Effect of injectable progestin-only contraceptives, depot medroxyprogesterone acetate and norethisterone enanthate, on cytokine production during T-cell activation.

PROBLEM: There is paucity of human data about the effects of depot medroxyprogesterone (DMPA) and norethisterone enanthate (Net-En) use on systemic immune function, which may have implications for reproductive tract infection susceptibility and transmissibility. We sought to evaluate the impact of injectable contraceptive use on T-cell responsiveness using T cells exposed in vivo and tested ex vivo. METHODS: Peripheral blood mononuclear cells were obtained from healthy, HIV-negative women after 30, 90 and 180 days of DMPA, norethisterone enanthate (Net-En) or copper intrauterine device (Cu-IUD) contraceptive use. Cells were stimulated ex vivo with phorbol myristate acetate and ionomycin, stained and analysed using flow cytometry. Mixed-effects linear models were used to evaluate change in proportions of T cells producing IFN-γ, TNF-α, IL-4 and IL-13. RESULTS: Compared with baseline, decreased proportions of IFN-γ-producing CD4+ and CD8+ T cells (p = .003, p = .006, respectively) and TNF-α-producing CD4+ and CD8+ T cells (p = .039, p = .034, respectively) were observed after 180 days of DMPA use. Decreased IL-4-producing CD4+ and CD8+ T cells (p = .045 and p = .024, respectively) were noted after 180 days of Net-En use. Decreased IL-4-producing CD4+ T cells were observed after 30 days (p = .035) and not after 180 days of DMPA use (p = .49). There were no changes in proportion of T cells producing IL-13 in DMPA users, nor any changes in IFN-γ, TNF-α and IL-13 in Net-En and Cu-IUD users. CONCLUSION: In vivo exposure of CD4+ and CD8+ T cells to typical pharmacologic concentrations of DMPA does not cause broad suppression to stimuli; however, depletion of specific cytokine-producing T cells may occur after prolonged DMPA use.

Zim CHIC: A cohort study of immune changes in the female genital tract associated with initiation and use of contraceptives.

PROBLEM: Contraceptive hormones are systemically active, potent, and likely to invoke biological responses other than known fertility regulation impacts. We hypothesized that initiation of depot medroxyprogesterone acetate (DMPA) would increase genital HIV-target-cells and soluble immune mediators compared with baseline and initiation of other contraceptive methods. METHOD OF STUDY: We collected cervical cytobrushes and cervicovaginal fluid from healthy Zimbabwean women aged 18-34 to assess immune cell populations, cytokines, and innate anti-HIV activity at baseline and after 30, 90, and 180 days use of DMPA (n = 38), norethisterone enanthate (n = 41), medroxyprogesterone acetate/estradiol cypionate (n = 36), levonorgestrel implant (n = 43), etonogestrel implant (n = 47), or copper intrauterine device (Cu-IUD) (n = 45). Cells were quantified by flow cytometry, cytokines were detected by multiplex assays, and innate anti-HIV activity was assessed by in vitro HIV challenge. RESULTS: Compared to baseline, the number of cervical HIV target cells (#CD4 cells P < .04 and #CD11c cells P < .04), the concentration of the inflammatory cytokine IL-1ß (P < .01), and the innate in vitro anti-HIV activity (P < .001) significantly decreased following DMPA initiation. In Cu-IUD users, genital HIV target cells increased (#CD4 cells P < .001, #CD4CCR5 cells P = .02, #CD4CD69 cells P < .001, #CD8CD69 P = .01, and #CD11c cells P = .003) at day 30 and resolved by day 180. IFN-γ (P < .001), IL-1ß (P < .001), IL-6 (P < .001), IL-8 (P < .001), IL-10 (P < .01), and RANTES (P < .001) were also significantly increased at day 30. Minimal alterations were observed following initiation of subdermal implantable contraceptives. CONCLUSIONS: This head-to-head study compared six contraceptives and found increased HIV target cells and cervical inflammation temporally associated with Cu-IUD initiation. Use of hormonal contraception, including DMPA, did not increase cervical HIV target cells or inflammation. Clinical Trial Number: NCT02038335.

