Reproductive and endocrinological effects of Benign Prostatic Hyperplasia and finasteride therapy in dogs

Benign prostatic hyperplasia (BPH) is one of the most important reproductive disorders in aging dogs. Therapeutic measures include orchiectomy and pharmacological treatment, leading to reduction of prostate volume and clinical signs. One of the most common drugs used in BPH treatment is finasteride, but data regarding its possible side effects are scarce. Thus, the aim of this study was to evaluate the effects of BPH and short-term (2 months) finasteride therapy on clinical, endocrinological, and reproductive parameters in dogs. Dogs were allocated into four experimental groups: Non-affected (n = 5), BPH (n = 5), Non-Affected-Finasteride (n = 5) and BPH-Finasteride (n = 5) groups. Dogs were evaluated monthly during 2 months by a complete breeding soundness examination, B-mode ultrasound and Doppler ultrasonography of the testicular artery, hormonal profile (testosterone, estrogen and dihydrotestosterone) and oxidative profile of the prostatic fluid. After 2 months, dogs were gonadectomized and testicles were subjected to histologic analysis. Finasteride treatment reduced dihydrotestosterone concentrations, without negative influence on semen quality and also reverted testicular hemodynamics changes of BPH. On the other hand, BPH was accompanied by significant changes in testosterone and estrogen concentrations and semen quality, mainly related to sperm kinetics alterations. In conclusion, BPH dogs have important hormonal and sperm alterations, however, short-term finasteride treatment (2 months) was able to reduce overall effects of BPH, thus representing a method of therapy for BPH treatment.

www.nature.com/scientificreports/ azoospermia [21][22][23] . In addition, Vidigal et al. 24 showed reduction in spermatogenesis rate and seminiferous tubules atrophy in hamsters, indicating a possible irreversible and negative effect of finasteride therapy. In dogs, decreased sperm quality was only observed after long term treatment with finasteride 4 . On the other hand, Iguer-Ouada; Verstegen 25 , observed increased sperm concentration whilst prostate reduction with finasteride treatment. Hence, the effects of both BPH and subsequent finasteride treatment on sperm output and quality should be analyzed by clinical trials. The obtained knowledge may allow for the safe use of stud dogs in reproductive programs while these dogs are under the finasteride therapy for BPH. In this context, the purpose of this study was to evaluate the influence of BPH and/or finasteride therapy on sperm features, testicular morpho-function and hormonal profile in dogs.

Results
No statistical interaction among BPH, finasteride treatment and time-points (0, 30 and 60 days) was observed.
After 30 days of finasteride treatment, all BPH-Finasteride dogs fully recovered from the initial clinical signs (tenesmus, hematospermia, hematuria or dysuria). Dogs of the BPH Group remained clinically stable, without any further complications.
Regardless of the experimental group, all testicular samples (right and left testicles) presented mild to moderate cellularity of the seminiferous tubules, with scarce areas of focal high cellularity. Seminiferous tubules were filled with acellular eosinophilic amorphous material in the absence of inflammatory infiltrate or blood. All the evaluated groups were classified as score 0, that means, no lesions were observed.  www.nature.com/scientificreports/ No differences between groups were verified for the oxidative status of the prostatic fluid (Table 2). No timepoint differences (Day 0, Day 30 and Day 60; P > 0.05) were observed throughout the experiment.

Discussion
In the present study, we evaluated the influence of benign prostatic hyperplasia and/or treatment with finasteride in dogs, by means of a complete breeding soundness examination. Benign prostatic hyperplasia is a homologous condition in men and dogs, and the most commonly diagnosed prostatic disorder in aged and intact dogs 26,27 . Although castration is the treatment of choice, drug treatment with finasteride, a synthetic inhibitor of enzyme 5α-reductase, is one of the feasible medical alternatives for breeding dogs 17,28 .
Our data show that dogs with BPH have lower testosterone and estrogen concentration compared to unaffected dogs, as well as positive correlation between testosterone concentration and sperm kinects (total and progressive sperm motility). In fact, testosterone is the most important male hormone, acting on spermatogenesis, spermiogenesis and epididymal maturation 29 . Also, an appropriate hormonal environment is necessary for adequate spermatogenesis, including estrogen synthesis by the action of aromatase on Sertoli cells 30 . Thus, decreased concentration of testosterone and estrogen in dogs with BPH may directly affect ejaculate quality 13,29 . Thus, we assume that spermatogenesis dysfunction in BPH dogs is an indirect consequence of the hormone imbalance rather than a consequence of testicular morphological alterations, since no remarkable histological findings were observed that can justify the significant reduction in sperm concentration and higher percentage of minor sperm defects. Additionally, alterations of sperm kinetics in BPH dogs can be related to the decreased secretory function of Leydig cells.
