Int Urol Nephrol DOI 10.1007/s11255-015-1037-6
UROLOGY - ORIGINAL PAPER
Correlation between the androgen receptor status of the appendix testis and the efficacy of human chorionic gonadotropin treatment in undescended testis Gyula Drabik1 · Tamás Józsa2 · Olga Ruzsnavszky3 · Kornél Kistamás4 · Beatrix Dienes4 · Ilona Kovacs4 · Mátyás Benyó1 · Tibor Flaskó1
Received: 30 April 2015 / Accepted: 13 June 2015 © Springer Science+Business Media Dordrecht 2015
Abstract Purpose To compare the androgen receptor (AR) status of the appendix testis (AT) in congenital undescended and retractile testes. Materials and methods Twenty-four appendix testes (AT) were removed from 21 boys to detect AR expression by immunohistochemistry and immunofluorescence staining. Group 1 (n = 3) includes ATs from three patients with unilateral and group 2 (n = 6) with bilateral congenital undescended testis. All patients with bilateral form had been previously treated with human chorionic gonadotropin (hCG) without an acceptable effect. Group 3 (n = 12) includes ATs collected from 12 boys with acquired undescended testis or retractile testicle. Group 4 (n = 3) includes ATs from three young adults who received hCG treatment for undescended testis in their childhood and underwent open testicular biopsy to investigate infertility. Further seven ATs were collected to detect AR mRNA using RT-PCR analysis. Results Both immunohistochemistry and immunofluorescence staining of ATs showed AR expression in 100 % of the cases in groups 3 and 4 (12/12 and 3/3), but there was no visible AR expression in groups 1 and 2 (0/3 and
* Gyula Drabik [email protected] 1
Department of Urology, Faculty of Medicine, University of Debrecen, Nagyeredi krt. 98, Debrecen 4032, Hungary
2
Division of Urology, Department of Pediatric Surgery, Astrid Lindgren Children’s Hospital, Karolinska University Hospital, Stockholm, Sweden
3
Department of Physiology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary
4
Division of Pathology, Kenézy Gyula Hospital, Debrecen, Hungary
0/6); however, RT-PCR analysis revealed mRNA expression of AR both in congenital undescended and in retractile testicles. Conclusions The presence of AR in the epithelial cells of AT in patients with retractile testicle and its absence in patients with congenital undescended testis can be a possible cause of the effectiveness of hormonal treatment in retractile testis and its inefficacy in patients with congenital undescended testis. Keywords Androgen receptor · Appendix testis · hCG treatment · Undescended testis
Introduction The appendix testis (AT), also called Morgagni hydatid, is the vestigial remnant of the Mullerian duct [1]. According to our recent knowledge, that has no function and undergoes physiological involution in late adulthood. Several studies have determined the occurrence of AT which varies from 62 to 92 %, depending on the age of the investigated population [2–4]. Previous articles described significant differences in the occurrence [3] and the anatomical structure of vascular fibrous core [5] of AT in cryptorchidism. The presence of androgen receptor (AR) in the AT of the descended testes and acquired undescended testes and its absence in patients with congenital undescended testis (CUDT), which have been detected by immunostaining and immunofluorescence labelling, suggest that AT might play a role in the process of testicular descent [6]. The recently recommended treatment of undescended testis is the surgical therapy conducted between 6 and 12–18 months [7, 8], and the indication of the hormonal treatment is still debated. Since the introduction of human
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chorionic gonadotropin (hCG) treatment in the 1930s [9], several studies and meta-analyses have described the usefulness of this therapy and most of them do agree that its overall efficacy is about 20 %, less if retractile testes were excluded and higher for distal UDT [10–12]. Despite this poor result, the indication of hCG therapy has been widely accepted in the treatment of UDT over the past decades, and although recent studies highlight its immediate and late side effects (most notably, apoptotic loss of spermatogonia and reduction in the size of the testes in adulthood) [13, 14], the primary or postoperative hormonal therapies have still remained recommended in certain selected cases of UDT [15, 16]. For the further examination of the possible role of AT, we aimed to investigate AR expression in patients who received hCG therapy for the various forms of UDT. However, the protein-level expression of AR was demonstrated in our previous studies [6, 17], mRNA expression of AR has never been examined, and the question whether the absence of AR protein in the epithelial layer of AT in patients with congenital UDT is a result of some focal epithelial damage or of the lack of mRNA expression is still open.
