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Vulvodynia and fungal association: A preliminary report
Medical Hypotheses 81 (2013) 228–230
Contents lists available at SciVerse ScienceDirect
Medical Hypotheses journal homepage: www.elsevier.com/locate/mehy
Vulvodynia and fungal association: A preliminary report G. Ventolini ⇑, S.E. Gygax 1, M.E. Adelson 2, D.R. Cool 3 Medical School, Texas Tech University Health Sciences Center – Permian Basin, 800 W 4th Street, Odessa, TX 79763, United States
a r t i c l e
i n f o
Article history: Received 30 January 2013 Accepted 26 April 2013
a b s t r a c t Vulvodynia (vulvar pain syndrome) is a chronic multifactorial disease affecting almost 13 million women in the USA and can lead to morbidity and a reduced quality of life. We hypothesize that an initial microbiological insult in the vagina causes modifications in the biological vaginal milieu and/or an alteration on the lactobacilli flora. The vaginal milieu responds to the insult by developing an inflammatory reaction with abnormal cytokine production. These hypotheses were tested quantifying vaginal lactobacillus and cytokines, in patients with vulvodynia compared to matched healthy controls. Our preliminary data suggest a vaginal flora alteration and an immunological response involving Candida in patients with vulvodynia. Ongoing studies will assist us to clarify these findings. Ó 2013 Elsevier Ltd. All rights reserved.
Introduction Vulvodynia (vulvar pain syndrome) is a chronic, heterogeneous, and multifactorial disease affecting as many as 13 million women in the USA and can lead to morbidity and a reduced quality of life [1]. While recent research activities have created a foundation for understanding the etiology and pathology of this condition, our knowledge is largely incomplete. As in other chronic pain disorders, even the basic biological processes involved in vulvodynia are unclear and further investigation of these processes is vital for constructing a substantive knowledge base. It has been reported that an initial inflammatory response may result in peripheral and central pain sensitization [2]. Cytokines are known to be among the biochemical mediators of inflammation [3].
The Hypothesis We hypothesize that an initial microbiological insult in the vagina (possibly involving a bacteria or fungi) causes modifications in the biological vaginal milieu and/or an alteration on the lactobacilli flora. The vaginal milieu responds to the insult by developing an inflammatory reaction characterized by abnormal cytokine production [4]. ⇑ Corresponding author. Tel.: +1 432 335 5113; fax: +1 432 335 5104. E-mail address: [email protected] (G. Ventolini). Ph.D. Director, Femesis, Women’s Health Research Center - Hamilton, New Jersey. Ph.D. Vice President, Genesis Biotechnology Group - Hamilton, New Jersey. 3 Ph.D., Professor Director, Proteome Analysis Laboratory at Boonshoft School of medicine at Wright State University, Dayton, Ohio. 1 2
0306-9877/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mehy.2013.04.043
These hypotheses were tested in the vaginal milieu using microbiology and cytokines, in a population of patients affected by vulvodynia compared to matched healthy controls.
Evaluation of the hypothesis methods In a double blinded study we collected vaginal samples for bacterial flora analysis and cytokines, between day 22 and 28 of the menstrual period, in patients with no previous sexual intercourse for 3 days, on no medications or contraceptives. A real-time PCR (qPCR) assay was used to determine the presence and relative concentration of the vaginal flora. The qPCR assays identified vaginal Lactobacillus spp. bacteria including Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus iners, and Lactobacillus jensenii. Additionally, it detected the presence of facultative anaerobic bacteria (Gardnerella vaginalis, Atopobium vaginae, BVAB2, Megasphaera spp., Eggerthella spp., Prevotella spp., Ureaplasma urealyticum, Ureaplasma parvum, Mycoplasma hominis, and Mycoplasma genitalium). qPCR analysis of gene transcripts was accomplished with a BioRad iCycler RealTime PCR machine, 2 Taqman Master Mix. For RNA preparation, samples were processed using the TRIzolâ (Invitrogen, Carlsbad, CA) method [Ropp, 2008]. Primer probe sets were designed in house with the software packages Primer ExpressTM v2.0 (Applied Biosystems) and Beacon Designer v2.0 (PREMIER Biosoft International). RealTime PCR data was analyzed using the comparative DDCt method (2 DDCt) [Livak, 2002]. Statistical significance was determined using a two-tailed paired t-test of the sample groups for each gene of interest. Cytokines were analyzed using a multiplex kit for 27 different cytokines. For each specimen, 50 ll of sample was incubated with magnetic beads in a Bio-Rad BioPlex 96 well plate from the kit and
