DOI: 10.1111/1471-0528.13115
Review article
www.bjog.org
The vaginal microbiome, vaginal anti-microbial defence mechanisms and the clinical challenge of reducing infection-related preterm birth SS Witkin Division of Immunology and Infectious Diseases, Department of Obstetrics and Gynecology, Weill Cornell Medical College, New York, NY, USA Correspondence: SS Witkin, Department of Obstetrics and Gynecology, Weill Cornell Medical College, 525 East 68th Street, Box 35, New York, NY 10065, USA. Email [email protected] Accepted 18 July 2014. Published Online 15 October 2014.
Ascending bacterial infection is implicated in about 40–50% of preterm births. The human vaginal microbiota in most women is dominated by lactobacilli. In women whose vaginal microbiota is not lactobacilli-dominated anti-bacterial defence mechanisms are reduced. The enhanced proliferation of pathogenic bacteria plus degradation of the cervical barrier increase bacterial passage into the endometrium and amniotic cavity and trigger preterm
myometrial contractions. Evaluation of protocols to detect the absence of lactobaciili dominance in pregnant women by self-measuring vaginal pH, coupled with measures to promote growth of lactobacilli are novel prevention strategies that may reduce the occurrence of preterm birth in low-resource areas. Keywords Bacterial vaginosis, D-lactic acid, lactobacilli, L-lactic acid, preterm birth, vaginal microbiome.
Please cite this paper as: Witkin SS. The vaginal microbiome, vaginal anti-microbial defence mechanisms and the clinical challenge of reducing infection-related preterm birth. BJOG 2014; DOI: 10.1111/1471-0528.13115.
Introduction Preterm birth, delivery before 37 weeks of gestation, is the major cause of neonatal mortality and morbidity and the risk is inversely related to gestational age at birth. The underlying aetiologies that precipitate this event also negatively affect maternal health. The World Health Organization estimates that about 15 million babies were born preterm in 2010.1 Prematurity is a major contributor to infant death, greater than malaria, AIDS and tuberculosis combined. Those preterm babies that survive have an elevated chance of subsequently developing major health problems. Women in low-income countries are at greatest risk for preterm birth. One of the United Nation’s millennium development goals is to reduce by two-thirds the under-5 mortality rate by 2015.2 This clearly cannot be achieved without reducing the rate of preterm deliveries. There is a striking need to identify lowcost, low-technology and safe protocols that are applicable to women in low-resource settings that will reduce the rate of premature delivery. Even in the most developed countries, the majority of attempts to prevent a preterm delivery take place after the initiation of premature myometrial contractions or evidence of infection. Protocols are needed to predict which asymptomatic pregnant women are at risk for
ª 2014 Royal College of Obstetricians and Gynaecologists
adverse events that impact their wellbeing and the subsequent development of preterm labour. The start of preventive treatment before the initiation of preterm labour, especially in areas with limited medical resources, has not been forthcoming and remains a major outstanding problem in obstetrics.
Primary and secondary prevention Primary and secondary prevention encompasses strategies to assess asymptomatic pregnant women for potential factors that increase susceptibility for a subsequent premature delivery and to introduce measures to reduce this risk. There are currently several identified risk factors for preterm birth in which effective interventions can reduce the likelihood of its occurrence (Table 1). The cessation of cigarette smoking, providing an adequate diet, treatment of asymptomatic bacteriuria and a reduction in environmental stress by modifying the work and home environment result in the prolongation of gestation.3–5 Women with a previous preterm birth or who have a short cervical length are at elevated risk for preterm birth and measures such as increased monitoring, bed rest or introduction of a cervical cerclage can lower their risk.6 Recently, treatment with
1
Witkin
Table 1. Primary prevention measures to reduce the risk of induction of a preterm birth Stop smoking Provide adequate nutrition Reduce environmental stress Treatment of asymptomatic bacteriuria Treatment of periodontal disease Increased bed rest, cerclage or progesterone supplementation for women with a previous preterm birth or short cervix
progesterone has been shown to be effective in most studies to reduce the risk of a preterm delivery in women with a short cervix or with a history of preterm birth.7 However, testing for urinary tract infections and interventions based on diet modification, stress reduction, previous pregnancy history or cervical length are not available in many lowresource areas. Other factors known to contribute to the aetiology of preterm birth that are not amenable to primary prevention include the presence of functional polymorphisms in genes present in the mother and/or fetus that determine the direction and magnitude of proinflammatory immune responses8 and decidual haemorrhage due to known or unknown causes.9 There are conflicting reports as to whether the haematogenous dissemination of bacteria to the amniotic cavity from women with periodontal disease increases the rate of preterm birth and whether appropriate dental treatment can improve pregnancy outcome.10 A consistent observation is the racial difference in the rate of preterm birth. Even after controlling for socioeconomic and lifestyle variables, the preterm birth rate and neonatal mortality in the USA are higher in black, than in white women.11,12 Furthermore, maternal infection has been shown to be associated with preterm delivery in black women to a greater extent than in white women.13 Evidence has been presented that genetic variants that were selected during evolution as being favourable to birth outcome among black women living in areas with prevalent endogenous infections may place them at a disadvantage and increase susceptibility to infection-related preterm birth following their relocation to regions with a low infection rate.14 This reinforces the notion that gene–environment interactions are critical for influencing disease acquisition and subsequent consequences.8 An environmental trigger will only be a risk factor for pathology if the affected individual has a less than appropriate genetic repertoire to defend against the particular insult.
