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).

Variability and quantification of serum medroxyprogesterone acetate levels.

Quantification of serum progestin levels in clinical contraceptive studies is now routinely performed to understand progestin pharmacokinetics and to correct for unreliable self-reporting of contraceptive use by study participants. Many such studies are focussed on the three-monthly progestin-only intramuscular (IM) injectable contraceptive depot medroxyprogesterone acetate (DMPA-IM). Methods commonly used to measure serum MPA levels include liquid chromatography coupled to mass spectrometry (LC/MS) and radioimmunoassay (RIA); however, RIA methods have not been used in recent years. We review the available literature and find that these methods vary widely in terms of use of organic solvent extraction, use of derivitization and choice of organic solvent and chromatography columns. There is a lack of standardization of LC/MS methodology, including a lack of detailed extraction protocols. Limited evidence suggests that RIA, without organic solvent extraction, likely over-estimates progestin levels. Maximum MPA concentrations in the first two weeks post-injection show wide inter-individual and inter-study variation, regardless of quantification method used. Standardization of quantification methods and sampling time post-injection is required to improve interpretation of clinical data, in particular the side effects arising at different times depending on the pharmacokinetic profile unique to injectable contraceptives.

Pharmacokinetics, metabolism and serum concentrations of progestins used in contraception.

Many different forms of hormonal contraception are used by millions of women worldwide. These contraceptives differ in the dose and type of synthetic progestogenic compound (progestin) used, as well as the route of administration and whether or not they contain estrogenic compounds. There is an increasing awareness that different forms of contraception and different progestins have different side-effect profiles, in particular their cardiovascular effects, effects on reproductive cancers and susceptibility to infectious diseases. There is a need to develop new methods to suit different needs and with minimal risks, especially in under-resourced areas. This requires a better understanding of the pharmacokinetics, metabolism, serum and tissue concentrations of progestins used in contraception as well as the biological activities of progestins and their metabolites via steroid receptors. Here we review the current knowledge on these topics and identify the research gaps. We show that there is a paucity of research on most of these topics for most progestins. We find that major impediments to clear conclusions on these topics include a lack of standardized methodologies, comparisons between non-parallel clinical studies and variability of data on serum concentrations between and within studies. The latter is most likely due, at least in part, to differences in intrinsic characteristics of participants. The review highlights the importance of insight on these topics in order to provide the best contraceptive options to women with minimal risks.

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.

Differential metabolism of clinically-relevant progestogens in cell lines and tissue: Implications for biological mechanisms.

Steroid hormones regulate a variety of physiological processes, including reproductive function, and are widely used in hormonal therapy. Synthetic progestogens, or progestins, were designed to mimic progesterone (P4) for use in contraception and hormonal replacement therapy in women. Medroxyprogesterone acetate (MPA) and norethisterone (NET) are the most widely used injectable contraceptives in the developing world, while other progestins such as levonorgestrel (LNG), etonogestrel (ETG) and nestorone (NES) are used in or being developed for other forms of contraception. As concerns remain about the most appropriate choice of progestin and dosage, and the associated side-effects, the mechanisms and biological effects of progestins are frequently investigated in various in vitro mammalian cell line and tissue models. However, whether progestogens are differentially metabolised in different cell types in vivo or in vitro is unknown. For nine mammalian cell lines commonly used to investigate progestogen mechanisms of action, we developed and validated an ultra-high performance supercritical fluid chromatography-tandem mass spectrometry (UHPSFC-MS/MS) protocol for simultaneously quantifying the metabolism of the above-mentioned steroids. We show for the first time that, while 50-100% of P4 was metabolised within 24 h in all cell lines, the metabolism of the progestins is progestin- and cell line-specific. We also show that MPA and NET are significantly metabolised in human cervical tissue, but to a lesser extent than P4. Taken together, our findings suggest that differential progestogen metabolism may play a role in cell-specific therapeutic and side-effects. Relative affinities for binding to steroid receptors as well as potencies, efficacies and biocharacters for transcriptional activity of progestins, relative to P4, are most frequently determined using some of the cell lines investigated. Our results, however, suggest that differential metabolism of progestins and P4 may confound these results. In particular, metabolism may under-estimate the receptor-mediated intrinsic in vitro binding and dose-response values and predicted endogenous physiological effects of P4.