Basic science
DOI: 10.1111/1471-0528.13072 www.bjog.org
Differential expression of lactic acid isomers, extracellular matrix metalloproteinase inducer, and matrix metalloproteinase-8 in vaginal fluid from women with vaginal disorders J Beghini,a,b IM Linhares,a,c PC Giraldo,b WJ Ledger,a SS Witkina a
Division of Immunology and Infectious Diseases, Department of Obstetrics and Gynecology, Weill Cornell Medical College, New York, NY, USA b Department of Gynecology and Obstetrics, University of Campinas, Campinas, Sao Paulo, Brazil c Department of Gynecology and Obstetrics, University of Sao Paulo Medical School, Sao Paulo, Brazil Correspondence: Dr SS Witkin, Department of Obstetrics and Gynecology, Weill Cornell Medical College, 525 East 68th Street, New York, NY 10065, USA. Email [email protected] Accepted 15 July 2014. Published Online 8 September 2014.
Objective Do metabolites in vaginal samples vary between women
with different vaginal disorders. Design Cross-sectional study. Setting Campinas, Brazil. Sample Seventy-seven women (39.9%) with no vaginal disorder,
52 women (26.9%) with vulvovaginal candidiasis (VVC), 43 women (22.3%) with bacterial vaginosis (BV), and 21 women (10.9%) with cytolytic vaginosis (CTV). Method Concentrations of D- and L-lactic acid, extracellular
matrix metalloproteinase inducer (EMMPRIN), and matrix metalloproteinase-8 (MMP-8), and the influence of Candida albicans on EMMPRIN production by cultured vaginal epithelial cells, were determined by enzyme-linked immunosorbent assay (ELISA). Associations were determined by the Mann–Whitney Utest and by Spearman’s rank correlation test. Main outcome measures Metabolite levels and their correlation
with diagnoses.
were elevated in CTV (P = 0.0116). EMMPRIN and MMP-8 concentrations were elevated in VVC (P < 0.0001). EMMPRIN and L-lactic acid concentrations (P ≤ 0.008), but not EMMPRIN and D-lactic acid, were correlated in all groups. EMMPRIN also increased in proportion with the ratio of L- to D-lactic acid in controls and in women with BV (P ≤ 0.009). Concentrations of EMMPRIN and MMP-8 were correlated in controls and women with VVC (P ≤ 0.0002). Candida albicans induced EMMPRIN release from vaginal epithelial cells. Conclusions Vaginal secretions from women with BV are deficient
in D- and L-lactic acid, women with VVC have elevated EMMPRIN and MMP-8 levels, and women with CTV have elevated L-lactic acid levels. These deviations may contribute to the clinical signs, symptoms, and sequelae that are characteristic of these disorders. Keywords Bacterial vaginosis, cytolytic vaginosis, D-lactic acid,
extracellular matrix metalloproteinase inducer, L-lactic acid, matrix metalloproteinase-8, vulvovaginal candidiasis.
Results Vaginal concentrations of D- and L-lactic acid were reduced from control levels in BV (P < 0.0001); L-lactic acid levels Please cite this paper as: Beghini J, Linhares IM, Giraldo PC, Ledger WJ, Witkin SS. Differential expression of lactic acid isomers, extracellular matrix metalloproteinase inducer, and matrix metalloproteinase-8 in vaginal fluid from women with vaginal disorders. BJOG 2015;122:1580–1585.
