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Keywords:

  • diabetes;
  • Infertility;
  • neuropathy;
  • seminal vesicles;
  • tadalafil

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Conflict of interest
  9. Author contributions
  10. References

We have previously reported that infertile patients with diabetes mellitus (DM) have a particular ultrasound features of the seminal vesicles (SV) characterized by higher fundus-to-body ratio and lower pre- and post-ejaculatory difference in body antero-posterior diameter (APD). Based on these premises the aim of the present study was to investigate possible ultrasound SV changes in infertile patients with DM and diabetic neuropathy (DN), after prolonged administration of tadalafil (TAD) (a specific phosphodiesterase-5 inhibitor). To accomplish this, 20 infertile patients with symptomatic DN and erectile dysfunction were selected and arbitrarily divided into two groups which were assigned to: daily administration of 5 mg TAD for 3 months (Group A) (n = 10) and administration of placebo (Group B) (n = 10). All patients underwent to scrotal and prostate-vesicular transrectal ultrasound evaluation and semen analysis (Laboratory Manual for the Examination and Processing of Human Semen, WHO, 2010) before and after treatment. The following SV US parameters were recorded: (i) body APD; (ii) fundus APD; (iii) parietal thickness of the right and left SVs; and (iv) number of polycyclic areas within both SVs. We then calculated the following parameters: (i) fundus/body (F/B) ratio; (ii) difference of the parietal thickness between the right and the left SV and (iii) pre- and post-ejaculatory APD difference. In addition, we also evaluated the SV ejection fraction. Group A patients showed a significant reduction in F/B ratio and higher pre- and post-ejaculatory body SV APD difference compared with baseline or Group B after 3 months. These patients showed also a significant increase in SV ejection fraction and a significant improvement of the total sperm count, progressive motility, seminal levels of fructose, leucocytes and ejaculate volume. In conclusion, these results suggest that infertile DM patients with DN and erectile dysfunction had an improvement of ultrasound features suggestive of diabetic neuropathy after daily treatment with low doses of TAD.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Conflict of interest
  9. Author contributions
  10. References

Recently, we reported that the seminal vesicles (SV) of infertile patients with diabetes mellitus (DM) have a peculiar ultrasound characteristics (La Vignera et al., 2011a) which may result from diabetic neuropathy (DN) (La Vignera et al., 2011b) and reflect glycaemic control (La Vignera et al., 2011c). Moreover, evidence is accumulating that type 5 phosphodiesterase inhibitors (PDE5i) may be used for the treatment of DN (Wang et al., 2011; Patil et al., 2004; Ali & Rakkah, 2007; Kamenov, 2011) and that these molecules can modify some functional features in the SV (Ückert et al., 2011; Orhan et al., 2006; Uckert et al., 2007; Gajar et al., 2007; Uckert et al., 2009; Birowo et al., 2010). Therefore, the aim of this study was to investigate possible changes in the ultrasound (US) characteristics of the SV of infertile patients with DM and DN, after prolonged administration of tadalafil, a specific PDE5i. To accomplish this, 20 infertile patients with symptomatic DN and erectile dysfunction were selected.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Conflict of interest
  9. Author contributions
  10. References

Twenty infertile diabetic patients [36.0 ± 4.0 years (range 30–44 years)] with DN and erectile dysfunction were selected consecutively for this study:

Characteristics of diabetes

In all cases enrolled type 2 diabetes mellitus with mean disease duration of 7.0 ± 3.0 years and mean glycated haemoglobin of 8.1 ± 2.2%. At the time of the enrolment the treatment administered to all patients was oral hypoglycaemic [metformin (11 patients), metformin and sulfonylureas (six patients) metformin and thiazolidinediones (three patients)]. All patients examined had symptomatic DN, stage 2 according to the Dyck's classification (Dyck, 1988; Vinik et al., 2003). The mean duration of infertility was 19 ± 5 months. In all cases there was a episodic history of erectile dysfunction over the last 6 months [mean value reported to administration of 5-item version (Rosen et al., 1999) of International Index of Erectile Function (IIEF-5):14 ± 2].

