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ORIGINAL RESEARCH |
From the Departments of Clinical Microbiology, Immunology, and Clinical Science, Obstetrics and Gynecology, Umeå University, S-901 85 Umeå, Sweden.
Address reprint requests to: Marie-Louise Hammarström, PhD, Umeå University, Department of Immunology, SE-901 85 Umeå, Sweden; E-mail: marie-louise.hammarstrom{at}climi.umu.se.
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ABSTRACT |
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 ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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METHODS: Vaginal biopsies were collected during the follicular and luteal phases in regularly menstruating women (controls) and in women using combined oral contraceptives, depot-medroxyprogesterone acetate injections, or levonorgestrel implants. Fifteen healthy women (aged 20–34 years) were enrolled in each group. Biopsies were analyzed in a blinded manner. Epithelial thickness was estimated by morphometry. Immune cells were analyzed by immunomorphometry with cell-type-specific monoclonal antibodies.
RESULTS: The epithelium of controls harbored 241 ± 35 leukocytes (CD45+ cells) per mm2 (mean ± 1 standard error of the mean), and the thickness was 261 ± 16 µm. T lymphocytes (CD3+) dominated, and cytotoxic or suppressor T cells (CD8+) were more frequent than T helper cells (CD4:CD8 ratio: 0.7 ± 0.1). Macrophages (CD68+) constituted the second-largest population, followed by Langerhans cells (CD1a+). B cells, natural killer cells, monocytes, and granulocytes were generally absent. There were no significant differences between the follicular and luteal phases. The epithelium was significantly thicker in all three groups that used hormonal contraceptive (333 ± 9 µm) compared with controls, and it exhibited superficial hyperplasia. The frequency of intraepithelial leukocytes (CD45+) was increased in depot-medroxyprogesterone acetate (P < .001) and levonorgestrel implant users (P < .04). In depot-medroxyprogesterone acetate users, this was explained by an increased frequency of the CD8+ T lymphocyte subset.
CONCLUSION: Hormonal contraceptives induce hyperplasia of the vaginal epithelium. The significant changes in the intraepithelial leukocyte population in depot-medroxyprogesterone acetate and levonorgestrel implant users most probably reflect altered local immune capacity.
The vagina is a critical portal of entry for most sexually transmitted diseases (STDs) in women.1,2 Two decades has passed since the beginning of the acquired immunodeficiency syndrome (AIDS) epidemic, but the immune mechanism operating in the vagina is still poorly understood, and efforts to control the human immunodeficiency virus (HIV)-1 worldwide epidemic have had limited success. The HIV-1 epidemic continues to grow, particularly in the developing world.3,4
Studies in rhesus macaques have shown that subcutaneous progesterone administration by implants resulted in vaginal epithelial thinning and increased simian immunodeficiency virus vaginal transmission.5 Administration of depot-medroxyprogesterone acetate to mice was shown to increase herpes simplex virus transmission.6 Worldwide, hundreds of millions of women use hormonal contraceptives, including oral contraceptive (OC) pills and long-term–acting, progestin-based methods (eg, depot-medroxyprogesterone acetate injections and subdermal levonorgestrel-containing implants).7 Given their widespread use by women during their sexually active years, any modification of hormonal contraceptives on risk of acquisition of HIV-1 and other STDs could have implications for the HIV-1 epidemic. Epidemiologic studies support an association between depot-medroxyprogesterone acetate or OC use and contraction of STDs.8–11
The aim of this study was to evaluate whether commonly used steroid hormone contraceptives modifies the immune protection components of the human vaginal epithelium. Little is known about the frequency and cellular composition of intraepithelial immune cells in normal human vaginal epithelium, and even less is known about variations during the menstrual cycle or the influence of the use of steroid hormone contraceptives on these parameters. Therefore, we estimated the composition and frequency of intraepithelial immune cells in the vaginal epithelium in the follicular and luteal phases of the menstrual cycle of healthy women not using steroid hormone contraceptives and compared these with the estimated frequencies of immune cells in the vaginal epithelium of OC, depot-medroxyprogesterone acetate injection, and levonorgestrel subdermal implant users. The influence of steroid hormone use on the thickness of the vaginal epithelium was also analyzed.
