Reversibility of Vasalgel™ male contraceptive in a rabbit model

Long-acting reversible contraception (LARC) methods are recommended for pregnancy prevention because of their potential for high effectiveness and low burden to users. LARC options available to women (IUDs and implants) have been associated with high levels of continuation [1]. While LARCs often cost more initially, they are among the most cost effective birth control methods given their high efficacy rate and duration of use, especially when considering the costs of unintended pregnancy [2, 3].

Having a variety of new fertility control options for men and women would accommodate people of different ages and life stages [4]. The availability, variety and effectiveness of LARCs for women have had a significant impact on reducing the unintended pregnancy rate [5]. It is likely that similar options for men would also have positive impacts on reproductive health. However, currently there are no LARCs for men. Vasectomy, while long-acting, is generally considered permanent due to difficulties of reversal, the expense, and lower success rate in restoring fertility [6]. Other options (condoms, withdrawal) are short-term solutions with high pregnancy rates for typical use [7].

Research on male contraception has revolved around both hormonal and nonhormonal methods that suppress sperm production, disrupt sperm maturation or function, or alter sperm transport or motility [8]. A promising advancement is Vasalgel, a high molecular weight polymer of styrene-alt-maleic acid (SMA acid) being developed as a LARC for men. The device is injected into the vas deferens to block sperm from transiting through the vas deferens. The implant remains in a soft gel-like state, with the ability to adapt to the interior lumen of the vas deferens and minimize any accommodation of the vas to the presence of the material. It forms a hydrogel which allows transit of water soluble molecules but not larger structures such as spermatozoa.

Initial research on rabbits has confirmed that Vasalgel provides a rapid onset of azoospermia with durability over a 12 month test period [9]. The study also confirmed the efficacy of the SMA acid in Vasalgel as compared to styrene-maleic anhydride (SMA anhydride), which is the basis of a similar product called RISUG that has been studied in India for over three decades [10]. Vasalgel’s composition gives it important advantages for large-scale manufacturing and product stability which should facilitate regulatory approval.

The purpose of the present study was to evaluate the efficacy of a sodium bicarbonate injection into the vasa deferentia to reverse Vasalgel-induced azoospermia in rabbits.

A total of twelve vasa deferentia were successfully flushed with 2–10 ml of sodium bicarbonate solution (average 6.8 ± 4.70 ml). One vas in each of two rabbits in the Vasalgel 80 group, was not readily accessible or identifiable and could not be flushed. The flushing medium resulted in off-gassing of CO₂ and the observation of some foam as it exited at the insertion site, flowing around the outside of the catheter. Minor distention of the vas deferens was observed during flushing in two subjects.

The average number of baseline semen samples collected prior to implantation was 2.4 ± 0.5. Baseline sperm concentration average prior to the administration of the test devices was 221 ± 46 × 10⁶ sperm/ml with 69.4 ± 10.2% motility and 40.9 ± 6.0% forward progression. Animals were azoospermic prior to the reversal procedure. Semen collection commenced 14 days after the reversal procedure and first semen samples obtained on average at 18.9 ± 5.5 days post-reversal and continued for an average of 22.6 ± 3.3 weeks. Eighty percent of attempted semen collections were successful and the number of analyzable collections was 15.9 ± 6.3 per subject during the post-reversal period.

Spermatozoa were observed in the ejaculates of all subjects at an average of 23.7 ± 9.3 days after the reversal procedure. Measurable sperm concentration was evident at the first sperm collection in four subjects (two Vasalgel 100 and two Vasalgel 80), with one as early as 12 days post-reversal. Sperm concentration was measurable in the other three rabbits after several additional collections. Sperm concentrations were initially higher in the first collections after the reversal procedure in several animals and also higher compared to the baseline (average 301.9 ± 57.2 × 10⁶ sperm/ml). Motility averaged 62.3 ± 9.6% after reversal. Forward progression averaged 5.3 ± 0.9% after reversal, increasing from less than 3% for all subjects immediately after reversal to more than 11% for all subjects at the last sample.

