The injectable contraceptives depot medroxyprogesterone acetate and norethisterone enanthate substantially and differentially decrease testosterone and sex hormone binding globulin levels: A secondary study from the WHICH randomized clinical trial.

HIV acquisition risk with norethisterone (NET) enanthate (NET-EN) is reportedly less than for depo-medroxyprogesterone acetate intramuscular (DMPA-IM). We investigated the effects of these progestin-only injectable contraceptives on serum testosterone and sex hormone binding globulin (SHBG) levels, since these may play a role in sexual behavior and HIV acquisition. The open-label WHICH clinical trial, conducted at two sites in South Africa from 2018-2019, randomized HIV-negative women aged 18-40 years to 150 mg DMPA-IM 12-weekly (n = 262) or 200 mg NET-EN 8-weekly (n = 259). We measured testosterone by UHPLC-MS/MS and SHBG by immunoassay in matched pairs of serum samples collected at baseline (D0) and at peak serum progestin levels at 25 weeks post initiation (25W) (n = 214-218 pairs). Both contraceptives substantially decreased, from D0 to 25W, the total testosterone [DMPA-IM D0 0.560, 25W 0.423 nmol/L, -24.3% (p < 0.0001); NET-EN D0 0.551, 25W 0.253 nmol/L, -54.1%, (p < 0.0001)], SHBG [DMPA-IM D0 45.0, 25W 32.7 nmol/L, -29.8% (p < 0.0001); NET-EN D0 50.2, 25W 17.6 nmol/L, -65.1% (p < 0.0001)], and calculated free testosterone levels [DMPA-IM D0 6.87, 25W 5.38 pmol/L, -17.2% (p = 0.0371); NET-EN D0 6.00, 25W 3.70, -40.0% (p < 0.0001)]. After adjusting for change from D0, the total testosterone, SHBG and calculated free testosterone levels were significantly higher for DMPA-IM than NET-EN (64.9%, p < 0.0001; 101.2%, p < 0.0001; and 38.0%, p = 0.0120, respectively). The substantial and differential decrease in testosterone and SHBG levels does not explain our previous finding of no detected decrease in risky sexual behavior or sexual function for DMPA-IM or NET-EN users from D0 to 25W. Medroxyprogesterone (MPA) and NET are androgenic and are both present in molar excess over testosterone and SHBG concentrations at 25W. Any within or between contraceptive group androgenic effects on behavior in the brain are likely dominated by the androgenic activities of MPA and NET and not by the decreased endogenous testosterone levels. The clinical trial was registered with the Pan African Clinical Trials Registry (PACTR 202009758229976).

Misreporting contraceptive use and the association of peak study progestin levels with weight and BMI among women randomized to the progestin-only injectable contraceptives DMPA-IM and NET-EN.

Progestin-only injectable contraceptives, mainly depo-medroxyprogesterone acetate intramuscular (DMPA-IM), are the most widely used contraceptive methods in sub-Saharan Africa. Insufficient robust data on their relative side-effects and serum concentrations limit understanding of reported outcomes in contraception trials. The WHICH clinical trial randomized HIV-negative women to DMPA-IM (n = 262) or norethisterone enanthate (NET-EN) (n = 259) at two South African sites between 2018-2019. We measured serum concentrations of study and non-study progestins at initiation (D0) and peak serum levels, one week after the 24-week injection [25 weeks (25W)], (n = 435) and investigated associations between study progestin levels, and BMI and weight of participants. Peak median serum concentrations were 6.59 (IQR 4.80; 8.70) nM for medroxyprogesterone (MPA) (n = 161) and 13.6 (IQR 9.01; 19.0) nM for norethisterone (NET) (n = 155). MPA was the most commonly quantifiable non-study progestin at D0 in both arms (54%) and at 25W in the NET-EN arm (27%), followed by NET at D0 in both arms (29%) and at 25W in the DMPA-IM arm (19%). Levonorgestrel was quantifiable in both arms [D0 (6.9%); 25W (3.4%)], while other progestins were quantifiable in ≤ 14 participants. Significant negative time-varying associations were detected between MPA and NET concentrations and weight and BMI in both contraceptive arms and a significant increase was detected for peak serum progestin concentrations for normal weight versus obese women. Contraceptive-related reported outcomes are likely confounded by MPA, more so than NET, with reported DMPA-IM effects likely underestimated, at sites where DMPA-IM is widely used, due to misreporting of contraceptive use before and during trials, and 'tail' effects of DMPA-IM use more than six months before trial enrolment. Peak serum levels of MPA and NET are negatively associated with BMI and weight, suggesting another source of variability between trial outcomes and a potential increase in side-effects for normal weight versus overweight and obese women. Trail registration: The clinical trial was registered with the Pan African Clinical Trials Registry (PACTR 202009758229976).

Glucocorticoids and medroxyprogesterone acetate synergize with inflammatory stimuli to selectively upregulate CCL20 transcription.