Materials and methods Patient selection and collection of samples A total of 31 ATs were used for immunochemistry, immunofluorescence staining and reverse transcription polymerase chain reaction (RT-PCR). Operations were carried out in the Department of Pediatric Surgery of Institute of Pediatrics and Department of Urology, University of the Medical and Health Science Center, University of Debrecen (MHSCUD). Written informed consent was obtained from the parents or guardians before the children entered the study. The study protocol was approved by the Ethics Committee of MHSCUD. The following classification was used: testes that have not passed down into the scrotum were considered as CUDT; the testis that was originally found in a scrotal position but subsequently, along with a longitudinal growth, ascended out of the scrotum was considered as acquired undescended testis (AUDT); testis that can descend fully into the scrotum but moves freely up into the inguinal canal was considered as retractile testis (RT). Twenty-four ATs were collected from 21 patients to detect AR expression with immunohistochemistry and immunofluorescence staining. Nine ATs were collected from boys who underwent orchidopexy because of CUDT (age 12 months to 5 years, mean 26 months). Three patients had unilateral CUDT without previous
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hCG treatment (group 1). Another six ATs (group 2) were removed from three patients (age 14–22 months, mean 18 months) who had been treated with hCG (Choragon, Ferring Pharmaceuticals Ltd, Copenhagen, Denmark, dosage: 500 IU twice a week for 5 weeks) without an acceptable result. Group 3 (n = 12, age 21 months to 10 years, mean 39 months) includes ATs which were removed from patients who were operated on because of AUDT, which were previously followed up because of RT, or RT which became hypoplastic during the follow-up period. Group 4 (n = 3, age 23–26 years, mean age 24 years) comprises ATs of three young adults who were treated successfully with hCG because of undescended testis in their childhood (data from patients anamnesis, without documentation) and underwent open testicular biopsy for the investigation of infertility. Further seven ATs were removed for mRNA detection of ARs with RT-PCR analysis, three from patients with undescended testis and four from patients with retractile testis (AUDT). Method of immunohistochemistry and laser scanning confocal microscopy The technique which was described in detail in our previous work [6] was completed with counterstain. Summarizing this, an immunohistochemical staining procedure was performed in which formalin-fixed, paraffin-embedded tissue sections of the excised AT were labelled for ARs with mouse monoclonal antihuman AR antibody (clone F39.4.1, BioGenex, San Ramon, CA, USA). ARs were labelled and visualization was carried out using diaminobenzidine (DAB). Brown staining for AR of the investigated cells was considered as a positive immunoreaction. After being incubated with primary antibody, the sections were stained with the fluorescein isothiocyanate (FITC)-conjugated goat antimouse secondary antibody (1:500, Vector Laboratories, Burlingame, CA, USA). Sections were counterstained with 4,6-diamidino-2-phenylindole (DAPI, Vector Laboratories). Receptors were imaged using LSM 510 laser scanning confocal microscope (Zeiss, Oberkochen, Germany) with a 63Ą water immersion objective (1.2 NA; Zeiss). Green fluorescence staining for AR was considered as a positive reaction. RT‑PCR analysis For RT-PCR analysis, appendices were stored in RNAlater solution at −70 °C. Total RNA was isolated from the appendices with Trizol reagent. We sonicated the samples in 1 ml Trizol reagent three times for 10 s each with half maximal output and power. The sequence of primer pair was as follows: forward primer: 5′-CAG GAA AGC GAC
Int Urol Nephrol
Fig. 1 Protein expression of AR demonstrated by immunohistochemistry and immunofluorescence staining of ATs. A representative specimen was taken from a patient with AUDT (group 3). a Brown staining in the tissue sections in the epithelial surface of AT shows the
AR expression with immunohistochemistry (magnification: ×40). b Green fluorescence signals show the AR expression by confocal laser microscopy (bar 30 µm)
TTC ACC G-3′, reverse primer: 5′-GAG TCA TCC CTG CTT CAT AAC AT-3′. PCR were allowed to proceed in a final volume of 50 μL (containing 1 μL forward and reverse primers, 1 μL dNTP [200 μM], and 5 units of Promega GoTaq® DNA polymerase in 1× reaction buffer) in a programmable thermocycler (Eppendorf Mastercycle, Netheler, Hinz GmbH, Hamburg, Germany) with the following settings: for 2 min at 95 °C for initial denaturation followed by repeated cycles of denaturation at 94 °C for 1 min, primer annealing for 60 s at an optimized temperature, and extension at 72 °C for 1 min 30 s. After the final cycle, further extension was allowed to proceed for another 10 min at 72 °C. PCR products were analysed using a 1.5 % agarose gel. The gel was stained with 0.01 % ethidium bromide solution for 30 min and observed in UV light.