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MATERIALS AND METHODS |
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ABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Women eligible for one of the contraceptive-user groups or the control group were sequentially recruited during a 17-month period. The study inclusion criteria were that women be healthy and informed, aged 20–34 years, and sexually active, with vaginal intercourse one to three times per week. Women in all four study groups were not allowed to have intercourse during the last week before biopsy sampling as a precaution against mechanical or immunologic influence on the vaginal mucosa from condoms or semen. Exclusion criteria were significant macroscopic vaginal pathology, current pelvic inflammatory disease, vaginal bleeding of unknown etiology, pregnancy, known or suspected thrombocytopenia or other bleeding disorders, known or suspected malignant disease, treatment with hormonal or other investigational drug other than the contraceptive steroids under study in this project during the previous 8 weeks, any systemic or vaginal medications other than the contraceptive during the last 2 weeks before sampling, or use of an intrauterine device. For steroid contraceptive groups, current use of barrier methods (mechanical or chemical) in addition to the reported hormonal method was also an exclusion criterion.
Participants were recruited on a voluntary basis, selected for age homogeneity between the groups, and interviewed by telephone regarding fulfillment of inclusion criteria, after which an appointment for the first visit was made. Included subjects attended the clinic twice, in the follicular phase (days 8–13) and the luteal phase (days 20–25 after first day of the last menstrual period) of a normal menstrual cycle (control group) and with the same interval among users of the hormonal contraceptive methods studied.
At the first visit, subjects were interviewed concerning demographic, medical, reproductive, and contraceptive history, including history of any previously diagnosed STD during their lifetime. Data were collected on coded, standardized forms. Weight and height were measured for calculation of body mass index (BMI; kg/m2). At both visits, serum samples for determination of concentrations of estradiol (E2) (detection limit 72 pmol/L) and progesterone (detection limit 0.6 nmol/L) were taken and analyzed at the Department of Clinical Chemistry, Umeå University Hospital, with a competitive immunoassay (Immulite 1 [Diagnostic Products Corporation, Los Angeles, CA]). The criterion for ovulation was set at a serum progesterone concentration of at least 16 nmol/L in the luteal phase.12–14 Urine samples were taken at both visits and analyzed for exclusion of pregnancy with a one-step, dip-stick test (OriPreg [Orion Diagnostica, Espoo, Finland], detection limit 50 IU human chorionic gonadotropin/mL). A gynecologic examination was performed for evaluation of any clinical signs of exclusion criteria and for subsequent tissue sampling.
Four biopsies were taken from each woman, two in the follicular and two in the luteal phase of the normal menstrual cycle (control group) or with the same interval in the hormonal contraceptive user groups. The order of sampling (ie, first biopsy in follicular or luteal phase) was randomized to avoid errors due to possible influences of biopsy sampling on the vaginal environment. A biopsy forceps (Hartmann, Stille, Germany) was used to obtain two full-thickness biopsies of vaginal mucosa, approximately 4 x 8 mm, from the lateral vaginal fornices at positions "4:30" and "10:30" at the first visit and at positions "1:30" and "7:30" at the second visit. At each occasion, one of the biopsies was immediately put in ice-cold tissue culture medium RPMI1640 supplemented with 3.5% sucrose, 0.4% human serum albumin, antibiotics, and amphotericin B, then oriented and embedded in OCT [Tissue-Tek O.C.T. Compound, Sakura, Zoeterwoude, The Netherlands] compound, snap frozen by immersion into isopentane, precooled in liquid nitrogen, and stored at -70C until use. The second biopsy was immediately fixed by immersion in 10% formalin in phosphate-buffered saline (pH 7.2) for 12–24 hours at room temperature, dehydrated, oriented, and embedded in paraffin. Local anesthesia in any form was not applied. A ferric sulfate paste was applied at the site of biopsy as needed. In most subjects the bleeding was negligible. Tissue samples were coded and analyzed in a blinded manner.