Vaginal anti-microbial defences The human vagina is lined with many layers of stratified squamous epithelial cells. This epithelium undergoes a rapid turnover. It has been estimated that the top layer is
2
exfoliated into the vaginal lumen approximately every 4 hours.15 This desquamation serves a protective function as microorganisms that have recently gained entry into the vagina and have adhered to this top layer of epithelial cells are prevented from remaining associated with the epithelium. A similar function is served by the presence of mucin on the epithelial cell surface. The vaginal epithelial cells also contribute directly to anti-microbial immunity. They are active components of the mucosal innate immune system and capable of sensing the presence of pathogens. Vaginal epithelial cells can be stimulated to release a number of anti-microbial peptides into the vaginal lumen including secretory leucocyte protease inhibitor, human epididymis protein 4, human defensin-5, human b-defensins 1 and 2, cathelicidin and lactoferrin.16 Vaginal epithelial cells also release proinflammatory cytokines that recruit phagocytic cells to enter the vaginal epithelial layers as well as the vaginal lumen and activate these cells to engulf microbial pathogens.16
Anti-microbial immunity in pregnancy Maternal immune responses to infection during pregnancy are unique and differ from defence mechanisms operative in nonpregnant women.17 There is an emphasis on tolerance mechanisms, i.e. minimising the consequences of an infectious event, rather than resistance mechanisms, i.e. mounting an immune attack to destroy microbial invaders. Undoubtedly, because of the need for the mother to not reject her semi-allogeneic fetus, during gestation there is an increasing focus of the maternal immune system to be more tolerant to the presence of immune system activators and a corresponding decreased emphasis on mounting a high magnitude immune response. An unmodulated proinflammatory immune reaction to an infectious insult is associated with triggering the sequence of events leading to preterm birth.18 The increased presence of Lactobacillus spp. in the vagina during pregnancy as opposed to the nonpregnant state (see below), appears to be one mechanism that becomes increasingly dominant during gestation to modulate maternal responses to potentially pathogenic bacteria at this site.