Interestingly, high percentage of sperm beat cross-frequency (BCF) was observed in dogs with BPH, suggesting a kinetic pattern similar to premature sperm hyperactivation 31 . The excessive production of reactive oxygen species (ROS), changes in pH and osmolarity of the seminal fluid are common causes of premature   www.nature.com/scientificreports/ hyperactivation 32 . However, oxidative stress and ROS generation are considered to occur only in severe and chronic prostatic disorders, with loss of prostatic parenchyma integrity 11,15 . In fact, we could not find significant differences in oxidative stress (TBARS) and antioxidant activity (SOD and GPx) within experimental groups. Thus, sperm kinetical alterations (i.e. BCF) are possibly related to altered biochemical characteristics of the prostatic fluid in dogs with BPH, such as ionic and binding protein conten 14,33,34 . Furthermore, we can also attribute the high percentage of sperm defects in BPH dogs to biochemical changes of the prostatic fluid, i.e., post-ejaculatory sperm alterations 13,14 . It is therefore possible to infer that biochemical changes of the prostatic fluid in BPH promote structural damage of post-ejaculated sperm cells. Interestingly, BPH dogs had lower estrogen and testosterone concentrations compared to the Non-Affected Group, despite being within the normal range for adult dogs 35 . However, low testosterone concentrations are responsible for the neoformation of androgenic receptors in the prostate 36 , thus increasing testosterone action. On the other hand, non-affected dogs treated with finasteride had lower testosterone and DHT concentrations compared to the Non-treated Group. Thus, we assume that 5α-reductase blockage led to inhibition of testosterone conversion into DHT, ultimately resulting in negative feedback of Leydig cells by accumulative concentrations of testosterone 37 .
It is interesting to point out that finasteride treatment caused reduction of testicular artery blood volume (pulsatility index) and increase in blood flow velocity (time average maximum velocity), compared to untreated dogs. Therefore, DHT seems to act as a regulator of testicular vascular homeostasis, since low rates of testicular artery pulsatility index (PI) are related to efficiency in spermatogenesis 1 . In fact, according to Bergh et al. 38 even a slight reduction in blood flow of the testicular artery may affect the initial process of spermatogenesis. Thus, we can suggest that the analysis of testicular artery PI can be used as a complementary marker of effective therapeutic effect during the course of finasteride treatment in BPH dogs.
Even though Vidigal et al. 24 observed changes in testicular parenchyma of finasteride treated hamsters, our results could not prove such assertive in short-term treated dogs. However, we believe that 2 month course of finasteride therapy was not a sufficient period to promote significant testicular and seminal changes, similarly to the results of Iguer-Ouada; Verstegen 25 . Thus, in this respect, semen use from finasteride treated dogs is feasible for reproductive purposes and can be recommended during the first 2 months of therapy, which is a period enough for the assuagement of clinical signs and reduction in prostate size 19 .
In conclusion, dogs with benign prostatic hyperplasia present a hormonal imbalance, leading to seminal changes related mostly to sperm kinetics. On the other hand, the 2 months duration of finasteride therapy decreases hormonal profile of dihydrotestosterone and testosterone, resulting in vascular alteration of the testicular artery without promoting additional significant changes in sperm quality.

Materials and methods
Animals and experimental study. The current study was approved by the Bioethics Committee of the School of Veterinary Medicine and Animal Science-University of São Paulo (protocol number 7122171213). All experiments were performed in accordance with relevant guidelines and regulations.
Twenty dogs of several breeds, with body weights ranging from 10.2 to 29.5 kg and aged 5 to 13 years were selected and equally divided into four experimental groups according to BPH diagnosis and finasteride treatment. Presumptive diagnosis of BPH was based on clinical signs, prostatic secretion assessment and prostatic biometry by B-mode ultrasound 1,5 . The most common clinical signs of BPH dogs were hematospermia, hematuria, pollakiuria, dysuria and tenesmus, besides marked prostatomegaly. In addition, macroscopic examination of the prostatic fluid revealed reddish color and blood cells content. After examination, dogs were then randomly assigned to four experimental groups, composing a 2 × 2 factorial experimental design, considering BPH and finasteride therapy as the main experimental factors: To assure the appropriate sample size, an analysis was conducted with the SAS Power and Sample Size 12 (SAS Institute Inc., Cary, NC, EUA). A retrospective analysis of the data indicated there was a power of 0.99, which is considered an acceptable statistical power (at least 0.8). Hence, a minimum of 5 dogs per group were sufficient to demonstrate significant differences in the data. To ensure that the age of the dogs was not a determinant factor for the seminal analysis, dogs were equally distributed among groups. The mean age (± SEM) in each experimental group was as follow: 7 ± 1.2 years for the Non-affected group, 10 ± 1.9 years for the BPH group, 9 ± 2.1 years for the BPH-Finasteride group and 7 ± 1.4 years for the Non-affected-Finasteride group, with no significant statistical difference among groups.
