Identification of bacteriophages in the vagina of pregnant women: a descriptive study.

OBJECTIVE: To determine the presence and identity of extracellular bacteriophage (phage) families, genera and species in the vagina of pregnant women. DESIGN: Descriptive, observational cohort study. SETTING: São Paulo, Brazil. POPULATION: Pregnant women at 21-24 weeks' gestation. METHODS: Vaginal samples from 107 women whose vaginal microbiome and pregnancy outcomes were previously determined were analysed for phages by metagenomic sequencing. MAIN OUTCOME MEASURES: Identification of phage families, genera and species. RESULTS: Phages were detected in 96 (89.7%) of the samples. Six different phage families were identified: Siphoviridae in 69.2%, Myoviridae in 49.5%, Microviridae in 37.4%, Podoviridae in 20.6%, Herelleviridae in 10.3% and Inviridae in 1.9% of the women. Four different phage families were present in 14 women (13.1%), three families in 20 women (18.7%), two families in 31 women (29.1%) and one family in 31 women (29.1%). The most common phage species detected were Bacillus phages in 48 (43.6%), Escherichia phages in 45 (40.9%), Staphylococcus phages in 40 (36.4%), Gokushovirus in 33 (30.0%) and Lactobacillus phages in 29 (26.4%) women. In a preliminary exploratory analysis, there were no associations between a particular phage family, the number of phage families present in the vagina or any particular phage species and either gestational age at delivery or the bacterial community state type present in the vagina. CONCLUSIONS: Multiple phages are present in the vagina of most mid-trimester pregnant women. TWEETABLE ABSTRACT: Bacteriophages are present in the vagina of most pregnant women.

Amniotic fluid lactic acid and matrix metalloproteinase-8 levels at the time of fetal surgery for a spine defect: association with subsequent preterm prelabour rupture of membranes.

OBJECTIVE: In utero fetal surgery to correct incomplete closure of the spinal cord lessens the extent of permanent damage but is associated with preterm prelabour rupture of membranes (PPROM). We determined whether compounds in amniotic fluid collected at the time of surgery predicted subsequent development of PPROM. DESIGN: Prospective study. SETTING: Hospitals in Sao Paulo, Brazil. POPULATION: Twenty-four consecutive pregnant women at 24-26 weeks of gestation seen between February and October 2017 with a singleton pregnancy underwent in utero surgery to correct an open spinal defect in their fetus. METHODS: Amniotic fluid was tested for lactic acid, matrix metalloproteinase 2 (MMP-2), MMP-8, MMP-9 and interleukin-6 (IL-6) by enzyme-linked immunosorbent assay. Clinical data were collected after completion of all laboratory studies. MAIN OUTCOME MEASURE: Amniotic fluid concentration of compounds in women with or without PPROM. RESULTS: Preterm prelabour rupture of membranes occurred in seven (29.2%) women. There were no differences in maternal age, gravidity, parity, race, history of caesarean sections or fetal gender between women with or without PPROM. Length of surgery, days of wound healing and length of hospital stay were also indistinguishable. The median concentrations of MMP-8 (1.7 versus 0.6 ng/ml; P = 0.0041) and lactic acid (7.1 versus 5.9 mm; P = 0.0181) were higher in women with PPROM. The amniotic fluid MMP-8 level was also negatively correlated with gestational age at delivery (Spearman r = -0.4217, P = 0.0319). CONCLUSION: Differences in susceptibility to develop PPROM are present before fetal surgery. An increase in anaerobic glycolysis, evidenced by the intra-amniotic lactic acid level, may enhance MMP-8 production and weaken maternal and fetal membranes. TWEETABLE ABSTRACT: Matrix metalloproteinase-8 and lactic acid in amniotic fluid predict preterm prelabour rupture of membranes.

Differential expression of lactic acid isomers, extracellular matrix metalloproteinase inducer, and matrix metalloproteinase-8 in vaginal fluid from women with vaginal disorders.

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 U-test and by Spearman's rank correlation test. MAIN OUTCOME MEASURES: Metabolite levels and their correlation with diagnoses. RESULTS: Vaginal concentrations of D- and L-lactic acid were reduced from control levels in BV (P < 0.0001); L-lactic acid levels 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.

Silibinin attenuates oxidative metabolism and cytokine production by monocytes from preeclamptic women.

