Catherine A. Chappell

Studying the effects of reproductive hormones and bacterial vaginosis on the glycome of lavage samples from the cervicovaginal cavity

The cervicovaginal fluid (CVF) coating the vaginal epithelium is an important immunological mediator, providing a barrier to infection. Glycosylation of CVF proteins, such as mucins, IgG and S-IgA, plays a critical role in their immunological functions. Although multiple factors, such as hormones and microflora, may influence glycosylation of the CVF, few studies have examined their impact on this important immunological fluid. Herein we analyzed the glycosylation of cervicovaginal lavage (CVL) samples collected from 165 women under different hormonal conditions including: (1) no contraceptive, post-menopausal, (2) no contraceptive, days 1-14 of the menstrual cycle, (3) no contraceptive, days 15-28 of the menstrual cycle, (4) combined-oral contraceptive pills for at least 6 months, (5) depo-medroxyprogesterone acetate (Depo-Provera) injections for at least 6 months, (6) levonorgestrel IUD for at least 1 month. Glycomic profiling was obtained using our lectin microarray system, a rapid method to analyze carbohydrate composition. Although some small effects were observed due to hormone levels, the major influence on the glycome was the presence of an altered bacterial cohort due to bacterial vaginosis (BV). Compared to normal women, samples from women with BV contained lower levels of sialic acid and high-mannose glycans in their CVL. The change in high mannose levels was unexpected and may be related to the increased risk of HIV-infection observed in women with BV, as high mannose receptors are a viral entry pathway. Changes in the glycome were also observed with hormonal contraceptive use, in a contraceptive-dependent manner. Overall, microflora had a greater impact on the glycome than hormonal levels, and both of these effects should be more closely examined in future studies given the importance of glycans in the innate immune system.

The Effects of Hormones and Vaginal Microflora on the Glycome of the Female Genital Tract: Cervical-Vaginal Fluid

In this study, we characterized the glycome of cervical-vaginal fluid, collected with a Catamenial cup. We quantified: glycosidase levels; sialic acid and high mannose specific lectin binding; mucins, MUC1, MUC4, MUC5AC, MUC7; and albumin in the samples collected. These data were analyzed in the context of hormonal status (day of menstrual cycle, hormonal contraception use) and role, if any, of the type of the vaginal microflora present. When the Nugent score was used to stratify the subjects by microflora as normal, intermediate, or bacterial vaginosis, several important differences were observed. The activities of four of six glycosidases in the samples from women with bacterial vaginosis were significantly increased when compared to normal or intermediate women: sialidase, P = <0.001; α-galactosidase, P = 0.006; β-galactosidase, P = 0.005; α-glucosidase, P = 0.056. Sialic acid binding sites as measured by two lectins, Maackia amurensis and Sambucus nigra binding, were significantly lower in women with BV compared to women with normal and intermediate scores (P = <0.0001 and 0.008 respectively). High mannose binding sites, a measure of innate immunity were also significantly lower in women with BV (P = <0.001). Additionally, we observed significant increases in MUC1, MUC4, MUC5AC, and MUC7 concentrations in women with BV (P = <0.001, 0.001, <0.001, 0.02 respectively). Among normal women we found that the membrane bound mucin MUC4 and the secreted MUC5AC were decreased in postmenopausal women (P = 0.02 and 0.07 respectively), while MUC7 (secreted) was decreased in women using levonorgestrel-containing IUDs (P = 0.02). The number of sialic acid binding sites was lower in the postmenopausal group (P = 0.04), but the number of high mannose binding sites, measured with Griffithsin, was not significantly different among the 6 hormonal groups. The glycosidase levels in the cervical-vaginal mucus were rather low in the groups, with exception of α-glucosidase activity that was much lower in the postmenopausal group (P<0.001). These studies present compelling evidence that the vaginal ecosystem responds to the presence of different vaginal microorganisms. These effects were so influential that it required us to remove subjects with BV for data interpretation of the impact of hormones. We also suggest that certain changes occurring in vaginal/cervical proteins are due to bacteria or their products. Therefore, the quantitation of vaginal mucins and lectin binding offers a new method to monitor bacteria-host interactions in the female reproductive tract. The data suggest that some of the changes in these components are the result of host processing, such as the increases in mucin content, while the microflora is responsible for the increases in glycosidases and the decreases in lectin binding. The methods should be considered a valid marker for insult to the female genital tract.

