Objective
The primary target cells for the human immunodeficiency virus (HIV) infection in the genital tract are CD4 T cells that express CCR5 on the surface. Alterations in genital tract T cells that express CCR5 could impact HIV acquisition risk. We hypothesized that, when compared with baseline, the use of a hormonal intrauterine device (IUD) would alter HIV target cells (primarily CCR5+ CD4 cells) in the female genital tract more than a nonhormonal IUD.
Study Design
Thirty-four healthy HIV-negative women aged 18-40 years who were seeking an IUD for contraception were assigned randomly to receive a levonorgestrel IUD or a copper T380A IUD. A parallel group of 8 control women who did not need contraception was also enrolled. Genital tract mucosal immune cell populations that were collected by cervical cytobrush and endometrial biopsy before and 2 months after IUD placement were analyzed by flow cytometry. Mean differences in cell number and percent that expressed receptors from baseline to follow-up examination were evaluated with the use of paired Student t tests.
Results
Neither IUD altered the number of T cells within the upper and lower genital tracts. Levonorgestrel IUD users had a decrease in T cells that expressed the HIV coreceptor CCR5 in the endometrium and cervix after 2 months of use compared with baseline. There was a decrease in activated endometrial T cells in levonorgestrel IUD users and a decrease in activated cervical T cells in copper IUD users after 2 months of IUD use, compared with baseline.
Conclusion
Women who use IUDs have reduced expression of the CCR5 HIV coreceptor on T cells in the endometrium and cervix compared with expression before IUD placement. These findings suggest that susceptibility to HIV infection would not be increased by IUD use.
Combating the spread of the human immunodeficiency virus (HIV) is a major global goal, achievement of which could be accelerated by the eventual development of highly effective dual protection methods that prevent both sexual acquisition of HIV and unwanted pregnancy. There is currently a tension between HIV prevention and family planning because emerging data suggest that some hormonal contraceptives, particularly injectable progestins, may increase the risk of HIV acquisition and transmission. Long-acting reversible contraceptives, which include intrauterine devices (IUDs) and implants, are more effective than combined oral contraceptive pills (COCs) and depot medroxyprogesterone acetate (DMPA), and offer a significant reduction or complete elimination of systemic exposure to exogenous hormones, compared with these methods. IUDs are the most commonly used reversible contraceptive worldwide and are regaining popularity in the United States; currently approximately 8.5% of women in the United States who use contraception choose an IUD. Observational data from women who live in high HIV incidence areas have included few IUD users. Thus, the available data cannot provide reliable estimates of HIV acquisition risk associated with IUD use.
HIV acquisition and sexual transmission are dependent on the immune environment of the female genital tract and may be under hormonal regulation. Endogenous sex hormones vary through the menstrual cycle, and exogenous hormonal exposure occurs commonly with contraceptive use, which may influence mucosal immune cellular populations.
Mucosal CD4 T lymphocytes in the vagina and cervix are thought to be the primary targets for sexual transmission of HIV to women. HIV infects discrete subsets of CD4 T cells, which express phenotypic receptors and coreceptors that are necessary for HIV to gain intracellular access. Antigen-presenting cells, such as mucosal dendritic cells, monocytes and macrophages (CD14+ cells) may help transport HIV from the surface to underlying target cells.
CCR5 is expressed on genital tract T cells and is the predominant target coreceptor for initial HIV infection. CCR5 expression is enhanced by sex hormones as ex vivo studies have demonstrated stimulation of CCR5 within explanted cervical tissue when incubated in media containing progesterone. In vivo, increased CCR5 expression in the setting of increased progesterone may contribute to the observed increased risk of HIV acquisition during pregnancy. However, there are limited data on lymphocyte changes with use of COCs and DMPA ; there are no data on genital-tract immune cell populations within reproductive tract mucosa of women who use other contraceptive methods, including IUDs.
We hypothesized that genital-tract immune cell populations, particularly CCR5+ T cells, would be increased from baseline 2 months after the initiation of intrauterine contraception with a levonorgestrel IUD (LNG-IUD) that contained 52 mg of LNG more than after the initiation of use of a nonhormonal copper IUD (Cu-IUD). We hypothesized that concentrated progestin exposure in the genital tract would recruit HIV target cells to the area. We tested this hypothesis by examining immune cellular populations in upper and lower genital tract samples that were obtained immediately before and 2 months after IUD insertion in healthy women who were assigned randomly to receive an LNG-IUD or Cu-IUD. Because IUDs are placed directly into the uterus, we hypothesized that the greatest impact on genital lymphocytes would be observed in T cells that were recovered from endometrial biopsy specimens.