was not detected either with immunohistochemistry or with immunofluorescence staining in groups 1 and 2 (0/3 and 0/6). Its image captured in a CUDT patient is demonstrated by Fig. 2a, b. Our examinations revealed mRNA expression of AR with RT-PCR analysis in all samples taken from CUDT (3/3) and AUDT patients (4/4) (Fig. 3).
Results AR positivity was observed as a brown staining of the cells of the surface epithelium and gland-like structures in immunohistochemistry sections. Stromal cells were stained variably. Immunofluorescence labelling showed a green, nuclear and cytoplasmic staining in the cells of the epithelial surface and gland-like structures. The protein level expression of AR was demonstrated using immunohistochemistry (Fig. 1a) and immunofluorescence staining (Fig. 1b) in the tissue sections of ATs from all patients with AUDT (group 3, 12/12) and from all patients who underwent open testicular biopsy during infertility examinations (group 4, 3/3). AR expression
Discussion There are several forms of hormone therapies which are known and used in the treatment of undescended testis, such as primary, neoadjuvant and postoperative hormonal treatments, but the role of these treatments in the therapy of cryptorchidism is still controversial. The most recent guidelines AUA 2014 [18] and EAU 2013 [19] do not support the use of hormonal therapy to induce testicular descent as a primary treatment; however, there are a few countries where this therapy is frequently used and recommended [16, 20]. Although there are no long-term results available regarding the use of preoperative neoadjuvant and postoperative LHRH or hCG administration to improve infertility, the first examinations described their favourable effect on germ cell maturation and/or sperm production [21–23]. Drugs used for hormonal treatment include androgens (testosterone), hCG, gonadotropin-releasing hormone (GnRH), follicle-stimulating hormone (FSH) and human menopausal gonadotropin (hMG), or their combinations. The first introduced and most frequently used hormonal therapy for undescended testes was hCG, which stimulates the Leydig cells of the testicle to produce testosterone. From the
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Fig. 2 Lack of protein expression of AR demonstrated by immunohistochemistry and immunofluorescence staining of AT in a specimen which was taken from a patient with CUDT (group 1). a The absence of brown staining in the epithelial surface of AT confirms the lack of
Fig. 3 mRNA expression of AR with RT-PCR analysis in all specimens taken from CUDT and AUDT patients
1930s to the early 2000s, hCG administration was a widely accepted treatment method in cryptorchidism. The first descriptions about its harmful side effects, i.e. transient inflammatory changes in the testes, germ cell apoptosis and decreased testicular volume in adulthood [13, 24], led to the decreased popularity of hCG treatment. The Nordic consensus on treatment of UDT was the first to publish on the contraindication of the hormonal treatment for undescended testis, due to its poor results and adverse effects [7]. HCG therapy was used in the treatment of bilateral UDT in the Departments of Paediatric Surgery and Urology of MHSCUD up to 2009. Our investigations on the AR status of AT included examinations of Morgagni hydatids which were collected from patients who were operated on because of the various disorders of testicular descent. After a few years, we have completed our study with examinations of ATs which were collected from young adults who underwent successful hormonal treatment in their
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AR expression by immunohistochemistry in CUDT patients (magnification: ×40). b The absence of green fluorescence signal by confocal laser microscopy (bar 30 µm) confirms the lack of AR expression in CUDT patients
childhood because of bilateral undescended testis and who had open testis biopsy as a part of infertility investigations. Although several studies have previously reported on the protein expression of AR in Morgagni hydatids [6, 17, 25], whether the absence of these receptors in patients with CUDT is ascribed to the failure of mRNA expression or is a consequence of other local disturbances is yet unclear. In this study, we employed RT-PCR analysis to examine the mRNA expression of ARs in ATs taken from CUDT and AUDT patients and could demonstrate mRNA expression of ARs in all the examined specimens. These results do support that absence of ARs in the epithelial layer of AT in CUDT patients is a result of some local effects. The presence of AR protein in the epithelial layers of ATs showed a strong correlation with the different forms of undescended testes and with the efficacy of hCG treatment. Investigations revealed the absence of AR expression in specimens which were removed from patients with CUDT (group 1) and the presence in patients with AUDT (group 3). We confirmed the absence of AR protein in patients treated with hCG (group 2) without any satisfactory results and its presence in those young adults who underwent successful hCG treatment in their childhood which resulted in testicular descent (group 4). Although the exact detailed mechanism of hCG treatment is still unclear and the relation between AT and testicular descent is not yet identified, our findings comply with the observation that 72.7–100 % of retractile testes respond to hCG administration [26], while only 20 % of the undescended testes descend with hCG therapy. HCG treatment may have a limited role in the evaluation of undescended testis in patients younger than 2 years [12].