Introduction Bacterial vaginosis (BV) and vulvovaginal candidiasis (VVC) are the first and second most frequent vulvovaginal disorders in women of reproductive age.1 BV is characterised by alterations in the vaginal bacterial microbiota. Lactobacillus species become greatly reduced in number, whereas concentrations
1580
of Gardnerella vaginalis, Mycoplasma hominis, and grampositive anaerobic rods are greatly elevated.2 VVC is mostly caused by an overgrowth of Candida albicans, although other Candida species are sometimes involved. There is typically no change in the composition of the vaginal microbiota, and vaginal pH remains unchanged.3 Two additional common microbial vaginal disorders are trichomoniasis and cytolytic
ª 2014 Royal College of Obstetricians and Gynaecologists
Altered metabolites in vaginal disorders
vaginosis (CTV). CTV is a disorder characterised by a large increase in the number of vaginal Lactobacilli, lysis of vaginal epithelial cells, absence of inflammation, and symptoms of discharge and burning, and/or itching.4,5 The mechanisms responsible for the initiation and persistence of each of these disorders remain incompletely determined. Recent studies on interactions between the vaginal microbiota and vaginal epithelial cells have provided an enhanced appreciation of the role of individual components in maintaining vaginal wellbeing. L-Lactic acid is produced by vaginal epithelial cells, Lactobacilli, and other lactic acid-producing bacteria.6 In addition to maintaining vaginal acidity, L-lactic acid participates in a number of immunological activities. It activates the Th17 subclass of T lymphocytes,7 induces vaginal epithelial cells to release pro-inflammatory cytokines in the presence of synthetic viral RNA,8 modulates dendritic cell activation,9 and is a potent inhibitor of the bacteria associated with BV.10 D-lactic acid is produced almost exclusively by bacteria. Of the four most prevalent species of vaginal Lactobacilli—Lactobacillus crispatus, Lactobacillus iners, Lactobacillus gasseri, and Lactobacillus jensenii—all but L. iners produce D-lactic acid.11 The function(s) of D-lactic acid in the vagina has scarcely been investigated. A recent report from our laboratory suggests that the ratio of L- to D-lactic acid in vaginal fluid may regulate the local production of extracellular matrix metalloproteinase inducer (EMMPRIN) by vaginal epithelial cells.11 EMMPRIN is an essential co-factor for monocarboxylate transporter-1, the receptor responsible for the regulation of intracellular lactic acid levels and prevention of acid-mediated cell death.12 EMMPRIN is also a potent inducer of matrix metalloproteinase-8 (MMP-8).13 MMP-8 is capable of degrading the cervical plug and, thereby, allowing bacteria to gain access to the upper genital tract.14 In this article we characterise the vaginal concentrations of D- and L-lactic acid, EMMPRIN, and MMP-8 in women with BV, VVC, and CTV, and compare the results with the levels present in women with no vaginal disorder. We then hypothesise on the influence of these findings on the pathogenesis and possible novel treatment of each disorder.
Methods Subjects A total of 209 premenopausal sexually active women attending the outpatient genital infections clinic in the Department of Obstetrics and Gynecology at Campinas University between May and November 2013 were eligible for analysis. Exclusion criteria included menses, pregnancy, current use of antibiotics, antifungal medications, corticosteroids, or any other immunosuppressive medication or any vaginal product, and women whose last vaginal sexual
ª 2014 Royal College of Obstetricians and Gynaecologists
intercourse was 1 45.5 ≥1 abortion 11.7 Contraception (%) Pill 53.2 Condom 9.1 IUD 2.6 Other/none 31.2 Recent sex partners (last 6 months) (%) 0 11.7 1 85.7 >1 2.6 Average no. vaginal intercourse 9.5 (6.1) per month (SD) Smoker (%) 13
1582
Vulvovaginal candidiasis n = 52
Bacterial vaginosis n = 43
Cytolytic vaginosis n = 21
28 (7.3) 69.2 10.3 (4.2)
29 (8.6) 50.0 9.2 (3.1)
32 (9.0) 71.4 9.5 (3.7)
55.8 21.1 23.1 15.4
30.9 16.7 52.4 19.1
52.4 19.0 28.8 9.5
57.8 19.2 1.9 19.2
50.0 7.1 7.1 35.8
47.7 19.0 0 28.5
7.7 82.7 9.6 8.8 (5.8)
7.1 85.8 7.1 10.1 (6.8)
9.5 80.9 4.8 6.0 (5.4)
19.2
28.6
14.3
ª 2014 Royal College of Obstetricians and Gynaecologists
Altered metabolites in vaginal disorders
Table 2. Concentration of vaginal metabolites in healthy controls and women with bacterial vaginosis, vulvovaginal candidiasis, and cytolytic vaginosis Assay
Diagnosis
L-Lactic
acid
D-Lactic
acid
EMMPRIN
MMP-8
Controls BV VVC CTV Controls BV VVC CTV Controls BV VVC CTV Controls BV VVC CTV
Median value (range) 0.11 mmol/l 0.02 mmol/l 0.13 mmol/l 0.31 mmol/l 0.03 mmol/l
DOI: 10.1111/1471-0528.13072 www.bjog.org
Differential expression of lactic acid isomers, extracellular matrix metalloproteinase inducer, and matrix metalloproteinase-8 in vaginal fluid from women with vaginal disorders J Beghini,a,b IM Linhares,a,c PC Giraldo,b WJ Ledger,a SS Witkina a
Division of Immunology and Infectious Diseases, Department of Obstetrics and Gynecology, Weill Cornell Medical College, New York, NY, USA b Department of Gynecology and Obstetrics, University of Campinas, Campinas, Sao Paulo, Brazil c Department of Gynecology and Obstetrics, University of Sao Paulo Medical School, Sao Paulo, Brazil Correspondence: Dr SS Witkin, Department of Obstetrics and Gynecology, Weill Cornell Medical College, 525 East 68th Street, New York, NY 10065, USA. Email [email protected] Accepted 15 July 2014. Published Online 8 September 2014.
