We have previously demonstrated spermicidal activity of LL-37 antimicrobial peptide on mouse/human sperm and its contraceptive effects in female mice. With its microbicidal action against Neisseria gonorrhoeae, LL-37 warrants development into a multipurpose prevention technology (MPT) agent for administering into the female reproductive tract (FRT). However, it is important to verify that multiple administrations of LL-37 do not lead to damage of FRT tissues and/or irreversible loss of fecundity. Herein, we transcervically injected LL-37 (36 µM-10× spermicidal dose) into female mice in estrus in three consecutive estrous cycles. A set of mice were sacrificed for histological assessment of the vagina/cervix/uterus 24 h after the last injection, while the second set were artificially inseminated with sperm from fertile males 1 week afterwards, and then monitored for pregnancy. Mice injected with PBS in parallel were regarded as negative controls, whereas those injected with vaginal contraceptive foam (VCF, available over the counter), containing 12.5% nonoxynol-9, served as positive controls for vaginal epithelium disruption. We demonstrated that the vagina/cervix/uterus remained normal in both LL-37-injected and PBS-injected mice, which also showed 100% resumption of fecundity. In contrast, VCF-injected mice showed histological abnormalities in the vagina/cervix/uterus and only 50% of them resumed fecundity. Similarly, LL-37 multiply administered intravaginally caused no damage to FRT tissues. While our results indicate the safety of multiple treatments of LL-37 in the mouse model, similar studies have to be conducted in non-human primates and then humans. Regardless, our study provides an experimental model for studying in vivo safety of other vaginal MPT/spermicide candidates.

With an unplanned pregnancy rate of 50% or more in many countries, there is an urgent need for contraceptives that are more accessible and acceptable. To meet the growing demand for new contraceptives, ZabBio developed ZB-06, a vaginal film containing HC4-N, a human contraceptive antibody that inactivates sperm.
This study aimed to assess the potential contraceptive activity of the ZB-06 film using a surrogate assessment for contraceptive efficacy, the postcoital test. We also assessed clinical safety of film use among healthy heterosexual couples. Serum, cervical mucus, and vaginal fluid HC4-N antibody concentrations and sperm agglutination potency were determined after single film use. Changes in the concentration of soluble proinflammatory cytokines and vaginal Nugent score after film use were measured as subclinical safety endpoints.
This was a phase 1, first-in-woman, open-label, proof-of-concept, postcoital test and safety study.
A total of 20 healthy women were enrolled in the study, and 8 heterosexual couples completed all study visits. The product was safe for both female participants and their male sexual partners. The postcoital test performed on ovulatory cervical mucus at baseline (no product use) revealed a mean of 25.9 (±30.6) progressively motile sperm per high-power field. After use of a single ZB-06 film before intercourse, this number dropped to 0.04 (±0.06) progressively motile sperm per high-power field (P<.0001). At the follow-up postcoital test visit approximately 1 month later (no product use), a mean of 47.4 (±37.4) progressively motile sperm per high-power field was observed, indicating contraceptive reversibility.
A single dose of the ZB-06 film applied before intercourse was safe and met efficacy surrogate benchmarks of excluding progressively motile sperm from ovulatory cervical mucus. These data indicate that ZB-06 is a viable contraceptive candidate warranting further development and testing.