Silibinin is a polyphenolic plant flavonoid with anti-inflammatory properties. The present study investigated the effect of silibinin on oxidative metabolism and cytokine production - tumor necrosis factor-alpha (TNF-α), interleukin (IL)12, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, IL-10, and transforming growth factor beta (TGF-ß1) - by peripheral blood monocytes (PBM) from preeclamptic pregnant women. It is a case-controlled study involving women with preeclampsia (PE, n = 30) compared with normotensive pregnant (NT, n = 30) and with non-pregnant (NP, n = 30) women. Monocytes were obtained and cultured with or without silibinin (5 µM or 50 µM) for 18 h. Superoxide anion (O2-) and hydrogen peroxide (H2O2) release were determined by specific assays, and cytokine levels were determined by immunoenzymatic assays (ELISA). Monocytes from preeclamptic women cultured without stimulus released higher levels of O22, H2O2 and TNF-α, and lower levels of IL-10 and TGF-ß1 than did monocytes from NT and NP women. Treatment in vitro with silibinin significantly inhibited spontaneous O2- and H2O2 release and TNF-α production by monocytes from preeclamptic women. The main effect of silibinin was obtained at 50 µM concentration. Thus, silibinin exerts anti-oxidative and anti-inflammatory effects on monocytes from preeclamptic pregnant women by inhibiting the in vitro endogenous release of reactive oxygen species and TNF-α production.

PP061. The role of heat shock protein 60 and 70 in early- and late-onset preeclampsia differentiation.

INTRODUCTION: Preeclampsia is a human pregnancy-specific syndrome characterized by the onset of hypertension and proteinuria. These manifestations may occur before the 34th week of gestation or from this period on, being denominated early-onset or late-onset preeclampsia respectively. The etiology of both disorders seems to differ qualitatively; therefore, different strategies of prevention and treatment must be studied. OBJECTIVES: The aim of the present study is to determine whether the plasma levels of heat-shock proteins Hsp60 and Hsp70 as well as specific antibodies anti-Hsp60 and anti-Hsp70 may differentiate early-onset from late-onset preeclampsia. METHODS: We evaluated 175 pregnant women with PE (55 early-onset PE and 120 late-onset PE). Plasma was obtained from peripheral blood and Hsp60, Hsp70 as well as anti-Hsp60 and anti-Hsp70 antibody levels were determined by enzyme immunoassay. Uric acid levels were also determined in the plasma of patients. For statistical analyses, the Mann-Whitney U-test and the Spearman rank order correlation were applied with significance level set at 5%. RESULTS: Hsp70 levels obtained from early-onset PE group were significantly higher than the late-onset PE women and showed positive correlation with uric acid (r=0.4547; p=0.0028). The Hsp60 production was similar in both groups. Our results also indicate that there was no significant difference of anti-Hsp60 and anti-Hsp70 antibody levels between women with early- and late-onset PE. However,these antibody levels were high,indicating a strong relationship with the production of HSP60 and Hsp70 protein. CONCLUSION: Association between levels of Hsp70 and uric acid in plasma of patients with early-onset PE seems to reflect the oxidative stress in this group of patients. This study provides evidence that Hsp70 determination may be utilized to assess the differentiation between early- and late-onset PE. FINANCIAL SUPPORT: FAPESP 2010/09241-2.

Interleukin-1 receptor antagonist gene polymorphism and circulating levels of human immunodeficiency virus type 1 RNA in Brazilian women.

Interleukin-1 receptor antagonist (IL-1ra) gene polymorphisms in 83 human immunodeficiency virus (HIV)-seropositive women were evaluated. Fourteen of the subjects (16.9%) were homozygous for IL-1ra allele 2 (IL-1RN*2). These women had a lower median level of HIV RNA than did women homozygous for allele 1 (IL-1RN*1) (P = 0.01) or heterozygous for both alleles (P = 0.04). Among 46 subjects not receiving antiretroviral treatment, HIV levels were also reduced in IL-1RN*2 homozygous individuals (P < 0.05). There was no relation between IL-1ra alleles and CD4 levels.

Differentiation between women with vulvovaginal symptoms who are positive or negative for Candida species by culture.