Int Urol Nephrol
Conclusions The presence of AR in the epithelial cells of ATs shows correlation with the efficacies of hormonal treatment in patients with undescended testis and can be a possible cause of the effectiveness of hCG treatment in RT and its inefficacy in patients with CUDT. Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest. Ethical standard All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent Written informed consent was obtained from the parents or guardians before the children entered the study. The study protocol was approved by the Ethics Committee of MHSCUD.
References 1. Atkinson GO Jr, Patrick LE, Ball TI Jr, Stephenson CA, Broecker BH, Woodard JR (2009) The normal and abnormal scrotum in children: evaluation with color Doppler sonography. AJR 158:613–617 2. Johnson KA, Dewbury KC (1996) Ultrasound imaging of the appendix testis and appendix epididymis. Clin Radiol 51:335–337 3. Józsa T, Csízy I, Kutasy B, Cserni T, Flaskó T (2008) Decreased incidence of appendix testis in cryptorchidism with intraoperative survey. Urol Int 80:317–320 4. Sahni D, Jit I, Joshi K, Sanjeev S (1996) Incidence and structure of the appendices of the testis and epididymis. J Anat 189:341–348 5. Tostes GD, Costa SF, de Carvalho JP, Costa WS, Sampaio FJ, Favorito LA (2013) Structural analysis of testicular appendices in patients with cryptorchidism. Int Braz J Urol 39:240–247 6. Józsa T, Dienes B, Telek A, Hargitai Z, Pór A, Kiss C (2008) Differential expression of androgen and estrogen receptor of appendix testis in patients with descended and undescended testes. Int J Urol 15:171–174 7. Ritzén EM, Bergh A, Bjerknes R, Christiansen P, Cortes D, Haugen SE, Jörgensen N, Kollin C, Lindahl S, Läckgren G, Main KM, Nordenskjöld A, Rajpert-De Meyts E, Söder O, Taskinen S, Thorsson A, Thorup J, Toppari J, Virtanen H (2007) Nordic consensus on treatment of undescended testes. Acta Paediatr 96:638–643 8. Hutson JM, Balic A, Nation T, Southwell B (2010) Cryptorchidism. Semin Pediatr Surg 19:215–224 9. Schapiro B (1957) 25 Years of hormonal treatment of cryptorchidism. Harefuah 15(53):198–199
10. Pyörälä S, Huttunen NP, Uhari M (1995) A review and metaanalysis of hormonal treatment of cryptorchidism. J Clin Endocrinol Metab 80:2795–2799 11. Henna MR, Del Nero RG, Sampaio CZ, Atallah AN, Schettini ST, Castro AA et al (2004) Hormonal cryptorchidism therapy: systematic review with metanalysis of randomized clinical trials. Pediatr Surg Int 20:357–359 12. Miller OF, Stock JA, Cilento BG, McAleer IM, Kaplan GW (2003) Prospective evaluation of human chorionic gonadotropin in the differentiation of undescended testes from retractile testes. J Urol 169:2328–2331 13. Dunkel L, Taskinen S, Hovatta O, Tilly JL, Wikstrom S (1997) Germ cell apoptosis after treatment of cryptorchidism with human chorionic gonadotropin is associated with impaired reproductive function in the adult. J Clin Invest 100:2341–2346 14. Ong C, Hasthorpe S, Hutson JM (2005) Germ cell development in the descended and cryptorchid testis and the effects of hormonal manipulation. Pediatr Surg Int 21:240–254 15. Hadziselimovic F, Zivkovic D (2007) Is the prohibition of hormonal treatment for cryptorchidism, as suggested by the Nordic consensus group, justifiable? Acta Paediatr 96:1368–1369 16. Abacı A, Çatlı G, Anık A, Böber E (2013) Epidemiology, classification and management of undescended testes: does medication have value in its treatment? J Clin Res Pediatr Endocrinol 5:65–72 17. Józsa T, Telek A, Kutasy B, Benyó M, Csanádi G, Kovács I, Balla G, Flaskó T, Csernoch L, Kiss C (2009) Effect of hydrocele on appendix testis in children. Asian J Androl 11:741–745 18. Kolon TF, Herndon CD, Baker LA, Baskin LS, Baxter CG, Cheng EY, Diaz M, Lee PA, Seashore CJ, Tasian GE, Barthold JS (2014) American Urological Association Evaluation and treatment of cryptorchidism: AUA guideline. J Urol 192:337–345 19. Tekgül