Impact of Bacterial Vaginosis, as Assessed by Nugent Criteria and Hormonal Status on Glycosidases and Lectin Binding in Cervicovaginal Lavage Samples

The objective of this study was to evaluate the impact of hormonal status and bacterial vaginosis (BV) on the glycosidases present and glycosylation changes as assessed by lectin binding to cervicovaginal lavage constituents. Frozen cervicovaginal lavage samples from a completed study examining the impact of reproductive hormones on the physicochemical properties of vaginal fluid were utilized for the present study. In the parent study, 165 women were characterized as having BV, intermediate or normal microflora using the Nugent criteria. The presence of glycosidases in the samples was determined using quantitative 4-methyl-umbelliferone based assays, and glycosylation was assessed using enzyme linked lectin assays (ELLA). Women with BV had elevated sialidase, α-galactosidase, β-galactosidase and α-glucosidase activities compared to intermediate or normal women (P<0.001, 0.003, 0.006 and 0.042 respectively). The amount of sialic acid (Sambucus nigra, P = 0.003) and high mannose (griffithsin, P<0.001) were reduced, as evaluated by lectin binding, in women with BV. When the data were stratified according to hormonal status, α-glucosidase and griffithsin binding were decreased among postmenopausal women (P<0.02) when compared to premenopausal groups. These data suggest that both hormonal status and BV impact the glycosidases and lectin binding sites present in vaginal fluid. The sialidases present at increased levels in women with BV likely reduce the number of sialic acid binding sites. Other enzymes likely reduce griffithsin binding. The alterations in the glycosidase content, high mannose and sialic acid binding sites in the cervicovaginal fluid associated with bacterial vaginosis may impact susceptibility to viruses, such as HIV, that utilize glycans as a portal of entry.

The effect of menopause on the innate antiviral activity of cervicovaginal lavage

OBJECTIVE Reproductive hormones are known to impact innate mucosal immune function of the lower genital tract. Our objectives were to determine the effect of hormonal status on intrinsic antiviral (herpes simplex virus [HSV]-1, HSV-2, and human immunodeficiency virus [HIV]-1) activity of cervicovaginal lavage (CVL). STUDY DESIGN CVL was collected from 165 asymptomatic women including postmenopausal women (n = 29); women not on contraception in days 1-14 (n = 26) or days 15-28 (n = 27) of the menstrual cycle; and women using the levonorgestrel intrauterine device (n = 28), depot medroxyprogesterone acetate (n = 28), or combined oral contraceptives (n = 27). The anti-HSV-1/-2 and the anti-HIV-1 activity of the CVL were measured using plaque assays and the Jurkat-Tat-CCR5 assay, respectively. RESULTS CVL from all of the groups had modest antiviral activity. Anti-HIV-1 activity was decreased in CVL from postmenopausal women when compared to premenopausal women (11% vs 34%, P = .002). However, there was no difference in anti-HIV-1 activity among premenopausal women regardless of phase of menstrual cycle or contraceptive use. Anti-HIV-1 activity was associated with the protein content of the CVL (r = 0.44, P < .001). There was no difference in anti-HSV-1 or -2 activity by hormonal group. CONCLUSION Menopause is associated with decreased innate HIV-1 activity in the lower genital tract, suggesting that factors in the vaginal fluid could play a role in increased susceptibility of HIV-1 infection in postmenopausal women. Hormonal contraceptive use, menopause, and phase of menstrual cycle did not have a measurable impact on the intrinsic anti-HSV-1 or -2 activity.

Synthetic osmotic dilators with adjunctive misoprostol for same-day dilation and evacuation: a randomized controlled trial ☆ ☆☆ ★ ★★