Objective Do metabolites in vaginal samples vary between women
with different vaginal disorders. Design Cross-sectional study. Setting Campinas, Brazil. Sample Seventy-seven women (39.9%) with no vaginal disorder,
52 women (26.9%) with vulvovaginal candidiasis (VVC), 43 women (22.3%) with bacterial vaginosis (BV), and 21 women (10.9%) with cytolytic vaginosis (CTV). Method Concentrations of D- and L-lactic acid, extracellular
matrix metalloproteinase inducer (EMMPRIN), and matrix metalloproteinase-8 (MMP-8), and the influence of Candida albicans on EMMPRIN production by cultured vaginal epithelial cells, were determined by enzyme-linked immunosorbent assay (ELISA). Associations were determined by the Mann–Whitney Utest and by Spearman’s rank correlation test. Main outcome measures Metabolite levels and their correlation
with diagnoses.
were elevated in CTV (P = 0.0116). EMMPRIN and MMP-8 concentrations were elevated in VVC (P < 0.0001). EMMPRIN and L-lactic acid concentrations (P ≤ 0.008), but not EMMPRIN and D-lactic acid, were correlated in all groups. EMMPRIN also increased in proportion with the ratio of L- to D-lactic acid in controls and in women with BV (P ≤ 0.009). Concentrations of EMMPRIN and MMP-8 were correlated in controls and women with VVC (P ≤ 0.0002). Candida albicans induced EMMPRIN release from vaginal epithelial cells. Conclusions Vaginal secretions from women with BV are deficient
in D- and L-lactic acid, women with VVC have elevated EMMPRIN and MMP-8 levels, and women with CTV have elevated L-lactic acid levels. These deviations may contribute to the clinical signs, symptoms, and sequelae that are characteristic of these disorders. Keywords Bacterial vaginosis, cytolytic vaginosis, D-lactic acid,
extracellular matrix metalloproteinase inducer, L-lactic acid, matrix metalloproteinase-8, vulvovaginal candidiasis.
Results Vaginal concentrations of D- and L-lactic acid were reduced from control levels in BV (P < 0.0001); L-lactic acid levels Please cite this paper as: Beghini J, Linhares IM, Giraldo PC, Ledger WJ, Witkin SS. Differential expression of lactic acid isomers, extracellular matrix metalloproteinase inducer, and matrix metalloproteinase-8 in vaginal fluid from women with vaginal disorders. BJOG 2015;122:1580–1585.