OBJECTIVE: To investigate whether clinical criteria could differentiate between women with vulvovaginitis who were culture positive or negative for vaginal Candida species. METHODS: Vulvovaginal specimens were obtained from 501 women with a vaginal discharge and/or pruritus. Clinical information and wet mount microscopy findings were obtained. All specimens were sent to a central laboratory for species identification. RESULTS: A positive culture for Candida species was obtained from 364 (72.7%) of the specimens. C. albicans was identified in 86.4% of the positive cultures, followed by C. glabrata in 4.5%, C parapsilosis in 3.9%, C. tropicalis in 2.7% and other Candida species in 1.4%. Women with a positive Candida culture had an increased utilization of oral contraceptives (26.1% vs. 16.8%, p = 0.02) and antibiotics (8.2% vs. 0.7%, p = 0.001), and were more likely to be pregnant (9.1% vs. 3.6%, p = 0.04) than the culture-negative women. Dyspareunia was more frequent in women without Candida (38.0% vs. 28.3%, p = 0.03) while vaginal erythema (p = 0.01) was more common in women with a positive Candida culture. CONCLUSIONS: Although quantitative differences were observed, the presence of vaginal Candida vulvovaginitis cannot be definitively identified by clinical criteria.

Ureaplasma urealyticum colonization in the vaginal introitus and cervix of human immunodeficiency virus-infected women.

Ureaplasma urealyticum colonization was examined in paired cervical and introital specimens from 56 untreated HIV-seropositive women. Specimens were tested for U. urealyticum by polymerase chain reaction (PCR). Peripheral blood was examined for CD4 lymphocyte counts and HIV RNA concentration. U. urealyticum was detected in the cervix of 38 (69.1%) women. Introital U. urealyticum was present in 16 (28.6%) women, all of whom were cervical-positive. Cervical motion pain was present in 40.0% of women with U. urealyticum in the introitus and cervix, 23.8% of those with only cervical U. urealyticum and 5.9% of women negative for this organism (P=0.03). There was no relation between U. urealyticum colonization and CD4 lymphocyte count. However, 64.3% of women with introital U. urealyticum had circulating HIV RNA levels > 10,000 copies per ml as compared with 28.6% of women with only cervical U. urealyticum and 7.1% of women negative for this organism (P < 0.01).

Circulating heat shock proteins in women with a history of recurrent vulvovaginitis.

OBJECTIVE: Predisposing factors influencing recurrences of bacterial vaginosis (BV) or vaginitis from Candida remain unidentified for most women. As a component of studies to determine host susceptibility factors to genital tract infections in women, we measured expression of the 60-kDa and 70-kDa heat shock proteins (hsp60 and hsp70, respectively) in the circulation of women with or without a history of recurrent BV or candidal vaginitis and with or without a current lower genital tract infection. Heat shock protein expression is associated with a down-regulation of pro-inflammatory immune responses that would inhibit microbial infection. METHOD: The investigators measured hsp60 and hsp70, antibodies to these proteins, the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha), and the anti-inflammatory cytokine interleukin-10 (IL-10) in sera by ELISA. The study population consisted of 100 women who attended a gynecology clinic in Campinas, Brazil. Of those, 55 had a history of recurrent vulvovaginitis (RV), while 45 were controls with no such history. Only women who were asymptomatic for at least 1 month were studied. RESULTS: Although all were asymptomatic, clinical and microbiological examination revealed that five of the women with a history of RV and two controls had a current candidal vaginal infection; 16 RV patients and 12 controls had BV; and six RV patients had both BV and candidiasis. Twenty-eight RV patients and 31 controls had no clinical or microbiological detectable vaginal infection. Among the RV patients, hsp60 and hsp70 were more prevalent in those with current BV (40.9% and 50.0%, respectively) or a candidal infection (45.5% and 54.5%) than in women with no current infection (21.4% and 17.9%). In the women with no history of RV, BV was not associated with a high prevalence of hsp60 (8.3%) or hsp70 (8.3%). Interleukin-10 and TNF were not more prevalent in vaginitis patients or controls with a current candidal infection or BV than in uninfected subjects. CONCLUSION: The high prevalence of circulating hsp60 and hsp70 in women with a history of RV and current BV or vaginal candidiasis, but not in women with no history of RV, suggests that differences in heat shock protein induction may be related to susceptibility to recurrent vaginal infections.

The 60 kDa heat shock protein in human semen: relationship with antibodies to spermatozoa and Chlamydia trachomatis.