OBJECTIVE This study aims to evaluate buccal misoprostol as an adjunct to synthetic osmotic dilators for same-day dilation and evacuation (D&E). STUDY DESIGN A randomized, double-blinded, placebo-controlled trial of women 16 0/7 to 20 6/7 weeks gestation desiring D&E was used. Participants received synthetic osmotic cervical dilators (Dilapan-S®) at least 4 h prior to D&E and were randomized to 400mcg buccal misoprostol or placebo 3 h preoperatively, stratified by gestational age. The primary outcome was operative time with 36 participants needed to detect a 4-min difference (two-sided α=0.05, 80% power). Secondary outcomes included total procedure time, patient and provider acceptability, baseline cervical dilation and complications. RESULTS Twenty-nine women were enrolled (misoprostol n=14, placebo n=15) and mean operative time was similar between the groups (11.1 vs. 13.5 min, respectively, p=.17). Complications were nonsignificantly more common for participants ≥19 weeks compared to <19 weeks (22% vs. 9%, p=.62) and those who received placebo compared to misoprostol (27% vs. 7%, p=.33). Two serious adverse events in the placebo group prompted early study closure for safety and futility. Placebo participants had longer overall procedure times (24 vs. 18 min, p=.03) and less cramping preoperatively (p<.01) but similar results for other secondary outcomes compared to those receiving misoprostol. Women strongly preferred same-day cervical preparation (98%). CONCLUSIONS Adjunctive buccal misoprostol may not decrease operative times but may decrease complications when combined with synthetic osmotic dilators for cervical preparation for same-day D&E procedures. IMPLICATIONS Although the trial was halted early and underpowered to make conclusions about the primary outcome, complication frequency and type warrant caution for use of synthetic osmotic dilators alone for cervical preparation for same-day D&E at ≥19 weeks gestation.

Efavirenz decreases etonogestrel exposure: a pharmacokinetic evaluation of implantable contraception with antiretroviral therapy

Objectives: The primary objective of this study was to characterize the pharmacokinetics of etonogestrel (ENG) released from a contraceptive implant in Ugandan women living with HIV who were receiving efavirenz (EFV) or nevirapine (NVP)-based antiretroviral therapy (ART), compared with ART-naive women over 24 weeks. Design: Nonrandomized, parallel-group study with three arms: ART-naive, NVP, or EFV-based ART (N = 20/group). Methods: Sparse pharmacokinetic sampling of ENG, NVP, or EFV were performed at screening, entry, and then 1, 4, 12, and 24-week postimplant insertion. The primary endpoint was ENG concentrations at week 24, compared between the ART-naive group and each ART group, using geometric mean ratio (GMR) with 90% confidence intervals. Results: Sixty participants competed the 24-week study and data from 58 participants are included; one participant each was excluded from the NVP group and EFV group because of a sample processing error and ART nonadherence, respectively. At week 24, geometric mean ENG was 362, 341, and 66 pg/ml in the ART-naive, NVP, and EFV groups, respectively [GMR: NVP : ART-naive 0.94 (0.90–1.01); EFV : ART-naive 0.18 (0.17–0.20)]. NVP and EFV concentrations were lower at week 24 compared to preimplant [NVP: geometric mean 5.7 versus 6.8 mg/l, respectively, GMR 0.84 (0.83–0.85); EFV: geometric mean 3.6 versus 4.9 mg/l, respectively, GMR 0.73 (0.69–0.80)]. Conclusion: After 24 weeks of combined use, ENG exposure was 82% lower in women using EFV-based ART compared with ART-naive women. In contrast, NVP did not significantly impact ENG exposure. These results raise concerns about reduced effectiveness of implantable contraception for women taking EFV-based ART.

Hepatitis C Virus Screening Among Children Exposed During Pregnancy

Despite the increasing prevalence of HCV infection among pregnant women, most exposed infants are not screened for perinatal acquisition of the HCV. OBJECTIVES: Because of the opioid epidemic, hepatitis C virus (HCV) infection is increasing among pregnant women, resulting in an increased risk of perinatal transmission and HCV infection among children. Our primary objectives in this study were to determine the prevalence of HCV among pregnant women and the frequency of pediatric HCV screening. METHODS: A population-based, retrospective cohort of pregnant women who delivered between 2006 and 2014 was identified and classified as HCV infected or HCV uninfected by billing codes. Infant records linked to the HCV-infected pregnant women were identified and queried for HCV tests and the receipt of well-child services, which were defined as the presence of hemoglobin and/or lead testing at or after 9 months of age. RESULTS: Between 2006 and 2014, 1043 (1.2%) HCV-infected pregnant women delivered, and the HCV prevalence increased by 60%. HCV-infected women were more likely to be <30 years of age (67% vs 53%; P < .001), white (93% vs 72%; P < .001), insured by Medicaid (77% vs 29%; P < .001), and have opiate use disorder (68% vs 1%; P < .001) than HCV-uninfected women. Infants born to HCV-infected women were more likely to be preterm (<37 weeks’ gestation; 22% vs 10%; P < .001) and of low birth weight (<2500 g; 23% vs 8%; P < .001). Among 1025 HCV-exposed infants with available pediatric records, 323 (31%) received well-child services, and among these, only 96 (30%) were screened for HCV. CONCLUSIONS: Despite the increased HCV prevalence among pregnant women and the risk of perinatal HCV transmission, HCV-exposed infants are not adequately screened, and many pediatric HCV infections remain undetected.

