Validating the Non-Human Primate as a Model for Testing Non-Hormonal Contraception

Leo Han M.D. MPH^1,2 Rachel Looney¹, Shan Yao¹, Jeffrey T. Jensen M.D. MPH^1,2,

<11. Oregon National Primate Research Center, Division of Reproductive and Developmental Sciences, Portland, OR

< !2. Oregon Health & Science University, Department of Obstetrics and Gynecology, Portland, OR

Abstract Text: Research programs focused on nonhormonal approaches to contraceptive drug discovery such as sperm targeting, lower tract barriers, tubal interruption, and male contraception, share a common hurdle in transition from in vitro to in vivo proof of concept studies as we lack validated surrogate markers in animal studies, short of a fertility trial. The non-human primate (NHP) may provide the most relevant and direct translational model given the homologous structure and function of the lower reproductive tract anatomy and the presence of menstrual cycles. However, common clinical surrogates such as post-coital testing and clinical mucus scores have not been well studied in the NHP model. 
We used the rhesus macaque (Macaca mulatta) to develop a pre-clinical model for studying endocervical mucus changes and to perform post-coital testing. To do this we administered antide (subcutaneous injection, 0.075 mg/kg) for ovarian suppression followed by estrogen replacement [estradiol (E2) sub-cutaneous implants (50 mg/kg) or estradiol valerate (E2V) (intramuscular (IM) injection, 2.5-5 mg)] and a progestogen replacement (oral, ethinyl estradiol 0.03 mg/ levonorgestrel 0.15 mg) to recreate mid cycle and luteal phase hormonal condition. We measured E2 levels following hormonal treatment and assessed corresponding clinical mucus scores (spinnbarkeit, ferning & viscosity, each on a 0-3 scale, with a 9-point total). Finally, we performed post-coital testing using vaginal insemination of rhesus sperm (~100 million/ml) and assessed for progressively motile sperm in an endocervical mucus sample.
We found that spinnbarkeit, ferning and viscosity changed in response to hormonal conditions in a direction like women. We did not find cellularity to be a useful metric as the mucus was highly cellular in all hormonal conditions. IM injection of E2V resulted in higher peak E2 levels (2.5 mg injection: mean E2 1015±SD 437 pg/mL; 5 mg injection: 2526±498 pg/mL) compared to subcutaneous implants (117±37 pg/mL) as well as more fertility-favorable cervical mucus changes (2.5 mg injection: mean mucus score 6.4±SD 1.5; implant: 4.6±2.3). Administration of an oral contraceptive containing levonorgestrel sufficiently reversed mucus changes in all animals despite persistent E2 elevation (0.6±1.0).  Finally, post-coital testing demonstrated passage of motile sperm ( >20+ progressively motile/LPF) with E2V only conditions and absence of sperm following LNG treatment.
In conclusion, we found that our artificial hormonal replacement model in rhesus macaque produced cervical mucus that correlated with expected cycle-related changes. The artificial cycle allows investigators to time sample collection with strict control over the hormonal milieu.   Post-coital testing is a useful method for evaluating contraceptives that are aimed at preventing sperm from ascending through the cervical canal at mid-cycle.