Effect of experimentally induced Escherichia coli epididymo-orchitis and ciprofloxacin treatment on rat spermatogenesis

Authors


  • Authors' contribution: AD (20%); PT (20%); FFÖ (20%); SS (10%); AF (10%); TT (20%).

Tufan Tarcan md, phd, Marmara University Hospital, Department of Urology, Tophanelioglu cd. 13-15, Altunizade (34662), Istanbul, Turkey. Email: tufan@marmara.edu.tr

Abstract

Abstract:  We investigated the effects of epididymo-orchitis and ciprofloxacin on rat testicular histology and spermatogenesis. The control group underwent left orchiectomy. The second group received oral ciprofloxacin (150 mg/kg/day) for 10 days. Escherichia coli (106 cfu/mL, 0.1 mL) was injected into the proximal right ductus deferens in the third group. The fourth group received ciprofloxacin treatment 48 h after E. coli inoculation. In groups 3 and 4, bilateral orchiectomy was performed 14 days after the challenge. In healthy rats, ciprofloxacin caused recognizable histological damage associated with a mild decrease in testicular volume and sperm concentration. Infected testicles in groups 3 and 4 revealed severe histological damage associated with severe testicular atrophy and impaired spermatogenesis that were more significant in infected rats which received ciprofloxacin treatment. Contralateral testicles in these animals showed similar histopathological changes to a lesser extent. The results of our study suggest a gonadotoxic potential for ciprofloxacin and this potential in humans should be addressed with further studies.

Introduction

Orchitis is an inflammatory lesion of the testicle that generally occurs after retrograde inoculation of common urinary pathogens, which access the epididymis by retrograde propagation through the vas deferens, and is commonly referred as epididymo-orchitis (EO). Chronic inflammatory changes due to orchitis alter both sperm number and quality; therefore, EO is considered as an important cause of spermatogenetic arrest and testicular atrophy.1

Fluoroquinolones are the first-choice antimicrobial drugs for the treatment of EO and ciprofloxacin is one of the most widely used fluoroquinolones.2In vivo genotoxicity studies suggest ciprofloxacin as safe for therapeutic use.2 However, other studies have demonstrated ciprofloxacin to significantly impair both testicular function and structure.3 In this study, we investigated the effects of ciprofloxacin treatment on testicular morphology and spermatogenesis in a rat EO model.

Methods

Animal model

All protocols were approved by our Institutional Ethics Committee for the Care and Use of Experimental Animals. Twenty-four male Sprague–Dawley rats (6 months old, 300–350 g) were divided into four groups: Control group (group 1) did not undergo any intervention. Group 2 received oral ciprofloxacin (obtained in powder form from Dr Reddy's Laboratory, Pradsh, India) via a stomach tube in once daily doses of 150 mg/kg bodyweight for 10 days, treatment dose prepared to imitate the human therapeutic dose.3 EO was induced in group 3 by injecting 0.1 mL Escherichia coli suspension (106 cfu/mL, ATCC 25922, Oxoid, Hampshire, UK) into the epididymal direction of the proximal right vas deferens as described previously.4 In the EO + ciprofloxacin group (group 4), ciprofloxacin treatment (same as group 2) was started 48 h after E. coli inoculation.

Testicular size and tissue sampling

Under aseptic conditions, all rats were anesthetized and bilateral testes were exposed via a midline vertical incision of the scrotum. Only left orchiectomy was performed in the control and antibiotic groups one sperm cycle (14 days) following the last ciprofloxacin dose. Bilateral orchiectomy was performed in the EO and EO + ciprofloxacin groups one sperm cycle after the E. coli inoculation and last antibiotic dose, respectively. Thus, a total of six specimen groups were formed from four rat groups. Dimensions of each testis were measured and removed testicles were transected into two halves. Testicular volumes were calculated by using the empiric formula of Lambert (length × width × depth × 0.71)5 with the aid of an orchidometer. Values for each group were given as means ± SE. For each testis, one half was placed in 10% formaldehyde solution while the remaining half was placed in a tissue culture tube containing modified human tubule fluid (modified HTF, Irvine Scientific, Santa Ana, CA, USA) that was kept at 37°C. Spermatogenesis was assessed within 1 h of tissue sampling.