All dogs were evaluated monthly during 2 months (Day 0, Day 30 and Day 60) by a complete breeding soundness examination, beginning at the first day of finasteride therapy or at the day of presumptive diagnosis of BPH.
After 60 days, bilateral orchiectomy was performed and testicles were subjected to histological analysis. www.nature.com/scientificreports/ Seminal evaluation. On days 0, 30 and 60, a semen sample was collected from each dog by digital manipulation of the penis. We collected only a single semen sample from each dog before ultrasonographic analysis, due to a remarkable influence of ejaculation on prostate vascularization as attested by Alonge et al. 40 . After the semen collection, the sperm-rich fraction was immediately analyzed for the following methods: Volume of the sperm-rich fraction was measured using a graded test tube (mL). Sperm count (number of spermatozoa per mL) was analyzed through a haemocytometer counting chamber. For the assessment of sperm morphology, the eosin/nigrosin stain was performed. Briefly, 5 μL of semen and 5 μL of the previously prepared stain were placed in a glass slide and smeared. Sperm analysis was performed under light microscopy (Nikon, Eclipse E200, Japan) at × 1,000 magnification by counting 200 cells. Sperm morphological abnormalities were classified as major, minor and total, according to Barth and Oko 41 . In order to rule out a possible concurrent prostatitis, sperm smears were also analyzed for the presence of bacteria or leukocyte.
In order to analyze the integrity of sperm plasma and acrosome membrane, semen were analyzed by flow cytometry using the BD FACSCalibur equipment (Becton Dickinson, East Rutherford, NJ, USA). Sperm cells were previously stained with fluorescent probes according to the specific analysis, using a fixed sperm concentration of 188,000 cells diluted in 37.5 μL of TALP (Tyrode`s Albumin Lactate and Pyruvate). For the analysis of plasma and acrossomal membrane integrity, propidium iodide fluorescent probe (PI; 6 μM in TALP) was combined with Psyllium agglutinin conjugated with fluorescence isothiocyanate (FITC-PSA; 100 μg/mL of 1% sodium azide). The mixed probe solution was added to diluted sperm and incubated at 37 °C for 5 min. Subsequently, 300 μL of TALP was added and analyzed by flow cytometry, according to the protocol previously described 43,44 . For the analysis of sperm mitochondrial potential, 1 μL of 50 μg/mL JC-1 probe (5,5′, 6,6′ tetrachloro-1,1,3,3′-tet raethylbenzimidazolylcarbocyanine iodide) was added to diluted sperm. Samples were incubated at 37 °C for 10 min and then 300 μL of TALP were added and analyzed by flow cytometry, according to protocol previously described 45 .
Oxidative profile of the prostatic fluid. Measurement of thiobarbituric acid reactive substances (TBARS) was performed to indirectly analyze lipid peroxidation, as previously developed by Ohkawa et al. 46 . To precipitate proteins, 300 μL of prostatic fluid and 600 μL of 10% trichloroacetic acid (TCA, Sigma Aldrich®) were mixed and centrifuged (20,817×g for 15 min at 5 °C). After centrifugation, 800 μL of the supernatant and 800 μL of 1% thiobarbituric acid (TBA, Sigma Aldrich®) in 0.05 N sodium hydroxide were placed into glass tubes, boiled in water bath (100 °C) for 10 min, and subsequently cooled in ice bath (0 °C) to stop the chemical reaction. The TBARS were then quantified using a spectrophotometer (Ultrospec 3300 pro®) at a wavelength of 532 nm. The results were compared to a standard curve previously prepared with a standard solution of malondialdehyde (MDA). The lipid-peroxidation index was described as nanograms of TBARS/mL.
