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Johns Hopkins University School of Medicine and Bloomberg School of Public Health, Baltimore, Maryland
Montefiore Medical Center and the Albert Einstein College of Medicine, Bronx, New York
Brown University, Providence, Rhode Island
Wayne State University, Detroit, Michigan
Centers for Disease Control and Prevention, Atlanta, Georgia
The impact of demographic characteristics, phase of the menstrual cycle, use of hormonal contraceptives, and concomitant lower genital-tract infections on cervicovaginal inflammatory cells was assessed in 967 women, 654 of whom were infected with human immunodeficiency virus type 1 (HIV-1). Cervicovaginal lavage (CVL) fluid was evaluated for total white blood cell (WBC), polymorphonuclear leukocyte, and monocyte counts. HIV-1 infection was not associated with statistically significant differences in numbers of inflammatory cells in CVL fluid except in 1 groupHIV-1infected women with Chlamydia trachomatis infection had a 0.43 log10 higher WBC count than their HIV-uninfected, chlamydia-positive counterparts (P = .04). Younger age and use of progesterone-based hormonal contraceptives were independently associated with increased numbers of inflammatory cells in CVL fluid. A 0.150.2 log10 increase in inflammatory cells was seen in black versus white and Hispanic women after adjustment for known potential confounders. Progesterone-based contraceptives, younger age, and race have an independent effect on cervicovaginal inflammatory cells.
Worldwide, heterosexual transmission is still the most common route of spreading HIV-1 [1]. Most HIV-1 infections in men are thought to have resulted from exposure to the virus in vaginal secretions [2]. Proviral HIV-1 DNA, in addition to both cell-free and cell-associated RNA, has been found in cervicovaginal secretions [35]. The source of the virus is still unclear: studies have shown that monocytes, Langerhans cells, and CD4 lymphocytes, which are found in cervicovaginal secretions, may be responsible [6, 7]. A more recent study by Ellerbrock et al. [8] suggested that cellular replication within vaginal secretions occurs independently of that in the blood compartment.
Attempts to correlate HIV-1 loads in cervicovaginal secretions and in the blood compartment have yielded mixed results: some studies have shown a positive correlation [913], but others have reported no correlation between the 2 compartments [1416]. Some of those differences can be attributed to differences in the techniques used to detect HIV-1 in the lower genital-tract compartment and the significantly higher short-term variations in HIV-1 load in the genital-tract compartment than in that of blood [17].
The effect of sex hormones on susceptibility to sexually transmitted infections (STIs) has been debated for the past several decades [18]. With regard to HIV-1, some studies have suggested that a menstrual phasedependent variation in HIV load exists [1921], but others have not [22]. Several other studies have suggested that progesterone increases the risk of HIV-1 acquisition [23, 24]. Studies in macaques have demonstrated the role of progesterone implants in enhancing the transmission of simian immunodeficiency virus (SIV) and the protective role of estrogen in that setting [25, 26].
The presence of cervicovaginal inflammation has been shown to increase the HIV-1 load in both cervical and vaginal secretions [27, 28]. In one study, cervical inflammation and ulceration increased by 10,000-fold the amount of HIV-1 present in vaginal secretions [29]. The associations between the risk of HIV-1 acquisition and inflammatory STIs, such as gonorrhea and chlamydia [30], further support the role of inflammation. Other than the presence of cervicovaginal infections, the use of surfactant-based spermicides [31], and vaginal douching [32], little is known about predisposing factors to cervicovaginal inflammation.
Our aim was to define other covariates that might affect the distribution and presence of cervicovaginal inflammatory cells, and we evaluated whether these variables differed in those patients who were infected with HIV. We focused on demographic characteristics, behavioral characteristics (douching and condom use), phase of the menstrual cycle, use of hormonal contraceptives, and the presence of lower genital-tract infections; we then compared the numbers and distribution of inflammatory cells measured in cervicovaginal lavage (CVL) fluid between HIV-infected and -uninfected women.
SUBJECTS, MATERIALS, AND METHODS
Population.