S, Dogan HS, Hoebeke P, Kocvara R, Nijman JM, Radmayr C, Stein R (2013) Recommendations for cryptorchidism. EAU Guidelines on Pediatric Urology. http://uroweb.org/ wp-content/uploads/22-Paediatric-Urology_LR.pdf 20. Mathers MJ, Sperling H, Rübben H, Roth S (2009) The undescended testis: diagnosis, treatment and long-term consequences. Dtsch Arztebl Int 106:527–532 21. Schwentner C, Oswald J, Kreczy A, Lunacek A, Bartsch G, Deibl M, Radmayr C (2005) Neoadjuvant gonadotropin-releasing hormone therapy before surgery may improve the fertility index in undescended testes: a prospective randomized trial. J Urol 173(974–977):22 22. Hadziselimović F, Herzog B (1997) Treatment with a luteinizing hormone-releasing hormone analogue after successful orchiopexy markedly improves the chance of fertility later in life. J Urol 158:1193–1195 23. Huff DS, Snyder HM 3rd, Rusnack SL, Zderic SA, Carr MC, Canning DA (2001) Hormonal therapy for the subfertility of cryptorchidism. Horm Res 55:38–40 24. Hjertkvist M, Bergh A, Damber JE (1988) HCG treatment increases intratesticular pressure in the abdominal testis of unilaterally cryptorchid rats. J Androl 9:116–120 25. Samnakay N, Cohen RJ, Orford J, King PA, Davies RJ (2003) Androgen and oestrogen receptor status of the human appendix testis. Pediatr Surg Int 19:520–524 26. Metin A, Kayigil O, Ahmed SI (2005) The efficacy of human chorionic gonadotropin in retractile testis. Neuro Endocrinol Lett 26:39–42
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UROLOGY - ORIGINAL PAPER
Correlation between the androgen receptor status of the appendix testis and the efficacy of human chorionic gonadotropin treatment in undescended testis Gyula Drabik1 · Tamás Józsa2 · Olga Ruzsnavszky3 · Kornél Kistamás4 · Beatrix Dienes4 · Ilona Kovacs4 · Mátyás Benyó1 · Tibor Flaskó1
Received: 30 April 2015 / Accepted: 13 June 2015 © Springer Science+Business Media Dordrecht 2015
Abstract Purpose To compare the androgen receptor (AR) status of the appendix testis (AT) in congenital undescended and retractile testes. Materials and methods Twenty-four appendix testes (AT) were removed from 21 boys to detect AR expression by immunohistochemistry and immunofluorescence staining. Group 1 (n = 3) includes ATs from three patients with unilateral and group 2 (n = 6) with bilateral congenital undescended testis. All patients with bilateral form had been previously treated with human chorionic gonadotropin (hCG) without an acceptable effect. Group 3 (n = 12) includes ATs collected from 12 boys with acquired undescended testis or retractile testicle. Group 4 (n = 3) includes ATs from three young adults who received hCG treatment for undescended testis in their childhood and underwent open testicular biopsy to investigate infertility. Further seven ATs were collected to detect AR mRNA using RT-PCR analysis. Results Both immunohistochemistry and immunofluorescence staining of ATs showed AR expression in 100 % of the cases in groups 3 and 4 (12/12 and 3/3), but there was no visible AR expression in groups 1 and 2 (0/3 and
* Gyula Drabik [email protected] 1
Department of Urology, Faculty of Medicine, University of Debrecen, Nagyeredi krt. 98, Debrecen 4032, Hungary
2
Division of Urology, Department of Pediatric Surgery, Astrid Lindgren Children’s Hospital, Karolinska University Hospital, Stockholm, Sweden
3
Department of Physiology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary
4
Division of Pathology, Kenézy Gyula Hospital, Debrecen, Hungary
0/6); however, RT-PCR analysis revealed mRNA expression of AR both in congenital undescended and in retractile testicles. Conclusions The presence of AR in the epithelial cells of AT in patients with retractile testicle and its absence in patients with congenital undescended testis can be a possible cause of the effectiveness of hormonal treatment in retractile testis and its inefficacy in patients with congenital undescended testis. Keywords Androgen receptor · Appendix testis · hCG treatment · Undescended testis