Is 17BIPHE2, an engineered cathelicidin antimicrobial peptide with low susceptibility to proteases, a better spermicide in cervicovaginal fluid (CVF) than its parental peptides, LL-37 and GF-17?
At the same mass concentration, 17BIPHE2 exhibited the highest spermicidal activity on human sperm resuspended in CVF-containing medium.
LL-37 and its truncated peptide GF-17 exert both spermicidal and microbicidal activities, although they are prone to proteolytic degradation in body fluids.
Spermicidal activities of 17BIPHE2 were evaluated in vitro in mouse and human sperm, both resuspended in medium, and then on human sperm incubated in CVF-containing medium; in the latter condition, the spermicidal activity and peptide stability in CVF of 17BIPHE2 were compared with that of LL-37 and GF-17. The in vivo contraceptive effects of 17BIPHE2 and the reversibility thereof were then assessed in mice. Finally, in vitro microbicidal effects of 17BIPHE2 on Neisseria gonorrhoeae were determined.
Sperm motility and plasma membrane integrity were assessed by videomicroscopy and exclusion of Sytox Green, a membrane-impermeable fluorescent dye, respectively. Successful in vitro fertilization (IVF) was determined by the presence of two pronuclei in oocytes following their coincubation with capacitated untreated or 17BIPHE2-treated sperm. Sperm alone or with 17BIPHE2 were transcervically injected into female mice and successful in vivo fertilization was indicated by the formation of two-cell embryos 42-h postinjection, and by pregnancy through pup delivery 21-25 days afterwards. Peptide intactness was assessed by immunoblotting and HPLC. Reversibility of the contraceptive effects of 17BIPHE2 was evaluated by resumption of pregnancy of the female mice, pretranscervically injected with 17BIPHE2, following natural mating with fertile males. Minimum inhibitory/bactericidal concentrations of 17BIPHE2 on N. gonorrhoeae were obtained through microdilution broth assay.
At the same mass concentration, 17BIPHE2 was a more effective spermicide than LL-37 or GF-17 on human sperm resuspended in CVF-containing medium, with the spermicidal concentration of 32.4 µM. This was mainly due to lower susceptibility of 17BIPHE2 to CVF proteases. Importantly, the reproductive tract of mouse females treated three times with 32.4 µM 17BIPHE2 remained normal and their fecundity resumed after stopping 17BIPHE2 treatment.
For ethical reasons, the inhibitory effects of 17BIPHE2 on fertilization and pregnancy cannot presently be performed in women. Also, while our study has proven the effectiveness of 17BIPHE2 as a spermicide for mouse and human sperm in vitro, dosage formulation (e.g. in hydrogel) of 17BIPHE2 still needs to be developed to allow 17BIPHE2 to remain in the vagina/uterine cavity with controlled release for its spermicidal action.
Since 17BIPHE2 also exerted bactericidal activity against N. gonorrhoeae at its spermicidal concentration, it is a promising candidate to be developed into a vaginal multipurpose prevention technology agent, thus empowering women against unplanned pregnancies and sexually transmitted infections.
This work was supported by the Canadian Institutes of Health Research (PJT 173268 to N.T.). There are no competing interests to declare.
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To summarize the currently available phase I and II clinical trials of the effects of nonoxynol-9 (N-9) on human sperm structure and functions.
A systematic review and meta-analysis aiming to evaluate the spermicidal activity of N-9 on motility, was conducted in PubMed, EMBASE, and Cochrane databases by 10 March 2021. The counted numbers of progressive motile (PR) sperm in cervical mucus and the vanguard sperm penetration distances were analyzed. Other effects on sperm structures and physiological activities were reviewed as well.
In the pooled results, percentages or counted numbers of PR sperm decreased after the treatment of N-9. Vanguard sperm penetration distance was shortened in treated groups. N-9 has been confirmed to damage the structures of sperm, as well as other organelles like acrosome and mitochondria. The physiological activities such as generation of reactive oxygen species, superoxide dismutase activity, acrosin activity, and hemizona binding were all inhibited in the reviewed studies.
N-9 has several impacts on sperm owing to its potency in reducing sperm motility and cervical mucus penetration, as well as other functional competencies.
Nonoxynol-9 (N-9) has been used worldwide as a spermicide to kill sperm for more than 60 years but can cause side effects including vaginal irritation and can increase the rate of contraceptive failure. A detailed analysis of published literature aiming to evaluate the spermicidal activity of N-9 on sperm was carried out. In the pooled results, N-9 reduced the number of active sperm and the distance they traveled. It also caused damage to the structures of sperm and to the way the sperm acted and interacted with the egg. In conclusion, N-9 impacts on sperm in a number of ways that lead to sperm death and dysfunction.

