Evaluation of the novel vaginal contraceptive agent PPCM in preclinical studies using sperm hyaluronan binding and acrosome status assays

Abstract

Background: Polyphenylene carboxymethylene (PPCM) sodium salt is a promising multipurpose technology for prevention of both sexually transmitted infections (STIs) and pregnancy. In preclinical studies, PPCM has demonstrated significant (1) antimicrobial activity against several important viral and bacterial pathogens and (2) contraceptive activity associated with premature acrosome loss.

Objective: To further evaluate a vaginal antimicrobial compound as a contraceptive agent in preclinical studies utilizing a repurposed hyaluronan binding assay (HBA).

Materials and methods: Semen samples containing either neat semen or washed spermatozoa were treated with increasing concentrations of PPCM or calcium ionophore A23187 (positive control). Sperm inactivation was measured by two methods: (1) double acrosome staining (AS), and (2) a hyaluronan binding assay (HBA^® ). Percentage of inactivated sperm was compared between untreated control sperm and those treated with PPCM or A23187.

Results: PPCM had a significant (p < 0.05) and dose-dependent effect on sperm inactivation in both assays, with HBA detecting a higher proportion of inactivated sperm than AS. PPCM did not affect sperm motility and exhibited equivalent responses in the neat and washed samples.

Discussion: Both HBA and AS confirmed that spermatozoa were rapidly inactivated at PPCM concentrations likely present in the vagina under actual use conditions and in a time-frame comparable to in vivo migration of spermatozoa out of seminal plasma into cervical mucus.

Conclusion: PPCM vaginal gel may provide contraceptive protection as well as help with STI prevention. HBA may be a sensitive and much needed biomarker for sperm activity in future contraceptive development.

Keywords: acrosome reaction; human spermatozoa; hyaluronan binding assay; polyphenylene carboxymethylene; vaginal contraceptive.

FIGURE 1

Comparison of sperm inactivation by PPCM measured by both the HBA (red bars) and the AS assay (black bars). Washed human spermatozoa (20 million/ml) were resuspended in BWW, divided into aliquots, and incubated with increasing concentrations of PPCM for 15 min. Control groups without PPCM were incubated in BWW (negative control) or with Ca++ ionophore A23187 (positive control). The “% Unbound to Hyaluronan” represents the fraction of spermatozoa inactivated by PPCM or A23187 and unable to bind to the hyaluronan‐coated slide. The “% Acrosome Reaction” represents the inactivated spermatozoa with premature acrosome loss caused by PPCM or A23187. Both assays demonstrated dose‐dependent sperm inactivation (contraceptive activity) after exposure to PPCM. The HBA exhibited a higher sensitivity for PPCM‐induced hyaluronan binding loss with a maximal effect at the 100 µg/ml dose resulting in 80% binding reduction, comparable to A23187. The AS assay at its peak showed maximal activity of only 27% acrosome‐reacted sperm, comparable to A23187 effects. Bars represent the mean ± SEM, n = 6/treatment group. #p < 0.01 PPCM at 100, 10, 1.0, and 0.1 versus no PPCM in the HBA assay. *p < 0.01 PPCM at 100, 10, 1.0, 0.1 µg/ml versus no PPCM in the AS assay. (Reprinted by permission of the J Biol Reprod, Oxford University Press. Weitzel, North, Waller, 2020, 103[2], 299‐309)

FIGURE 2

Time course analysis of 10 µg/ml PPCM inhibition of sperm binding in the HBA used neat semen and washed spermatozoa. The HBA activity of spermatozoa incubated for 1, 5, 10, and 15 min in BWW (negative control), the Ca++ ionophore (Ion) A23187 (positive control) and PPCM (10 µg/ml) was examined in split semen specimens (n = 5); one sperm portion remained in seminal plasma (neat) while the second portion was washed free of seminal plasma and resuspended in BWW. Bars represent the mean ± SEM

FIGURE 3

Analysis of high‐dose PPCM effects on sperm binding in the HBA over time. Spermatozoa in neat semen were exposed to BWW (negative control), the Ca++ ionophore (Ion) A23187 (positive control) and increasing doses of PPCM (100, 1000, and 10,000 µg/ml) and spermatozoa bound to hyaluronan‐coated slides were quantified at for 1, 5, 10, and 15 min. Bars represent the mean ± SEM, n = 3

FIGURE 4

Effect of high‐dose PPCM exposure over time on sperm motility (%). Unwashed spermatozoa exposed to BWW (negative control), the Ca++ ionophore (Ion) A23187 (positive control) and increasing doses of PPCM (100, 1000, and 10,000 µg/ml) were assessed for % motility at 20 min, 2, 6, and 18 h after exposure. Sperm motility slowly declined in all exposed spermatozoa over time at the same rate, with no significant differences between treatment groups at each time point. Bars represent the mean ± SEM, n = 3 semen samples