The patients enrolled were arbitrarily divided into two groups according to the treatment assigned:

  • Group A comprised 10 diabetic patients treated with daily doses of tadalafil (5 mg) for 3 months. The mean age of this group was 38.0 ± 4.0 years (range 33–42 years). These patients had a mean body mass index (BMI) of 26.6 ± 3.0 kg/m2 (range 25–30 kg/m2) and a mean value of IIEF-5 score of 15.0 ± 2.0.
  • Group B comprised 10 diabetic patients treated with placebo (one capsule containing 0.500 g of l-arginine) for 90 consecutive days. The mean age of this group was 36.0 ± 4.0 years (range 30–43 years). These patients had a mean BMI of 27.6 ± 5.8 kg/m2 (range 24–31 kg/m2) and a mean value of IIEF-5 score of 15.0 ± 1.0.

All enrolled patients during the first visit received a diary to report the possible adverse effects in the course of the treatment.

All patients were subjected to the following evaluation: physical examination, semen analysis according to most recent guidelines (WHO, 2010) and scrotal and prostate-vescicular transrectal ultrasound evaluation. Semen analysis and ultrasound evaluation were repeated after 3 months of treatment.

Main exclusion criteria

  • Ultrasound findings of epididymal and/or prostato-vesicular obstruction (La Vignera et al., 2008);
  • Alcaline pH of the semen;
  • Ultrasound and/or microbiological criteria of male accessory gland infections;
  • Endocrine disorders: hypogonadism (total testosterone <3 ng/mL or 10.4 nmol/L) (Dandona & Rosenberg, 2010), hyperprolactinemia (prolactin >25 ng/mL) (Walsh & Pullan, 1997) and hypothyroidism (TSH >4.5 μU/mL) (Cooper, 2001).The serum concentrations of these patients are shown in Table 1. Endocrine factors can affect the quality of ejaculation (Corona et al., 2011), and we therefore arbitrarily chose to exclude these patients to avoid factors stasis in the seminal vesicles.
  • Presence of sexual dysfunction different from erectile dysfunction.
Table 1. Serum hormone concentrations of the patients enrolled in this study
GroupsTSH (mUI/mL)FSH (mUI/mL)LH (mUI/mL)Testosterone (ng/mL)17β-Estradiol (pg/mL)Prolactin (ng/mL)AbTg (UI/mL)AbTPO (UI/mL)
  1. FSH: Follicle-stimulating hormone, LH: Luteinzing hormone, AbTg: Thyreoglobulin antibodies, AbTPO: Thyreoperoxidase antibodies, TSH: Thyroid-stimulating hormone.

  2. The reference intervals: TSH = 0.3–4.2 mUI/mL, LH = 1.6–9.0 mUI/mL, FSH = 2.0–12.0 mUI/mL, 17β-Estradiol = 8.0–43.0 pg/mL, Total testosterone = 2.8–8.0 ng/mL, prolactin = 4.0–15.0 ng/mL, Thyroglobulin antibody (AbTg) = <100.0 mUI/mL and Thyroid peroxidase antibody (AbTPO) = 0.0–34.0 mUI/mL.

Group A1.7 ± 1.43.8 ± 3.23.7 ± 2.25.3 ± 1.711.0 ± 15.09.0 ± 2.034.0 ± 12.016.0 ± 4.0
Group B1.9 ± 1.13.7 ± 2.33.3 ± 3.05.7 ± 1.316.0 ± 13.09.0 ± 3.029.0 ± 14.018.0 ± 7.0

Other exclusion criteria

Cigarette smoking, alcohol consumption, occupational chemical exposure, fever or drug use within 3 months prior to enrolment in this study, azoospermia, testicular volume <15 mL (Sakamoto et al., 2008) and past or present cryptorchidism or varicocoele.