Materials and Methods
We performed a randomized study of women who were initiating intrauterine contraception plus a parallel control group of women who were not at risk of pregnancy because of heterosexual abstinence or previous surgical sterilization. The primary objective was to assess the impact of IUD initiation on T cells in the upper and lower genital tract. The University of Pittsburgh Institutional Review Board approved this study. All participants were enrolled at the Center for Family Planning Research, Magee-Womens Hospital of the University of Pittsburgh Medical Center and signed informed consent before study participation.
Forty-two women, 18-40 years old, were enrolled; 34 women were seeking an IUD for contraception, and 8 women who were not seeking contraception comprised an observational control group. Eligible women were healthy, HIV negative, and nonpregnant and had regular menstrual cycles. All enrolled study participants were free of genital tract infection on screening examination, including rapid testing for Trichomonas vaginalis (OSOM; Sekisui Diagnostics, Lexington, MA), yeast vaginitis, symptomatic bacterial vaginosis by Amsel’s criteria, and abnormal inflammation (>10 white blood cells/high-power field on wet mount). Women were excluded if within 60 days of enrollment they (1) used any hormonal or intrauterine contraceptive, (2) were pregnant or breastfeeding, (3) underwent any genital tract procedure (including biopsy), (4) were diagnosed with any genital tract infection, and/or (5) had a new sexual partner. Exclusion criteria included the use of DMPA within 10 months of enrollment; the use of oral or vaginal antibiotics, oral or vaginal steroids, or any vaginal product except tampons (such as spermicide, microbicide, douche, antifungal, steroid, or hormone) within 30 days of enrollment; a contraindication to IUD use or an allergy to any component of the IUDs; or a previous malignancy of the cervix or uterus. Women in the control group had to be not at risk of pregnancy, which was defined as heterosexually abstinent or surgically sterile.
Screening also included urine pregnancy testing, collection of blood to rule out HIV infection, and collection of cervical swabs for detection of Neisseria gonorrhoeae and Chlamydia trachomatis by nucleic acid amplification testing (Gen-Probe, San Diego, CA). One participant was found to be ineligible after enrollment because of chlamydial infection, and a second participant withdrew from the study after IUD insertion; both were replaced to maintain the targeted sample size.
Participants were enrolled immediately after screening if eligible that day or returned for enrollment on a day when no vaginal bleeding was present. Day of menses at the time of enrollment was recorded. Participants were asked to refrain from any vaginal or anal intercourse for 1 week before sample collection at both visits. The 34 women who were seeking an IUD for contraception were assigned randomly 1:1 to receive either an LNG-IUD (Mirena; Bayer HealthCare Pharmaceuticals, Wayne, NJ) or copperT380A IUD (ParaGard; Teva Pharmaceuticals, Sellersville, PA). At the time of random assignment, the study investigator opened the next sequentially numbered, opaque, sealed envelope that contained the group assignment of LNG-IUD or Cu-IUD. A statistician who was not involved with the clinical conduct of the study prepared the envelopes using computer-generated random allocations in permutated blocks. The IUD was inserted per standard clinical practice at the enrollment visit immediately after the collection of all study samples. All laboratory personnel were masked to clinical status of participants, including random assignment to IUD type.
Genital tract samples were collected at enrollment and at 8-week follow-up visits. Endocervical specimens were obtained with a cytobrush (CooperSurgical Inc, Trumbull, CT) that was inserted into the cervical os and rotated 360 degrees; the cytobrush was placed in 4-mL Roswell Park Memorial Institute (RPMI)–1640 medium (Mediatech Inc, Manassas, VA) that was supplemented with 25 mmol/L HEPES, L-glutamine, and 10% fetal bovine serum (tRPMI). The ectocervix and endocervix were cleansed with chlorhexidine solution (Hibiclens; Mölnlycke Health Care, Norcross, GA) and dried with a sterile swab. Endometrial aspiration biopsy specimens (Pipelle; CooperSurgical Inc) were obtained; care was taken not to touch the aspirator to the vaginal walls or the ectocervix. Adequacy of the sample was assessed visually by the clinician who was obtaining the biopsy. Endometrial samples were transported in the aspirator to the laboratory and extruded under sterile conditions. All samples were transported to the laboratory for processing within 30 minutes of collection.