The HIV Research and Epidemiology Study (HERS) was a prospective, multicenter cohort study of 871 HIV-infected women and 439 demographically matched, HIV-uninfected women, 1655 years old, who were recruited between 1993 and 1995. A detailed description of participant selection and methods is available elsewhere [33, 34]. In brief, the participants were interviewed every 6 months for a total of 14 visits; interviews covered medical history, medications, reproductive history, contraceptive use, douching practices, and illicit-drug use. Blood, oral, vaginal, and cervical samples were obtained for a variety of laboratory tests. The study sample for the present analysis consisted of all women who had at least 1 follow-up visit and had both menstrual history data and CVL fluid available. Because STIs could act as confounders for cervical inflammation, only the first 5 visits for the participants were evaluated in the analysis. Women who had undergone a hysterectomy were excluded. Informed consent was obtained from all participants in HERS at each of the 4 recruitment sites, and human-experimentation guidelines of the US Department of Health and Human Services were followed in the conduct of clinical research.
Hormonal data.
All patients were asked about the starting date of their last menstrual period (LMP). The cycle phases were divided into follicular (days 010), periovulatory (days 1116), and luteal (days 1735) phases. At the time of CVL, it was noted whether a participant was actively bleeding, and the data were used to adjust for the follicular phase, because active menstrual bleeding could independently influence the number of inflammatory cells in CVL fluid. Women who were not receiving hormonal contraceptives, who were not pregnant, and who reported an absence of menses for >60 days were considered to be amenorrheic. A subset of women (n = 236) also provided a menstrual calendar at 1 of their visits. In a separate analysis, the correlation between the self-reported LMP and the date inscribed in the menstrual calendar was >90%. Data on hormonal contraceptives were obtained at every patient encounter. Data for current use of oral contraceptive pills, levonorgestrel implants (Norplant), and medroxyprogesterone acetate injections (Depo-Provera) were obtained. In view of the potential carryover effects of some of these drugs, data on their use during the preceding 6 and 12 months were also obtained. Patients who reported the use of either medroxyprogesterone acetate injections or levonorgestrel implants during the previous 6 months but not at the current visit were labeled as current users. This would ensure that the direction of any potential bias would be toward the null.
Genital-tract infections.
Laboratory workers were masked to the patients' clinical information and the results of other laboratory tests. Neisseria gonorrhoeae infection was diagnosed by use of culture. Endocervical swabs for N. gonorrhoeae were streaked on Thayer-Martin agar plates within 15 min of collection. Gram staining and an oxidase test of characteristic colonies were performed 48 h after inoculation. Chlamydia trachomatis infection was diagnosed by use of endocervical specimens by use of an EIA (Pathfinder Chlamydia EIA Microplate; Bio-Rad Laboratories), according to the manufacturer's guidelines. Trichomonas vaginalis infection was diagnosed by use of a wet preparation obtained from a vaginal swab that had been immediately placed in 10 mL of sterile saline. Bacterial vaginosis was diagnosed by use of Amsel's criteria (3 of 4 of abnormal vaginal discharge, pH >4.7, the presence of clue cells on a wet mount of vaginal secretions, and a positive "whiff test"). Vulvovaginal candidiasis (VVC) was diagnosed by use of a definition of positive KOH smear for yeast or positive culture for yeast and 1 of the following: abnormal vaginal discharge, vulvovaginal edema, or vulvovaginal erythema. The presence of genital ulcer disease (GUD) was documented by the clinician at the time that the CVL was performed. Finally, results of the twice-yearly Pap smears were also available and were categorized as normal, atypical squamous cells of undetermined significance, low-grade squamous intraepithelial lesion (SIL), high-grade SIL, or squamous-cell carcinoma. All infections were diagnosed at the time that CVL was performed.
CVL specimens.
The procedure was standardized across all sites: 10 mL of normal saline was instilled in the vaginal canal and recovered by use of a syringe. The fluid was evaluated for total white cell (WBC) count and the percentage of polymorphonuclear lymphocytes (PMNs) and monocytes, by use of automated cell counters. The results are reported as the log10 of the number of cells per cubic milliliter of CVL fluid. The CVL specimens were not tested for HIV-1 loads, and no flow-cytometric analysis on the cell populations was performed.
Miscellaneous.