Histopathology

Tissue specimens were fixed with 10% formaldehyde and processed routinely for embedding in paraffin. Sections of 5 µm thick were stained with hematoxylin–eosin (HE) and with Masson's trichrome. Stained sections were then examined under an Olympus BX51 photomicroscope (Tokyo, Japan). Histopathological assessment of the tissues was based on a slight modification of parameters described by Ludwig et al.4 Microscopic scoring was done by experienced histologists who were unaware of the study group examined. Scores were given as 0, none; 1, mild; 2, moderate; and 3, severe for each criterion, using the semiquantitative scale as follows: (i) degeneration of epithelium; (ii) inflammatory cell infiltration; (iii) tubular atrophy; (iv) vasocongestion; and (v) interstitial fibrosis. The microscopic score of each tissue was calculated as the sum of the scores given to each criterion and at least five microscopic areas were examined to score each specimen.

Calculation of sperm concentration

Specimens in modified HTF were dispersed in a Petri-box with the tip of a needle subsequently vortexed for 30 s, and were centrifuged at 540 g for 20 min. A drop of this homogenous mixture was placed in a Makler chamber. Calculation of sperm concentration was determined by counting the numbers of spermatozoa in 10 boxes which revealed the concentration in millions/mL, and expressed as means ± SE for each group.

Analysis of data

Testicular volumes, histological variables and sperm concentrations were analyzed by a one-way anova test with the aid of a computerized software. For multiple comparisons, a Tukey post-hoc test was used. A P-value of ≤ 0.05 was accepted as statistically significant.

Results

Forty-eight hours after inoculation, all infected animals developed clinical symptoms of acute EO in the right scrotum. Local clinical findings ranged from a mild to moderate edema of the scrotum to severe enlargement and erythema. The left scrotum usually had no clinical signs of inflammation. These signs were vanished in all animals 10 days after bacterial inoculation.

Histopathological findings

Histopathological findings in the examined groups are summarized in Table 1. The control group revealed a regular seminiferous tubule and germinal epithelial cell morphology (Fig. 1a). The ciprofloxacin group showed severe degeneration of germinal epithelium, moderate tubular atrophy and vasocongestion (Fig. 1b). In the EO group, severe degeneration of germinal epithelial cells, tubular atrophy and vasocongestion, moderate inflammation and mild interstitial fibrosis were observed in the infected right testis (Fig. 1c). Interstitial fibrosis was seen to progress and the other findings did not show regression in the infected testicles of animals with EO which received ciprofloxacin (Fig. 1d). In the contralateral testicles of the EO group, moderate tubular atrophy and epithelial degeneration, mild inflammation and interstitial fibrosis, and severe vasocongestion were observed (Fig. 1e). Germinal epithelium was severely degenerated in the contralateral testicles of the EO + ciprofloxacin group (Fig. 1f ). Interstitial fibrosis was not observed and inflammation showed regression, although tubular atrophy and vasocongestion were still present in these specimens.

Table 1.  Histological criteria of testicular injury with respect to specimens of study groups have been demonstrated
SpecimensEpithelial degenerationInflammatory cell infiltrationTubular atrophyVasocongestionInterstitial fibrosisTotal score
  • Significant difference from specimen 1 (< 0.05).

  • Significant difference from specimen 2 (< 0.05).

  • §

    Significant difference from specimen 3 (< 0.05).

  • Significant difference from specimen 4 (< 0.05).

  • ††

    Significant difference from specimen 5 (< 0.05).

  • ‡‡

    Significant difference from specimen 6 (< 0.05).