229
G. Ventolini et al. / Medical Hypotheses 81 (2013) 228–230
washed 3 times with BioPlex wash buffer. A multiplexed standard curve was generated for each of the cytokines on the same plate as the samples. The data was normalized to protein concentration using the Bradford protein assay. The plate was analyzed on the BioPlex 200 machine using appropriate detector settings and data exported to an Excel spreadsheet for further analysis by 2-way ANOVA. Microbiological fungal cultures were performed in Sabouraud’s medium. The patient group included females ages 18–60 diagnosed with vulvodynia and currently symptomatic. The control included asymptomatic healthy women without any vulvovaginal condition matched to the study group for age, race and parity. The control group was identified among patients who attend our same university affiliated clinical private practice for their physical examination. Patients willing to participate were given informed consent and agreed to sign and follow the protocol. Exclusion criteria: patients ages <18 or >60, patients having being surgically treated for vulvodynia, or currently on therapy and in remission from vulvodynia, patients diagnosed with cancer or having any medical condition that was not under control including diabetes mellitus, hypertension, collagen disease, hemoglobinopathy, renal insufficiency, depression, anxiety, psychosis and panic attacks. Patients who had neurological or psychiatric conditions interfering with the protocol including post-traumatic stress disorder, Munchausen, eating disorders, tokophobia, alcohol and/ or drug addiction. Additionally patients with fibromyalgia, levator ani syndrome, pelvic floor tension myalgia, interstitial cystitis, endometriosis and irritable bowel syndrome were also excluded. Moreover pregnant patients and patients unable to follow the protocol or unwilling to participate were also excluded. The protocol included the following visits: (1) Exhaustive history and comprehensive assessment of environmental factors, topical vulvovaginal irritants and co morbid conditions acknowledged in the exclusion criteria. Additionally, psychosocial contributing factors, daily stressors, pain assessment other associated symptoms including previous sexual experiences and current sexual activity. (2) Detailed physical and pelvic examination with diagnosis confirmation was done and samples were obtained for cytokines, vaginal lactobacillus and facultative anaerobic bacterial from vaginal secretions and specimens for vaginal fungal cultures. (3) Results were discussed with the patients.
Table 1 Cytokine analysis of vulvosecretions in vulvodynia. Cytokine
Control (ng/ml)
Vulvodynia (ng/ml)
Change
IL-1ra Il-1b Il-2 IL-4 Il-6 Il-7 Il-8 Il-9 Il-10 Il-12 Il-13 Il-15 Il-17 Eotaxin FGF basic G-CSF GM-CF IFN-g IP-10 MCP-1 (MCAF) MIP-1a PDGF-b Mip-1b RANTES TNF-a VEGF
OOR OOR OOR 1.12 ± 0.01 18.80 ± 1.08 6.31 ± 0.73 OOR 10.71 ± 2.06 11.44 ± 1.37 181.49 ± 8.39 23.93 ± 2.72 OOR 1.32 ± 0.07 OOR OOR 27.13 ± ND 2947.25 ± 29 531.56 ± 39.41 16850.00 ± 0 93.11 ± 0 3.66 ± 0 74.02 ± 15 173.32 ± 2.73 74.78 ± 0.78 OOR OOR
OOR OOR OOR 1.87 ± 0.24 18.85 ± 1.05 12.54 ± 2.15 OOR 43.60 ± 10.25 7.26 ± 1.19 52.12 ± 7.67 26.78 ± 5.16 OOR 44.92 ± 3.02 OOR OOR 36.71 ± 5.035 3396.60 ± 241 1518.25 ± 320 5912.52 ± 326.8 65.63 ± 2.59 41.99 ± 9.21 235.47 ± 47.0 24.00 ± 2.64 158.68 ± 33.64 OOR OOR
– – – Increase None Increase – Increase Decrease Decrease None – Increase – – None Increase? Increase Decrease? Decrease? Increase? Increase Decrease Increase – –
OOR, out of range (usually below threshold). ND, not enough samples in range to determine. Data represents mean ± SEM.