Female partners

Gynaecological evaluation had previously excluded the main functional and/or mechanical causes of female infertility.

Measurement of glycated haemoglobin (HbA1C)

HbA1c was determined by high-pressure liquid chromatography [Bio-Rad, Hercules, CA, USA, model Variant II. Reference ranges are as follows: 3.7–6.2% and up to 48 (nmol/mol)].

Measurement of serum hormone concentrations

The hormone assays were performed by electrochemiluminescence with a Hitachi-Roche device (Cobas 6000; Roche Diagnostics, Indianapolis, IN, Hercules, CA, USA). The reference intervals are reported in Table 1.

Ultrasound evaluation

The testicular and epididymal regions were carefully assessed with the help of scrotal US and with a 7.5 MHz linear transducer. The prostate-vesicular region was assessed with rectal US using a 7.5 MHz biplan biconvex transducer (Esaote GPX Megas, Genova, Italy). All patients underwent ultrasound evaluation before and after ejaculation, after sexual abstinence of 4 days.

For the aim of this study were evaluated the same ultrasound parameters of the seminal vesicles previously described in our study (La Vignera et al., 2011a,b,c).

For the first time, we also evaluated another ultrasound parameter, the so-called ‘SV ejection fraction’, calculated as [(SV volume before ejaculation − SV volume after ejaculation)/SV volume before ejaculation] × 100 (Lotti et al., 2012). This article describes also how to calculate the initial SV volume (Lotti et al., 2012).

The measurements were repeated twice and the results were expressed as a mean in the final report. For a measurement below 1 mm, the calculations were performed on ultrasound paper with a millimetric measurement system.

The protocol was approved by the Institutional Review Board, and informed written consent was obtained from each patient and control subject.

Seminal fructose determination

Fructose has been measured in semen using a spectrophotometric method and was assessed 1 h after ejaculation using the Mann resorcinol and HCl method (Mann & Lutwak-Mann, 1976).

Statistical analysis

Results are reported as mean ± SEM throughout the study. The data were analysed by one-way analysis of variance (anova) followed by the Duncan's multiple range test. Statistical analysis was performed using spss 9.0 for Windows (spss Inc., Chicago, IL, USA). A p value less than 0.05 was accepted as statistically significant.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Conflict of interest
  9. Author contributions
  10. References

The characteristics of the two groups appeared comparable without significant differences on the following parameters: age, body mass index, glycated haemoglobin and testosterone levels (Tables 1 and 2). Therefore, we did not include these covariates, which could affect SV parameters, in the statistical analysis.

Table 2. Characteristics of diabetes of the patients enrolled in this study
GroupsType of diabetesDuration of diseasesHbA1C (%)Treatment
  1. OHA: Oral hypoglycaemic agents.

Group AType 28.3 ± 2.18.5 ± 2.0OHA
Group BType 26.9 ± 2.17.8 ± 2.6OHA

Group A patients showed a significant improvement (p < 0.05) of erectile dysfunction after treatment (mean value of IIEF 5 score = 20.0 ± 3.0) compared with baseline. Group B patients showed no significant increase in this value (mean value of IIEF 5 score = 16.0 ± 2.0).

In addition, there were no significant differences among the groups as far as testicular size, prostate volume and epididymal head and tail sizes by scrotal ultrasound scan (Table 3).

Table 3. Scrotal and prostate ultrasound parameters of the patients enrolled in this study
GroupsProstate volume (cm3)Testicular volume (mL)Epididymal head size (mm)Epididymal tail size (mm)
Group A34.0 ± 6.018.0 ± .3.09.0 ± 3.04.0 ± 4.0
Group B35.5 ± 4.020.0 ± 4.08.0 ± 4.04.0 ± 3.0

There were no cases of withdrawal from study. The following more frequent adverse effects were detected in the course of the therapy: back pain (2.0%), nasopharyngitis (1.1%), dyspepsia (1.1%), headache (1.2%) and myalgia (2.0%).