Bacteria-induced preterm birth Bacterial infection is recognised as a major factor in the induction of preterm birth and neonatal morbidity and mortality. A percentage of 40–50% is often given as the fraction of cases of premature delivery that may be due to infection.19 Recent studies using culture-independent gene amplification technology to detect bacteria in pregnant women have suggested that this number can perhaps be even higher.20 Bacteria can enter the amniotic cavity and infect the fetus via several routes. The reported association
ª 2014 Royal College of Obstetricians and Gynaecologists
Reducing infection-related preterm birth
between bacteria in the oral cavity and preterm birth10 indicates that there is haematogenous dissemination of bacteria to the fetus. The detection of bacteria in the uterine cavity of healthy nonpregnant women implies that bacteria are present at this site before conception in some women.21 These microbes can subsequently become reactivated or increase in invasiveness following pregnancy initiation. However, it is generally assumed that the majority of bacterial infections that affect pregnancy outcome are due to the migration of bacteria from the vagina through the cervix and into the uterus, where they invoke deciduitis and chorioamnionitis or, alternatively, penetrate the chorionic and amniotic membranes and invade the amniotic cavity and the fetus.18 Once the amniotic membranes have been breached the bacteria trigger a sequential series of responses—proinflammatory cytokine induction, release of prostaglandins, myometrial contractions—that result in the premature expulsion of the fetus. This mechanism has probably evolved to protect the mother, and possibly also the fetus, from prolonged exposure to pathogenic bacteria. In situations where the vaginal microbiota of pregnant women is not dominated by lactobacilli there is an increased association with infection-related preterm birth and maternal and neonatal morbidity. The condition known as bacterial vaginosis (BV) has received the most attention in this regard,22 although other conditions known as aerobic vaginitis (AV)22 and intermediate flora22 have also been cited as predisposing factors. BV refers to an alteration in the composition of the vaginal microbiota whereby lactobacilli are either greatly reduced in number or totally absent and there is a large increase in concentration of anaerobic and facultative bacteria. The composition of the vaginal bacterial populations may differ between individual women with a diagnosis of BV but in most cases Gardnerella vaginalis, Prevotella and Bacteroides spp., and Mycoplasma hominis are present in high concentrations. In AV, lactobacilli are replaced predominantly by Staphylococcus spp., group B streptococci, Escherichia coli and enterococci. Intermediate flora represents a situation that is intermediate between a Lactobacillus-dominant microbiota and one dominated by BV-related bacteria. There are areas on a Gram-stained slide that are dominated by lactobacilli and other areas that are dominated by BV-related microorganisms.
The human vaginal microbiome The vaginal microbiome in women is unique and differs from that of any other species, including nonhuman primates.23 The vaginal pH of animals most commonly used in laboratory experiments—mice, rats and rabbits—is near neutral and the vaginal microbiome is not dominated by lactobacilli. Similarly, the vaginal concentration of glycogen and lactic acid, as well as of Lactobacillus spp., is greatly
ª 2014 Royal College of Obstetricians and Gynaecologists
reduced in nonhuman primates compared with women.24 This necessarily brings into question the relevance of nonhuman models for the study of female genital tract infectious and noninfectious disorders. It also leads to a fascinating question: why has the human vaginal microbiome developed its unique composition during the course of evolution and how do these alterations provide optimum protection for the human female against infection during pregnancy? The composition of the vaginal bacterial microbiota is different in pregnant women from that present in women who are not pregnant.25,26 Rising estrogen levels during gestation result in increased vaginal glycogen deposition. This favours the proliferation of lactobacilli and so a vaginal microbiota dominated by lactobacilli increases in frequency and is more stable during pregnancy; non-lactobacillus genera are less often detected.
Vaginal lactic acid Whatever is the explanation for the evolutionary dominance of Lactobacillus species in the vagina, it is clear that this change coincides with vaginal health.27 Recent studies have identified multiple mechanisms whereby L-lactic acid promotes wellbeing (Table 2). Specifically lactic acid, and not other related acidic compounds, inhibits the growth of bacteria associated with BV as well as HIV.28 Hence, lactic acid has a unique role in promoting the dominance of microbes with a low pathogenic potential. Lactic acid is also becoming increasingly recognised as a component of immune defence. In the presence of a synthetic analogue of double-stranded viral RNA, lactic acid potentiates the production of protective pro-inflammatory cytokines by vaginal epithelial cells.29 Lactic acid also promotes activation of the T helper type 17 subclass of helper T lymphocytes,30 stimulates dendritic cell maturation31 and induces interferon-c production.32 Each of these activities enhances activation of anti-microbial innate or acquired immunity. Lactic acid-producing bacteria are unique in their production of both the D- and L-chiral isomers of lactic acid. In contrast, mammalian cells produce L-lactic acid almost exclusively.22 The unique production of the D-lactic acid