Introduction The appendix testis (AT), also called Morgagni hydatid, is the vestigial remnant of the Mullerian duct [1]. According to our recent knowledge, that has no function and undergoes physiological involution in late adulthood. Several studies have determined the occurrence of AT which varies from 62 to 92 %, depending on the age of the investigated population [2–4]. Previous articles described significant differences in the occurrence [3] and the anatomical structure of vascular fibrous core [5] of AT in cryptorchidism. The presence of androgen receptor (AR) in the AT of the descended testes and acquired undescended testes and its absence in patients with congenital undescended testis (CUDT), which have been detected by immunostaining and immunofluorescence labelling, suggest that AT might play a role in the process of testicular descent [6]. The recently recommended treatment of undescended testis is the surgical therapy conducted between 6 and 12–18 months [7, 8], and the indication of the hormonal treatment is still debated. Since the introduction of human
13
chorionic gonadotropin (hCG) treatment in the 1930s [9], several studies and meta-analyses have described the usefulness of this therapy and most of them do agree that its overall efficacy is about 20 %, less if retractile testes were excluded and higher for distal UDT [10–12]. Despite this poor result, the indication of hCG therapy has been widely accepted in the treatment of UDT over the past decades, and although recent studies highlight its immediate and late side effects (most notably, apoptotic loss of spermatogonia and reduction in the size of the testes in adulthood) [13, 14], the primary or postoperative hormonal therapies have still remained recommended in certain selected cases of UDT [15, 16]. For the further examination of the possible role of AT, we aimed to investigate AR expression in patients who received hCG therapy for the various forms of UDT. However, the protein-level expression of AR was demonstrated in our previous studies [6, 17], mRNA expression of AR has never been examined, and the question whether the absence of AR protein in the epithelial layer of AT in patients with congenital UDT is a result of some focal epithelial damage or of the lack of mRNA expression is still open.
Materials and methods Patient selection and collection of samples A total of 31 ATs were used for immunochemistry, immunofluorescence staining and reverse transcription polymerase chain reaction (RT-PCR). Operations were carried out in the Department of Pediatric Surgery of Institute of Pediatrics and Department of Urology, University of the Medical and Health Science Center, University of Debrecen (MHSCUD). Written informed consent was obtained from the parents or guardians before the children entered the study. The study protocol was approved by the Ethics Committee of MHSCUD. The following classification was used: testes that have not passed down into the scrotum were considered as CUDT; the testis that was originally found in a scrotal position but subsequently, along with a longitudinal growth, ascended out of the scrotum was considered as acquired undescended testis (AUDT); testis that can descend fully into the scrotum but moves freely up into the inguinal canal was considered as retractile testis (RT). Twenty-four ATs were collected from 21 patients to detect AR expression with immunohistochemistry and immunofluorescence staining. Nine ATs were collected from boys who underwent orchidopexy because of CUDT (age 12 months to 5 years, mean 26 months). Three patients had unilateral CUDT without previous
