Medroxyprogesterone acetate

For vaginal infections, mice were pre-treated starting two days pre-infection with i.p. injections of streptomycin sulfate (2.4 mg) and vancomycin hydrochloride (0.6 mg) diluted in PBS, and trimethoprim sulfate (0.04 g/L) in drinking water to suppress vaginal microbiota as previously described [18 (link)]. Concurrent hormone injections of 0.5 mg water-soluble 17β-estradiol (Sigma) dissolved in PBS were also administered every other day, starting two days before N. gonorrhoeae infection, to synchronize the mice in estrus [21 (link)]. Low-passage clinical isolates of N. gonorrhoeae [19 (link)] were lawn-streaked on GC agar (Difco) supplemented with IsoVitalex and VCNT antibiotics (BD) overnight, washed and prepared in PBS⁺⁺ (Life Technologies), and either 10 μL of PBS⁺⁺ or 1 × 10⁸ CFU N. gonorrhoeae in 10 μL of PBS⁺⁺ was introduced vaginally with a pipette. One month after vaginal infection, mice were transcervically inoculated with either 1 × 10⁸ CFU N. gonorrhoeae or 20 μL of PBS using a blunt 25-gauge needle (Sai Infusion Technologies), as previously described [19 (link)]. Five days prior to transcervical infection, mice were subcutaneously injected with 2 mg medroxyprogesterone acetate (Pfizer) to synchronize mice to diestrus stage.

Pregnant ewes received intramuscular (IM) medroxyprogesterone acetate (150 mg; Pfizer, Australia) at 120 days’ gestational age (GA; term ~150 d). Ultrasound-guided intra-amniotic (IA) injection of lipopolysaccharide (LPS;4 mg; 2 mg/mL; Escherichia coli 055:B5; Sigma-Aldrich, NSW, Australia; n = 10) or saline (n = 9) was performed at 126 days’ GA. This dose of LPS results in a well characterized fetal inflammatory response that peaks 48 h after injection [23 (link)]. Ewes received an intramuscular (IM) injection of betamethasone (5.7 mg/dose; Celestone, Merck Sharp & Dohme Pty Ltd, Australia) 48 h and 24 h prior to planned cesarean section delivery at 128 days’ gestational age (GA; term ~150 d), commencing 6 hours after IA LPS injection.

Medroxyprogesterone acetate (Pfizer, Surrey, UK) was used to induce withdrawal menstruation. Kisspeptin-10 was custom synthesized under GMP standards (Bachem GmBH, Weil am Rhein, Germany) (George et al., 2011 (link)), and 1 mg kisspeptin-10 was dissolved in 5 ml sterile normal (0.9%) saline immediately before infusion. The syringe and line for infusion were first coated for 30 min with kisspeptin-10 to minimize peptide loss from adherence to the plastic. Sterile normal saline was infused as vehicle. The NK3Ra MLE4901 (previously termed AZD4901, Astra-Zeneca, Macclesfield, UK) was administered orally at 40 mg twice daily. This dose of MLE4901 reduced LH secretion in normal women and in women with PCOS (George et al., 2016 (link); Skorupskaite et al., 2016 (link)).