Table 2. Biological properties of L-lactic acid Inhibition of growth of bacteria associated with bacterial vaginosis Inhibition of HIV Potentiation of proinflammatory cytokine production by vaginal epithelial cells in response to viral double-stranded RNA Activation of the T helper type 17 lymphocyte subset. Stimulation of dendritic cell maturation Induction of interferon-c production
3
Witkin
isomer by some strains of lactobacilli enhances protection against microbial invasion of the upper genital tract.33 Matrix metalloproteinase 8 (MMP-8) is known to decrease the integrity of the cervical os and permit passage of bacteria into the endometrium. The inducer of MMP-8, extracellular matrix metalloproteinase inducer (EMMPRIN), is produced by vaginal epithelial cells and its concentration in vaginal secretions is dependent upon the relative levels of 33 D- and L-lactic acid. High D-lactic acid production limits EMMPRIN concentrations, and so MMP-8 levels, and minimises MMP-8-induced cervical changes. Interestingly, while Lactobacillus crispatus, Lactobacillus gasseri and Lactobacillus jensenii are all producers of D-lactic acid, Lactobacillus iners lacks the gene coding for D-lactate dehydrogenase and so cannot synthesise D-lactic acid.33 While the clinical implications of this observation remain to be determined, L. iners is often present in association with BV-related vaginal bacteria.34,35 In a recent study of the vaginal microbiome in healthy reproductive-age women, vaginal concentrations of L-lactic acid were only slightly higher when Lactobacillus species were dominant than when other bacteria dominated. This was not surprising as the vaginal epithelial cells are also a source of L-lactic acid.27 In contrast, D-lactic acid levels were significantly higher when L. crispatus was the dominant vaginal bacterium than when L. iners (P < 0.0001) or Gardnerella (P = 0.0002) dominated the vaginal microbiota. Importantly, levels of L-lactic acid (P < 0.0001) and the ratio of L- to D-lactic acid (P = 0.0060), but not concentrations of D-lactic acid, were correlated with the EMMPRIN concentration. Moreover, vaginal concentrations of EMMPRIN and MMP-8 levels were highly correlated (P < 0.0001).33 The most likely explanation for these observations is that L-lactic acid induction of EMMPRIN production is necessary to regulate intracellular lactic acid concentrations and maintain cell viability. Vaginal epithelial cells36 and uterine fibroblasts37 have been shown to produce EMMPRIN. In addition, the vaginal concentrations of EMMPRIN and MMP-8 were also correlated, suggesting that MMP-8 may be induced as a secondary consequence of this induction. MMP-8 may aid in exfoliation and maintenance of the epithelial cell layer. While the concentration of D-lactic acid by itself was unrelated to vaginal EMMPRIN levels, an increase in the concentration of D-lactic acid relative to the L-lactic acid level correlated significantly with a decrease in EMMPRIN concentration. As vaginal EMMPRIN is an inducer of MMP-8, the D- to L-lactic acid ratio in the vagina would also influence MMP-8 production at this site. Two of the major bacterial biotypes associated with bacterial vaginosis, namely Gardnerella and L. iners, appear to be either very poor D-lactic acid producers or capable of readily degrading this isomer. This provides a plausible mechanism for the observation of an
4
association between vaginal MMP-8 levels and bacterial vaginosis.38 The paucity or absence of vaginal D-lactic acid due to a relative scarcity of lactobacilli other than L. iners results in elevated local EMMPRIN levels and, hence to high concentrations of MMP-8. This facilitates breakdown of the extracellular matrix and more readily allows bacterial migration past the endocervix and into the uterus. This will increase susceptibility to infection-related preterm birth. In addition, MMP-8 by itself has been implicated in rupture of the fetal membranes.39
Identification of women with a lactobacilli-deficient vaginal microbiota and low-cost treatment to reduce susceptibility to preterm birth The detection of a vaginal pH ≥4.7 in women of reproductive age is strongly indicative of a vaginal microbiota deficient or lacking in lactobacilli. Therefore, it might be advantageous for pregnant women to be screened or to undergo a self-test for their vaginal pH. Several studies have used a pH indicator stick or a specially constructed glove or a panty liner for self-measurement of vaginal pH.40–44 An elevated pH detects BV with high sensitivity but the specificity is low. Other conditions such as AV, the predominance of intermediate flora or trichomoniasis also elevate vaginal pH. The pioneering studies of Hoyme and Saling have clearly demonstrated that self-detection of an elevated vaginal pH at early pregnancy stages followed by treatment with antibiotics or probiotic lactobacilli in cases where the absence or scarcity of lactobacilli or an elevated vaginal pH exists significantly reduces the rate of preterm birth.45 However, antibiotic treatment of BV in pregnant women remains controversial. A recent Cochrane review concluded that while antibiotics can eradicate BV it did not reduce the risk of a subsequent preterm birth.46 Another systematic review, reporting that treatment of women with clindamycin at
Review article
www.bjog.org
The vaginal microbiome, vaginal anti-microbial defence mechanisms and the clinical challenge of reducing infection-related preterm birth SS Witkin Division of Immunology and Infectious Diseases, Department of Obstetrics and Gynecology, Weill Cornell Medical College, New York, NY, USA Correspondence: SS Witkin, Department of Obstetrics and Gynecology, Weill Cornell Medical College, 525 East 68th Street, Box 35, New York, NY 10065, USA. Email [email protected] Accepted 18 July 2014. Published Online 15 October 2014.