Peripheral total WBC counts; corresponding percentages of neutrophils, PMNs, and monocytes; and, in HIV-seropositive women, HIV-1 load, CD4+ cell count, and use of antiretroviral therapy were documented. Because most of these samples were obtained before 1995, very few patients were receiving highly active antiretroviral therapy. Urine pregnancy tests were also obtained at each visit. Patients were asked whether they had used vaginal douching preparations within 48 h before their clinic visit, and the data were documented for all visits. Information on numbers of sex partners and condom use during the preceding 6 months was also obtained at each visit. For the analysis, the reported rate of condom use was trichotomized as "always" (100% condom use with main sex partner), "never" (0% condom use with main sex partner), and "sometimes" (inconsistent condom use with main sex partner).
Statistics.
Generalized estimating equations for longitudinal data were used for modeling. In each analysis, the correlation of the outcome over time was plotted by use of variograms, and the appropriate correlation structure was used in each model (a uniform correlation structure pervaded). Robust estimates for the SEs are reported. Statistically significant univariate predictors of each of the outcome measures were included in the final models; additionally, other variables that were not significant in the univariate analysis but were thought to be biologically relevant were also included. Interaction terms among HIV status, race, concomitant STIs, and use of hormonal contraceptives were generated and were reported if they were statistically significant. Normally distributed continuous variables were compared by use of t tests; the 2 test was used to compare categorical variables, and the K-sample equality of medians test was used to compare median values. McNemar's test was used to compare categorical observations with repeated measures. Two-sided P values were calculated, and those <.05 were assumed to indicate significance. STATA was used for all analyses (version 8; STATA).
RESULTS
Of the 1310 women in HERS, 967 (73.8%) were deemed to be eligible, having had no hysterectomy and at least 1 visit with both menstrual-cycle data and CVL fluid available. Of these, 654 (67.6%) were infected with HIV-1. The 967 women made 3914 visits, with a median number of 3/participant (no difference by HIV status). Baseline demographic characteristics of participants were similar by HIV status (table 1). As shown in table 2, HIV-positive women were significantly more likely to have visits with GUD and VVC present (4.5% vs. 2.3% and 9.5% vs. 6.2%, respectively) and with an abnormal Pap smear (28% vs. 10%).
Demographic characteristics.
In both univariate (not shown) and multivariate models (table 3), race was a significant independent predictor of inflammatory cells in CVL fluid. On average, the total WBC count in CVL fluid for black women was 0.15 log10 higher than that in white women (P = .02) and 0.19 log10 higher than that in Hispanic women (P < .001). Similar findings were seen for both CVL PMN and CVL monocyte counts. Increasing age resulted in a significant decrease of 0.01 log10 WBC and PMN counts per 1-year increment. Although age was significant as a univariate predictor of differences in monocyte count (-0.01 log10 cells/mL; P = .01), it was not predictive in the multivariate model (P = .9).
HIV status.
In the multivariate models, there were no significant differences in log10 CVL WBC, PMN, or monocyte counts between HIV-positive and -negative women (0 log10 cells/mL, P = .97; -0.05 log10 cells/mL, P = .5; and -0.11 log10 cells/mL, P = .3, respectively). In further analyses of the degree of immunosuppression, women with a CD4 count <50 cells/mm3, compared with those with a CD4 count >200 cells/mm3, showed no significant difference in log10 WBC, PMN, or monocyte counts (0.04 log10 cells/mL, P = .7; -0.02 log10 cells/mL, P = .9; and -0.08 log10 cells/mL, P = .7, respectively). In the univariate models, HIV infection (independent of CD4 and HIV load) was associated with significantly higher log10 WBC, PMN, and monocyte counts (0.1 log10 cells/mL, P = .02; 0.12 log10 cells/mL, P = .01; and 0.11 log10 cells/mL, P = .04, respectively), but, in analyses that examined the degree of immunosuppression, neither CD4 count nor HIV load was predictive in any of the multivariate models.
Hormonal status.
In general, the phase of the menstrual cycle and the use of oral contraceptive pills were not associated with a significant difference numbers of inflammatory cells in CVL fluid in either the univariate or multivariate analyses (data not shown). Pregnancy was associated in univariate analysis with a 0.37, 0.44, and 0.35 log10 decrease in total WBC, PMN, and monocyte counts, respectively (P < .001 for all associations), compared with nonpregnant subjects. The association was no longer significant in the multivariate model. Women who presented with amenorrhea, however, were likely to have more inflammatory cells in their CVL fluid than women in the follicular phase of the menstrual cycle (table 3). In both the univariate and multivariate analyses, the use of levonorgestrel implants were associated with higher log10 numbers of WBCs, PMNs, and monocytes in the CVL fluid (tables 3 and 4). Finally, in the univariate analysis, the use of medroxyprogesterone acetate injections were associated an increased log10 total WBC, PMN, and monocyte counts, but the association did not remain significant in the multivariate models (table 4).