Ultrasound evaluation

Before treatment, all ultrasound SV parameters did not show any statistically significant difference (Table 4). After treatment Group A patients showed a significant reduction in F/B ratio compared with baseline (p < 0.05) and lower value of this parameter compared with Group B patients (p < 0.05). Moreover, they also showed a higher pre- and post-ejaculatory difference of the body SV APD compared with baseline or with the other group of diabetic patients (p < 0.05) (Table 4).

Table 4. Ultrasound evaluation of seminal vesicles of the patients enrolled in this study
GroupsBody APD (mm)Fundus APD (mm)F/B ratioParietal thickness (mm)Parietal differences (mm)Number of polycyclical areasPre- and post-ejaculatory APD difference (mm)SV ejection fraction (%)
  1. APD: Antero-posterior diameter, F/B: Fundus-to-body ratio, SV: Seminal vesicles.

  2. a

    p < 0.05 vs. baseline.

  3. b

    p < 0.05 vs. the other group after treatment.

At baseline
Group A14.0 ± 3.039.0 ± 6.02.8 ± 3.00.86 ± 0.200.78 ± 0.102.0 ± 1.00.40 ± 0.0623.0 ± 4.0
Group B14.0 ± 2.040.0 ± 8.02.9 ± 4.00.88 ± 0.300.80 ± 0.101.0 ± 2.00.36 ± 0.1223.0 ± 5.0
After treatment
Group A12.0 ± 2.028.0 ± 7.0ab2.3 ± 4.0ab0.84 ± 0.300.80 ± 0.301.0 ± 1.01.2 ± 1.1ab30.0 ± 7.0ab
Group B13.0 ± 3.040.0 ± 10.03.0 ± 3.00.88 ± 0.300.82 ± 0.201.0 ± 2.00.38 ± 0.1023.0 ± 6.0

Finally, after treatment Group A patients showed a significant increase in SV ejection fraction (p < 0.05) compared with baseline or Group B (p < 0.05).

Sperm parameters

After the treatment, Group A patients showed a significant improvement of the following parameters: total sperm number, progressive motility, fructose seminal levels, leucocytes and volume of ejaculate. These parameters improved significantly (p < 0.05) compared with baseline or Group B after therapy (Table 5).

Table 5. Semen analysis of the patients enrolled in this study
GrSp. concentr. (106 mL)Total sp. number (106 ejacul.)Progr. motility (%)Total num. progr. motile sperm.Normal forms (%)Total num. normal sperm.Leucocytes (106 m/L)Ejaculate volume (mL)Biochemical fructose (mg %)
  1. a

    p < 0.05 vs. baseline.

  2. b

    p < 0.05 vs. the other group after treatment.

At baseline
A12.0 ± 8.019.2 ± 6.416.0 ± 6.03.07 ± 0.913.0 ± 6.02.49 ± 0.81.0 ± 0.51.6 ± 0.8150.7 ± 30.0
B13.0 ± 5.024.7 ± 4.014.0 ± 4.03.34 ± 0.515.0 ± 4.03.70 ± 0.61.1 ± 0.51.9 ± 0.8162.2 ± 20.0
After treatment
A13.5 ± 9.037.8 ± 18.0ab25.0 ± 9.0ab9.45 ± 4.5 ab14.6 ± 6.55.51 ± 2.51.5 ± 1.4ab2.8 ± 1.6ab206.0 ± 45.0ab
B15.0 ± 9.024.0 ± 2.416.0 ± 6.03.84 ± 0.313.0 ± 6.03.12 ± 0.11.0 ± 0.71.7 ± 0.6166.6 ± 22.2

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Conflict of interest
  9. Author contributions
  10. References

Temporary sexual disorders resulting after diagnosis and during the treatment are common in couples with fertility problems (Wischmann, 2010). Erectile dysfunction is common in patients with type 2 DM with important consequences on the psychological well-being and reproductive function (Isidro, 2012), and PDE5i are the preferred therapy for most men with organic erectile dysfunction who do not have a specific contraindication to their use and for the patients with sufficient nerve function to induce the erection (Isidro, 2012; Fode et al., 2012).

