Comparative Analysis of Progesterone Secretion and Plasma Proteome Across the Pregnant and Non-Pregnant Luteal Phase of the Koala (Phascolarctos cinereus)

Background: Koalas are a vulnerable marsupial species with unique reproductive traits. Efforts to develop assisted breeding technologies have been hindered by a limited understanding of maternal recognition of pregnancy and physiological changes induced by the foeto-placental unit. Differences in the reproductive physiology of pregnant and non-pregnant koalas were examined to investigate the possibility of maternal recognition and identify potential pregnancy and/or embryonic loss biomarkers. Methods: Koalas were separated into three groups: pregnant (n = 4 cycles from three females), mated but non-parturient (n = 4 cycles from three females), and gonadotropin-releasing hormone (GnRH) agonist-treated females (n = 7). Plasma was collected on day of mating/GnRH injection (D0) and on multiple subsequent days. Progesterone concentrations were measured by enzyme immunoassay, and plasma proteomes were analysed using filter-aided sample preparation followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), employing sequential window acquisition of all theoretical fragment ion spectra. Results: Ovulation induction mechanisms influenced peri-ovulatory progesterone secretion (pregnant 40.7 ± 3.3 ng/mL vs. GnRH-treated 15.7 ± 1.8 ng/mL), with no significant differences in progesterone that occurred later in the luteal phase. LC-MS/MS identified 158 proteins, representing the first koala plasma proteome. Leucine-rich α-2-glycoprotein (LRG1) was significantly elevated at D2 in pregnant females compared to GnRH-treated females, and pregnant D9 and D19. In pregnant females, fibronectin (FN1) was significantly more abundant at D19 compared to D9 but not significantly different between treatments. Conclusions: These preliminary findings provide foundational data for further investigation into maternal recognition and pregnancy/embryonic loss in koalas.