Serial 5-µm-thick cryostat sections were cut and mounted on poly-L-lysine-pretreated microscopic slides and processed for immunoperoxidase staining. Sections were air-dried and fixed in acetone for 5 minutes at -20°C, dried, and then processed at room temperature. After washing in 0.02 mol/L phosphate-buffered saline (pH 7.3), endogenous peroxidase activity was blocked by incubation in phosphate-buffered saline containing 0.003% hydrogen peroxide and 1 mmol/L sodium azide. Unspecific binding sites were blocked by incubation with 0.4% bovine serum albumin. Thereafter, the sections were incubated with relevant monoclonal antibody for 60 minutes, followed by horseradish peroxidase-conjugated F(ab')2 fragments of sheep antimouse immunoglobulin (Jackson ImmunoResearch Laboratories, West Grove, PA). Sections were developed with 0.05% 3,3'-diaminobenzidine tetrahydrochloride and 0.03% hydrogen peroxide in 0.05 mol/L Tris-hydrochloric acid buffer (pH 7.6) and counterstained with Mayer hematoxylin.
Negative and positive controls were included in every staining procedure. Sections incubated with isotype- and concentration-matched monoclonal antibody specific for Aspergillus niger glucose oxidase served as negative controls. Sections incubated with anti-CD45 monoclonal antibody served as positive controls. A limited number of vaginal samples were taken from volunteers outside the controlled study. Sections of these samples were stained in parallel at each occasion as additional positive controls.
Monoclonal antibodies used and their main specificities are listed in Table 1
. The optimal concentration for each monoclonal antibody was determined by titration in immunohistochemistry on tissue sections of frozen vaginal mucosa and palatine tonsils. B cells were analyzed with a mixture of the anti-CD19, anti-CD20, and anti-CD22 monoclonal antibodies.
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Serial sections of formalin-fixed, paraffin-embedded biopsies were cut at 5-µm thickness perpendicular to the epithelial surface, mounted on poly-L-lysine-pretreated microscopic slides, and stained with hematoxylin and eosin. The average epithelial thickness in each tissue sample was determined from measurements of epithelial thickness in five randomly chosen microscopic fields that showed a full-thickness epithelial segment with a x 100 magnification. In each microscopic field, the basal membrane of the epithelium measured was oriented vertically on the microscope-connected screen; thus, the vertical length of each epithelial segment corresponded to the constant height of the image on the screen, measuring 600 µm in the actual magnification. The epithelial segment was outlined with the cursor. The enclosed area of each segment was calculated by computerized morphometry, and the average epithelial thickness was calculated as the ratio of the estimated epithelial area and the constant length of the epithelial segment. Determination of epithelial thickness was performed by one investigator (AI) using coded slides.
The study was approved by the Scientific and Ethical Review Group of the Human Reproduction Program at the World Health Organization and by the Research Ethics Committee of the Medical Faculty, Umeå University, Sweden. Oral and written, informed consent was received from all study participants.
All data were entered into a database in Microsoft Excel 2000 (Microsoft, Redmond, WA) for storage and subsequently exported to SPSS 11.0 (SPSS, Chicago, IL) and GraphPad Prism 3.02 (GraphPad Software, San Diego, CA) for statistical analysis and graphic representation. Comparisons between the follicular and the luteal phases within the control group were performed with two-tailed Student paired t test for normally distributed data and two-tailed Wilcoxon signed rank test for non-parametric data. Normality was evaluated by F test. Differences between the control and hormonal contraceptive user groups in frequencies of intraepithelial immune cells, epithelial thickness, steroid hormone serum levels, age, and BMI were analyzed with one-way analysis of variance, with Dunnett post hoc test, or test for linearity. Differences in sociodemographic parameters were analyzed with Fisher exact test. Differences with a P value of .05 or less were considered statistically significant. Within the control group, results are given as mean plus or minus standard deviation, and when comparing the mean values of groups based on mean values for each individual and parameter, results are given as mean plus or minus standard error of the mean.
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RESULTS |
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ABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The control group and the hormonal contraceptive user groups each comprised 15 women. The women were 20–34 years old, and there were no significant differences between the four study groups concerning age homogeneity. Similarly, there was no significant difference between the groups with respect to BMI. None of the women had detectable levels of human chorionic gonadotropin in urine at either of the two visits. As expected, the frequency of women with no previous pregnancy was highest in the OC user group (11 of 15) and lowest in the depot-medroxyprogesterone acetate user group (two of 15), whereas the frequency of parous women was highest in the depot-medroxyprogesterone acetate user group (12 of 15) and lowest in the OC user group (three of 15). Six women were smokers; they were all hormonal contraceptive users. One subject in the depot-medroxyprogesterone acetate user group had a compulsory school degree only (9 years), whereas the remaining subjects had senior high school degrees, and of these 61% had a university education.