In this study, we found that prolonged treatment with low doses of tadalafil (a selective PDE5i) commonly used in the treatment of erectile dysfunction, is associated with a change in ultrasound abnormalities detected in SV of infertile patients with DM and DN. Moreover, this treatment was associated with a relative improvement of sperm parameters, in particular was observed a significant increase in the volume of the seminal plasma associated with an increase in the total number of spermatozoa in the ejaculate. Finally, this treatment resulted in a significant increase in concentrations of leucocytes in the ejaculate.

There are no particular adverse effects reported during the course of the therapy, and the frequency of the main effects (back pain, nasopharyngitis, dyspepsia, headache and myalgia) was comparable with the results of another recent study (Porst et al., 2012).

Several studies on the use of PDE5i data for the treatment of DN have been published (Wang et al., 2011; Patil et al., 2004; Ali & Rakkah, 2007; Kamenov, 2011); the effects of these molecules on some functional aspects of the SV have been described (Ückert et al., 2011; Orhan et al., 2006; Uckert et al., 2007; Gajar et al., 2007; Uckert et al., 2009; Birowo et al., 2010). Moreover, there are some data on the effects of PDE5i administration on sperm parameters (Hellstrom et al., 2008; Pomara et al., 2007). Finally, some authors have debated the usefulness of these molecules for the treatment of premature ejaculation (Gökçe et al., 2011; Jannini et al., 2011).

On the contrary there are no data on the ultrasound characteristics of the SV after treatment with PDE5i in infertile diabetic patients with neuropathy, or changes on sperm parameters.

Recent evidence indicates that cyclic adenosine monophosphate and cyclic guanosine monophosphate (cGMP)-phosphodiesterase isoenzymes are involved in the control of secretory activity and efferent neurotransmission in the SV (Ückert et al., 2011) and that PDE5i can counteract the contraction of human SV mediated by alpha-adrenergic receptors and enhance the levels of cyclic nucleotides (Birowo et al., 2010), as well as inhibit electrical field stimulation-induced and spontaneous contractile activity of isolated human SV tissue (Uckert et al., 2009). Another aspect to consider is that the decreased nNOS (neuronal nitric oxide synthase)-cGMP system may play a crucial role in the pathogenesis of DN and, therefore the use of PDE5i may find a rational for the treatment of this condition (Patil et al., 2004). Moreover, it is known that hyperglycaemia substantially upregulates PDE5 expression and that the cGMP/PKG (protein kinase GI) signalling pathway activated by PDE5i mediates the beneficial effects of these molecules on diabetic peripheral neuropathy (Wang et al., 2011). Finally, a recent study suggests a chronic neuro-physiological effect of sildenafil (the first selective PDE5i used in the clinical practice) administration on male fertility profile exclusively in DN. This study showed an improvement in the entire smooth musculature of the reproductive tract altered because of the neuropathy which made them rigid leading to atonia of bladder and urethra. This resulted in partial or retrograde ejaculation associated with a decreased sperm motility (Ali & Rakkah, 2007). In our study, under the influence of tadalafil, the fundus diameter of the seminal vesicles is reduced from 39 to 28, a reduction by 25%, as this drug increases the bioavailability of cGMP which is a smooth muscle relaxant, we speculatively assume that the increase in ejection fraction is related to wall stiffness which is reduced by cGMP. However, to our knowledge, there are no other data in the literature regarding this aspect.

