Aswathi Jayaram

Influence of Vaginal Bacteria and d- and l-Lactic Acid Isomers on Vaginal Extracellular Matrix Metalloproteinase Inducer: Implications for Protection against Upper Genital Tract Infections

ABSTRACT We evaluated levels of vaginal extracellular matrix metalloproteinase inducer (EMMPRIN) and matrix metalloproteinase (MMP-8) in vaginal secretions in relation to the composition of vaginal bacterial communities and d- and l-lactic acid levels. The composition of vaginal bacterial communities in 46 women was determined by pyrosequencing the V1 to V3 region of 16S rRNA genes. Lactobacilli were dominant in 71.3% of the women, followed by Gardnerella (17.4%), Streptococcus (8.7%), and Enterococcus (2.2%). Of the lactobacillus-dominated communities, 51.5% were dominated by Lactobacillus crispatus, 36.4% by Lactobacillus iners, and 6.1% each by Lactobacillus gasseri and Lactobacillus jensenii. Concentrations of l-lactic acid were slightly higher in lactobacillus-dominated vaginal samples, but most differences were not statistically significant. d-Lactic acid levels were higher in samples containing L. crispatus than in those with L. iners (P < 0.0001) or Gardnerella (P = 0.0002). The relative proportion of d-lactic acid in vaginal communities dominated by species of lactobacilli was in concordance with the proportions found in axenic cultures of the various species grown in vitro. Levels of l-lactic acid (P < 0.0001) and the ratio of l-lactic acid to d-lactic acid (P = 0.0060), but not concentrations of d-lactic acid, were also correlated with EMMPRIN concentrations. Moreover, vaginal concentrations of EMMPRIN and MMP-8 levels were highly correlated (P < 0.0001). Taken together, the data suggest the relative proportion of l- to d-lactic acid isomers in the vagina may influence the extent of local EMMPRIN production and subsequent induction of MMP-8. The expression of these proteins may help determine the ability of bacteria to transverse the cervix and initiate upper genital tract infections. IMPORTANCE A large proportion of preterm births (>50%) result from infections caused by bacteria originating in the vagina, which requires that they traverse the cervix. Factors that influence susceptibility to these infections are not well understood; however, there is evidence that matrix metalloproteinase (MMP-8) is known to alter the integrity of the cervix. In this work, we show that concentrations of vaginal extracellular matrix metalloproteinase inducer (EMMPRIN) are influenced by members of the vaginal microbial community and concentrations of d- or l-lactic acid isomers in vaginal secretions. Elevated levels of d-lactic acid and the ratio of d- to l-lactic acid influence EMMPRIN concentrations as well as MMP-8 levels. Thus, isomers of lactic acid may function as signaling molecules that alter host gene expression and influence risk of infection-related preterm birth. A large proportion of preterm births (>50%) result from infections caused by bacteria originating in the vagina, which requires that they traverse the cervix. Factors that influence susceptibility to these infections are not well understood; however, there is evidence that matrix metalloproteinase (MMP-8) is known to alter the integrity of the cervix. In this work, we show that concentrations of vaginal extracellular matrix metalloproteinase inducer (EMMPRIN) are influenced by members of the vaginal microbial community and concentrations of d- or l-lactic acid isomers in vaginal secretions. Elevated levels of d-lactic acid and the ratio of d- to l-lactic acid influence EMMPRIN concentrations as well as MMP-8 levels. Thus, isomers of lactic acid may function as signaling molecules that alter host gene expression and influence risk of infection-related preterm birth.

Altered autophagy induction by sera from pregnant women with pre-eclampsia: a case–control study

Mechanisms leading to pre‐eclampsia remain incompletely defined. Autophagy is a conserved process necessary for cell survival under adverse conditions. We hypothesised that sera from women with healthy pregnancies and women with pre‐eclampsia differed in autophagy induction.

Human Epididymis Protein 4 and Secretory Leukocyte Protease Inhibitor in Vaginal Fluid Relation to Vaginal Components and Bacterial Composition

Human epididymis protein 4 (HE4) is a protease inhibitor and a recently identified serum biomarker for ovarian cancer. Properties of HE4 in the genital tract of healthy women have not been evaluated. We evaluated associations between HE4 and a second vaginal protease inhibitor, secretory leukocyte protease inhibitor (SLPI), with vaginal concentrations of innate immune mediators or proteases and with the types of vaginal bacterial communities. Vaginal secretions were collected from 18 healthy reproductive age women and assayed by enzyme-linked immunosorbent assay for concentrations of HE4, SLPI, kallikrein 5, cathepsin B, interleukin 1β (IL-1), IL-1 receptor antagonist (IL-1 ra), mannose-binding lectin (MBL), the inducible 70-kDa heat shock protein, and matrix metalloproteinase (MMP)-8. The species composition of vaginal bacterial communities in 16 women was characterized by sequencing amplicons derived from 16S bacterial ribosomal RNA genes. Correlations between any 2 assays were analyzed by the Spearman rank correlation tests. Differences in the concentrations of HE4 and SLPI, and between soluble components and vaginal community types, were analyzed by the Mann-Whitney U tests. Vaginal HE4 concentrations, but not SLPI levels, were positively correlated with levels of IL-1β (P = .0152), IL-1ra (P = .0061), MBL (P = .0100), and MMP-8 (P = .0315). The median vaginal HE4 level, as well as concentrations of MBL, IL-1β, IL-1ra, and MMP-8, was highest when Gardnerella vaginalis dominated a vaginal community. The association between HE4, elevated levels of proteases, immune mediators and high proportions of G vaginalis strongly suggests that HE4 is a component of the proinflammatory immune response in the female genital tract.