As illustrated in Fig. 1, some of the subjects in the Vasalgel 80 group had low initial sperm concentrations, but this observation is difficult to interpret due to high variation across individuals and over time as well as the small number of subjects. Two of the Vasalgel 80 subjects only had one vas deferens flushed. Of these, one had sperm present in the first semen sample and the other had sperm by the fifth sample. Statistically, the three sperm parameters (concentration, motility and forward progression) did not differ for the Vasalgel 100 vs. Vasalgel 80 (all p > 0.05). Therefore, all groups were combined for further statistical tests.

Statistical comparisons between the baseline sperm measurements and those obtained after the reversal procedure were conducted to determine whether the values had returned to normal. Sperm concentration was not significantly different between baseline and post-reversal (Wilcoxon Z = 1.01, p < 0.31) (see Fig. 2). Sperm motility was also not significantly different when comparing baseline to post-reversal (Wilcoxon Z = 1.25, p < 0.17) (Fig. 3). Forward progression was significantly lower after the reversal procedure (Wilcoxon Z = 2.37, p < 0.018) (Fig. 3) with a positive trendline (R ² = 0.90 (see Fig. 4)).

The normal large rabbit acrosome was not observed on spermatozoa during post reversal semen analysis. This condition was observed throughout the period of time in which sperm were obtained and analyzed post reversal.

Gross observations of the reproductive tract during necropsy indicated that the testes were normal in size and color. The prostate and epididymides appeared normal except one subject had a darkened epididymis on the right side and two subjects had a smaller than normal epididymis. Other gross observations of the vasa deferentia included adhesions, swelling or distention at the end near the epididymis (5/12) and thinning at the prostate end (8/12).

Twenty segments of vas deferens from five of the rabbits were examined (two segments from the treated left and right vas deferens from each animal). The vasa deferentia in the animals exhibited a variety of intraluminal changes (See Fig. 5a–f). Hydrogel residue was observed in 4 of 20 segments (See Fig. 5a, b and c). Two of these segments were from one animal with residue observed in segments from both vasa deferentia. The material appeared as a non-fibrillar homogenous amphophilic material. Frequently, clear fracturing was observed in this material and was interpreted as a processing artifact. In these animals, the mucosal epithelium was variably replaced by round to polygonal cells with abundant eosinophilic cytoplasm and round to oval nuclei. These cells often exhibited close association to each other and were interpreted as epithelioid macrophages. Similar material present in the lumen of the vas deferens of these animals was also observed in the surrounding interstitial connective tissue and interpreted as extraluminal hydrogel. The accumulation of this material was often arranged in a multifocal to coalescing nodular pattern and was associated with a surrounding rim of epithelioid macrophages and multinucleated giant cells (See Fig. 5f). This was a similar change to that seen in the non-reversed animals in the previous study [9].

In seven of the 20 segments, intraluminal fragmented material was observed with similar staining properties as the material seen in the intact device plugs. Some of these fragmented hydrogel regions contained scattered spermatozoa (See Fig. 5e). Nine of 20 segments exhibited moderate epithelial attenuation and flattening characterized by a cuboidal to squamous appearance. Segmental mucosal epithelial loss without inflammation was observed in some regions in this group. This epithelial change was not always associated with the presence of an intact plug or fragments of intraluminal device (i.e. was seen in segments of clear vas deferens). The remaining segments (7 of 20) exhibited intact epithelium that appeared columnar and frequently had a ciliated apical surface (See Fig. 5d).

The historical lack of focus on male contraceptive development entails cultural, financial and technical aspects [12]. Contraceptives have been aimed at women, who bear the responsibility for child-bearing and also traditionally for child-rearing. With societal shifts in gender roles and with modern paternity testing, men have taken on more parenting responsibility. Questions of trust in male partners to take birth control also continue to play into attitudes about the importance of developing male contraceptives, yet many commenters are not aware that as much as a quarter of the world’s population already relies on modern or traditional male methods. These changes are reflected in recent studies and surveys indicating that men are not only willing to share responsibility for contraception but demand greater reproductive control [8, 12]. Even with increased demand, male contraceptive development has still been challenged by liability issues, politics, religion and lack of public/private investment [4, 13]. While research has explored a number of different male contraceptive options, no commercially available LARCs for men are on the market.