Ascending bacterial infection is implicated in about 40–50% of preterm births. The human vaginal microbiota in most women is dominated by lactobacilli. In women whose vaginal microbiota is not lactobacilli-dominated anti-bacterial defence mechanisms are reduced. The enhanced proliferation of pathogenic bacteria plus degradation of the cervical barrier increase bacterial passage into the endometrium and amniotic cavity and trigger preterm
myometrial contractions. Evaluation of protocols to detect the absence of lactobaciili dominance in pregnant women by self-measuring vaginal pH, coupled with measures to promote growth of lactobacilli are novel prevention strategies that may reduce the occurrence of preterm birth in low-resource areas. Keywords Bacterial vaginosis, D-lactic acid, lactobacilli, L-lactic acid, preterm birth, vaginal microbiome.
Please cite this paper as: Witkin SS. The vaginal microbiome, vaginal anti-microbial defence mechanisms and the clinical challenge of reducing infection-related preterm birth. BJOG 2014; DOI: 10.1111/1471-0528.13115.
Introduction Preterm birth, delivery before 37 weeks of gestation, is the major cause of neonatal mortality and morbidity and the risk is inversely related to gestational age at birth. The underlying aetiologies that precipitate this event also negatively affect maternal health. The World Health Organization estimates that about 15 million babies were born preterm in 2010.1 Prematurity is a major contributor to infant death, greater than malaria, AIDS and tuberculosis combined. Those preterm babies that survive have an elevated chance of subsequently developing major health problems. Women in low-income countries are at greatest risk for preterm birth. One of the United Nation’s millennium development goals is to reduce by two-thirds the under-5 mortality rate by 2015.2 This clearly cannot be achieved without reducing the rate of preterm deliveries. There is a striking need to identify lowcost, low-technology and safe protocols that are applicable to women in low-resource settings that will reduce the rate of premature delivery. Even in the most developed countries, the majority of attempts to prevent a preterm delivery take place after the initiation of premature myometrial contractions or evidence of infection. Protocols are needed to predict which asymptomatic pregnant women are at risk for
ª 2014 Royal College of Obstetricians and Gynaecologists
adverse events that impact their wellbeing and the subsequent development of preterm labour. The start of preventive treatment before the initiation of preterm labour, especially in areas with limited medical resources, has not been forthcoming and remains a major outstanding problem in obstetrics.
Primary and secondary prevention Primary and secondary prevention encompasses strategies to assess asymptomatic pregnant women for potential factors that increase susceptibility for a subsequent premature delivery and to introduce measures to reduce this risk. There are currently several identified risk factors for preterm birth in which effective interventions can reduce the likelihood of its occurrence (Table 1). The cessation of cigarette smoking, providing an adequate diet, treatment of asymptomatic bacteriuria and a reduction in environmental stress by modifying the work and home environment result in the prolongation of gestation.3–5 Women with a previous preterm birth or who have a short cervical length are at elevated risk for preterm birth and measures such as increased monitoring, bed rest or introduction of a cervical cerclage can lower their risk.6 Recently, treatment with
1
Witkin
Table 1. Primary prevention measures to reduce the risk of induction of a preterm birth Stop smoking Provide adequate nutrition Reduce environmental stress Treatment of asymptomatic bacteriuria Treatment of periodontal disease Increased bed rest, cerclage or progesterone supplementation for women with a previous preterm birth or short cervix
progesterone has been shown to be effective in most studies to reduce the risk of a preterm delivery in women with a short cervix or with a history of preterm birth.7 However, testing for urinary tract infections and interventions based on diet modification, stress reduction, previous pregnancy history or cervical length are not available in many lowresource areas. Other factors known to contribute to the aetiology of preterm birth that are not amenable to primary prevention include the presence of functional polymorphisms in genes present in the mother and/or fetus that determine the direction and magnitude of proinflammatory immune responses8 and decidual haemorrhage due to known or unknown causes.9 There are conflicting reports as to whether the haematogenous dissemination of bacteria to the amniotic cavity from women with periodontal disease increases the rate of preterm birth and whether appropriate dental treatment can improve pregnancy outcome.10 A consistent observation is the racial difference in the rate of preterm birth. Even after controlling for socioeconomic and lifestyle variables, the preterm birth rate and neonatal mortality in the USA are higher in black, than in white women.11,12 Furthermore, maternal infection has been shown to be associated with preterm delivery in black women to a greater extent than in white women.13 Evidence has been presented that genetic variants that were selected during evolution as being favourable to birth outcome among black women living in areas with prevalent endogenous infections may place them at a disadvantage and increase susceptibility to infection-related preterm birth following their relocation to regions with a low infection rate.14 This reinforces the notion that gene–environment interactions are critical for influencing disease acquisition and subsequent consequences.8 An environmental trigger will only be a risk factor for pathology if the affected individual has a less than appropriate genetic repertoire to defend against the particular insult.