UNASSIGNED: Spermicides have been identified as a potentially attractive alternative to hormonal contraceptives and/or intrauterine devices. Thus, this study aimed evaluating the efficacy and local tolerance of benzalkonium chloride (BKC) and myristalkonium chloride (MKC) contained in Pharmatex® vaginal formulations and compare them with nonoxynol-9 (N-9), the most common active ingredient in topical vaginal contraceptives.
UNASSIGNED: Human normozoospermic samples were assessed for motility, viability, acrosome status and penetration ability after exposure to control, N-9 or different BKC and MKC doses for 0 and 10 minutes. Local tolerance on HeLa cells was evaluated by the Trypan-blue and MTT assays.
UNASSIGNED: Exposure to BKC and MKC reduced acrosome integrity while promoting total immobilisation and complete loss of sperm viability (p < .001, n = 15). Both compounds also compromised sperm penetration ability upon exposure (p < .001, n = 15). N-9 induced the same outcomes (p < .001, n = 15); nevertheless, it was more toxic to HeLa cells than BKC and MKC (p < .05, n = 14).
UNASSIGNED: BKC and MKC present strong in vitro spermicidal activity at lower doses than N-9 and were better tolerated after immediate exposure than N-9. Available Pharmatex® galenic formulations were as effective as products based on N-9.

Our primary objective was to evaluate whether new types of single-size diaphragms or cervical caps differ in prevention of pregnancy compared with older types of diaphragms, and whether different types of gels differ in their ability to prevent pregnancy. A secondary aim was to evaluate method discontinuation and complications.
A comprehensive search was conducted in PubMed, Embase and the Cochrane Library. The certainty of evidence was assessed according to the GRADE system.
Four randomised controlled studies were included in the assessment. When comparing the new and old types of female barrier contraceptives the 6-month pregnancy rate varied between 11%-15% and 8%-12%, respectively. More women reported inability to insert or remove the FemCap device (1.1%) compared with the Ortho All-Flex diaphragm (0%) (p<0.0306). Urinary tract infections were lower when using the single-size Caya, a difference of -6.4% (95% CI -8.9 to -4.09) compared with the Ortho All-Flex diaphragm. The 6-month pregnancy rate for acid-buffering gel and spermicidal nonoxynol-9 gel varied between 10% and 12%. The discontinuation rate was lower in women who used acid-buffering gel compared with nonoxynol-9 gel (risk ratio (RR) 0.77, 95% CI 0.68 to 0.97).
Pregnancy rates were generally high in women using female barrier contraceptives. There was no difference in the efficacy for pregnancy prevention between the new types of diaphragms and cervical caps and the older diaphragms. The new types of diaphragms and cervical caps resulted in fewer urinary tract infections. Acid-buffering gels did not differ from spermicidal nonoxynol-9 gels regarding pregnancies but seemed to be better tolerated.

Inhibition of the sperm transport process in the female reproductive tract could lead to infertility. We previously showed that a pan-serine protease inhibitor, 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), blocked semen liquefaction in vivo and resulted in a drastic decrease in the number of sperm in the oviduct of female mice. In this study, we used a mouse model to test the efficacy of AEBSF as a reversible contraceptive, a sperm motility inhibitor, and a spermicide. Additionally, this study evaluated the toxicity of AEBSF on mouse vaginal tissues in vivo and human endocervical cells in vitro. We found that female mice treated with AEBSF had significantly less pups born per litter as well as fertilization rates in vivo compared to the vehicle control. We then showed that AEBSF reduced sperm motility and fertilization capability in vitro in a dose-dependent manner. Furthermore, AEBSF also exhibited spermicidal effects. Lastly, AEBSF treatment in female mice for 10 min or 3 consecutive days did not alter vaginal cell viability in vivo, similar to that of the vehicle and non-treated controls. However, AEBSF decreased cell viability of human ectocervical (ECT) cell line in vitro, suggesting that cells in the lower reproductive tract in mice and humans responded differently to AEBSF. In summary, our study showed that AEBSF can be used as a prototype compound for the further development of novel non-hormonal contraceptives for women by targeting sperm transport in the female reproductive tract.