  • §§

    Significant difference from all specimens (< 0.05). It is important to note that EO and EO + ciprofloxacin induced a similar degree of testicular pathology in the site of bacterial inoculation. Ciprofloxacin treatment alone has also been shown to cause a substantial testicular injury.

1: Group 1, left testis0§§0§,,††,‡‡0§§0§§0§,††0§§
2: Group 2, left testis3 ± 0,0§,,††,‡‡2.1 ± 0.22,§2.7 ± 0.160§,††7.8 ± 0.38,§,††
3: Group 3, right testis2.9 ± 0.12,2.4 ± 0.26,,,‡‡2.9 ± 0.12,,,‡‡2.9 ± 0.121.3 ± 0.16§§12.4 ± 0.78,,,‡‡
4: Group 3, left testis2.3 ± 0.16§§1.1 ± 0.12§§2 ± 0,§2.5 ± 1.880.3 ± 0.16§,††8.2 ± 2.32,§,††
5: Group 4, right testis3 ± 0,2.3 ± 0.25,,,‡‡2.4 ± 0.182.5 ± 0.182.3 ± 0.16§§12.5 ± 0.77,,,‡‡
6: Group 4, left testis3 ± 0,1.3 ± 0.12§§2.1 ± 0.29,§2.6 ± 0.180§,††9 ± 0.59,§,††
Figure 1.

Control group: normal histological appearance of the seminiferous tubules and germinal epithelial cells (a). Ciprofloxacin group: severe degeneration of germinal epithelial cells (arrow, b). Epididymo-orchitis (EO) group, infected testis: severely degenerated germinal epithelial cells (arrow, c), inflammatory cell (neutrophil) infiltration (arrowhead, c), sloughing of the epithelial cells into the lumen (asterisk, c), and mild interstitial fibrosis (asterisk, inset, c). EO + ciprofloxacin group, infected testis: severely degenerated germinal epithelial cells (arrow, d) and inflammatory cell infiltration (asterisk, d) as well as progression of the interstitial fibrosis (asterisk, d, inset) as compared to EO group infected testis. EO group, non-infected testis: severe degeneration of germinal epithelial cells (arrow, e), mild inflammatory cell infiltration (arrowhead, e), and mild interstitial fibrosis (asterisk, inset, e). EO + ciprofloxacin group, non-infected testis: severely degenerated germinal epithelial cells (arrow, f ) have been demonstrated. (a–f ) HE stain, original magnification ×400. Insets of (c–e): Masson's trichrome staining, original magnification ×400.

Testicular volumes and sperm concentrations

Ciprofloxacin treatment in healthy rats caused a slight decrease in testicular volume and sperm concentration that was not significantly different from the control group and contralateral testicles of infected group animals (Table 2, P > 0.05 for all comparisons).

Table 2.  Mean testicular volumes and sperm concentrations calculated in each group after the completion of one sperm cycle (14 days) have been demonstrated
SpecimensMean sperm concentration (million/mL)Mean testicular volume (cc)
  • Significant difference from specimen 1 (< 0.05).

  • Significant difference from specimen 2 (< 0.05).

  • §

    Significant difference from specimen 3 (< 0.05).

  • Significant difference from specimen 4 (< 0.05).

  • ††

    Significant difference from specimen 5 (< 0.05).

  • ‡‡

    Significant difference from all specimens (< 0.05). It is to note that spermatogenetic impairment was most severe in the infected testicles after ciprofloxacin treatment followed by the infected testicles which were not treated.

