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 CO 2 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.

Fig. 1 Sperm concentration (x 106) following reversal for the seven rabbits showing the baseline average per group and trends for the two different devices. No significant difference between the devices was found Full size image

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)).

Fig. 2 Sperm concentration (x 106) during baseline and following reversal procedures. Sperm concentration returned to baseline levels following reversal. No significant difference between baseline and post-reversal Full size image

Fig. 3 Sperm motility and forward progression percentage at baseline and following reversal procedures. Average motility returned to baseline levels following device reversal. Forward progression was significantly lower following device reversal (p < 0.02) Full size image

Fig. 4 The percent of sperm showing forward progression after the device reversal for each rabbit. A linear trend line (y = 0.089x − 0.798, R 2 = 0.90) shows that the forward progression measures are increasing over time Full size image

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].

Fig. 5 Rabbit vas deferens examined after device reversal procedure. a Longitudinal section (100X magnification) of vas deferens, containing residual material appearing as homogenous luminal substance. The ★ depicts a layer of granulomatous inflammation which replaces the mucosal epithelial cells. Muscularis (M), vas deferens lumen (L). Bar = 100 μm. b Cross section (100X magnification) of vas deferens containing fragmented residual material. Muscularis (M), vas deferens lumen (L). Bar = 100 μm. c Longitudinal section (200X magnification) of vas deferens. Fragmented material is present in the lumen. The mucosal epithelium is attenuated in this photomicrograph (arrow). Muscularis (M), vas deferens lumen (L). Bar = 50 μm. d Longitudinal section (100X magnification) of vas deferens. The lumen is empty and tall columnar epithelium is present. Muscularis (M), vas deferens lumen (L). Bar = 100 μm. e Additional longitudinal section (200X magnification) of vas deferens. Clumps of spermatozoa are present in this image (arrows). Muscularis (M), vas deferens lumen (L). Bar = 50 μm. f Extraluminal adventital material with associated granulomatous inflammation (200X magnification). Arrows depict a multinucleated giant cells adjacent eosinophilic to amphophilic material in the interstitium. Bar = 50 μm Full size image

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).