Introduction Bacterial vaginosis (BV) and vulvovaginal candidiasis (VVC) are the first and second most frequent vulvovaginal disorders in women of reproductive age.1 BV is characterised by alterations in the vaginal bacterial microbiota. Lactobacillus species become greatly reduced in number, whereas concentrations
1580
of Gardnerella vaginalis, Mycoplasma hominis, and grampositive anaerobic rods are greatly elevated.2 VVC is mostly caused by an overgrowth of Candida albicans, although other Candida species are sometimes involved. There is typically no change in the composition of the vaginal microbiota, and vaginal pH remains unchanged.3 Two additional common microbial vaginal disorders are trichomoniasis and cytolytic
ª 2014 Royal College of Obstetricians and Gynaecologists
Altered metabolites in vaginal disorders
vaginosis (CTV). CTV is a disorder characterised by a large increase in the number of vaginal Lactobacilli, lysis of vaginal epithelial cells, absence of inflammation, and symptoms of discharge and burning, and/or itching.4,5 The mechanisms responsible for the initiation and persistence of each of these disorders remain incompletely determined. Recent studies on interactions between the vaginal microbiota and vaginal epithelial cells have provided an enhanced appreciation of the role of individual components in maintaining vaginal wellbeing. L-Lactic acid is produced by vaginal epithelial cells, Lactobacilli, and other lactic acid-producing bacteria.6 In addition to maintaining vaginal acidity, L-lactic acid participates in a number of immunological activities. It activates the Th17 subclass of T lymphocytes,7 induces vaginal epithelial cells to release pro-inflammatory cytokines in the presence of synthetic viral RNA,8 modulates dendritic cell activation,9 and is a potent inhibitor of the bacteria associated with BV.10 D-lactic acid is produced almost exclusively by bacteria. Of the four most prevalent species of vaginal Lactobacilli—Lactobacillus crispatus, Lactobacillus iners, Lactobacillus gasseri, and Lactobacillus jensenii—all but L. iners produce D-lactic acid.11 The function(s) of D-lactic acid in the vagina has scarcely been investigated. A recent report from our laboratory suggests that the ratio of L- to D-lactic acid in vaginal fluid may regulate the local production of extracellular matrix metalloproteinase inducer (EMMPRIN) by vaginal epithelial cells.11 EMMPRIN is an essential co-factor for monocarboxylate transporter-1, the receptor responsible for the regulation of intracellular lactic acid levels and prevention of acid-mediated cell death.12 EMMPRIN is also a potent inducer of matrix metalloproteinase-8 (MMP-8).13 MMP-8 is capable of degrading the cervical plug and, thereby, allowing bacteria to gain access to the upper genital tract.14 In this article we characterise the vaginal concentrations of D- and L-lactic acid, EMMPRIN, and MMP-8 in women with BV, VVC, and CTV, and compare the results with the levels present in women with no vaginal disorder. We then hypothesise on the influence of these findings on the pathogenesis and possible novel treatment of each disorder.
Methods Subjects A total of 209 premenopausal sexually active women attending the outpatient genital infections clinic in the Department of Obstetrics and Gynecology at Campinas University between May and November 2013 were eligible for analysis. Exclusion criteria included menses, pregnancy, current use of antibiotics, antifungal medications, corticosteroids, or any other immunosuppressive medication or any vaginal product, and women whose last vaginal sexual
ª 2014 Royal College of Obstetricians and Gynaecologists
intercourse was 1 45.5 ≥1 abortion 11.7 Contraception (%) Pill 53.2 Condom 9.1 IUD 2.6 Other/none 31.2 Recent sex partners (last 6 months) (%) 0 11.7 1 85.7 >1 2.6 Average no. vaginal intercourse 9.5 (6.1) per month (SD) Smoker (%) 13
1582
Vulvovaginal candidiasis n = 52
Bacterial vaginosis n = 43
Cytolytic vaginosis n = 21
28 (7.3) 69.2 10.3 (4.2)
29 (8.6) 50.0 9.2 (3.1)
32 (9.0) 71.4 9.5 (3.7)
55.8 21.1 23.1 15.4
30.9 16.7 52.4 19.1
52.4 19.0 28.8 9.5
57.8 19.2 1.9 19.2
50.0 7.1 7.1 35.8
47.7 19.0 0 28.5
7.7 82.7 9.6 8.8 (5.8)
7.1 85.8 7.1 10.1 (6.8)
9.5 80.9 4.8 6.0 (5.4)
19.2
28.6
14.3
ª 2014 Royal College of Obstetricians and Gynaecologists
Altered metabolites in vaginal disorders
Table 2. Concentration of vaginal metabolites in healthy controls and women with bacterial vaginosis, vulvovaginal candidiasis, and cytolytic vaginosis Assay
Diagnosis
L-Lactic
acid
D-Lactic
acid
EMMPRIN
MMP-8
Controls BV VVC CTV Controls BV VVC CTV Controls BV VVC CTV Controls BV VVC CTV
Median value (range) 0.11 mmol/l 0.02 mmol/l 0.13 mmol/l 0.31 mmol/l 0.03 mmol/l