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The women in the control group all had regular menstrual cycles; however, samples from four women were excluded from comparative analyzes between the follicular and luteal phase. Two subjects were excluded because their progesterone concentrations in the luteal phase did not indicate ovulation (less than 16 nmol/L). One subject was excluded because no determinations of serum E2 and progesterone concentrations were performed in the luteal phase. One subject was excluded because both biopsies were collected in the follicular phase of two successive cycles. The average serum progesterone concentration was 36 ± 16 nmol/L in the luteal phase and 3.0 ± 1.4 nmol/L (P < .001) in the follicular phase. The serum E2 concentrations were 360 ± 302 and 486 ± 248 pmol/L in the follicular and luteal phases, respectively.
The vaginal epithelium in controls consisted of a stratified squamous epithelium overlying a loose connective tissue (Figure 1a). The mean epithelial thickness was 280 ± 109 µm and 211 ± 57 µm in the follicular and luteal phases, respectively (n = 11). No statistically significant differences were seen between the follicular and luteal phases intraindividually (P > .05). Therefore, in subsequent analyses, all individuals in the control group were included, and the two biopsies from each individual were considered as duplicates, giving an average epithelial thickness of the vaginal epithelium of 261 ± 16 µm (n = 15).
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). Langerhans cells were scattered throughout the epithelium, often with dendrites spanning several epithelial cell layers (Figure 1i). Although CD45+ cells were present throughout the epithelium, the majority was located in the vicinity of the basal lamina facing the lamina propria and the connective tissue papillae (Figure 1d). T cells (CD3+ cells; Figure 1e), both CD4+ and CD8+ cells (Figures 1f,h), as well as tissue macrophages (CD68+ cells; Figure 1g) were commonly seen. Immunomorphometry analyses of total leukocytes (CD45+ cells) and the different immune cell types were performed separately on paired biopsies from the follicular and luteal phases. Statistical analyses comparing the results for the 11 women with verified ovulation showed that there was no significant difference between the follicular and luteal phases with regard to frequency of any of the types of intraepithelial immune cells analyzed. For all parameters analyzed, the mean value for the two biopsies from each individual was calculated and used for calculation of the mean for the entire group. The results are summarized in Figure 2. The frequency of intraepithelial CD45+ cells was 241 ± 35 cells per mm2 of vaginal epithelium. The majority consisted of T cells (CD3+ cells; 186 ± 34 cells per mm2), predominantly of T cytotoxic or suppressor phenotype (CD8+ cells; 109 ± 19 cells per mm2), followed by T helper cells (CD4+ cells; 61 ± 18 cells per mm2). Tissue macrophages were also frequent (CD68+ cells; 40 ± 11 cells per mm2). Langerhans cells (CD1a+ cells) were detected in eight of the 15 women and constituted a minor population (14 ± 9 cells per mm2). Few (less than 1 cell per mm2) granulocytes (CD15+ cells) and natural killer cells (CD57+ cells) were detected and only in occasional samples (n = 5 and 2, respectively). B cells (CD19+, CD20+, or CD22+ cells) and monocytes (CD14+ cells) were not detected in the vaginal epithelium of controls.
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Because there were no differences between the follicular and the luteal phases in the control group, mean values from the two biopsies of hormonal contraceptive users were used in comparative analyses between groups. In all three hormonal contraceptive user groups, the average thickness of the epithelium was significantly increased compared with controls (Figure 3a). This increase was most pronounced in OC and depot-medroxyprogesterone acetate users, with mean thicknesses of 344 ± 14 and 348 ± 16 µm, respectively. The increased epithelial thickness among the hormonal contraceptive users was mainly due to a distended peripheral portion of the epithelium constituting loose hyperplastic layers of cells (Figure 1b).
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, the immune-cell-frequency-to-epithelial-thickness function exhibited different linear regression coefficients in the four groups. To allow comparison between the groups, the values were normalized by calculating the number of positive cells present in a linear length of the epithelium tissue—the estimated frequency of marker-positive cells per square millimeter multiplied by the average epithelial thickness of the sample (number of positive cells per millimeter).