The presence of the 60 kDa heat shock protein (hsp60) in seminal fluid and its relationship to sperm autoimmunity or a localized immune response to Chlamydia trachomatis were examined. Semen from 64 male partners of infertile couples with no history of a chlamydial infection were investigated. Hsp60 was identified by an enzyme-linked immunosorbent assay (ELISA) using a monoclonal anti-hsp60 antibody bound to wells of a microtitre plate and a polyclonal anti-hsp60 antibody for detection. Antisperm antibodies on motile spermatozoa were detected by immunobead binding, while antichlamydial immuno-globulin (Ig) A and IgG in seminal fluid were identified by a commercial ELISA (SeroELISA: Savyon Diagnostics, Beer-Sheva, Israel). RNA was purified from isolated seminal round mononuclear cells and tested for hsp60-specific mRNA by a reverse transcription polymerase chain reaction ELISA. Hsp60 was present in semen from nine (14.1%) men, 12 (18.8%) men had antisperm autoantibodies. 16 (25.0%) were positive for antichlamydial IgA and 17 (26.6%) had detectable hsp60-specific mRNA. The presence of hsp60 in semen correlated with the occurrence of antichlamydial IgA (P = 0.0005), hsp60 mRNA (P = 0.04) and antisperm antibodies (P = 0.05). Thus, hsp60 was present in a soluble form in semen primarily in men with evidence of immune system activation within their genital tract. The role of hsp60 in promoting or inhibiting immune responses within the genital tract remains to be determined.

Autoimmunity to spermatozoa, asymptomatic Chlamydia trachomatis genital tract infection and gamma delta T lymphocytes in seminal fluid from the male partners of couples with unexplained infertility.

The relationship between an undetected, asymptomatic Chlamydia trachomatis genital tract infection, the concentration of gamma delta and alpha b T cells in semen and sperm autoimmunity was examined in 48 male partners of couples with unexplained infertility. Immunoglobulin A (IgA) antibodies to C. trachomatis were detected in seminal fluids from 14 (29.2%) of the men. Only four of these were positive for circulating anti-chlamydial IgA, suggesting that the stimulus for antibody production was within the genital tract. In contrast, four men were positive for anti-chlamydial IgG in their semen; all were also seropositive for anti-chlamydial IgG. T lymphocytes bearing the alpha beta and gamma delta antigen receptors were present in every semen sample. Men with seminal anti-chlamydial IgA, however, had significantly (P = 0.035) elevated semen gamma delta T cell concentrations (median 3100 cells/ml) than did men lacking this antibody (median 1400 cells/ml); concentrations of alpha beta T cells were comparable in both groups. Genital tract sperm autoimmunity, as shown by antibodies bound to motile ejaculated spermatozoa, was detected in 13 (27.1%) men. The presence of these antibodies was associated with elevated concentrations of both gamma delta (median 4200 versus 700 cells/ml) and alpha beta (median 5000 versus 850 cells/ml) T cells (P = 0.0002 and 0.0001 respectively). Men with antisperm antibodies only in their serum had seminal T cell concentrations comparable with men testing negative for antisperm antibodies. Anti-chlamydial IgA was identified in semen from four of 10 men with IgA bound to their spermatozoa and in none of the men with only spermatozoa-bound IgG.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of circulating gamma delta T-lymphocytes by autologous sperm from men sensitized to sperm.

The in vitro proliferative responses to sperm of T-lymphocytes bearing the alpha beta or gamma delta form of the antigen receptor were investigated. Peripheral blood mononuclear cells from five men without antisperm antibodies and eight men with antisperm antibodies both on sperm and in serum were incubated for 72 h in the presence of an equal concentration of autologous live sperm, freeze-thawed sperm and heat-killed sperm. Prior to incubation and after 72 h, aliquots were applied to Teflon coated microscope slides, which were subsequently incubated with monoclonal antibodies to the beta chain and delta chain of the human T-cell receptor. Urethral samples were cultured for bacteria and tested for Chlamydia trachomatis by direct staining and by ELISA. The four men with chlamydial infections had the highest concentrations of circulating gamma delta T-cells (P = 0.0008). The concentration of gamma delta T-cells from men with antisperm antibodies increased 245% over the buffer control in response to live sperm (P < 0.0001). Proliferation of gamma delta T-cells was also seen to a lesser extent in response to freeze-thawed (P = 0.002) and heat killed (P = 0.03) sperm. In contrast, gamma delta T-cells from men without antisperm antibodies proliferated only marginally (36%) in response to live sperm (P = 0.05) and were unresponsive to non-viable sperm. Only from men sensitized to sperm were alpha beta T-cells responsive, to a small extent, to live sperm (P = 0.04). Thus, in men with antisperm antibodies, peripheral gamma delta and alpha beta T-cells appeared to be sensitized to antigens on the surface of viable sperm. The immune response of gamma delta T-cells to sperm may be a useful in vitro system to examine the mechanism of gamma delta T-cell activation.