Vaginal anti-microbial defences The human vagina is lined with many layers of stratified squamous epithelial cells. This epithelium undergoes a rapid turnover. It has been estimated that the top layer is
2
exfoliated into the vaginal lumen approximately every 4 hours.15 This desquamation serves a protective function as microorganisms that have recently gained entry into the vagina and have adhered to this top layer of epithelial cells are prevented from remaining associated with the epithelium. A similar function is served by the presence of mucin on the epithelial cell surface. The vaginal epithelial cells also contribute directly to anti-microbial immunity. They are active components of the mucosal innate immune system and capable of sensing the presence of pathogens. Vaginal epithelial cells can be stimulated to release a number of anti-microbial peptides into the vaginal lumen including secretory leucocyte protease inhibitor, human epididymis protein 4, human defensin-5, human b-defensins 1 and 2, cathelicidin and lactoferrin.16 Vaginal epithelial cells also release proinflammatory cytokines that recruit phagocytic cells to enter the vaginal epithelial layers as well as the vaginal lumen and activate these cells to engulf microbial pathogens.16
Anti-microbial immunity in pregnancy Maternal immune responses to infection during pregnancy are unique and differ from defence mechanisms operative in nonpregnant women.17 There is an emphasis on tolerance mechanisms, i.e. minimising the consequences of an infectious event, rather than resistance mechanisms, i.e. mounting an immune attack to destroy microbial invaders. Undoubtedly, because of the need for the mother to not reject her semi-allogeneic fetus, during gestation there is an increasing focus of the maternal immune system to be more tolerant to the presence of immune system activators and a corresponding decreased emphasis on mounting a high magnitude immune response. An unmodulated proinflammatory immune reaction to an infectious insult is associated with triggering the sequence of events leading to preterm birth.18 The increased presence of Lactobacillus spp. in the vagina during pregnancy as opposed to the nonpregnant state (see below), appears to be one mechanism that becomes increasingly dominant during gestation to modulate maternal responses to potentially pathogenic bacteria at this site.
