Table 2 and Graph).
Comparing L-Lac concentrations before and after PFMT between groups, we observed similar mean values in controls and VVC patients (p=0.37) and lower in women with BV (p=˂0.0001) (Table 2). PFMT increased L-Lac concentrations significantly in controls (p=˂0.0001) and in VVC patients (p=˂0.0001), but there was no significant increase in BV patients (p=0.12) (Graph).
Initially, a more acid mean pH was seen in controls than in VVC and BV patients (p=0.0005 and ˂0.0001, respectively). After PFMT, there was no difference in mean pH between controls and VVC patients, BV patients mantained higher pH (p=0.0001). Analyzing the effect of PFMT on vaginal pH, there was an increase in vaginal pH in controls and in VVC patients (p=0.0001 and 0.0061, respectively). BV patients did not show a significant difference in pH after PFMT exercise (Table 2).
The mean temperature was initially lower in BV patients than in controls and
VVC patients (p=0.017 and 0.033 respectively). After intervention, the groups had similar temperatures, without any significant difference. PFMT did not influence vaginal
temperature. Changes in temperature in the BV, VVC and control groups were not
significant (Table 2).
Comparing PFMT (difference before and after exercise) in L-Lac and pH between groups, the effect was the same in the 80 women, both in controls and VVC patients (p=˃0,05). However, in the 3 groups the difference was significantly higher in the BV group (p˂0.01). There was no difference in vaginal temperature between groups regarding the PFMT effect (Table 2).
Discussion
L-Lac production in the vaginal flora is of extreme importance, owing to acidification, antimicrobial action and proinflammatory immune response in the presence of pathogenic agents (5, 6, 21, 22). BV patients have a lower L-Lac
concentration (4, 9) than controls, which favors the presence of bacteria. In VVC patients, L-Lac concentrations were equal to those observed in healthy women, explaining the acid pH (9).
Our data corroborate these results, since this study showed higher
concentrations of L-Lac isomers in the vaginal flora of women who had no vaginal alterations and in VVC patients, and a lower quantity was observed in the BV patients. However, this is the first study to observe an increase in L-Lac using pelvic floor physical therapy in women with and without vulvovaginal infections.
According to a classification proposed by the American College of Sports Medicine (ACMS), PFMT may be considered a resistance exercise, since it activates local metabolism and promotes an increase in muscle strength and resistance. It recruits both type 1 slow twitch fibers and type 2 fast twitch fibers. The PFM, with the elevator ani as the main muscle, are composed of 70% type 1 fibers (predominantly aerobic metabolism) and 30% type 2 fibers (predominantly anaerobic metabolism) with types IIa and IIb variations. Depending on training intensity, fibers type IIb can be converted into type IIa, which are more resistant to fatigue (23). Training leads to an increased number of mitochondria and capillary density (24).
Vaginal L-Lac is abundantly produced by lactobacillus crispatus and lactobacillus gasseri, and a lower amount is produced by lactobacillus jensenii and lactobacillus iners (4). It is produced in smaller proportions by epithelial cellular metabolism (2). This is an indication that perineal exercise most probably increased basal metabolism. Regional blood flow increased (24), creating favorable conditions for L-Lac production in women without vulvovaginal conditions and in VVC patients.
There was not a significant increase in acid production in patients with BV. The vaginal flora in these women had a lower quantity of lactobacillus. It is unknown whether this was the cause or the effect of BV. Therefore, the insignificant increase observed may be attributed to stimulation from cellular production. As previously mentioned, epithelial cells produce small amounts of L-Lac. Furthermore, the mean strength of muscle contraction was lower in BV patients than in women without vulvovaginal infections. This may interfere in L-Lac production, since less muscle fibers are recruited for contraction, and blood irrigation is decreased in the region (23).
Initial vaginal pH values corresponded to expected values. More acid values were observed in women without infections and VVC patients. It was more alkaline in BV, due to scarce amount of lactic acid, in concordance with previous studies (9).
There was a significant increase in pH in women without vulvovaginal conditions and VVC patients. With increased L-Lac, acidification of the vaginal environment is expected, decreasing pH. Two reasons may explain the increase in pH found.
The first reason is that vaginal exercises were performed by women who were not using lingerie. A previous study of pH measurement showed that a 10-min exposure of the female genitalia to air can immediately cause a twofold increase in vaginal pH, due to a decrease in CO2 pressure in the vaginal lumen (25). Another reason is that an increased vaginal lubrication generated by pelvic floor exercises, elevate transudation of blood plasma and mucus secretions, producing a more alkaline environment. Perineal exercise may improve vaginal lubrication (14), which is an additional physiological confirmation of the effect that occurs immediately after exercise.
In BV, an insignificant decrease in pH was observed and it was maintained above 5.0. Most probably, due to the already alkaline environment, factors including vaginal lubrication and decreased CO2 pressure, are not sufficient to elevate vaginal
pH in these women. A small increase in L-Lac may be the stimulus for a slight decrease in average pH value, which was not significant in either event.
In contrast, initially lower temperatures in BV patients, when compared to controls and VVC patients may be explained by the lower metabolism of BV, lack of lactobacillus and leukocytes. Exercise may increase vaginal temperature, resulting from increased local metabolism, which is observed in aerobic exercises that elevate body temperature (26). A rise in temperature did not occur, which was a positive sign. A high-temperature environment favors humidity and proliferation of microorganisms. In contrast, exercise promoted small insignificant changes benefitting homeostasis of the vaginal environment.
This was the first study to evaluate the influence of PFMT on pH, temperature, and vaginal L-Lac concentrations. Further studies are required to confirm this influence on long-term interventions. Physical therapy may be a natural, simple and cost-effective manner to stimulate improved control of vaginal homeostasis, and assist in the prevention and complementation of treatment for recurrent vaginal infections.
Limitations of this study were the number of women evaluated, particularly the number of BV patients. The influence of PFMT on L-Lac was observed. It would
also be important to understand the influence of exercise on D-lactic acid, in addition to other antimicrobial substances and inflammatory mediators.
In conclusion, PFMT exercises promote an immediate increase in L-Lac and pH concentrations in women with VVC and controls. However, it was not sufficient to modify the same parameters in BV patients in this study.
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