Moreover L. iners was present among patients with vulvodynia as well as in controls. In our sampled population it appears that L. iners does not appear to play any role in patients with vulvodynia. Preliminary studies on 13 patient samples (6 normal and 7 vulvodynia) were analyzed with the BioPlex bead based cytokine analysis system. Overall, twenty-seven cytokines were assayed. The Table 1 indicates the increase, decrease or no change for each of these. The vaginal samples obtained from patient with vulvodynia demonstrated an increase of almost 35 times interleukin 17 (IL-17) (P = 0.0001) and at the same time a decrease of 7-fold of the macrophage inflammatory protein 1 beta (Mip-Ib) and 3-fold IL-12 compare to matched controls. Four samples were excluded because of insufficient specimen.
Statistical analysis Discussion All of the results remained blind to the investigators until the sampling was completed. Computerized data collection, data tabulation and data analysis were accomplished by experienced clinical personnel and statisticians of the Department of Mathematics and Statistics at Wright State University. Significance level was set at 0.05 with an 80% power to show differences. The Institutional Review Board approved the study.
Results The 17 patients were divided in 3 groups: (a) patients with symptomatic vulvodynia; (b) patients with vulvodynia in remission; (c) patient with no history of vulvodynia (Controls). All the 17 patients had a negative culture for fungi and were negative by PCR for Atopobium, Megasphera and Leptotrichia. In our sampled population L. crispatus was found only in control patients. None of the patients with symptomatic vulvodynia or in remission had L. crispatus. (P = 0.02). Additionally L. gassery was found in patients with symptomatic or vulvodynia in remission.
Our preliminary data are suggesting the presence of a vaginal flora alteration in patients with vulvodynia. Is it only related to the presence of specific lactobacillus? Since there are different lactobacillus variations on the vaginal microflora was our finding just a coincidence or is it a more consistent result in all patients with vulvodynia [5]? It has been reported that nearly all of the L. crispatus produce hydrogen peroxide (H2O2) and that only 71% of L. gassery produce it [6]. L. crispatus promotes in pregnant patients the stability of the normal microflora and L. gassery and L. iners are more conductive to the occurrence of abnormal vaginal flora [7]. Additionally our preliminary results suggest an immunological response involving Candida in patients with vulvodynia. It has been reported that IL-17 is increased in patients with vaginal candidiasis reaching their pick 14 days after the infection; and that IL-12 and Mip-Ib are reduced in post-infection due to Candida albicans [8] Is it an initial infection by Candida the first vulvodynia event or it is the vulvovaginal colonization by Candida that initiates an immunological response resulting in vulvodynia?
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G. Ventolini et al. / Medical Hypotheses 81 (2013) 228–230
Conclusion Our preliminary data suggest a vaginal flora alteration and an immunological response involving Candida in patients with vulvodynia. Ongoing studies will assist us to clarify these findings. Conflicts of interest No conflicts of interest. References [1] Boardman LA. Sexual pain. Clin Obstet Gynecol 2009;52(4):682–90. [2] Bohm-Starke N. Medical and physical predictors of localized provoked vulvodynia. Acta Obstet Gynecol Scand 2010;98(12):1504–10.
[3] Omoigui S. The biochemical origin of pain: the origin of all pain is inflammation and the inflammatory response. Part 2 of 3-inflammatory profile of pain syndromes. Med Hypotheses 2007;69(6):1169–78. [4] Leclair CM. Differences in primary compared with secondary vestibulodynia by immunohistochemistry. Obstet Gynecol 2011;117(6):1307–13. [5] Gerber S. Immunological and genetic characterization of women with vulvodynia. J Med Life 2008;1(14):432–8. [6] Antonio May AD, The identification of vaginal Lactobacillus species and the demographic and microbiologic characteristics of women colonized by these species. In: Presented in part: international society for sexually transmitted disease research meeting, August 1995, New Orleans (abstract 207). [7] Verstraelen H. Longitudinal analysis of the vaginal microflora in pregnancy suggests that L. crispatus promotes the stability of the normal vaginal microflora and that L. gasseri and/or L. iners are more conducive to the occurrence of abnormal vaginal microflora. BMC Mircrobiol 2009;9:116. [8] Pietrella D, Rachini A, Pines M, Pandey N, Mosci P. Th17 cells and IL-17 in protective immunity to vaginal candidiasis. PLos One 2011;6(7). Epub 2011 Jul 27.