dl-Mandelic acid (MA), an alpha-hydroxycarboxylic acid, has been widely used as an intermediate of pharmaceutical and fine chemicals. Here, we evaluated the sperm-immobilizing activity of MA and its safety profiles. Spermatozoon motility was assessed by computer-aided sperm analysis, the integrity of the plasma membrane and. mitochondrial potential was assessed using fluorescein isothiocyanate-pisum sativum agglutinin and JC-1, respectively. The local tolerance of the MA-containing gel formulation was evaluated using a rabbit vaginal irritation test. We found that MA inhibited sperm motility and movement patterns in a concentration-dependent manner. Within 20 s, MA-induced spermatozoa immobilization occurred with a minimum effective concentration and a median effective concentration of 0.86 and 0.54 mg/mL, respectively. Plasma membrane disruptions of MA-treated spermatozoa were relatively mild, but mitochondrial depolarization occurred. Histopathological examination showed that MA exposure did not exert obvious effects on the integrity of spermatozoa membrane structures and only caused slight irritation to the rabbit vaginal epithelium. The vaginal irritation scores of the vehicle control and the nonoxynol -9 gel control groups were 1.38 ± 0.65 and 7.88 ± 1.67, respectively (p < 0.01), whereas those of the MA gel groups at 10, 20, and 40 mg/mL were 1.69 ± 1.04, 2.98 ± 0.77, and 4.35 ± 1.04 with p values of >0.05, >0.05, and <0.05 (vs. vehicle control), respectively, which were within the clinically acceptable range (<8). Therefore, our results confirmed that MA exhibited significant sperm-immobilizing effects and caused mild plasma membrane injury, suggesting that it has potential for development as a future non-surfactant spermicide.

Zinc pyrithione (ZPT), a zinc coordination complex, is used as an antimicrobial agent. This study investigated the molecular mechanisms underlying ZPT-induced spermatozoa immobilization by examining plasma membrane integrity, mitochondrial dysfunction, and the cAMP/PKA signaling pathway response. ZPT inhibited spermatozoa motility and movement patterns in a concentration-dependent manner. The 100% effective concentration (EC100) and median effective concentration (EC50) at which ZPT-induced spermatozoa immobilization at 20 s were 40 μmol/L and 16.19 μmol/L, respectively. ZPT did not significantly disrupt spermatozoa plasma membranes, but it exerted a strong and significant effect on the depolarization of mitochondria. In addition, ZPT exposure induced intracellular H+ accumulation and Ca2+ dissipation in spermatozoa, accompanied by suppression of the cAMP/PKA signaling pathway. Thus, ZPT induces spermatozoa immobilization without significant plasma membrane injury and so could be a candidate microbicidal spermicide.

Today, rape and sexual assault cases are mainly solved using evidence such as medical evidence or DNA analysis. Condom traces have been found to be present in 10% of assaulted women, when no DNA is found [1]. Numerous studies have emphasized the interest of analysing the composition of male condoms and their traces, and developing specific methods for the analysis of this type of evidence. However, transfer and persistence of condom traces in a specific matrix are rarely referenced. Therefore, forensic scientists have no complete knowledge of the trace and what could be expected in a real case. The purpose of this article is to review the literature addressing the composition of condoms and their traces as well as its influence on the transfer and persistence from a forensic point of view. Peer-reviewed literature, patents, professional literature, data from international administrations and international organisations\' reports have been used to track the composition and the problematics of transfer and persistence of condom traces. The results of this review show that the composition of male condoms and their traces are complex systems, with numerous compounds originating from the condom at the moment of the transfer and evolving over time according to specific persistence patterns. Although numerous types of analyses have already been proposed and tested for condom traces, forensic evidence considerations have not been fully studied yet. Considering the fact that sexual assaults without the detection of DNA are increasingly frequent, there is a definite medical and forensic need to improve our knowledge of the processes involved in the development of condom traces in order to better understand analytical results.