1: Group 1 left testis39.1 ± 2.65††1.1 ± 0.06§,††
2: Group 2, left testis30.8 ± 3.74††0.9 ± 0.07§,††
3: Group 3, right testis23.6 ± 3.24††0.5 ± 0.12,,,††
4: Group 3, left testis27.5 ± 5.50††0.9 ± 0.07§,††
5: Group 4, right testis4.0 ± 1.06‡‡0.1 ± 0.01‡‡
6: Group 4, left testis19.5 ± 4.310.8 ± 0.02††

Acute EO promoted a significant testicular atrophy in the infected side (Table 2, P < 0.05 compared to control and ciprofloxacin groups, and contralateral testicles of infected rats) associated with a substantial decrease in sperm concentration compared to the control group (23.6 ± 3.24 vs 39.1 ± 2.65 million/mL, P = 0.063). Contralateral testicle in the EO group revealed a slight decrease in testicular volume that was not statistically significant when compared to control and ciprofloxacin-treated animals (Table 2, P > 0.05 for all comparisons). Sperm concentration in the contralateral testicle of EO rats was comparable to that of the infected side (27.5 ± 5.5 vs 23.6 ± 3.24 million/mL), however, the difference was not statistically significant when compared to the control group (Table 2, P > 0.05).

There was a profound atrophy of the testis, volume of which was significantly less than all other specimens (Table 2, P < 0.05 for all comparisons) in the EO + ciprofloxacin group. Likewise, there was a significant decrease in the sperm concentration when compared to other specimens (Table 2, P < 0.05 for all comparisons). Contralateral testicles revealed a moderate decrease in testicular volume that was not significantly different from controls and contralateral testicles of ciprofloxacin-treated groups (Table 2, P > 0.05 for all comparisons), but a significant decrease in sperm concentration when compared to controls (Table 2, P < 0.05).

Discussion

Our results showed that retrograde inoculation of E. coli promoted a clinically recognizable acute orchitis with subsequent testicular damage in the rat. Histopathological findings were also encountered to a lesser extent in the contralateral testes, although local clinical signs of inflammation were not present initially. These findings were associated with a recognizable but statistically insignificant decrease in testicular volume and sperm concentration when compared to control testicles. Nevertheless, the histopathological evidence of contralateral testicular damage is an important finding which suggests that unilateral EO is not an isolated pathology and may adversely affect male fertility by several mechanisms. Although isolation of the pathogens from infected and non-infected testicles was not performed in our study, the present findings of contralateral damage in the absence of clinical infection are most likely due to involvement of immunological mechanisms.6 Recent published work data further suggest the involvement of other physiological mechanisms. For example, Kaya et al. has demonstrated experimental unilateral epididymitis to promote increased oxidative stress in both ipsilateral and contralateral testicle in the rat associated with a significant impairment of antioxidant status.7 Thus, further research is needed to define the physiological pathways involved in contralateral testicular insult following EO which may facilitate the development of new and more effective target therapies.

Fluoroquinolone drugs are the first-choice antimicrobial drugs for the treatment of EO.1 However, the results of our experimental study reveal that ciprofloxacin may have a potential to adversely affect testicular function. Subclinical inflammation observed in the ciprofloxacin group was associated with a mild decrease in testicular volume and sperm concentration that was not statistically significant when compared to controls. Furthermore, ciprofloxacin treatment did not reverse the histopathological findings of EO in the present study, and the most profound testicular findings were observed in the infected testicles of rats which received antibiotic treatment. Abd-Allah has similarly shown that ciprofloxacin treatment for 15 days in rats resulted in a marked reduction in sperm count and motility.3 These results are possibly due to an increased peroxide radical generation in the testis following ciprofloxacin treatment,8 which then induces DNA single-strand breaks and chromosomal aberrations as demonstrated by in vitro genotoxicity studies.9,10 Conversely, a few studies reveal that ciprofloxacin has neither clastogenic activity nor induces DNA damage when investigated in vivo, suggesting ciprofloxacin as a safe drug for human therapeutic use.2 In conclusion, our experimental results suggest a gonadotoxic potential of ciprofloxacin in the short term. Further studies that utilize larger animal populations and assess testicular functions at later time-point intervals are definitely needed.

Acknowledgments

The authors are grateful to Neval Elgörmüs phd, from the Department of Microbiology at Marmara University School of Medicine, for the invaluable help she provided during the preparation and injection of E. coli suspensions.

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