Findings and interpretation

Using a very robust sample size not often observed in this type of in vitro studies, we herein report for the first time that N-9, BKC and MKC at the concentrations used herein display spermicidal activity by interfering with human sperm motility, viability and acrosome status, besides strongly affecting sperm penetration ability in a matrix mimicking midcycle female cervical mucus. Indeed, it has long been reported that N-9, the most often used spermicide in vaginal contraceptives [5–7], is able to disrupt sperm plasma membrane and induce loss of acrosome integrity, completely immobilising spermatozoa and promoting viability loss upon immediate/short contact [31]. This non-ionic surfactant and the cationic surfactants BKC and MKC were found to have spermicidal activities that are not statistically different from each other, possibly acting above their critical micellar concentration (concentration above which surfactant molecules associate with phospholipids, creating ‘‘holes’’ and disrupting the cell membrane), thus producing detergent-unspecific cell damage. BKC and MKC were indeed able to produce a complete loss of motility and viability. Importantly, both BKC and MKC induced these effects at lower concentrations than N-9, which was also observed with all Pharmatex® galenic preparations tested (pessary, mini-pessary, capsule, cream and tablet).

Sperm acrosome integrity was also sharply affected by BKC and MKC, suggesting that even if no effects in sperm motility and viability were found, premature acrosome reaction would certainly prevent pregnancy. Indeed, no sperm cells possess fertilising ability in vivo if their acrosome reacts before being in the vicinity of the oocyte. Although it seems that N-9 may induce more acrosome reaction when compared with BKC and MKC at specific time points, this was not the case when galenic formulations were compared. Indeed, Pharmatex® and AMCAPHARM’s galenic preparations were found similar and a previous study performed by our group has inclusively showed that when compared with a formulation containing a lower quantity of N-9 (75 mg), Pharmatex® mini-pessary at this dose more severely affected acrosome integrity, inducing premature acrosome reaction [32], suggesting that this difference among N-9 and both BKC and MKC may not be so significant. Furthermore, attesting their great spermicidal activity in vitro, the Sander-Cramer test also revealed a complete immobilisation of spermatozoa upon exposure in all sperm samples used.

Moreover, if sperm cells enter the cervical mucus immediately upon ejaculation, instead of moving in the surrounding vaginal and cervical secretions, the vaginal contraceptive agent may not contact with spermatozoa, which may lead to a potential failure of the contraceptive method. We found that both sperm migration distance and sperm penetration ability were severely affected upon exposure. Importantly, while after exposure to the untreated control sperm migrated as expected, in both BKC and MKC groups sperm migration did not reach 0.5 cm in any timepoint addressed, and were not expected to migrate more after 10 minutes as they presented no motility at this point (data not shown). Indeed, this same pattern was obtained with N-9, showing the efficacy of these compounds as contraceptive agents even when dissolved in a viscous cervical mucus-mimicking matrix, which is not surprising since BKC has been reported to penetrate and thicken cervical mucus [12,33], explaining the short sperm migration distance and poor penetration ability. The effects of MKC on in vitro sperm penetration ability, which were not known up to now, seem to be similar to those observed for BKC.

The tolerance of any potential vaginal contraceptive to human cervicovaginal epithelium is another aspect that should be taken into consideration in this type of studies. While being tremendously effective in compromising human sperm function, N-9, BKC and MKC were found to have an impact on HeLa cells, as were Pharmatex® galenic preparations, an outcome attributed to the detergent nature of the compounds. As in sperm cells, the experiments performed with this suitable human adherent epithelial cell line that mimicks the vaginal/cervical epithelium [9,25] were performed in a great sample size, thus allowing no margin for data misinterpretations.

Indeed, many studies have reported that BKC can affect HeLa or other cells in vitro [17,34,35] as well as promoting vaginal damage/disruption in vivo. Using the MTT assay, which is a finer assay than the Trypan blue test and does not solely rely in the human eye, we were able to determine subtle but significant differences between active ingredients, with N-9 being more harmful to HeLa cells upon immediate contact. With regards to N-9, its toxicity is well described and allows no margin for errors, apparently being far worst than BKC and MKC. Despite the fact that AMCAPHARM’s nonoxinol 120 mg vaginal suppository has never been studied before, the high quantity of N-9 used in its formulation anticipated the findings obtained herein. Indeed, lower N-9 concentrations have shown to promote severe consequences for woman general and reproductive health [7], therefore explaining its use as a positive control in the present study.

Differences and similarities in relation to other studies

Both BKC and MKC were reported to have antimicrobial and antiviral properties [11–13], with spermicidal activity being also demonstrated by the former [11,12]. However, despite being used in vaginal formulations, no information on the toxicity of MKC was available and consistent in vitro studies addressing the efficacy and safety of a single exposure to both compounds were still lacking.

Although lacking studies on MKC, many have reported an adverse effect of BKC on HeLa or other cells in vitro [17,34,35], as well as promoting vaginal damage/disruption. Nevertheless, although in some cases some degree of vaginal/cervical irritation and de-epithelialization has occurred in vivo [12], associated or not with inflammation and some kind of discomfort, many women did not find these effects crucial to discontinue BKC treatment [18]. Furthermore, the acceptability of vaginal contraceptives containing BKC was shown to be better than that of N-9 [19,20]. Pregnancy rates were also considered moderate in contraceptives containing BKC [18], but mainly because of the lack of information and/or mistakes in the application of the spermicide rather than due to their poor contraceptive ability. Indeed, the success of local contraceptives not only depends on their complete/incomplete vaginal coverage but also on the information of users, their compliance with the usage rules and their personal motivation [3,14,15].

As observed in our study, N-9 was also found by others to induce cellular toxicity in vitro [36]. Along with this, genital irritation and epithelial disruption that can increase the risk of transmission of STIs has been reported. One such example was the study regarding the frequent use of N-9 by female commercial sex workers, in which a consistent association between genital lesions and HIV transmission was determined [37]. Studies in women using N-9 has often indicated genital irritation detected by colposcopy unrelated to symptoms [38]. In light of this information, and given that many studies also failed to demonstrate any benefit [39], clinical research into N-9 was suspended, leading to a recommendation by the WHO against its use [40].

Strenghts and weaknesses

Local vaginal contraception is perhaps the oldest method of fertility control. With minimal systemic involvement, it was also considered one of the safest methods of contraception. Indeed, the few papers available describing potential BKC systemic effects have shown no/negligible adverse reactions [15,41], and even its inadvertent use at the start of a pregnancy does not induce foetal malformation in humans nor passes to breast milk according to Liebert [15].

However, the lack of interest and innovation in this field coupled with the introduction of oral contraceptive pills and intrauterine devices in the 1960s made the available vaginal contraceptives somewhat outdated, resulting in poor efficacy and acceptability [1]. Nevertheless, local contraceptives can be preferred over other contraceptive methods by women after medical counselling [2,3,6]. Nowadays, FSRH supports the use of hormonal contraception and intrauterine devices to a large extent, but women may opt for vaginal contraceptives inclusively when rarer sexual intercourse occurs and/or when a decline in fertility has been associated. Furthermore, vaginal contraceptives are easy to use, available over the counter and, contrary to the male condom, woman-controlled. To this extent, Pharmatex® products may represent a more suitable alternative than N-9.

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