Conceived and designed the experiments: RM DNF MJK BCH. Performed the experiments: RM CC TF SK. Analyzed the data: RM TM MK SV. Contributed reagents/materials/analysis tools: DNF BCH. Wrote the paper: RM BCH.
The authors have declared that no competing interests exist.
Genital secretions collected from adult women exhibit
Anti-HSV and anti-
Biomarkers of genital mucosal innate immunity may differ substantially between sexually active adolescents and adult women. These findings warrant further study and may have significant implications for prevention of sexually transmitted infections in adolescent females.
Epidemiological studies consistently demonstrate that sexually transmitted infections (STI) disproportionately affect adolescent females
Genital tract secretions contain a wide array of innate host defense molecules and represent locally-produced mediators and transudate from the systemic circulation. These molecules include human neutrophil peptides 1–3 (HNP 1-3), lactoferrin, and lysozyme, produced primarily by immune cells; IgG and IgA, secreted by plasma cells, secretory leukocyte protease inhibitor (SLPI) and β defensins, which are secreted by epithelial cells and keratinocytes. These mediators may protect against infection but, paradoxically, may also recruit and activate immune cells that serve as targets for HIV infection
The extent of endogenous antimicrobial activity has not been described in genital secretions from adolescent females. We hypothesize that adolescents have altered genital mucosal host defenses compared to reproductively mature adults and conducted a cross-sectional analysis of antimicrobial activity and its potential mediators, including vaginal microbiota, in genital specimens collected from healthy, sexually active adolescent females aged 15–18 years. Outcomes for adolescents were compared to those for adult females who were participating in unrelated studies.
This study was approved by the Albert Einstein College of Medicine Institutional Review Board (IRB), and all clinical investigation was conducted according to the principles expressed in the Declaration of Helsinki. All participants provided written informed consent. This study involved screening for sexually transmitted infections and gynecologic care, for which minors have a legal right to confidentiality in New York State
Sexually active females between the ages of 15 and 18 years were recruited from the Bronx, New York, from December 2008 to May 2011 to complete a single study visit. Enrollment criteria included a reported history of vaginal intercourse. Exclusion criteria included pregnancy, breastfeeding, incident genital tract infection, or menstrual bleeding at the time of study visit; reported history of vaginal intercourse or use of douching products in the 48 hours prior to study visit; or use of oral or vaginal antimicrobial products in the seven days prior to study visit. In addition, specimens and data were collected from healthy, premenopausal adult women who were fewer than 50 years of age and who were participating in separate, IRB-approved genital mucosal immunity studies. Exclusion criteria for adult subjects included those listed above for adolescents, although some of the adult studies excluded women using hormonal contraception. Study screening procedures and specimen collection methods were identical for adolescent and adult subjects.
Urine was collected for pregnancy testing. Prior to speculum exam, vaginal pH was measured from a Dacron® swab of the lateral vaginal wall (Whatman pH paper, pH 3.8–5.5). A gynecological speculum examination was then performed. A Dacron® swab was rolled across the lateral vaginal wall and stored in 0.5 ml phosphate buffered saline (PBS) at −80°C for quantification of vaginal microbiota. Screening for
CVL specimens were transported to the laboratory on ice and subjected to centrifugation at 700 g for 10 minutes at 4°C. Supernatants were aliquoted and stored at −80°C.
The antimicrobial activity of each subject’s CVL sample against HSV-2 and
The protein concentration of each CVL specimen was quantified (Micro BCA Protein Assay, Pierce Biotechnology, Rockford, IL). Interleukin (IL)-1α, IL-1β, IL-6, IL-8, interferon (IFN)-γ, IFN-α2, IL-1 receptor antagonist (ra), macrophage inflammatory protein (MIP)-1α, MIP-1β, and regulated upon activation, normal T-cell expressed and secreted (RANTES) were measured by multiplex proteome array with beads from Chemicon (Billerca, MA), quantified with Luminex100 (Austin, TX), and analyzed with StarStation (Applied Cytometry Systems, Sacramento, CA). Concentrations that fell below the manufacturer’s minimal detection concentration (MMDC) were set at the midpoint between zero and the MMDC.
The following immune mediators were quantified by ELISA: SLPI (R & D Systems, Minneapolis, MN), lactoferrin (Calbiochem, San Diego, CA), lysozyme (Alpco Diagnostics, Salem, NH), HNP 1–3 (HyCult Biotechnology, Uden, The Netherlands), IgG and IgA (Cygnus Technologies, Southport, NC).
