Injection of the test material was successfully accomplished in all male subjects. The time to complete the implantation surgery decreased 33 % from the first five to the last five procedures (54.0 ± 21.6 vs. 36.0 ± 10.8 min ) as the surgeon gained experience identifying and injecting the small and fragile rabbit vasa deferentia. Extravasation of a small amount of test material occurred in four vasa deferentia and one became distended when the material was injected.

Twelve animals (n = 6 in each of the 100 % acid and 80:20 mix groups) from the original 15 were retained in the study. Three rabbits died postoperatively without precipitating illness, apparently from surgical trauma as the operative technique was being refined. In two of the subjects (one 100 % acid, one 80:20 mix), the reproductive tract became engorged with blood caudal to the injection site, involving a single vas deferens in one male and both testes, vasa deferentia and epididymides in the other male. There were no direct observations of inflammation due to the presence of Vasalgel within the vasa. All other organs appeared normal. The third male (100 % acid) was found dead three days after surgery with no obvious cause. All organs, including the reproductive tract, appeared normal and the incision site was healing normally. There were no direct observations of any inflammation or tissue damage due to the presence of Vasalgel. One of the twelve study subjects died six months following the implant surgery due to neck trauma unrelated to surgery or the presence of Vasalgel. The reproductive tract appeared normal and his data were included in analyses. See Table 1 for information on each subject.

Table 1 Number of semen samples and sperm concentration during baseline and after implantation of the test articles for each subject Full size table

A total of 264 semen samples were successfully collected (of 459 attempts) from the 12 subjects over the course of the study (Table 1). Most unsuccessful attempts were due to ejaculation outside the artificial vagina. An average of 2.6 ± 0.5 samples were collected per subject in the baseline condition and 19.4 ± 7.6 were collected per subject over the 12 months following implantation. Semen volume was 0.30 ± 0.07 ml during baseline and 0.76 ± 0.30 ml after implantation of the test article.

Significant decreases in sperm measurements occurred in all 12 animals following implant of the test article (Table 1). Sperm concentration during baseline was 233.1 ± 81.2 × 106 sperm/ml (range of individual averages 130.7 – 324.0 × 106 sperm/ml). Semen samples following implantation contained no measurable sperm using standard methods during the 12 month follow-up period, except for one rabbit with very low sperm concentrations measured in five of the first 13 semen samples but eventually provided semen samples with no spermatozoa present in the ejaculate. The average post-implantation sperm concentration from this animal was 0.24 × 106 sperm/ml.

At baseline, the mean percentage of motile sperm was 78.2 ± 6.8 and the mean percentage showing forward progression was 42.5 ± 7.2. Following implant, these parameters were zero in all samples.

Semen analysis also included qualitative observations of small numbers of sperm or fragments in the samples, which were too few to reliably count using standard methods. Of 233 samples collected after implantation with the contraceptive, three rabbits in the 80:20 mix group had very few sperm in their first semen sample (occurring at day 29, 35 and 64 post-implant) indicating that virtually all sperm were blocked.

There was no significant difference in the three baseline semen parameters for the two test articles (all p > 0.2). Statistical analysis of the impact of condition (baseline vs. implant) by test article (100 % vs 80:20 mix) indicated a significant condition effect with sperm concentration being significantly lower during the implant condition (F = 90.0, p < 0.001) (Fig. 1). The test article was not influential in this relationship since the test article and condition x test article interactions were not significant (p > 0.05). It was not possible to conduct a similar statistical test on the motility and forward progression because there was no variability in the implant condition (due to zero motility and progression values).

Fig. 1 Sperm concentration during baseline and implant conditions for the two test articles. Difference between conditions was significant (p < 0.001) Full size image

Histological evaluation of the vasa deferentia of six subjects was completed. The vas deferens in the one animal after three days of implant exposure exhibited an intraluminal accumulation of non-fibrillar eosinophilic to amphophilic homogenous material (Fig. 2a). The material accumulated in the lumen was devoid of inflammatory cells and epithelial cells. One animal exhibited a moderate accumulation of spermatozoa embedded in the material and the remaining animals evaluated exhibited scattered spermatozoa or no spermatozoa embedded in the intraluminal material. The mucosal epithelium in all animals demonstrated some degree of attenuation, taking on a cuboidal to flattened appearance in contrast to the ciliated columnar epithelium observed in regions devoid of intraluminal material. Segmental mucosal epithelial loss without inflammation was observed. In some treated animals, the mucosal epithelium was replaced by round to polygonal cells with abundant eosinophilic cytoplasm and round to oval nuclei (Fig. 2b,c). These cells often exhibited close association to each other and were interpreted as epithelioid macrophages. Larger cells were also present with similar cytoplasm but contained multiple round to oval nuclei (ranging from 2–10 nuclei within a cell), interpreted as multinucleated giant cells. This replacement of mucosal epithelium was observed in both a segmental and a circumferential manner in the treated animals.

Fig. 2 Rabbit ductus deferens containing SMA hydrogel. a Longitudinal section (100X magnification) of vas deferens containing hydrogel appearing as eosinophilic homogenous luminal material. Arrowheads depict the mucosal epithelium. Muscularis (M), vas deferens lumen (L). b Longitudinal section (400X magnification) of vas deferens containing hydrogel. Arrowheads depict the attenuated mucosal epithelium in this section. Scattered spermatozoa are present (arrows). Muscularis (M), vas deferens lumen (L). c Cross section (200X magnification) of vas deferens. A layer of epithelioid macrophages replaces the luminal epithelium in this section (★). Muscularis (M), vas deferens lumen (L). d Interstitial hydrogel with associated granulomatous inflammation (400X magnification). Arrows depict epithelioid macrophages and multinucleated giant cells surrounding eosinophilic to amphophilic material in the interstitium Full size image

Similar material present in the lumen of the vas deferens was also observed in the some surrounding interstitial connective tissue interpreted as extraluminal hydrogel (Fig. 2d). 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. The multinucleated giant cells observed in the interstitial space frequently exhibited higher nuclei numbers than those seen at the mucosal surface of the vas deferens. Both test articles (100 % SMA and 80:20 mix) were similar in the degree of epithelial attenuation present as well as the inflammation present at the mucosa and in the interstitium.

The histological observations did not appear to be dependent upon the duration of Vasalgel exposure.