Unique variation in genetic selection among Black North American women and its potential influence on pregnancy outcome.

We hypothesize that variations in the frequency of genetic polymorphisms, reflecting ancestral differences in living conditions and exposure to microorganisms, increase susceptibility to adverse pregnancy outcome among present day Black North American women. Striking differences were observed in the frequency of genetic variants between Black and White or Hispanic women in 5 genes (IL1RN, MBL2, PPARA, ATG16L1, CIAS1) associated with inflammation and anti-microbial immunity. The CIAS1 and IL1RN polymorphisms were associated with altered interleukin-1β serum levels; the MBL2 polymorphism resulted in a decreased serum mannose-binding lectin concentration. Gene polymorphisms associated with an alteration in innate immunity were most frequent in Black women. This may reflect an evolutionary selection in response to an ancient environment containing a high multitude of microorganisms, and may increase susceptibility of Black women to infection-associated preterm birth in the current North American environment.

Autophagy and female genital tract infections: new insights and research directions

Autophagy is a highly conserved process by which defective organelles, non‐functional proteins, and intracellular microorganisms become sequestered within structures called autophagosomes, which fuse with lysosomes and the engulfed components are degraded by lysosomal enzymes. In microbial autophagy degraded peptides are used to induce antigen‐specific acquired immunity. Viruses, bacteria, fungi, and protozoa have developed strategies to subvert autophagy and/or to use this process to promote their replication and persistence. This review details the mechanisms by which microorganisms that infect the female genital tract and/or are detrimental to pregnancy interact with this host defence mechanism. Based on an understanding of autophagy‐related pathological mechanisms, we propose new avenues for research to more effectively prevent and/or treat these infectious diseases.

The Vaginal Microbiome: New Findings Bring New Opportunities

Preclinical Research

Pregnancy History Influences the Level of Autophagy in Peripheral Blood Mononuclear Cells From Pregnant Women

Objective: Maternal immune responses are altered during pregnancy and differ between nulliparous and multiparous women. The influence of a prior gestation on autophagy in peripheral blood mononuclear cells (PBMCs) from pregnant women has not been determined and is the subject of this investigation. Methods: Peripheral blood mononuclear cells were isolated from 212 pregnant women and immediately lysed in the presence of protease inhibitors, and the extent of autophagy was determined by quantitation of the concentration of p62 (sequestosome-1) in the lysates by enzyme-linked immunosorbent assay (ELISA). In PBMCs, the p62 level is inversely related to the extent of autophagy. The level of the stress-inducible 70-kDa heat shock protein (hsp70), an inhibitor of autophagy, was also measured in the lysates by ELISA. Data were analyzed by the Spearman rank correlation, Mann-Whitney U test, or Kruskal-Wallis test, as appropriate. Results: The p62 concentration in PBMCs increased (autophagy decreased) with the number of previous live (P = .0322), preterm (P = .0143), or term (P = .0418) deliveries. The p62 level was lower (autophagy higher) in women with a prior spontaneous pregnancy loss but no deliveries as compared to women with their first conception (P = .0087). The intracellular hsp70 concentration correlated with the p62 level (P < .0001). Conclusion: Multiparity is associated with a reduced level of autophagy in PBMCs. Dysregulated autophagy might be one mechanism leading to spontaneous abortion in nulliparous women.

Association Between Neurotrophin 4 and Long-Chain Polyunsaturated Fatty Acid Levels in Mid-Trimester Amniotic Fluid

The omega-3 long-chain polyunsaturated fatty acid (LCPUFA) docosahexaenoic acid (DHA) and the omega-6 LCPUFA arachidonic acid (AA) are essential nervous system components that increase in concentration throughout gestation. The neurotrophins, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin 3 (NT3), and neurotrophin 4 (NT4) are small basic peptides crucial for fetal brain development. The DHA supplementation during pregnancy has been suggested to enhance neural development. We evaluated whether amniotic fluid DHA and AA concentrations correlated with intra-amniotic neurotrophin levels. Amniotic fluid, obtained at 15 to 19 weeks gestation from 62 women, was tested for BDNF, NGF, NT3, and NT4 by enzyme-linked immunosorbent assay. Concentrations of DHA and AA, and saturated and monounsaturated fatty acids, were determined by gas chromatography. Associations were analyzed by the Spearman rank correlation test. Median levels of AA and DHA were 2.3% and 1.3% of the total intra-amniotic fatty acids, respectively. Median neurotrophin levels (pg/mL) were 36.7 for NT3, 26.8 for BDNF, 5.2 for NT4, and 0.8 for NGF. Intra-amniotic NT4 and BDNF levels were correlated (P = .0016), while NT3 and NGF levels were unrelated to each other or to BDNF or NT4. Only NT4 was positively correlated with amniotic fluid DHA (P < .0001) and AA (P = .0003) concentrations. There were no associations between DHA, AA, or any neurotrophin and maternal age, gestational age at time of amniocentesis, amniocentesis indication, parity, or gestational age at delivery. Elevations in intra-amniotic NT4 with increasing levels of DHA and AA suggest that these LCPUFAs may specifically influence the extent of NT4-mediated fetal brain neurogenesis.