Bacteria-induced preterm birth Bacterial infection is recognised as a major factor in the induction of preterm birth and neonatal morbidity and mortality. A percentage of 40–50% is often given as the fraction of cases of premature delivery that may be due to infection.19 Recent studies using culture-independent gene amplification technology to detect bacteria in pregnant women have suggested that this number can perhaps be even higher.20 Bacteria can enter the amniotic cavity and infect the fetus via several routes. The reported association
ª 2014 Royal College of Obstetricians and Gynaecologists
Reducing infection-related preterm birth
between bacteria in the oral cavity and preterm birth10 indicates that there is haematogenous dissemination of bacteria to the fetus. The detection of bacteria in the uterine cavity of healthy nonpregnant women implies that bacteria are present at this site before conception in some women.21 These microbes can subsequently become reactivated or increase in invasiveness following pregnancy initiation. However, it is generally assumed that the majority of bacterial infections that affect pregnancy outcome are due to the migration of bacteria from the vagina through the cervix and into the uterus, where they invoke deciduitis and chorioamnionitis or, alternatively, penetrate the chorionic and amniotic membranes and invade the amniotic cavity and the fetus.18 Once the amniotic membranes have been breached the bacteria trigger a sequential series of responses—proinflammatory cytokine induction, release of prostaglandins, myometrial contractions—that result in the premature expulsion of the fetus. This mechanism has probably evolved to protect the mother, and possibly also the fetus, from prolonged exposure to pathogenic bacteria. In situations where the vaginal microbiota of pregnant women is not dominated by lactobacilli there is an increased association with infection-related preterm birth and maternal and neonatal morbidity. The condition known as bacterial vaginosis (BV) has received the most attention in this regard,22 although other conditions known as aerobic vaginitis (AV)22 and intermediate flora22 have also been cited as predisposing factors. BV refers to an alteration in the composition of the vaginal microbiota whereby lactobacilli are either greatly reduced in number or totally absent and there is a large increase in concentration of anaerobic and facultative bacteria. The composition of the vaginal bacterial populations may differ between individual women with a diagnosis of BV but in most cases Gardnerella vaginalis, Prevotella and Bacteroides spp., and Mycoplasma hominis are present in high concentrations. In AV, lactobacilli are replaced predominantly by Staphylococcus spp., group B streptococci, Escherichia coli and enterococci. Intermediate flora represents a situation that is intermediate between a Lactobacillus-dominant microbiota and one dominated by BV-related bacteria. There are areas on a Gram-stained slide that are dominated by lactobacilli and other areas that are dominated by BV-related microorganisms.
The human vaginal microbiome The vaginal microbiome in women is unique and differs from that of any other species, including nonhuman primates.23 The vaginal pH of animals most commonly used in laboratory experiments—mice, rats and rabbits—is near neutral and the vaginal microbiome is not dominated by lactobacilli. Similarly, the vaginal concentration of glycogen and lactic acid, as well as of Lactobacillus spp., is greatly
ª 2014 Royal College of Obstetricians and Gynaecologists
reduced in nonhuman primates compared with women.24 This necessarily brings into question the relevance of nonhuman models for the study of female genital tract infectious and noninfectious disorders. It also leads to a fascinating question: why has the human vaginal microbiome developed its unique composition during the course of evolution and how do these alterations provide optimum protection for the human female against infection during pregnancy? The composition of the vaginal bacterial microbiota is different in pregnant women from that present in women who are not pregnant.25,26 Rising estrogen levels during gestation result in increased vaginal glycogen deposition. This favours the proliferation of lactobacilli and so a vaginal microbiota dominated by lactobacilli increases in frequency and is more stable during pregnancy; non-lactobacillus genera are less often detected.
Vaginal lactic acid Whatever is the explanation for the evolutionary dominance of Lactobacillus species in the vagina, it is clear that this change coincides with vaginal health.27 Recent studies have identified multiple mechanisms whereby L-lactic acid promotes wellbeing (Table 2). Specifically lactic acid, and not other related acidic compounds, inhibits the growth of bacteria associated with BV as well as HIV.28 Hence, lactic acid has a unique role in promoting the dominance of microbes with a low pathogenic potential. Lactic acid is also becoming increasingly recognised as a component of immune defence. In the presence of a synthetic analogue of double-stranded viral RNA, lactic acid potentiates the production of protective pro-inflammatory cytokines by vaginal epithelial cells.29 Lactic acid also promotes activation of the T helper type 17 subclass of helper T lymphocytes,30 stimulates dendritic cell maturation31 and induces interferon-c production.32 Each of these activities enhances activation of anti-microbial innate or acquired immunity. Lactic acid-producing bacteria are unique in their production of both the D- and L-chiral isomers of lactic acid. In contrast, mammalian cells produce L-lactic acid almost exclusively.22 The unique production of the D-lactic acid