Contents lists available at SciVerse ScienceDirect
Medical Hypotheses journal homepage: www.elsevier.com/locate/mehy
Vulvodynia and fungal association: A preliminary report G. Ventolini ⇑, S.E. Gygax 1, M.E. Adelson 2, D.R. Cool 3 Medical School, Texas Tech University Health Sciences Center – Permian Basin, 800 W 4th Street, Odessa, TX 79763, United States
a r t i c l e
i n f o
Article history: Received 30 January 2013 Accepted 26 April 2013
a b s t r a c t Vulvodynia (vulvar pain syndrome) is a chronic multifactorial disease affecting almost 13 million women in the USA and can lead to morbidity and a reduced quality of life. We hypothesize that an initial microbiological insult in the vagina causes modifications in the biological vaginal milieu and/or an alteration on the lactobacilli flora. The vaginal milieu responds to the insult by developing an inflammatory reaction with abnormal cytokine production. These hypotheses were tested quantifying vaginal lactobacillus and cytokines, in patients with vulvodynia compared to matched healthy controls. Our preliminary data suggest a vaginal flora alteration and an immunological response involving Candida in patients with vulvodynia. Ongoing studies will assist us to clarify these findings. Ó 2013 Elsevier Ltd. All rights reserved.
Introduction Vulvodynia (vulvar pain syndrome) is a chronic, heterogeneous, and multifactorial disease affecting as many as 13 million women in the USA and can lead to morbidity and a reduced quality of life [1]. While recent research activities have created a foundation for understanding the etiology and pathology of this condition, our knowledge is largely incomplete. As in other chronic pain disorders, even the basic biological processes involved in vulvodynia are unclear and further investigation of these processes is vital for constructing a substantive knowledge base. It has been reported that an initial inflammatory response may result in peripheral and central pain sensitization [2]. Cytokines are known to be among the biochemical mediators of inflammation [3].
The Hypothesis We hypothesize that an initial microbiological insult in the vagina (possibly involving a bacteria or fungi) causes modifications in the biological vaginal milieu and/or an alteration on the lactobacilli flora. The vaginal milieu responds to the insult by developing an inflammatory reaction characterized by abnormal cytokine production [4]. ⇑ Corresponding author. Tel.: +1 432 335 5113; fax: +1 432 335 5104. E-mail address: [email protected] (G. Ventolini). Ph.D. Director, Femesis, Women’s Health Research Center - Hamilton, New Jersey. Ph.D. Vice President, Genesis Biotechnology Group - Hamilton, New Jersey. 3 Ph.D., Professor Director, Proteome Analysis Laboratory at Boonshoft School of medicine at Wright State University, Dayton, Ohio. 1 2
0306-9877/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mehy.2013.04.043
These hypotheses were tested in the vaginal milieu using microbiology and cytokines, in a population of patients affected by vulvodynia compared to matched healthy controls.
Evaluation of the hypothesis methods In a double blinded study we collected vaginal samples for bacterial flora analysis and cytokines, between day 22 and 28 of the menstrual period, in patients with no previous sexual intercourse for 3 days, on no medications or contraceptives. A real-time PCR (qPCR) assay was used to determine the presence and relative concentration of the vaginal flora. The qPCR assays identified vaginal Lactobacillus spp. bacteria including Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus iners, and Lactobacillus jensenii. Additionally, it detected the presence of facultative anaerobic bacteria (Gardnerella vaginalis, Atopobium vaginae, BVAB2, Megasphaera spp., Eggerthella spp., Prevotella spp., Ureaplasma urealyticum, Ureaplasma parvum, Mycoplasma hominis, and Mycoplasma genitalium). qPCR analysis of gene transcripts was accomplished with a BioRad iCycler RealTime PCR machine, 2 Taqman Master Mix. For RNA preparation, samples were processed using the TRIzolâ (Invitrogen, Carlsbad, CA) method [Ropp, 2008]. Primer probe sets were designed in house with the software packages Primer ExpressTM v2.0 (Applied Biosystems) and Beacon Designer v2.0 (PREMIER Biosoft International). RealTime PCR data was analyzed using the comparative DDCt method (2 DDCt) [Livak, 2002]. Statistical significance was determined using a two-tailed paired t-test of the sample groups for each gene of interest. Cytokines were analyzed using a multiplex kit for 27 different cytokines. For each specimen, 50 ll of sample was incubated with magnetic beads in a Bio-Rad BioPlex 96 well plate from the kit and