With regards to sperm parameters, this study was not comparable with the main data of the literature because these patients had a particular clinical characterization never examined to date in any other study. In fact the recent study of Hellstrom and colleagues is a double-blind, placebo-controlled, non-inferiority study on healthy men (or with mild erectile dysfunction) who were randomized to receive tadalafil 20 mg or placebo (Hellstrom et al., 2008), as well as the study by Pomara and colleagues (Pomara et al., 2007), is a prospective, randomized, double-blind, cross-over clinical investigation on sperm parameters after the administration of a single dose of tadalafil (20 mg). Probably, the increase in sperm motility is associated with increased levels of fructose (marker of vescicular function) (Gonzales, 2001) and the increase in the total sperm count is associated with increase in the semen volume, although on fructose and motility there are different data reported in the literature (Elzanaty, 2007). In fact, it is know that tadalafil amplifies the bioavailability of nitric oxide (NO) and in the experimental model the glandular NO production is a prerequisite for muscarinic fructose secretion in the seminal vesicle (Ehrén et al., 1997).

There is an apparent discrepancy between the moderate increase in the ejection fraction (35%) and the higher increase in ejaculate volume (75%). In our opinion this finding can be explained by the presence of other constituents of the ejaculate that are not a result of the ejection fraction of the seminal vesicles and their possible different secretory activity. Another apparent contradictory result is the difference between sperm concentration and the total number of spermatozoa. In this case, we believe that the increase in volume of ejaculate represent the main cause of this difference.

Finally, the increase in concentration of leucocytes in the semen does not have a clear pathophysiological interpretation. However, we may speculate that greater empting capacity of the SV is associated with a temporary leucocyte response such as occurs during their inflammation (Vicari, 1999). Therefore, in these patients the differential diagnosis with the male accessory gland infections (MAGI) (La Vignera et al., 2011d) should be very accurate because diagnosis of MAGI have clear criteria for the classification (La Vignera et al., 2011d) which are absent in infertile diabetic patients. Therefore, in these patients there could be a condition of SV pseudo-inflammation of the seminal vesicles. Finally, we observed a paradoxical increase in sperm motility associated with moderate leucocytospermia in these patients but also other authors had reported this association in other clinical models (Ziyyat et al., 2008; Barraud-Lange et al., 2011).

Another speculative explanation is the potential anti-inflammatory role of leucocytes in the semen in addition with cGMP and NO enhanced by the use of tadalafil. In fact it is known that at moderate levels (<106/mL), leucocytospermia is associated with improved sperm fertilization ability and pregnancy outcome (Barraud-Lange et al., 2011), and NO has been shown to modulate in vitro motility, viability and the acrosome reaction of spermatozoa (Revelli et al., 2001).

A last aspect concerns the use of an ultrasound parameter, the so-called ‘ejection fraction of the seminal vesicles’ (Lotti et al., 2012) that appears to increase significantly after prolonged treatment with tadalafil at low daily doses in patients with DM and DN. To our knowledge, there are no other data in the literature regarding the application of this parameter in unconventional categories of infertile patients.

Possible limitations of the study

The patients were arbitrary assigned to each group, therefore there was no randomization, however, the possible selection bias are limited by the presence of very homogeneous clinical features of the patients enrolled and not to have lost data during the study or the careful application of inclusion/exclusion criteria. In our opinion, the only real limitation of this study is the low number of patients, but they represent a particular and selected category of men in clinical practice.

In conclusion, this study suggests a potential use of tadalafil different from erectile dysfunction, emphasizing the positive aspects of a prolonged treatment for a selected category of infertile patients and in an unconventional form of DN.

Author contributions

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Conflict of interest
  9. Author contributions
  10. References

S. L., R. C., E. V. and A. E. C carried out the study, analysed the data and wrote the manuscript. F. L, V. F, G. M and M. M were involved in the study design, data management and analysis of the study.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Conflict of interest
  9. Author contributions
  10. References