Optimization of processing female genital tissue samples for lymphocyte analysis by flow cytometry.

PROBLEM: A variety of methods have been used to process cervical cytobrush and genital tissue for flow cytometric evaluation of immune cell populations. We sought to optimize genital tract specimen processing and to determine if blood could be used as a model for assessment of tissue processing methods. METHOD OF STUDY: Cervical cytobrushes, PBMCs, and genital tissue samples (cervical and endometrial biopsies) were subjected to varying processing conditions to characterize the effects on cell yields, lymphocyte viability, and surface receptors. We exposed PBMC and tissue specimens to varied collagenase types, concentrations, and exposure durations and cytobrushes to immediate vs delayed processing with/without vortexing. RESULTS: PBMCs and tissues exposed to varying enzymatic digestion conditions demonstrated stability of some cell surface receptors, including CD3+ , CD4+ , and CD8+ , while others, including CCR6+ , were cleaved when exposed to any concentration of collagenase B, or ≥0.25 mg/mL of collagenase D. We observed increased CD69 expression (marker of cell activation) after exposure to collagenase B. Neither a 2-hour delay in cytobrush processing nor vortexing at a setting of 50% for 30 seconds had significant impacts on viability or quantities of genital immune cells of interest. CONCLUSION: Although tissue digestion with collagenase D was sufficient to recover and analyze cells from endometrial biopsy specimens, cervical biopsy specimens required a limited exposure to collagenase B at 1 mg/mL to optimize cell yield and viability for cytometric analysis. PBMCs can be used as a model to assess the impact of tissue processing on co-receptor expression and to optimize methods prior to study implementation.

Depot medroxyprogesterone acetate and norethisterone enanthate differentially impact T-cell responses and expression of immunosuppressive markers.

PROBLEM: Injectable contraceptive use may impact immune cell responsiveness and susceptibility to infection. We measured responsiveness of T-cells from women before and after initiating depot medroxyprogesterone acetate (DMPA) or norethisterone enanthate (Net-En). METHOD OF STUDY: Peripheral blood mononuclear cells collected from women aged 18-34 years prior to, at steady state, and nadir concentrations after initiating DMPA (n = 30) or Net-En (n = 36) and from women initiating copper intrauterine device (CU-IUD; n = 32) were stimulated with phorbol myristate acetate and analyzed using flow cytometry. We evaluated percentage change in T-cells expressing programmed cell death-1 (PD-1) and cytotoxic T-lymphocyte associated protein-4 (CTLA-4). RESULTS: Compared to baseline, there were decreased numbers of CD4+CTLA4+ (P < .001) and CD8+CTLA4+ (P < .01) T-cells following ex vivo stimulation challenge at steady state DMPA concentrations and no differences at nadir concentrations (P = .781 and P = .463, respectively). In Net-En users, no differences in CD4+CTLA4+ T-cells at steady state (P = .087) and nadir concentrations (P = .217) were observed. DMPA users had fewer CD4+PD-1+ (P < .001) and CD8+PD-1+ (P < .001) T-cells at nadir concentrations. Number of CD4+PD-1+ and CD8+PD-1+ T-cells decreased at steady state concentration (P = .002 and P = .001, respectively) and at nadir concentrations after Net-En initiation (P < .001 and P < .001). In CU-IUD users, there were no changes in number of CD4+CTLA4+ (P = .426) and CD8+CTLA4+ (P = .169) and no changes in CD4+PD-1+ (P = .083) and CD8+PD-1+ (P = .936) compared to baseline. CONCLUSION: Activation of T-cells in response to ex vivo stimulation is suppressed at steady state DMPA concentration and resolves at nadir concentration, suggesting DMPA immunosuppressive effects may be transient.

Survival of vaginal microorganisms in three commercially available transport systems.