229
G. Ventolini et al. / Medical Hypotheses 81 (2013) 228–230
washed 3 times with BioPlex wash buffer. A multiplexed standard curve was generated for each of the cytokines on the same plate as the samples. The data was normalized to protein concentration using the Bradford protein assay. The plate was analyzed on the BioPlex 200 machine using appropriate detector settings and data exported to an Excel spreadsheet for further analysis by 2-way ANOVA. Microbiological fungal cultures were performed in Sabouraud’s medium. The patient group included females ages 18–60 diagnosed with vulvodynia and currently symptomatic. The control included asymptomatic healthy women without any vulvovaginal condition matched to the study group for age, race and parity. The control group was identified among patients who attend our same university affiliated clinical private practice for their physical examination. Patients willing to participate were given informed consent and agreed to sign and follow the protocol. Exclusion criteria: patients ages <18 or >60, patients having being surgically treated for vulvodynia, or currently on therapy and in remission from vulvodynia, patients diagnosed with cancer or having any medical condition that was not under control including diabetes mellitus, hypertension, collagen disease, hemoglobinopathy, renal insufficiency, depression, anxiety, psychosis and panic attacks. Patients who had neurological or psychiatric conditions interfering with the protocol including post-traumatic stress disorder, Munchausen, eating disorders, tokophobia, alcohol and/ or drug addiction. Additionally patients with fibromyalgia, levator ani syndrome, pelvic floor tension myalgia, interstitial cystitis, endometriosis and irritable bowel syndrome were also excluded. Moreover pregnant patients and patients unable to follow the protocol or unwilling to participate were also excluded. The protocol included the following visits: (1) Exhaustive history and comprehensive assessment of environmental factors, topical vulvovaginal irritants and co morbid conditions acknowledged in the exclusion criteria. Additionally, psychosocial contributing factors, daily stressors, pain assessment other associated symptoms including previous sexual experiences and current sexual activity. (2) Detailed physical and pelvic examination with diagnosis confirmation was done and samples were obtained for cytokines, vaginal lactobacillus and facultative anaerobic bacterial from vaginal secretions and specimens for vaginal fungal cultures. (3) Results were discussed with the patients.
Table 1 Cytokine analysis of vulvosecretions in vulvodynia. Cytokine
Control (ng/ml)
Vulvodynia (ng/ml)
Change
IL-1ra Il-1b Il-2 IL-4 Il-6 Il-7 Il-8 Il-9 Il-10 Il-12 Il-13 Il-15 Il-17 Eotaxin FGF basic G-CSF GM-CF IFN-g IP-10 MCP-1 (MCAF) MIP-1a PDGF-b Mip-1b RANTES TNF-a VEGF
OOR OOR OOR 1.12 ± 0.01 18.80 ± 1.08 6.31 ± 0.73 OOR 10.71 ± 2.06 11.44 ± 1.37 181.49 ± 8.39 23.93 ± 2.72 OOR 1.32 ± 0.07 OOR OOR 27.13 ± ND 2947.25 ± 29 531.56 ± 39.41 16850.00 ± 0 93.11 ± 0 3.66 ± 0 74.02 ± 15 173.32 ± 2.73 74.78 ± 0.78 OOR OOR
OOR OOR OOR 1.87 ± 0.24 18.85 ± 1.05 12.54 ± 2.15 OOR 43.60 ± 10.25 7.26 ± 1.19 52.12 ± 7.67 26.78 ± 5.16 OOR 44.92 ± 3.02 OOR OOR 36.71 ± 5.035 3396.60 ± 241 1518.25 ± 320 5912.52 ± 326.8 65.63 ± 2.59 41.99 ± 9.21 235.47 ± 47.0 24.00 ± 2.64 158.68 ± 33.64 OOR OOR
– – – Increase None Increase – Increase Decrease Decrease None – Increase – – None Increase? Increase Decrease? Decrease? Increase? Increase Decrease Increase – –
OOR, out of range (usually below threshold). ND, not enough samples in range to determine. Data represents mean ± SEM.