Table 2. Biological properties of L-lactic acid Inhibition of growth of bacteria associated with bacterial vaginosis Inhibition of HIV Potentiation of proinflammatory cytokine production by vaginal epithelial cells in response to viral double-stranded RNA Activation of the T helper type 17 lymphocyte subset. Stimulation of dendritic cell maturation Induction of interferon-c production
3
Witkin
isomer by some strains of lactobacilli enhances protection against microbial invasion of the upper genital tract.33 Matrix metalloproteinase 8 (MMP-8) is known to decrease the integrity of the cervical os and permit passage of bacteria into the endometrium. The inducer of MMP-8, extracellular matrix metalloproteinase inducer (EMMPRIN), is produced by vaginal epithelial cells and its concentration in vaginal secretions is dependent upon the relative levels of 33 D- and L-lactic acid. High D-lactic acid production limits EMMPRIN concentrations, and so MMP-8 levels, and minimises MMP-8-induced cervical changes. Interestingly, while Lactobacillus crispatus, Lactobacillus gasseri and Lactobacillus jensenii are all producers of D-lactic acid, Lactobacillus iners lacks the gene coding for D-lactate dehydrogenase and so cannot synthesise D-lactic acid.33 While the clinical implications of this observation remain to be determined, L. iners is often present in association with BV-related vaginal bacteria.34,35 In a recent study of the vaginal microbiome in healthy reproductive-age women, vaginal concentrations of L-lactic acid were only slightly higher when Lactobacillus species were dominant than when other bacteria dominated. This was not surprising as the vaginal epithelial cells are also a source of L-lactic acid.27 In contrast, D-lactic acid levels were significantly higher when L. crispatus was the dominant vaginal bacterium than when L. iners (P < 0.0001) or Gardnerella (P = 0.0002) dominated the vaginal microbiota. Importantly, levels of L-lactic acid (P < 0.0001) and the ratio of L- to D-lactic acid (P = 0.0060), but not concentrations of D-lactic acid, were correlated with the EMMPRIN concentration. Moreover, vaginal concentrations of EMMPRIN and MMP-8 levels were highly correlated (P < 0.0001).33 The most likely explanation for these observations is that L-lactic acid induction of EMMPRIN production is necessary to regulate intracellular lactic acid concentrations and maintain cell viability. Vaginal epithelial cells36 and uterine fibroblasts37 have been shown to produce EMMPRIN. In addition, the vaginal concentrations of EMMPRIN and MMP-8 were also correlated, suggesting that MMP-8 may be induced as a secondary consequence of this induction. MMP-8 may aid in exfoliation and maintenance of the epithelial cell layer. While the concentration of D-lactic acid by itself was unrelated to vaginal EMMPRIN levels, an increase in the concentration of D-lactic acid relative to the L-lactic acid level correlated significantly with a decrease in EMMPRIN concentration. As vaginal EMMPRIN is an inducer of MMP-8, the D- to L-lactic acid ratio in the vagina would also influence MMP-8 production at this site. Two of the major bacterial biotypes associated with bacterial vaginosis, namely Gardnerella and L. iners, appear to be either very poor D-lactic acid producers or capable of readily degrading this isomer. This provides a plausible mechanism for the observation of an
4
association between vaginal MMP-8 levels and bacterial vaginosis.38 The paucity or absence of vaginal D-lactic acid due to a relative scarcity of lactobacilli other than L. iners results in elevated local EMMPRIN levels and, hence to high concentrations of MMP-8. This facilitates breakdown of the extracellular matrix and more readily allows bacterial migration past the endocervix and into the uterus. This will increase susceptibility to infection-related preterm birth. In addition, MMP-8 by itself has been implicated in rupture of the fetal membranes.39
Identification of women with a lactobacilli-deficient vaginal microbiota and low-cost treatment to reduce susceptibility to preterm birth The detection of a vaginal pH ≥4.7 in women of reproductive age is strongly indicative of a vaginal microbiota deficient or lacking in lactobacilli. Therefore, it might be advantageous for pregnant women to be screened or to undergo a self-test for their vaginal pH. Several studies have used a pH indicator stick or a specially constructed glove or a panty liner for self-measurement of vaginal pH.40–44 An elevated pH detects BV with high sensitivity but the specificity is low. Other conditions such as AV, the predominance of intermediate flora or trichomoniasis also elevate vaginal pH. The pioneering studies of Hoyme and Saling have clearly demonstrated that self-detection of an elevated vaginal pH at early pregnancy stages followed by treatment with antibiotics or probiotic lactobacilli in cases where the absence or scarcity of lactobacilli or an elevated vaginal pH exists significantly reduces the rate of preterm birth.45 However, antibiotic treatment of BV in pregnant women remains controversial. A recent Cochrane review concluded that while antibiotics can eradicate BV it did not reduce the risk of a subsequent preterm birth.46 Another systematic review, reporting that treatment of women with clindamycin at