Transport systems are used to collect and maintain the viability of microorganisms. Two Amies media based transport systems, BD CultureSwab™ MaxV(+) Amies Medium without Charcoal (MaxV(+)) and Fisherfinest® with Amies gel Transport Medium without charcoal (Fisherfinest®) were compared to a Cary-Blair media based transport system, Starswab® Anaerobic Transport System (Starswab®), for their capacity to maintain the viability of 17 clinical microorganisms commonly isolated from the vagina (Lactobacillus crispatus, L. jensenii, L. iners, group B streptococci, Candida albicans, Escherichia coli, Enterococcus faecalis, Atopobium vaginae, Peptoniphilus harei, Mycoplasma hominis, Gardnerella vaginalis, Dialister microaerophilus, Mobiluncus curtisii, Prevotella amnii, P. timonensis, P. bivia, and Porphyromonas uenonis). Single swabs containing mixtures of up to five different species were inoculated in triplicate and held at 4 °C and room temperature for 24, 48, 72, and 96 h (h). At each time point, swabs were eluted into a sterile salt solution, serially diluted, inoculated onto selected media, and incubated. Each colony type was quantified and identified. A change in sample stability was reported as a ≥1 log increase or decrease in microorganism density from baseline. Overall, the viability of fastidious anaerobes was maintained better at 4 °C than room temperature. At 4 °C all three transport systems maintained the viability and prevented replication of C. albicans, E. faecalis, GBS, and E. coli. Microorganisms having a ≥1 log decrease in less than 24 h at 4 °C included A. vaginae, G. vaginalis, and P. uenonis in Starswab®, L. iners, A. vaginae, and P. amnii in MaxV(+), and A. vaginae, G. vaginalis, P. bivia, and P. amnii in Fisherfinest®. At 48 h at 4 °C, a ≥1 log decrease in concentration density was observed for P. harei and P. amnii in Starswab®, G. vaginalis, P. bivia and P. uenonis in MaxV(+), and L. iners, P. harei, P. timonensis, and P. uenonis in Fisherfinest®. Overall, at 4 °C the viability and stability of vaginal microorganisms was maintained better in the Cary-Blair based transport system (Starswab®) than in the two Amies based transport systems.

Changes in genital tract immune cell populations after initiation of intrauterine contraception.

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.

Survival of Trichomonas vaginalis in wet preparation and on wet mount.

OBJECTIVES: Microscopy is an insensitive method for detection of Trichomonas vaginalis, but is widely used because it is both rapid and inexpensive. Diagnosis of trichomoniasis by microscopy requires that motile forms be identified in vaginal fluid samples. However, microscopy cannot always be performed immediately after sample collection. The objective of this study was to assess the impact of sample storage at room temperature on trichomonad motility. METHODS: Vaginal swab samples from 77 women positive for T vaginalis infection were collected to determine the impact of storage on wet preparations (swabs in plastic tubes with saline) and wet mounts (samples placed onto a glass slide with a coverslip). Samples were read at 400× every 30 min for the first hour and then once per hour thereafter until there were no motile trichomonads observed. RESULTS: For wet preparations, motility was 100% at 30 min, 99% at 60 min and decreased by 3%-15% each subsequent hour, with samples having a lower density of trichomonads losing motility more quickly. Trichomonad motility diminished more rapidly in wet mounts compared with wet preparations, with a 20% decrement in motility in 60 min. CONCLUSIONS: These data suggest that vaginal fluid samples for diagnosis of trichomoniasis should be stored in saline rather than on microscope slides until they are examined under the microscope and samples should be evaluated by microscopy within an hour of collection. These findings also suggest that clinical sites which cannot perform microscopy within 1 h of sample collection should consider the use of other diagnostic tests.

Recalcitrance of bacterial vaginosis among herpes-simplex-virus-type-2-seropositive women.

AIM: The multifactorial etiology of bacterial vaginosis (BV) impedes development of effective treatment and prevention strategies. Herein, we evaluated the effects of herpes simplex virus type 2 (HSV-2), a suspected BV risk factor, on vaginal flora composition. MATERIALS AND METHODS: Correlations between HSV-2 infection and BV were prospectively explored among 12 HSV-2-seropositive women with asymptomatic BV who were asked to collect daily vaginal swab specimens for Gram stain analysis of vaginal flora and determination of HSV-2 shedding frequencies during the 1month before and after metronidazole therapy. RESULTS: Unlike prior longitudinal studies that reported rapid fluctuations in vaginal flora composition and frequent episodes of spontaneously resolving BV, we found that 99.4% (310/312) of vaginal smears collected before initiation of metronidazole were consistent with a diagnosis of BV. Effectiveness of metronidazole therapy was also much lower than previously reported in studies not restricting enrollment to HSV-2-seropositive women; we observed a BV recurrence rate of 89% in the first month after completion of therapy while the median time to this recurrence occurred only 14days after treatment. CONCLUSIONS: Our study demonstrates BV recalcitrance among HSV-2-infected women and provides additional evidence for a linkage between this chronic viral infection and abnormal vaginal flora. Additional work will be needed to define mechanisms responsible for this correlation and to determine if vaginal flora health of HSV-2-infected women is improved by medications that suppress HSV-2 shedding.