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hCG treatment (group 1). Another six ATs (group 2) were removed from three patients (age 14–22 months, mean 18 months) who had been treated with hCG (Choragon, Ferring Pharmaceuticals Ltd, Copenhagen, Denmark, dosage: 500 IU twice a week for 5 weeks) without an acceptable result. Group 3 (n = 12, age 21 months to 10 years, mean 39 months) includes ATs which were removed from patients who were operated on because of AUDT, which were previously followed up because of RT, or RT which became hypoplastic during the follow-up period. Group 4 (n = 3, age 23–26 years, mean age 24 years) comprises ATs of three young adults who were treated successfully with hCG because of undescended testis in their childhood (data from patients anamnesis, without documentation) and underwent open testicular biopsy for the investigation of infertility. Further seven ATs were removed for mRNA detection of ARs with RT-PCR analysis, three from patients with undescended testis and four from patients with retractile testis (AUDT). Method of immunohistochemistry and laser scanning confocal microscopy The technique which was described in detail in our previous work [6] was completed with counterstain. Summarizing this, an immunohistochemical staining procedure was performed in which formalin-fixed, paraffin-embedded tissue sections of the excised AT were labelled for ARs with mouse monoclonal antihuman AR antibody (clone F39.4.1, BioGenex, San Ramon, CA, USA). ARs were labelled and visualization was carried out using diaminobenzidine (DAB). Brown staining for AR of the investigated cells was considered as a positive immunoreaction. After being incubated with primary antibody, the sections were stained with the fluorescein isothiocyanate (FITC)-conjugated goat antimouse secondary antibody (1:500, Vector Laboratories, Burlingame, CA, USA). Sections were counterstained with 4,6-diamidino-2-phenylindole (DAPI, Vector Laboratories). Receptors were imaged using LSM 510 laser scanning confocal microscope (Zeiss, Oberkochen, Germany) with a 63Ą water immersion objective (1.2 NA; Zeiss). Green fluorescence staining for AR was considered as a positive reaction. RT‑PCR analysis For RT-PCR analysis, appendices were stored in RNAlater solution at −70 °C. Total RNA was isolated from the appendices with Trizol reagent. We sonicated the samples in 1 ml Trizol reagent three times for 10 s each with half maximal output and power. The sequence of primer pair was as follows: forward primer: 5′-CAG GAA AGC GAC
Int Urol Nephrol
Fig. 1 Protein expression of AR demonstrated by immunohistochemistry and immunofluorescence staining of ATs. A representative specimen was taken from a patient with AUDT (group 3). a Brown staining in the tissue sections in the epithelial surface of AT shows the
AR expression with immunohistochemistry (magnification: ×40). b Green fluorescence signals show the AR expression by confocal laser microscopy (bar 30 µm)
TTC ACC G-3′, reverse primer: 5′-GAG TCA TCC CTG CTT CAT AAC AT-3′. PCR were allowed to proceed in a final volume of 50 μL (containing 1 μL forward and reverse primers, 1 μL dNTP [200 μM], and 5 units of Promega GoTaq® DNA polymerase in 1× reaction buffer) in a programmable thermocycler (Eppendorf Mastercycle, Netheler, Hinz GmbH, Hamburg, Germany) with the following settings: for 2 min at 95 °C for initial denaturation followed by repeated cycles of denaturation at 94 °C for 1 min, primer annealing for 60 s at an optimized temperature, and extension at 72 °C for 1 min 30 s. After the final cycle, further extension was allowed to proceed for another 10 min at 72 °C. PCR products were analysed using a 1.5 % agarose gel. The gel was stained with 0.01 % ethidium bromide solution for 30 min and observed in UV light.
was not detected either with immunohistochemistry or with immunofluorescence staining in groups 1 and 2 (0/3 and 0/6). Its image captured in a CUDT patient is demonstrated by Fig. 2a, b. Our examinations revealed mRNA expression of AR with RT-PCR analysis in all samples taken from CUDT (3/3) and AUDT patients (4/4) (Fig. 3).