The pro-inflammatory cytokine, chemokine (C-C motif) ligand 20 (CCL20), is emerging as a therapeutic target for immune-based therapies. Cooperative regulation of CCL20 by glucocorticoids and progestins used in endocrine therapy and pro-inflammatory mediators could modulate immune function and affect disease outcomes. We show that glucocorticoids as well as medroxyprogesterone acetate (MPA), the progestin widely used in injectable contraception in sub-Saharan Africa, cooperate with pro-inflammatory mediators to upregulate CCL20 protein and/or mRNA in human peripheral blood mononuclear cells (PBMCs) and human cervical cell lines. Changes in CCL20 mRNA levels were shown to be synergistic, as assessed by Chou analysis, cell- and gene-specific and to involve transcriptional regulation, with a requirement for a nuclear factor kappa B (NF-κB) site and glucocorticoid receptor (GR) involvement. The novel results suggest a mechanism whereby MPA, like glucocorticoids, may impact inflammation both systemically and in the genital tract in patients using MPA and/or glucocorticoid therapy.

Reciprocal Modulation of Antiretroviral Drug and Steroid Receptor Function In Vitro.

Millions of women are exposed simultaneously to antiretroviral drugs (ARVs) and progestin-based hormonal contraceptives. Yet the reciprocal modulation by ARVs and progestins of their intracellular functions is relatively unexplored. We investigated the effects of tenofovir disoproxil fumarate (TDF) and dapivirine (DPV), alone and in the presence of select steroids and progestins, on cell viability, steroid-regulated immunomodulatory gene expression, activation of steroid receptors, and anti-HIV-1 activity in vitro Both TDF and DPV modulated the transcriptional efficacy of a glucocorticoid agonist via the glucocorticoid receptor (GR) in the U2OS cell line. In TZM-bl cells, DPV induced the expression of the proinflammatory interleukin 8 (IL-8) gene while TDF significantly increased medroxyprogesterone acetate (MPA)-induced expression of the anti-inflammatory glucocorticoid-induced leucine zipper (GILZ) gene. However, peripheral blood mononuclear cell (PBMC) and ectocervical explant tissue viability and gene expression results, along with TZM-bl HIV-1 infection data, are reassuring and suggest that TDF and DPV, in combination with dexamethasone (DEX) or MPA, do not reciprocally modulate key biological effects in primary cells and tissue. We show for the first time that TDF induces progestogen-independent activation of the progesterone receptor (PR) in a cell line. The ability of TDF and DPV to influence GR and PR activity suggests that their use may be associated with steroid receptor-mediated off-target effects. This, together with cell line and individual donor gene expression responses in the primary models, raises concerns that reciprocal modulation may cause side effects in a cell- and donor-specific manner in vivo.

Glucocorticoid-independent modulation of GR activity: Implications for immunotherapy.

Pharmacological doses of glucocorticoids (GCs), acting via the glucocorticoid receptor (GR) to repress inflammation and immune function, remain the most effective therapy in the treatment of inflammatory and immune diseases. Since many patients on GC therapy exhibit GC resistance and severe side-effects, much research is focused on developing more selective GCs and combination therapies, with greater anti-inflammatory potency. GCs mediate their classical genomic transcriptional effects by binding to the cytoplasmic GR, followed by nuclear translocation and modulation of transcription of target genes by direct DNA binding of the GR or its tethering to other transcription factors. Recent evidence suggests, however, that the responses mediated by the GR are much more complex and involve multiple parallel mechanisms integrating simultaneous signals from other receptors, both in the absence and presence of GCs, to shift the sensitivity of a target cell to GCs. The level of cellular stress, immune activation status, or the cell cycle phase may be crucial for determining GC sensitivity and GC responsiveness as well as subcellular localization of the GR and GR levels. Central to the development of new drugs that target GR signaling alone or as add-on therapies, is an in-depth understanding of the molecular mechanisms of GC-independent GR desensitization, priming and activation of the unliganded GR, as well as synergy and cross-talk with other signaling pathways. This review will discuss the information currently available on these topics and their relevance to immunotherapy, as well as identify unanswered questions and future areas of research.

Differential T-cell responses of semi-immune and susceptible malaria subjects to in silico predicted and synthetic peptides of Plasmodium falciparum.

Malaria remains a public health hazard in tropical countries as a consequence of the rise and spread of drug and insecticide resistances; hence the need for a vaccine with widespread application. Protective immunity to malaria is known to be mediated by both antibody and cellular immune responses, though characterization of the latter has been less extensive. The aim of the present investigation was to identify novel T-cell epitopes that may contribute to naturally acquired immune responses against malaria. Using the Microsoft software, Epitome™ T-cell peptide epitopes on 19 Plasmodium falciparum proteins in the Plasmodium Database (www.plasmodb.org.PlasmoDB 9.0) were predicted in-silico. The peptides were synthesized and used to stimulate peripheral blood mononuclear cells (PBMCs) in 14 semi-immune and 21 malaria susceptible subjects for interferon-gamma (IFN-γ) production ex-vivo. The level of IFN-γ production, a marker of T-cell responses, was measured by ELISPOT assay in semi-immune subjects (SIS) and frequently sick subjects (FSS) from an endemic zone with perennial malaria transmission. Of the 19 proteins studied, 17 yielded 27 pools (189 peptides), which were reactive with the subjects' PBMCs when tested for IFN-γ production, taking a stimulation index (SI) of ≥2 as a cutoff point for a positive response. There were 10 reactive peptide pools (constituting eight protein loci) with an SI of 10 or greater. Of the 19 proteins studied, two were known vaccine candidates (MSP-8 and SSP2/TRAP), which reacted both with SIS and FSS. Similarly the hypothetical proteins (PFF1030w, PFE0795c, PFD0880w, PFC0065c and PF10_0052) also reacted strongly with both SIS and FSS making them attractive for further characterization as mediators of protective immunity and/or pathogenesis.