Prostatic fluid was also subjected to the evaluation of antioxidant enzymes glutathione peroxidase (GPx) and superoxide dismutase (SOD) activity. Superoxide dismutase activity was assessed by indirect measurement of cytochrome c reduction by superoxide anion (O 2 -) through spectrometry at 550 nm wavelength at 25 °C for 5 min 30,31 . Glutathione peroxidase activity was measured by NADPH consumption, through the reaction between H 2 O 2 and reduced glutathione (GSH), which is catalyzed by GPx and measured through spectrometry at 340 nm wavelength at 37 °C for 100 min 47,48 . Testicular ultrasonographic examination. Ultrasound evaluation of the testicles (right and left) was performed using a Mindray® M5 ultrasound (Shenzhen, China) equipped with a 7.5 MHz linear transducer. Dogs were placed in dorsal recumbency and trans-scrotal evaluation was performed without sedation. Parenchyma echogenicity was analyzed by conventional B-mode and testicular biometry was performed, measuring testicular length (L) and width (W) of the right and left testicles. Testis volume (cm 3 ) was calculated considering the equation: L × W 2 × 0.71 49 .
Testicular blood perfusion and velocity of blood flow were analyzed by Doppler ultrasonography. Testicular arteries were scanned at the spermatic funiculum region 50 and pulsed-wave Doppler was used to characterize the waveform. Blood flow velocity parameters (peak systolic velocity-PSV-cm/s, end diastolic velocity-EDVcm/s, time average maximum velocity-TAMAX-cm/s) and hemodynamic indices (Resistance Index-RI, Pulsatility Index-PI, Systolic:Diastolic velocity-S/D) were automatically calculated by the equipment software using mathematical formulas or Pourcelot index. Blood sample volume was positioned in the center of the vessel and the insonation angle was adjusted (less than 60°). A total of 9 stable waves of each testicular artery were obtained to calculate the average of each variable.
Hormonal assay. Always during the morning period, blood samples were collected from the dogs by venous puncture into vacuum tubes without anticoagulant. The collection room was always maintained at 20 °C. Tubes were immediately centrifuged at 1,500×g for 10 min and serum was drawn-off and stored in microtubes at − 20 °C until analysis. www.nature.com/scientificreports/ Hormonal assays were performed with the use of commercial radioimmunoassay kits for quantitative measurement of testosterone and estrogen (Beckman Coulter®), previously validated for dogs 51,52 , and competitive ELISA kits for dihydrotestosterone assay 53 , previously validated for dog. For estradiol, the intra-assay coefficients were 7.15% (high intra-assay) and 3.45% (low intra-assay) and the limit detection was 1.12 pg/mL. For testosterone, the intra-assay coefficient was 4.52% and the limit detection was 0.01 ng/mL. For dihydrotestosterone, the limit detection was 6.58 pg/mL and the coefficient of variation ranged from 3.03% (low intra-assay) and 9.86% (high intra-assay). All reactions were performed in duplicate. Testicular histologic analysis. After orchiectomy, 0.5 cm fragments of testicular parenchyma were washed in 0.9% physiological solution and immediately fixed in 10% buffered formaldehyde solution for 24 h, as previously described 49,54 . Then, fragments were stored in 70% alcohol, and then embedded in paraffin. Histological sections of 0.5 μm were submitted to dewaxing following the standard protocol. Hematoxylin-Eosin stain (HE) was used to evaluate testicular histomorphological aspect 49 . A minimum of 2 microscopic slides were analyzed from each testis. Analysis of the testicular parenchyma was based on an arbitrary scale of 0 to 3 (0: no lesions; 1: superficial and focal changes; 2: general heterogeneous aspect of the parenchyma and tissue fibrosis; 3: loss of testicular morphology with reduction of testis size).
Statistical analysis. All data were evaluated using the SAS System for Windows (SAS Institute Inc., Cary, NC, USA). Effects of BPH, finasteride therapy and time-points (days 0, 30 and 60) and interactions between those factors were estimated by the repeated measures analysis of variance (Mixed Procedure of SAS). If no triple interactions (BPH X Finasteride X Time-points) occurred, the following interactions were considered: Timepoints X Finasteride, Time-points X BPH and Finasteride X BPH. If no significant interactions were observed, the effect of groups was analyzed merging all time-points and conversely, time-points were compared combining all groups taking into account the Bonferroni correction (Proc Glimmix). Otherwise, comparisons were performed taking into account both effects. Differences between BPH and finasteride therapy were analyzed using parametric and non-parametric tests, according to the residual normality (Gaussian distribution) and variance homogeneity. Whenever one of these assumptions was not respected data were transformed. When no transformations were successful, non-parametric tests were employed. Thus, differences between BPH and Finasteride therapy were analyzed using Student t test (parametric variables) and Wilcoxon test (nonparametric variables). Results were described as untransformed means ± SE. The significance level used was P < 0.05. Moreover, Spearman correlation was used to calculate the relationship between variables.