Stored vaginal swabs were subjected to DNA extraction as previously described, with one sham extraction control for every 12 vaginal swabs
Descriptive statistics (mean, standard deviation [SD], median, range, interquartile range [IQR]) were computed, and scatter plot graphical representations were generated. Continuous variables were compared by Student’s t test or the non-parametric equivalent. Categorical variables for each group were compared by Fisher’s exact test or by Chi squared test. Spearman’s correlation coefficients (SCC) were calculated to evaluate associations between continuous variables, and heat maps of SCC grouped by partitioning around medoids were generated. The data were clustered by an agglomerative approach, with the distance between two covariates defined as 1-SCC. Pairwise distances were calculated for all 18 variables. The first clustering was then determined by grouping together the variables with the most similar distance, with subsequent clusters determined by calculating the maximum pairwise distance within each cluster (complete linkage method).
In order to examine the relationship of
23 females between the ages of 15 and 18 were enrolled. Three subjects with asymptomatic chlamydia at enrollment were excluded from the analysis. Data from the remaining 20 adolescents (mean age ± standard deviation [SD] of 16.9±0.2 years) were compared to those collected from 54 adult females (mean age ± SD of 30.7±1.1 years, p<0.001) who had no evidence of incident genital tract infection (
Characteristic | Adolescents (n = 20) | Adults (n = 54) | P |
Age, mean ± standard deviation (SD) | 16.9±0.2 | 30.7±1.1 | <0.001 |
Race/ethnicity |
0 W, 3 B, 16 H, 1 O | 10 W, 24 B, 16 H, 4 O | 0.001 |
Vaginal pH, mean ± SD | 4.8±0.08 | 4.5±0.07 | 0.005 |
HSV seropositivity |
11 HSV-1, 1 HSV-2 | 36 HSV-1, 13 HSV-2 | 0.58, 0.1 |
Hormonal contraceptive use | 4 | 0 | 0.004 |
Current cigarette smoker | 1 | 2 | 1 |
H = Hispanic, B = black, W = white, O = other.
HSV serology results were available for 19 adolescent subjects.
CVL samples from adolescent subjects exhibited marked endogenous anti-HSV-2 activity and inhibited viral plaque formation by a median of 95.4% (range 12.6–100) (
For anti-HSV activity, Vero cells were infected with HSV-2(G) mixed 1∶1 with each CVL specimen or control buffer. Viral plaques were counted after 48 hours and percent inhibition calculated relative to control. For
In contrast to anti-HSV activity, endogenous anti-
Exclusion of the four adolescent subjects utilizing hormonal contraception did not significantly alter these results. Anti-HSV activity and anti-
SCC were grouped by partitioning around medoids, and a color-coded heat map was generated to examine potential associations between CVL endogenous antimicrobial activity (anti-HSV, anti-
The relationship between age and endogenous anti-
HSV inhibition (N = 74) | |||||
Mean ± SD 64.05±35.72 | Mean ± SD 59.33±29.61 | ||||
Variable | Mean (SD) or N (%) | ß (Standard error) | p | ß (Standard error) | p |
|
27.51 (9.24) | −0.69 (0.59) | 0.24 | 0.18 (0.50) | 0.72 |
|
|||||
|
10 (13.51) | Reference | Reference | ||
|
27 (36.49) | 0.37 (13.74) | 0.98 | −4.71 (11.62) | 0.69 |
|
32 (43.24) | 14.12 (13.79) | 0.31 | −12.18 (11.66) | 0.30 |
|
5 (6.76) | 21.91 (19.82) | 0.27 | −21.07 (16.76) | 0.21 |
|
47 (65.28) | −0.86 (9.51) | 0.93 | −3.64 (8.04) | 0.65 |
|
13 (18.06) | 5.11 (13.37) | 0.70 | −2.70 (11.30) | 0.81 |
|
4.57 (0.51) | −2.56 (8.22) | 0.76 | −12.54 (6.95) | 0.08 |