The immunomorphometry analyses revealed that the number of CD45+ cells (ie, total number of intraepithelial immune cells) was significantly increased in levonorgestrel implant and depot-medroxyprogesterone acetate users, compared with controls (P < .04 and < .001, respectively; Figure 4). The mean frequencies of CD4+ cells in levonorgestrel implant users and CD8+ cells in depot-medroxyprogesterone acetate users were higher than in controls, and CD8+ cells showed a statistically significant linear trend between controls and depot-medroxyprogesterone acetate users (P < .04; Figure 4). Furthermore, the mean ratio of CD4+ cells to CD8+ cells was higher in levonorgestrel implant users than in controls, whereas the reverse was true for depot-medroxyprogesterone acetate users (Figure 4). Interestingly, there was a statistically significant difference in the CD4:CD8 ratio between depot-medroxyprogesterone acetate users and levonorgestrel implant users (P < .01; Figure 4).
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DISCUSSION |
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The histology of the vaginal epithelium in normally menstruating women showed the typical features of non-keratinized squamous epithelium.1 The observed average thickness of 260 µm agrees well with values reported from earlier studies.1,15,16 We noted no statistically significant difference in epithelial thickness between the follicular and luteal phases of the menstrual cycle. One previous study addressed this issue by counting the number of cellular layers in the epithelium.17 A minute difference was seen between the follicular and luteal phases, with an average of 28 and 26 cellular layers, respectively. It seems unlikely that this small difference affects the ability of the epithelium to act as a mechanical barrier during the menstrual cycle. In all three groups of hormonal contraceptive users, we found a significant increase in the epithelial thickness, compared with non-users, and a consistent morphologic feature of a hyper-plastic distended appearance in the superficial epithelium. One previous study investigated the effect of long-term (2 to 3 years) use of depot-medroxyprogesterone acetate in this aspect and reported no significant change.15 No change and an approximately 20% thinning of the epithelium were reported in two studies on the effects of short-term use (within 3 months after one injection) of depot-medroxyprogesterone acetate.16,18 Morphologic changes were not reported. Most likely these discrepancies with our results have methodologic explanations. To minimize manipulation of the tissue, we did not use local anesthetics and did not pin-mount the samples. In the previous studies, both procedures were used.15–18 We determined the average epithelial thickness by morphometry analysis of a large surface area (at least 0.3 mm2 epithelium), whereas the other groups measured the height of the epithelium at a small number of randomly chosen locations. In any event, use of exogenous progestins is not associated with a dramatic thinning of vaginal epithelium in humans, which was observed in rhesus macaques and speculated to be an important component in their increased susceptibility to vaginally inoculated simian immunodeficiency virus.5,19 The increased epithelial thickness observed in hormonal contraceptive users in this study does not necessarily indicate an increased immune protective barrier. The morphologic change towards distention and hyperplasia might even indicate the opposite. The hyperplastic epidermal changes in dermatoses (eg, psoriasis vulgaris, eczema) are associated with increased susceptibility to secondary infections.20,21
The vaginal epithelium in controls harbored a small but significant population of immune cells (ie, on average 240 CD45+ cells per mm2 or 65 when calculated as CD45+ cells per millimeter linear length of the vaginal epithelial tissue. The majority of these were CD8+ T lymphocytes. This cell type commonly dominates within human mucosal epithelia.22–25 The role of intraepithelial CD8+ cells in immune protection of mucosa has yet to be determined, but surveillance by cytotoxicity against virus-infected cells has been suggested. CD4+ T lymphocytes constituted the second largest population of vaginal intraepithelial immune cells. Other types of analysis are required to determine whether these are helper cells for antibody production, cell-mediated immune responses, or even regulatory T cells with suppressive effects. However, their front-line position makes them potential targets for vaginal transmission of HIV infection. The third most common cell type was tissue macrophages, probably acting as scavengers and a first line of defense against bacterial attacks. Langerhans cells were detected in 50% of the controls and at low cell densities when present. This was unexpected because presence of Langerhans cells was reported in previous studies.15–17,26 There are some notable differences between these studies and ours. We estimated the frequency of Langerhans cells by counting intraepithelial CD1a-positive distinct cell bodies, occasionally associated with dendrites. Two studies used CD1a as a marker.17,26 In one, solitary dendrites were also counted,17 and in the other, tissue from women undergoing hysterectomy was used.26 In this case there is a possible risk for influence of age (34–54 years) as well as of the underlying diseases necessitating surgery. Two studies used the S100 marker, which might detect a cell population partly different from CD1a.15,16