A male LARC has the potential to decrease the rate of unintended pregnancies, provide men with additional options to control their reproduction, and offer couples an alternative when female contraceptive methods are problematic. Vasalgel is a promising male LARC, and studies in rabbits prove that Vasalgel not only provides rapid and durable contraception [9], but also a return of spermatozoa in the ejaculate shortly after undergoing a reversal procedure with a sodium bicarbonate solution.

In the current study the vasa deferentia of Vasalgel-induced azoospermic rabbits were flushed with a sodium bicarbonate solution to reestablish patency for the transport of spermatozoa. Bicarbonate solutions have been used in previous studies to reverse a similar hydrogel in the rat model [14, 15]. DMSO was not considered for dissolving the hydrogel plug since it could cause increased inflammation during prolonged contact with the vas deferens [16].

Spermatozoa concentrations returned to normal after the reversal procedure. The initially higher sperm concentrations were probably due to increased epididymal sperm reserves resulting from long term blockage of sperm transit [17, 18]. Motility also returned to baseline levels following reversal, but forward progression was significantly lower in the post-reversal period. A trend of increasing forward progression values over time indicated that a slow recovery process was occurring.

The normal time for spermatogenesis and transit to epididymal reserves is about 40 days [19]. Transit time from the testes through the epididymis is about 10 days [19, 20]. Thus, the post reversal semen collections were performed after a minimum of 2.5 cycles of sperm production to ensure newly produced spermatozoa were being ejaculated.

A lack of normal acrosomes on the spermatozoa was observed throughout the post reversal semen evaluations. Previous studies of other hydrogels indicate residual material in the vas lumen may also affect the spermatozoa during transit down the vas [21–23]. The decrease in normal forward progression and lack of normal acrosomes may indicate compromised epididymal and/or vasal support of sperm maturation and transport which can lead to reduced or complete infertility. Although the reversal process successfully re-established the transport of spermatozoa into the ejaculate, the ejaculated spermatozoa have not returned to normal.

The impact of the post-reversal sperm characteristics on pregnancy rates is unknown in this study since mating with females was not included in the study protocol. Previous work in artificial insemination in rabbits indicated that about 10 million motile sperm per sample were required for pregnancy [24]. In our study, rabbits averaged 187 million motile sperm with 16.0 million sperm showing forward progression. We would expect mating success based solely on sperm concentration and motility. However, the reduced forward progression and the lack of a normal acrosome would negatively impact fertility. Normal forward progression and acrosomes are important predictors of fertility. The forward progression recovery trend provides evidence that the return to completely normal spermatozoa may occur over a longer period of time and restore fertility.

The contact of spermatozoa with residual material during transit through the vas deferens may have negatively impacted morphology and functionality of the spermatozoa as observed in previous studies with a similar material [21–23]. Fertility in man would most likely require a return of all semen parameters to within normal ranges.

The vasal epithelium appeared to be returning to normal following the reversal procedure. Although some areas were observed with epithelial attenuation and flattening, most of the vasal epithelium appeared to have an intact structure with normal columnar appearance and a ciliated apical surface.

Nonetheless, all animals had spermatozoa present in the semen following the reversal procedure to confirm the reestablishment of patency in the vasa deferentia.

The blockage of sperm transit for 14 months following Vasalgel implantation was rapidly reversed through an intravasal injection of sodium bicarbonate. Sperm counts returned to normal in all of the subjects over the period of observation. Post-reversal concentration of sperm and sperm motility percent returned to baseline levels during the six-month follow up, with the reduced sperm forward progression exhibiting a recovering trend. Normal acrosomes were not observed over the post reversal evaluation period. Further investigations are needed to determine if a longer period of time would allow complete recovery of the vasal structures or if improved flushing procedures are needed to completely remove the Vasalgel leading to the return of normal sperm morphology and forward progression.

Study results support continued development of Vasalgel as a non-hormonal LARC for men.