Behavioral characteristics.
A self-report of vaginal douching within the 48 h preceding the clinic visit was not associated with a statistically significant change in numbers of inflammatory cells in either the univariate or multivariate models. Similarly, the use of condoms was not associated with changes in the number of cervicovaginal inflammatory cells in the univariate analyses. In the univariate analysis, women who reported having had >5 male sex partners during the 6 months preceding their current visit had a 0.21-log10 increase in total WBC count in their CVL fluid, compared with women who reported having had no sex partners during that time interval. The association was not significant in the multivariate model.
DISCUSSION
The size and prospective nature of the HERS cohort made it an ideal population in which to examine the relationship among CVL inflammatory cells, HIV-1 infection, concurrent STIs, and sex hormones. We found that HIV-1 status, irrespective of the degree of immunosuppression, was not associated with a significant difference in the number of inflammatory cells in CVL fluid after adjustment for other potential confounders. This finding supports a similar report by Cohn et al. [35] of 70 US and Thai women; they found no significant correlation between CD4 cell count and vaginal epidermal inflammatory cell counts according to tissue biopsy. In our analysis that compared the inflammatory cell content of HIV-positive and -negative women who had a concomitant STI, we found only 1 significant difference in inflammatory cells between the groups: women who were HIV positive and were concomitantly infected with chlamydia had higher total WBC counts in their CVL fluid than their HIV-1negative counterparts. However, the mechanism for what appears to be an HIV-mediated increase in inflammation in the setting of concomitant C. trachomatis infection is unclear. Paradoxically, a trend in the opposite direction was seen for gonorrhea, but the small numbers of incident cases of gonorrhea preclude any meaningful interpretation of the data, and the trend was not significant. We have noted, here, the relatively low incidence rates of both gonorrhea and chlamydia infections; both the older age of our cohort and the possibly safer sexual practices adopted by these seropositive and high-risk seronegative women may account for these findings.
The association in our data of cervicovaginal inflammation with hormones is interesting. There appears to be a positive correlation between various progesterone-containing hormonal contraceptive preparations and the numbers of cervicovaginal inflammatory cells (table 4) in CVL fluid. An intriguing hypothesis is that progesterone independently increases the recruitment of inflammatory cells in the cervicovaginal area, which potentially leads to the promotion of HIV-1 transmission and acquisition. In a study of macaques, progesterone implants enhanced the vaginal transmission of SIV, possibly by thinning the vaginal epithelium, although other mechanisms were not explored in that study [36]. To date, one prospective study has suggested that progesterone independently enhances the risk of HIV transmission [37]. Although these findings have been tempered by other studies that found no significant association [38], the issue remains of critical importance in view of the millions of women worldwide who use progesterone-based agents as a method of contraception. The nonsignificant association of inflammation with the use of oral contraceptives in the present study is difficult to interpret because of the lack of information about the specific composition of these pills. Combined oral contraceptives (estradiol and a progestin) are most commonly used, but progesterone-only pills are also available. In addition, although the amount of ethinyl estradiol (the most common estrogen used) tends to be fairly constant in combined pills, these differ in their progestin formulations (both type of progestin and dose), making the interpretation of the data uncertain.
The increase in the number of inflammatory cells in women who were amenorrheic raises questions about the specific mechanism involved. Unfortunately, the underlying cause of the amenorrhea was not systematically evaluated in these patients, so linking the increased number of inflammatory cells to a specific abnormality was not possible.
Two demographic characteristics independently predicted differences in the degree of cervicovaginal inflammation measured in CVL fluid. First, a correlation between increasing age and a decrease in the number of inflammatory cells was consistently seen in both HIV-infected and -uninfected women. Whether age has a direct effect on the immune system that leads to a decreased recruitment of inflammatory cells or whether age exerts an indirect influence via its effects on sex hormones is unclear. The second, and more intriguing, characteristic is race/ethnicity: in the present study, black women had higher total WBC, PMN, and monocyte counts in their cervicovaginal canals than did either white or Hispanic women, after adjustment for known confounders. This finding is most striking when one considers it in the context of the epidemiological data available regarding race/ethnicity and HIV: of all races, blacks have the highest incidence and prevalence rates of HIV [39, 40]. Although behavioral and socioeconomic factors have contributed to this finding, studies that have controlled for these factors have still shown racial disparity [41]. Recent studies have shown racial differences in CC chemokine receptor 5 haplotypes, which offers one biological hypothesis to explain some of the racial differences seen [42]. Although our data cannot prove causation, they may offer an additional mechanism that could account for these racial disparities.