Moreover L. iners was present among patients with vulvodynia as well as in controls. In our sampled population it appears that L. iners does not appear to play any role in patients with vulvodynia. Preliminary studies on 13 patient samples (6 normal and 7 vulvodynia) were analyzed with the BioPlex bead based cytokine analysis system. Overall, twenty-seven cytokines were assayed. The Table 1 indicates the increase, decrease or no change for each of these. The vaginal samples obtained from patient with vulvodynia demonstrated an increase of almost 35 times interleukin 17 (IL-17) (P = 0.0001) and at the same time a decrease of 7-fold of the macrophage inflammatory protein 1 beta (Mip-Ib) and 3-fold IL-12 compare to matched controls. Four samples were excluded because of insufficient specimen.
Statistical analysis Discussion All of the results remained blind to the investigators until the sampling was completed. Computerized data collection, data tabulation and data analysis were accomplished by experienced clinical personnel and statisticians of the Department of Mathematics and Statistics at Wright State University. Significance level was set at 0.05 with an 80% power to show differences. The Institutional Review Board approved the study.
Results The 17 patients were divided in 3 groups: (a) patients with symptomatic vulvodynia; (b) patients with vulvodynia in remission; (c) patient with no history of vulvodynia (Controls). All the 17 patients had a negative culture for fungi and were negative by PCR for Atopobium, Megasphera and Leptotrichia. In our sampled population L. crispatus was found only in control patients. None of the patients with symptomatic vulvodynia or in remission had L. crispatus. (P = 0.02). Additionally L. gassery was found in patients with symptomatic or vulvodynia in remission.
Our preliminary data are suggesting the presence of a vaginal flora alteration in patients with vulvodynia. Is it only related to the presence of specific lactobacillus? Since there are different lactobacillus variations on the vaginal microflora was our finding just a coincidence or is it a more consistent result in all patients with vulvodynia [5]? It has been reported that nearly all of the L. crispatus produce hydrogen peroxide (H2O2) and that only 71% of L. gassery produce it [6]. L. crispatus promotes in pregnant patients the stability of the normal microflora and L. gassery and L. iners are more conductive to the occurrence of abnormal vaginal flora [7]. Additionally our preliminary results suggest an immunological response involving Candida in patients with vulvodynia. It has been reported that IL-17 is increased in patients with vaginal candidiasis reaching their pick 14 days after the infection; and that IL-12 and Mip-Ib are reduced in post-infection due to Candida albicans [8] Is it an initial infection by Candida the first vulvodynia event or it is the vulvovaginal colonization by Candida that initiates an immunological response resulting in vulvodynia?
230
G. Ventolini et al. / Medical Hypotheses 81 (2013) 228–230
Conclusion Our preliminary data suggest a vaginal flora alteration and an immunological response involving Candida in patients with vulvodynia. Ongoing studies will assist us to clarify these findings. Conflicts of interest No conflicts of interest. References [1] Boardman LA. Sexual pain. Clin Obstet Gynecol 2009;52(4):682–90. [2] Bohm-Starke N. Medical and physical predictors of localized provoked vulvodynia. Acta Obstet Gynecol Scand 2010;98(12):1504–10.
[3] Omoigui S. The biochemical origin of pain: the origin of all pain is inflammation and the inflammatory response. Part 2 of 3-inflammatory profile of pain syndromes. Med Hypotheses 2007;69(6):1169–78. [4] Leclair CM. Differences in primary compared with secondary vestibulodynia by immunohistochemistry. Obstet Gynecol 2011;117(6):1307–13. [5] Gerber S. Immunological and genetic characterization of women with vulvodynia. J Med Life 2008;1(14):432–8. [6] Antonio May AD, The identification of vaginal Lactobacillus species and the demographic and microbiologic characteristics of women colonized by these species. In: Presented in part: international society for sexually transmitted disease research meeting, August 1995, New Orleans (abstract 207). [7] Verstraelen H. Longitudinal analysis of the vaginal microflora in pregnancy suggests that L. crispatus promotes the stability of the normal vaginal microflora and that L. gasseri and/or L. iners are more conducive to the occurrence of abnormal vaginal microflora. BMC Mircrobiol 2009;9:116. [8] Pietrella D, Rachini A, Pines M, Pandey N, Mosci P. Th17 cells and IL-17 in protective immunity to vaginal candidiasis. PLos One 2011;6(7). Epub 2011 Jul 27.