Prevention of Perinatal Transmission of Human Immunodeficiency Virus

The reproductive health needs of all women of childbearing age should routinely address effective and appropriate contraception, safer sex practices, and elimination of alcohol, illicit drugs and tobacco should pregnancy occur. Combined antepartum, intrapartum, and infant antiretroviral (ARV) prophylaxis are recommended because ARV drugs reduce perinatal transmission by several mechanisms, including lowering maternal viral load and providing infant pre- and post-exposure prophylaxis. Scheduled cesarean delivery at 38 weeks with IV AZT decreases the risk of perinatal transmission if the HIV RNA is greater than 1000 copies/mL or if HIV levels are unknown near the time of delivery. Oral AZT should generally be given for at least 6 weeks to all infants perinatally exposed to HIV to reduce perinatal transmission of HIV.

Use of an endotracheal tube for surgical abortion complicated by a leiomyomatous uterus: A case report

BackgroundAbnormal uterine anatomy, especially leiomyomas, can significantly impact the difficulty and potential morbidity of surgical uterine evacuation. To avoid hysterotomy and/or hysterectomy, limited evidence exists to guide surgical uterine evacuation when pregnancy tissue is inaccessible with routine instruments.Case presentationA 41-year-old G4P1021 African American woman at 14 4/7 weeks’ gestation was referred for surgical-induced abortion in the setting of an enlarged leiomyomatous uterus. Two large opposing leiomyomas at the internal cervical os rendered pregnancy tissue inaccessible with routine gynecologic surgical instruments. With ultrasound guidance, an endotracheal tube was connected to routine electric suction and utilized to complete uterine evacuation.ConclusionsWith distorted or markedly enlarged uterine anatomy rendering pregnancy tissue inaccessible with routine surgical instruments, the minimally invasive use of an endotracheal tube may aid completion of uterine evacuation for surgical uterine evacuation.

Heterogeneity of HIV-1 replication in ectocervical and vaginal tissue ex vivo

In clinical trials evaluating HIV-1 prevention products, ex vivo exposure of mucosal tissue to HIV-1 is performed to inform drug levels needed to suppress viral infection. Understanding assay and participant variables that influence HIV-1 replication will help with assay implementation. Demographic and behavioral data were obtained from 61 healthy women aged 21-45. Paired cervical tissue (CT) and vaginal tissue (VT) biopsies were collected and treated with HIV-1BaL or HIV-1JR-CSF, washed, and cultured. On days 3, 7, and/or 11, culture supernatant was collected, and viral replication was monitored by p24 ELISA. Tissue was extracted at study end, and HIV-1 relative RNA copies were determined by polymerase chain reaction. Cumulative p24 and RNA were log-transformed and analyzed using a linear mixed model, t-test, and an intraclass correlation coefficient (ICC). HIV replication was similar between CT and VT for each virus, but HIV-1BaL had 1.5 log10 and 0.9 log10 higher levels of p24 than HIV-1JR-CSF in CT and VT, respectively (p < .001), which correlated with HIV-1 relative RNA copies. Cumulative p24 and RNA copies in both tissues demonstrated low intraperson correlation for both viruses (ICC ≤0.513 HIV-1BaL; ICC ≤0.419 HIV-1JR-CSF). Enrollment into previous clinical studies in which genital biopsies were collected modestly decreased the HIV-1BaL cumulative p24 for CT, but not for VT. To improve the ex vivo challenge assay, viruses should be evaluated for replication in mucosal tissue before study implementation, baseline mucosal tissue is not needed if a placebo/no treatment group is included within the clinical trial, and previous biopsy sites should be avoided.