Incidence and epidemiology of Streptococcus pseudoporcinus in the genital tract.

Streptococcus pseudoporcinus, a beta-hemolytic microorganism first isolated from the female gastrourinary tract in 2006, cross-reacts with serogrouping kits for group B Streptococcus (GBS) and could be misidentified in the laboratory. The epidemiologic characteristics of this species have not been reported previously, but this organism is thought to be rare. Paired vaginal and rectal samples were collected from 663 nonpregnant women enrolled in a phase II clinical vaccine trial of a GBS type III capsular polysaccharide-protein conjugate vaccine, and isolates initially identified as S. pseudoporcinus were collected for further testing. A total of 120 isolates of S. pseudoporcinus were recovered from 36 unique individuals with 5.4% of 663 women having this organism recovered at least once during follow-up. All of these isolates cross-reacted with a commercially available GBS serogrouping kit. Women colonized with isolates confirmed as S. pseudoporcinus by genotypic and phenotypic methodologies were compared to women who were not colonized to determine whether there were any significant factors associated with acquisition of S. pseudoporcinus. Acquisition of S. pseudoporcinus vaginally and/or rectally was 36 per 846.0 women-years of follow-up for an annual incidence of 4 per 100 woman-years of follow-up. Acquisition of S. pseudoporcinus was independently associated with black women, being 30 to 40 years of age, recent Trichomonas vaginalis infection, primary or recurrent genital herpes, having bacterial vaginosis by Nugent criteria, and having had two or more male sexual partners since the last visit. This study suggests that S. pseudoporcinus is not rare, especially among black women, and could be misidentified as GBS.

Quantitative survival of aerobic and anaerobic microorganisms in Port-A-Cul and Copan transport systems.

Transport media should preserve the viability and stability of microorganisms in clinical specimens. In this study, the Port-A-Cul transport system and the Copan transport system without charcoal, both designed to preserve anaerobes, were evaluated. Dacron swabs were inoculated with two combinations of facultative and anaerobic organisms typically found in vaginal swab samples. Combination I contained Candida albicans, Escherichia coli, Enterococcus spp., group B streptococci, Lactobacillus crispatus, and Staphylococcus aureus. Combination II contained Lactobacillus iners, Peptoniphilus asaccharolyticus, Mycoplasma hominis, Prevotella bivia, Prevotella corporis, Porphyromonas asaccharolytica, Mobiluncus curtisii, Peptostreptococcus anaerobius, and Gardnerella vaginalis. Duplicate swabs were placed into the two transporters and held for 24, 48, 72, and 96 h at 4 and 24 degrees C. Both transporters maintained the viability of organisms better at 4 degrees C than at 24 degrees C. Prevotella bivia and Prevotella corporis had a loss of viability in both transporters at both temperatures. However, at 24 degrees C, there was a significantly greater loss of viability for Mycoplasma hominis, Prevotella bivia, Prevotella corporis, and Peptoniphilus asaccharolyticus when the organisms were stored in Copan transport medium than when they were stored in Port-A-Cul transport medium for 96 h (P < 0.002). Some organisms proliferated in the transport media, but when transporters were held at 24 degrees C for 96 h, a significantly greater increase in the concentrations of group B streptococci and Candida albicans, Escherichia coli, and Enterococcus spp. organisms in Copan medium than in Port-A-Cul medium was observed (P < 0.002). At room temperature, the Port-A-Cul system is superior to the Copan system with respect to the preservation of fastidious microorganisms and the prevention of the proliferation of facultative organisms.