The median CVL total protein concentration was significantly reduced in adolescents compared to adults (112.5 vs. 237.5 µg/ml, p<0.001) (
Mediator | Median (Interquartile range) | Adolescents (n = 20) | Adults (n = 54) | p |
Protein | µg/ml CVL | 112.5 (75–239) | 237.5 (149.5–386) | <0.001 |
HNP 1–3 | pg/ml CVL | 16,688 (5,067–44,093) | 35,616 (11,619–246,735) | 0.08 |
pg/µg protein | 117.5 (69.2–285.8) | 76.8 (21.9–366.7 | 0.16 | |
Lactoferrin | pg/ml CVL | 393.5 (244.5–787.3) | 474.5 (174–1,741) | 0.45 |
pg/µg protein | 3.5 (2–5.6) | 2 (0.7–6.4) | 0.29 | |
Lysozyme | pg/ml CVL | 115 (73–368.3) | 126 (80.9–651.3) | 0.29 |
pg/µg protein | 1.2 (0.8–1.6) | 0.8 (0.4–1.7) | 0.11 | |
SLPI | pg/ml CVL | 99,843 (28,680–286,468) | 237,988 (77,939–464,119) | 0.03 |
pg/µg protein | 609.1 (243.5–1,937) | 919.8 (388.8–2,012) | 0.28 | |
IgG | ng/ml CVL | 1,429 (1,046–1,686) | 6,684 (3,473–21,236) | <0.001 |
ng/µg protein | 12.5 (8.9–19.1) | 33.6 (14.4–64.8) | <0.001 | |
IgA | ng/ml CVL | 365.4 (84–3–805.3) | 1,104 (502.5–2,460) | <0.001 |
ng/µg protein | 2.4 (0.6–5.6) | 4.8 (2.5–8.6) | 0.04 |
The absolute concentrations of the six quantified antimicrobial peptides were lower in adolescents. However, the protein-corrected CVL concentrations of HNP 1–3, lysozyme, and lactoferrin were greater in adolescent subjects, although these trends did not reach statistical significance. Similarly, the protein-corrected concentrations of cytokines IL-6 and IL-1α were significantly higher in adolescent CVL specimens (p<0.001 and p = 0.005, respectively), as was the median concentration of IL-1ra (p<0.001) (
Mediator | Median (Interquartile range) | Adolescents (n = 20) | Adults (n = 54) | p |
IL-6 | pg/ml CVL | 5.8 (3.1–11.2) | 11.4 (2.7–22.3) | 0.21 |
pg/µg protein | 0.05 (0.02–0.08) | 0.03 (0.01–0.1) | <0.001 | |
IL-1â | pg/ml CVL | 3.4 (1.8–11.8) | 9.6 (1.5–2,122) | 0.14 |
pg/µg protein | 0.03 (0.02–0.07) | 0.05 (0.01–13.3) | 0.6 | |
IL-1á | pg/ml CVL | 73 (28.6–146.4) | 44.7 (19–97.3) | 0.19 |
pg/µg protein | 0.6 (0.1–1.4) | 0.18 (0.1–0.4) | 0.005 | |
IL-1ra | pg/ml CVL | 8,857 (5,771–11,410) | 7,819 (5,203–9,772) | 0.39 |
pg/µg protein | 80.5 (31.4–111.9) | 30.7 (16.6–52.5) | <0.001 | |
IFNã | pg/ml CVL | 1.04 (0.13–1.5) | 1.1 (0.5–2.2) | 0.61 |
pg/µg protein | 0.01 (0.001–0.02) | 0.004 (0.002–0.01) | 0.24 | |
IL-8 | pg/ml CVL | 213.5 (76.9–1,408) | 317.1 (97.9–1,078) | 0.56 |
pg/µg protein | 2 (0.7–5.8) | 1.6 (0.4–4) | 0.38 | |
RANTES | pg/ml CVL | 2 (0.5–5.5) | 2.3 (1–4.6) | 0.57 |
pg/µg protein | 0.01 (0.01–0.04) | 0.01 (0.004–0.02) | 0.15 | |
MIP-1á | pg/ml CVL | 3.5 (1.8–12.4) | 9.3 (4.5–18.2) | 0.08 |
pg/µg protein | 0.04 (0.02–0.09) | 0.04 (0.02–0.08) | 0.75 | |
MIP-1â | pg/ml CVL | 2.9 (2.3–11.7) | 7.6 (2.3–20.9) | 0.06 |
pg/µg protein | 0.03 (0.02–0.06) | 0.03 (0.02–0.09) | 0.63 |
In contrast, IgG and IgA remained significantly lower in adolescent CVL even after protein correction (p<0.001 for IgG, p = 0.04 for IgA), with a 50% reduction of the protein-corrected concentrations observed in adults. The concentration of antimicrobial and anti-inflammatory SLPI was also lower in adolescent CVL, although the difference was no longer statistically significant after protein correction. The difference between adolescent and adult protein-corrected concentrations of CVL IL-6 was no longer significant after exclusion of the four adolescents receiving hormonal contraception (p = 0.89), but comparison of the remaining mediators was not significantly altered by the loss of these subjects.