The results show no significant fluctuation between the follicular and luteal phases concerning the frequency and composition of intraepithelial immune cells. One previous human study has addressed the same question with use of part of the panel of this study.17 Their results are given as number of positive cells per high-power field and hence are difficult to compare with ours. However, the reported consistency throughout the menstrual cycle, the relative proportion of CD4+ to CD8+ cells, the presence of macrophages, and the absence of B cells agree well with our findings. Similarly, the frequency and composition of intraepithelial immune cells did not change at different stages of the cycle in rhesus macaques.27
We observed a significant association between use of depot-medroxyprogesterone acetate and the prevalence of intraepithelial immune cells (CD45+ cells). The analysis of immune cell subtypes strongly suggests that this increase is caused by a selective expansion in the number of CD8+ cells. Immune cells isolated from human vaginal mucosa were shown to have functional cytolytic machinery that can be triggered via the T cell receptor–CD3 complex,28 which suggest the presence of active cytotoxic T cells or virus-specific CD8+ memory T cells.29 Simian immunodeficiency virus–specific CD8+ intraepithelial lymphocytes were demonstrated in the vagina of simian immunodeficiency virus–infected rhesus macaques,30 and vaginal administration of attenuated herpes simplex virus produced protective immunity in the vagina of mice.6 Thus, it is an interesting possibility that the increased number of CD8+ cells in depot-medroxyprogesterone acetate users reflects a history of frequent viral infections. In this context, it is noteworthy that the E2 concentration in serum was very low in the depot-medroxyprogesterone acetate user group. A role for E2 in promoting immune protection against viral infection of the vagina has been suggested in animal models.6,31
The prevalence of intraepithelial immune cells was also significantly increased in levonorgestrel implant users. This group exhibited slightly increased numbers of CD4+ cells compared with controls and had a significantly higher CD4:CD8 ratio than depot-medroxyprogesterone acetate users. Thus, it seems likely that the increase in intraepithelial immune cells in this case is due to expansion of CD4+ cell subtype. Their role in local defense is unclear.
Langerhans cells have been suggested as major targets for HIV infection32,33 and to become rapidly infected after intravaginal inoculation of simian immunodeficiency virus in rhesus macaques.34 We found no significant changes in the frequency of Langerhans cells in hormonal contraceptive users. This is in agreement with previous studies on the effect of short-term16 and long-term15 use of depot-medroxyprogesterone acetate. However, one study reported an increase in Langerhans cells after intravaginal administration of progesterone.35
Although the study population was relatively small, with 15 subjects in each group, significant differences were shown in several aspects. The site of the biopsy sampling in our study is an area of the lower genital tract exposed to infectious agents, and the results suggest changes in the immune protection capacity at this site. Additional, in-depth studies should be conducted to define the effect of depot-medroxyprogesterone acetate injections and levonorgestrel implants on the vaginal immune system and to determine whether use of hormonal contraceptives increases the risk of contracting STDs.
In conclusion, use of any of the three hormonal contraceptives was associated with morphologic changes of the vaginal epithelium. Depot-medroxyprogesterone acetate and levonorgestrel implant use (ie, use of long-term-acting, progestin-only contraceptives) was associated with changes in frequency and composition of intraepithelial immune cells, whereas no such changes were observed in OC users. Thus, of the three hormonal contraceptives studied, low-dose levonorgestrel-combined pills seem to affect the immune protection components of the vaginal epithelium least.
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Footnotes |
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The authors thank Dr. Hans Stenlund, Department of Epidemiology, Umeå University, for expert advice in statistical matters and Dr. Thomas Höckenström, MD, Department of Pathology, Umeå University Hospital, for expert evaluation of epithelial morphology.
doi:10.1016/S0029-7844(03)00618-5
Received October 28, 2002. Received in revised form March 17, 2003. Accepted April 17, 2003.
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Significant linear trend; P < .05.