Menstrual cycle-dependent alterations in glycosylation: a roadmap for defining biomarkers of favorable and unfavorable mucus.

OBJECTIVE: To understand glycosylation of endocervical proteins at different times throughout the menstrual cycle in naturally cycling women and in women using hormonal or non-hormonal contraceptive methods, in order to characterize biochemical fingerprints of favorable and unfavorable cervical mucus. DESIGN: Lectin/antibody-probed protein blot analysis of endocervical mucus samples collected onto ophthalmologic sponges (wicks) from two groups: a longitudinal cohort of naturally cycling women at three time points in their menstrual cycles (discovery cohort), and a cross-sectional cohort of women on hormonal or non-hormonal contraceptive methods (validation cohort). SETTING: Participants were recruited from the San Francisco Bay Area from 2010 to 2016. PATIENT(S): Women with regular cycles not using hormonal or intrauterine device (IUD) contraceptives were recruited for the longitudinal cohort (n = 8). Samples from women using levonorgestrel-containing combined oral contraceptives (n = 16), levonorgestrel containing IUDs (n = 14), copper IUDs (n = 17), depo-medroxyprogesterone acetate (DMPA) (n = 15), and controls (n = 13) were used for validation. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Detection of specific glycosylation patterns on lectin/antibody probed protein blots. RESULT(S): Two lectins (Lens culinaris agglutinin and Lycopersicon esculentum [tomato lectin]), and the antibody MECA-79 demonstrated consistent cycle-dependent changes in protein binding. The glycan-binding patterns of the levonorgestrel-containing contraceptives were generally similar to each other and to those from women in the luteal phase. The DMPA samples showed slightly different binding patterns. CONCLUSION(S): We identified molecular signatures of unfavorable mucus from women in the luteal phase and on hormonal contraceptives. Further characterization of these biomarkers may be useful in contraceptive development and in evaluation of infertility.

Reproductive tract immune cells from pregnant women or those using depot medroxyprogesterone acetate show no excess susceptibility to HIV-1: Results of an ex vivo fusion assay.

INTRODUCTION: Ex vivo fusion assays offer an efficient method for studying HIV-1 entry associated with contraceptive use and pregnancy outside of cohort studies of HIV-1 incidence. METHODS: We measured ex vivo HIV-1 fusion to cervical or endometrial immune cells from three groups of women: pregnant, non-pregnant not using hormonal or intrauterine contraception, and using depot medroxyprogesterone acetate (DMPA). RESULTS AND CONCLUSIONS: There was no excess susceptibility to HIV-1 fusion of cells from pregnant women or DMPA users compared to controls. Although the number of target cells in endometrium was higher in DMPA users compared to controls, HIV-1 fusion was lower. IMPLICATIONS: In ex vivo assays, HIV-1 showed no enhanced fusion to cervical immune cells from pregnant women or DMPA users compared to controls, and lower fusion to endometrial immune cells from DMPA users. This assay is useful for studying hormonal and contraceptive effects on HIV-1 entry into reproductive tract immune cells.

Differential Effects of the Hormonal and Copper Intrauterine Device on the Endometrial Transcriptome.

The contraceptive effectiveness of intrauterine devices (IUDs) has been attributed in part to a foreign body reaction in the endometrium. We performed this study to better understand mechanisms of action of contraceptives of by studying their effects on endometrial and cervical transcriptomes. We collected endometrial and cervical biopsies from women using the levonorgestrel-releasing intrauterine system (LNG-IUS, n = 11), copper intrauterine device (cu-IUD, n = 13) or levonorgestrel-containing combined oral contraceptives (COC, n = 12), and from women not using contraceptives (control group, n = 11). Transcriptional profiling was performed with Affymetrix arrays, Principal Component Analysis and the bioconductor package limma. In endometrial samples from cu-IUD users, there were no genes with statistically significant differential expression compared to controls. In LNG-IUS users, 2509 genes were differentially expressed and mapped predominantly onto immune and inflammatory pathways. The cervical samples showed no statistically significant differential gene expression compared to controls. Hormonal and copper IUDs have significantly different effects on the endometrial transcriptome, with the LNG-IUS transcriptome showing pronounced inflammation and immune activation compared to controls whereas the cu-IUD transcriptome was indistinguishable from luteal phase endometrium. These findings argue against a foreign body reaction as a common mechanism of action of IUDs.

Effects of the levonorgestrel-containing intrauterine device, copper intrauterine device, and levonorgestrel-containing oral contraceptive on susceptibility of immune cells from cervix, endometrium and blood to HIV-1 fusion measured ex vivo.

Globally, HIV/AIDS is a leading cause of morbidity worldwide among reproductive-aged cisgender women, highlighting the importance of understanding effects of contraceptives on HIV-1 risk. Some observational studies suggest there may be an increased risk of HIV-1 acquisition among women using the long-acting injectable progestin contraceptive, depo-medroxyprogesterone acetate. The potential mechanism of this susceptibility is unclear. There are few data on the role of the upper female reproductive tract in HIV-1 transmission, and the mechanisms of HIV-1 infection are likely to differ in the upper compared to the lower reproductive tract due to differences in tissue composition and variable effects of sex steroids on mucosal immune cell distribution and activity. In this study, we measured the susceptibility of mucosal immune cells from the upper female reproductive tract to HIV-1 entry using the virion-based HIV-1 fusion assay in samples from healthy female volunteers. We studied 37 infectious molecular clones for their ability to fuse to cells from endometrial biopsies in three participants and found that subtype (B or C) and origin of the virus (transmitted founder or chronic control) had little influence on HIV-1 fusion susceptibility. We studied the effect of contraceptives on HIV-1 susceptibility of immune cells from the cervix, endometrium and peripheral blood by comparing fusion susceptibility in four groups: users of the copper intrauterine device (IUD), levonorgestrel-containing oral contraceptive, levonorgestrel-containing IUD and unexposed controls (n = 58 participants). None of the contraceptives was associated with higher rates of HIV-1 entry into female reproductive tract cells compared to control samples from the mid-luteal phase.

Progestin-Containing Contraceptives Alter Expression of Host Defense-Related Genes of the Endometrium and Cervix.

Epidemiological studies indicate that progestin-containing contraceptives increase susceptibility to HIV, although the underlying mechanisms involving the upper female reproductive tract are undefined. To determine the effects of depot medroxyprogesterone acetate (DMPA) and the levonorgestrel intrauterine system (LNG-IUS) on gene expression and physiology of human endometrial and cervical transformation zone (TZ), microarray analyses were performed on whole tissue biopsies. In endometrium, activated pathways included leukocyte chemotaxis, attachment, and inflammation in DMPA and LNG-IUS users, and individual genes included pattern recognition receptors, complement components, and other immune mediators. In cervical TZ, progestin treatment altered expression of tissue remodeling and viability but not immune function genes. Together, these results indicate that progestins influence expression of immune-related genes in endometrium relevant to local recruitment of HIV target cells with potential to increase susceptibility and underscore the importance of the upper reproductive tract when assessing the safety of contraceptive products.