Results AR positivity was observed as a brown staining of the cells of the surface epithelium and gland-like structures in immunohistochemistry sections. Stromal cells were stained variably. Immunofluorescence labelling showed a green, nuclear and cytoplasmic staining in the cells of the epithelial surface and gland-like structures. The protein level expression of AR was demonstrated using immunohistochemistry (Fig. 1a) and immunofluorescence staining (Fig. 1b) in the tissue sections of ATs from all patients with AUDT (group 3, 12/12) and from all patients who underwent open testicular biopsy during infertility examinations (group 4, 3/3). AR expression
Discussion There are several forms of hormone therapies which are known and used in the treatment of undescended testis, such as primary, neoadjuvant and postoperative hormonal treatments, but the role of these treatments in the therapy of cryptorchidism is still controversial. The most recent guidelines AUA 2014 [18] and EAU 2013 [19] do not support the use of hormonal therapy to induce testicular descent as a primary treatment; however, there are a few countries where this therapy is frequently used and recommended [16, 20]. Although there are no long-term results available regarding the use of preoperative neoadjuvant and postoperative LHRH or hCG administration to improve infertility, the first examinations described their favourable effect on germ cell maturation and/or sperm production [21–23]. Drugs used for hormonal treatment include androgens (testosterone), hCG, gonadotropin-releasing hormone (GnRH), follicle-stimulating hormone (FSH) and human menopausal gonadotropin (hMG), or their combinations. The first introduced and most frequently used hormonal therapy for undescended testes was hCG, which stimulates the Leydig cells of the testicle to produce testosterone. From the
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Fig. 2 Lack of protein expression of AR demonstrated by immunohistochemistry and immunofluorescence staining of AT in a specimen which was taken from a patient with CUDT (group 1). a The absence of brown staining in the epithelial surface of AT confirms the lack of
Fig. 3 mRNA expression of AR with RT-PCR analysis in all specimens taken from CUDT and AUDT patients
1930s to the early 2000s, hCG administration was a widely accepted treatment method in cryptorchidism. The first descriptions about its harmful side effects, i.e. transient inflammatory changes in the testes, germ cell apoptosis and decreased testicular volume in adulthood [13, 24], led to the decreased popularity of hCG treatment. The Nordic consensus on treatment of UDT was the first to publish on the contraindication of the hormonal treatment for undescended testis, due to its poor results and adverse effects [7]. HCG therapy was used in the treatment of bilateral UDT in the Departments of Paediatric Surgery and Urology of MHSCUD up to 2009. Our investigations on the AR status of AT included examinations of Morgagni hydatids which were collected from patients who were operated on because of the various disorders of testicular descent. After a few years, we have completed our study with examinations of ATs which were collected from young adults who underwent successful hormonal treatment in their
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Int Urol Nephrol
AR expression by immunohistochemistry in CUDT patients (magnification: ×40). b The absence of green fluorescence signal by confocal laser microscopy (bar 30 µm) confirms the lack of AR expression in CUDT patients
childhood because of bilateral undescended testis and who had open testis biopsy as a part of infertility investigations. Although several studies have previously reported on the protein expression of AR in Morgagni hydatids [6, 17, 25], whether the absence of these receptors in patients with CUDT is ascribed to the failure of mRNA expression or is a consequence of other local disturbances is yet unclear. In this study, we employed RT-PCR analysis to examine the mRNA expression of ARs in ATs taken from CUDT and AUDT patients and could demonstrate mRNA expression of ARs in all the examined specimens. These results do support that absence of ARs in the epithelial layer of AT in CUDT patients is a result of some local effects. The presence of AR protein in the epithelial layers of ATs showed a strong correlation with the different forms of undescended testes and with the efficacy of hCG treatment. Investigations revealed the absence of AR expression in specimens which were removed from patients with CUDT (group 1) and the presence in patients with AUDT (group 3). We confirmed the absence of AR protein in patients treated with hCG (group 2) without any satisfactory results and its presence in those young adults who underwent successful hCG treatment in their childhood which resulted in testicular descent (group 4). Although the exact detailed mechanism of hCG treatment is still unclear and the relation between AT and testicular descent is not yet identified, our findings comply with the observation that 72.7–100 % of retractile testes respond to hCG administration [26], while only 20 % of the undescended testes descend with hCG therapy. HCG treatment may have a limited role in the evaluation of undescended testis in patients younger than 2 years [12].
Int Urol Nephrol
Conclusions The presence of AR in the epithelial cells of ATs shows correlation with the efficacies of hormonal treatment in patients with undescended testis and can be a possible cause of the effectiveness of hCG treatment in RT and its inefficacy in patients with CUDT. Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest. Ethical standard All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent Written informed consent was obtained from the parents or guardians before the children entered the study. The study protocol was approved by the Ethics Committee of MHSCUD.
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