For both adolescents and adults, anti-HSV activity correlated positively and significantly with CVL concentrations of HNP 1–3 (ρ = 0.54 adolescents, ρ = 0.31 adults), lysozyme (ρ = 0.78 adolescents, ρ = 0.42 adults), lactoferrin (ρ = 0.3 for adolescents and adults) and IL-8 (ρ = 0.63 adolescents, ρ = 0.45 adults). The anti-HSV activity also correlated positively and significantly with IL-1β, MIP-1β, and IL-6 (ρ = 0.61, 0.58, 0.39, respectively) among adolescents and with IL-1α (ρ = 0.48) among adults. Anti-HSV activity did not correlate significantly with CVL IgG in either group. In contrast to anti-HSV activity, anti-
Associations between immune mediators were also analyzed by Spearman’s correlation coefficients. HNP 1–3 correlated positively with lysozyme (ρ = 0.74 adolescents, 0.7 adults), lactoferrin (ρ = 0.73 adolescents, 0.56 adults), IL-1β (ρ = 0.39 adolescents, 0.4 adults), IL-6 (ρ = 0.52 adolescents, 0.54 adults), IL-8 (ρ = 0.73 adolescents, 0.68 adults), MIP-1β (ρ = 0.58 adolescents, 0.55 adults) and protein (ρ = 0.47 adolescents, 0.5 adults).
Heat maps were generated to represent correlation coefficients and the degree of relatedness between anti-
SCC were grouped by partitioning around medoids, and a color-coded heat map was generated to examine potential associations between CVL endogenous antimicrobial activity (anti-HSV, anti-
The modest but significant increase in vaginal pH observed in adolescent subjects, combined with the lower levels of endogenous activity against
Adolescents | Adults | p | Adolescents | Adults | p | |
(n = 20) | (n = 24) | (n = 20) | (n = 24) | |||
No. PCR+ | No. PCR+ | DNA copies/swab, median | DNA copies/swab, median | |||
|
12 | 13 | 0.77 | 4.2×103 | 2.8×104 | 0.31 |
|
8 | 14 | 0.36 | 62.5 | 3.4×104 | 0.004 |
In addition, extracted DNA from vaginal swabs collected from adolescent subjects was available for quantification of
To our knowledge, this is the first study to examine genital secretion antimicrobial activity and its potential mediators in adolescent females. We found that CVL collected from a cohort of sexually active adolescents had significantly more endogenous anti-HSV activity relative to older women. This finding correlated with higher concentrations of specific cytokines and antimicrobial proteins (lactoferrin, lysozyme and HNP 1–3) in adolescent CVL, although for some cytokines and chemokines, the CVL concentrations were low and the differences between groups modest, thus the physiological impact of any differences noted is unclear. In contrast, adolescents exhibited lower CVL levels of SLPI, IgG, and IgA, reduced CVL anti-
A multivariable linear regression model identified a non-significant association between increasing anti-HSV inhibition and decreasing age, which could suggest that age was a surrogate marker for a more significant variable not assessed in our study, such as cervical ectopy or immune cell populations. Chemokine, cytokine, and antimicrobial peptide secretion may differ substantially between ectocervical, endocervical, and uterine microenvironments. For example, endocervical and uterine cell lines secrete significant levels of the lymphocyte and macrophage inflammatory protein 3 alpha (MIP-3α) in response to inflammatory stimuli
Importantly, the results of this study suggest several potential mechanisms that could contribute to genital mucosal immune activation and an increased risk of HIV and STI acquisition and transmission among adolescents. For adolescents, SCC and heat map analyses identified a tight, “clustering” association between anti-HSV activity, HNP 1–3, lysozyme, lactoferrin, IL-1β, and IL-8. An increase in antimicrobial factors such as HNP 1–3, lysozyme, and lactoferrin, released by degranulating neutrophils recruited in response to IL-8
The lower overall protein levels in adolescent CVL merit further study, as the specific proteins measured represent <2% of the total protein. IgG and IgA were significantly lower in adolescents, both as absolute CVL and protein-corrected concentrations. A prior study of three adolescent females identified steeper peri-ovulatory drops in cervical IgG relative to the nadir observed in sexually mature women
In contrast to the increased anti-HSV activity, we observed lower endogenous anti-
Larger studies with rigorous adolescent-to-adult matching for race, ethnicity, and hormonal contraceptive use are needed to address the limitations of this study. These studies should include sampling throughout a menstrual cycle, cervical cytobrush evaluation of immune cell populations, more complete analyses of vaginal microbiota, and assessment of intercurrent HPV and HSV shedding. Prospective studies are necessary to delineate the potential relationship between endogenous antimicrobial activity and risk for STI acquisition.
The results of this cross-sectional analysis, however, suggest several key differences between the adolescent and adult genital tract environment. We speculate that these differences may translate to an increased
The authors thank the Montefiore Medical Center Adolescent Medicine and Adolescent AIDS Programs for assistance in subject recruitment and the Center for AIDS Research at the Albert Einstein College of Medicine and Montefiore Medical Center.