The present study has several limitations. First, HIV-1 loads in the CVL fluid were not available. Peripheral HIV-1 loads and CD4 cell counts were used to adjust for the degree of immunosuppression in the analysis, but this may be misleading, because it is now reasonably well accepted that plasma and genital HIV loads are 2 separate compartments. It is unclear, however, whether inflammation is responsible for higher HIV loads or whether higher HIV loads are responsible for inflammation. It seems reasonable to assume that both processes may occur. In addition, multiple studies have shown no correlation between the concentration of sex hormones and either plasma or genital HIV loads. These data suggest that, although we may not have controlled for such a confounding effect in the HIV subanalysis, this should have no bearing on the hormone, STI, and demographic analyses. Moreover, if we are to assume that genital HIV-1 loads are completely independent of the degree of immunosuppression as manifested by both CD4 cell counts and peripheral HIV loads, then there is no reason to believe that the bias is differential across our HIV-infected stratum. If, on the other hand, genital and peripheral HIV loads are indeed correlated, then adjustment by use of the peripheral data should be adequate. Residual confounding likely is small and should have minimal effect on the results of the analysis. It would also have been interesting to evaluate lymphocyte subsets in our analysis, to determine whether the same covariates influence the CD4, CD8, and B cell populations as influence the total WBC, PMN, and monocyte populations.
A second limitation is that diagnostic methods for C. trachomatis and T. vaginalis detection may not have been ideal, given that EIAs have a lower sensitivity than nucleic acidbased tests and that cultures for trichomoniasis have a better yield than wet preparations. Any misclassification of outcome would have biased our results toward the null. A third concern is that the number of patients who were receiving progesterone-based contraceptives was relatively small; thus, the power to detect a possibly significant effect of medroxyprogesterone acetate was limited. Future studies are needed on this important topic. Finally, it is important to note that no threshold for abnormal inflammatory cell counts in CVL fluid has been established. It seems reasonable to assume that the amount of inflammation seen with causes of cervicitis such as gonorrhea or chlamydia infections might be a reasonable indicator of biological significance. Anderson et al. [43] used 106 WBCs/total CVL fluid as a definition for lower genital-tract inflammation in women.
In summary, although HIV-1 status does not seem to play a major role, both demographic characteristics and progesterone-based agents appear to modify the numbers of inflammatory cells in the cervicovaginal environment. This raises interesting questions regarding the risks of HIV transmission in blacks and in women who use progesterone-based contraceptive agents.
HIV EPIDEMIOLOGY RESEARCH STUDY (HERS) GROUP
Robert S. Klein, Ellie Schoenbaum, Julia Arnsten, Robert D. Burk, Penelope Demas, and Andrea Howard (Montefiore Medical Center and the Albert Einstein College of Medicine, Bronx, NY); Paula Schuman, Jack Sobel, Suzanne Ohmit, William Brown, Michael Long, Wayne Lancaster, and Jose Vazquez (Wayne State University School of Medicine, Detroit, MI); Anne Rompalo, David Vlahov, and David Celentano (Johns Hopkins University School of Medicine, Baltimore, MD); Charles Carpenter, Kenneth Mayer, Susan Cu-Uvin, Timothy Flanigan, Joseph Hogan, Valerie Stone, Karen Tashima, and Josiah Rich (Brown University School of Medicine, Providence, RI); Ann Duerr, Lytt I. Gardner, Chad Heilig, Scott D. Holmberg, Denise J. Jamieson, Janet S. Moore, Ruby M. Phelps, Dawn K. Smith, and Dora Warren (Centers for Disease Control and Prevention, Atlanta, GA); and Katherine Davenny (National Institute of Drug Abuse, Bethesda, MD).
Acknowledgments
We thank all the HIV Epidemiology Research Study participants and Pangaja Paramsothy, for her help in editing the manuscript.
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