Endometrial biopsy specimens were weighed and then washed 3-4 times with phosphate-buffered saline solution without calcium and magnesium (PBS; Mediatech Inc). The biopsy specimens were minced with the use of sterile scissors and placed in digest buffer that contained 20 mL RPMI-1640, 1 mg/mL collagenase D (Roche Ltd, Nutley, NJ), and 2000 U/mL DNase I (0.5 μL/mL; New England Biosciences, Ipswich, MA). Agitation of the tissue in digest buffer was limited to 15 minutes at approximately 300 rpm at 37°C to maintain cell surface marker integrity. The transport vial that contained the endocervical cytobrush was vortexed and washed with tRPMI to dislodge cells from the cytobrush. Both biopsy and cytobrush-collected cells were filtered through a 40-μm nylon cell sieve (Becton Dickenson, Franklin Lakes, NJ) to obtain single cell suspensions.
Endometrial biopsy specimens additionally underwent density gradient centrifugation to remove dead cells, red blood cells, and other debris. The digested and filtered endometrial cells were resuspended in 5 mL 36% Histopaque (Sigma-Aldrich, St. Louis, MO) in RPMI then layered over 4.5 mL undiluted Histopaque and under 500 μL PBS. This tube was centrifuged at 600 g for 30 minutes with no brake; lymphocytes were recovered from the interface. Recovered cells of both specimen types were then washed by centrifugation in RPMI and resuspended in 1 mL PBS. Using Trypan blue stain (Sigma-Aldrich) exclusion criteria, viable cell yields were obtained manually with a hemocytometer.
Cell suspensions were adjusted to 1 × 10 6 cells/mL in PBS; 1 mL was stained for viability with LIVE/DEAD Fixable Aqua Dead Cell Stain (Invitrogen, Carlsbad, CA) and incubated for 25 minutes at room temperature protected from light. Cells were washed once with 1 mL of flow cytometry staining buffer (FACs; eBioscience, San Diego, CA) by centrifugation for 5 minutes at 400 g and stained with fluorochrome-conjugated antibodies (BD Biosciences, San Jose, CA) specific for the following cell surface markers: CD45 (FITC), CD3 (PerCP), CD8 (APC-H7), CD4 (PacificBlue), CD195 (CCR5)(APC), CD69 (PE), and CD14 (PE-Cy7). Cells were incubated for 25 minutes at room temperature protected from light, washed with 1 mL FACs buffer by centrifugation for 5 minutes at 400 g , and fixed in 1% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA). Stained samples were stored at 4°C, and flow cytometric analysis was conducted no later than 24 hours after fixation.
Lymphocyte populations were analyzed using an LSR II flow cytometer (BD Biosciences). To compensate for spectral overlap, single color compensation was applied specific for each fluorochrome-conjugate used. T-cell populations were identified with the use of forward and side scatter, and fluorescent-minus-one controls were used to assist in defining gate positions. Two senior laboratory technicians who were trained in advanced flow cytometry independently reviewed and agreed on the gating parameters for each sample.
The sample size for this study was calculated based on available published data that indicated the mean percent expression of CCR5 on cervical CD4 cells among women not using contraception was 48 ± 4%, with a standard deviation of ±7% for women who used COCs. Assuming the standard deviation of the mean change in the percent expression would be no greater than ±8%, a sample size of 10 would have 90% power to detect at least a 20% difference (set to be >1 standard deviation for mean change) in the percent expression of CCR5 on CD4 measured before and 2 months after IUD placement, with the use of a paired t -test evaluated at the .05 2-sided significance level. The enrollment target was increased to 16 participants per group to account for potential loss to follow-up evaluation, postrandomization ineligibility, inadequate specimen quality, and the plan to evaluate additional cell populations.
Data were analyzed with FACS DIVA software (version 6.2; BD Biosciences) and FlowJo software (version 10.0.5; Tree Star Inc, Ashland, OR). The gating strategy for all populations can be seen in Figure 1 . Cell numbers from biopsy specimens were normalized per gram of tissue. Cytobrush-collected cells were reported as “cells per cytobrush.” The cell numbers quantified were log 10 transformed for analysis and presentation. Expression of CCR5 and CD69 was reported as the percentage of parent population that expressed these cell surface markers. Each participant served as her own control by the use of the baseline visit as the control normal and assessment of change at the 2-month follow-up visit. Statistical analysis was performed with SPSS statistical software (version 20.0; IBM Corporation, Armonk, NY), and statistical tests were evaluated at the 2-sided .05 significance level. Differences in enrollment characteristics between the groups were assessed with 1-way analysis of variance and Kruskal-Wallis and Fisher exact tests, where appropriate. Differences in levels of expression from baseline to follow-up were evaluated with paired Student t tests.
