Acquired cryptorchidism: More harm than thought?

Accepted Manuscript Acquired cryptorchidism - more harm than thought? Martin Promm, Josef Schrptor, Claudia Neissner, Fabian Eder, Annette Schrsner, Wolfgang H. Ridis PII:

S1477-5131(16)30034-1

DOI:

10.1016/j.jpurol.2016.04.010

Reference:

JPUROL 2173

To appear in:

Journal of Pediatric Urology

Received Date: 18 January 2016 Accepted Date: 29 April 2016

Please cite this article as: Promm M, Schrptor J, Neissner C, Eder F, Schrsner A, Ridis WH, Acquired cryptorchidism - more harm than thought?, Journal of Pediatric Urology (2016), doi: 10.1016/ j.jpurol.2016.04.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT ACQUIRED CRYPTORCHIDISM - MORE HARM THAN THOUGHT? Martin Promm1, Josef Schröder2, Claudia Neissner1, Fabian Eder2, Annette Schröder1 and Wolfgang H. Rösch1 1) Klinik St. Hedwig, University Medical Center of Regensburg, Department of Paediatric Urology, Regensburg, GERMANY - 2) University Medical Center of Regensburg, Department of Pathology, Regensburg, GERMANY

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INTRODUCTION The acquired cryptorchidism (AC) has been recognized as a subgroup of undescended testes (UDT). There is growing evidence that the compromising effect equals that of congenital UDT (cUDT). We performed an extensive histological examination of biopsies taken from AC patients in a prospective study.

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PATIENTS AND METHODS From 08/2013 to 12/2014, 21 boys (3-12 years of age) underwent a testicular biopsy during orchiopexy for AC. Patient and family histories were taken. The amount of germ cells (GC) per tubule (T), as well as the amount of adult dark spermatogonia (Ad-S) per T were determined by resin semithin sections examination. We also scanned the samples for signs of malformation. Immunohistochemical stains (PLAP, OCT 4) as markers for atypical germ cells were performed.

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RESULTS Four (19%) boys were born prematurely, 2 (9.5%) were small for gestational age (SGA). Nine (43%) boys had a positive family history of UDT. The median of GC/T was 1.06 in boys < 9 years and 0.60 in boys ≥ 9 years. The median of Ad-S/T was 0.02 in boys < 9 years and 0.01 in boys ≥ 9 years. There were no signs for malformation and no atypical cells. PLAP and OCT 4 stains were negative in all specimens.

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CONCLUSIONS Prematurity, SGA and a positive family history appear to be predictors for AC. Extensive histopathological examination of AC revealed a significant reduction of germ cell count and fertility markers, comparable to that in cUDT. The alterations were more severe in boys older than 9 years. Whether or not this is caused by a possibly longer duration of inguinal position is unclear, but this finding suggests that routine checks of testicular position throughout childhood are needed and that there is a cause for continued efforts in educating parents and primary care physicians regarding AC. Current data supports the notion of surgical correction once the diagnosis is made.

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ACCEPTED MANUSCRIPT Introduction The undescended testis (UDT) is one of the most common abnormalities in boys. It is associated with impaired fertility and an increased incidence of testicular cancer [1- 3].

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The term acquired cryptorchidism (AC) describes a testicle above scrotal position, which has previously been descended. This entity has been considered very rare for a long time, but nowadays is widely accepted as rather common, and may contribute to the often criticized “too late” orchiopexy [3-7]. Hack et al. found more than 50% of their patients undergoing orchiopexy had AC [5]. Several terms to describe this entity have been found, e.g. ascended testis, late maldescent, or acquired cryptorchidism (AC), the latter being the most commonly used. It is important to distinguish the AC from the retractile testis, which may be found in inguinal position from time to time, but can easily be maneuvered down into the scrotum and then remains there.

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The age, in which AC occurs varies and is clinically often difficult to pinpoint, however, early school age appears to be typical. To date, the cause of AC is unknown. Several theories for the occurrence of AC are being considered, including anatomical, hormonal and/or neurophysiological causes [4, 8, 9]. Equally unclear is whether or not the late ascension is harmful, however, there is growing evidence that the deleterious effect of AC is comparable to that of primary UDT [4, 10, 11, 12].

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Patients and methods

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In order to investigate the histological changes and the fertility prognosis in our patients, we designed a prospective cohort study and performed extensive histological examinations of biopsies, collected from patients with AC.

From August 2013 to December 2014, the parents of all boys who were presented for surgical correction of AC were offered to participate in a prospective cohort study.

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Boys who underwent preoperative hormonal therapy, previous inguinal surgery, or suffered from syndromes associated with UDT were excluded from the study. In all boys, it was documented that the testicle was found in scrotal position during previous scheduled pediatric routine checkups, which include assessment of testicular position at each visit (table 1). All parents had their child’s documentation of the exam results in a personal booklet, which was reviewed. A total of 23 boys met the criteria, of which 21 accepted to take part in the study. In 17 boys (81 %) AC was first diagnosed by a pediatrician and in 4 boys (19 %) by a family physician before referral to our institution. The patient history was taken, including birth weight and gestational age, and family history of cryptorchidism and testicular cancer. The age at which the testis was first diagnosed in ascended position was inquired.

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ACCEPTED MANUSCRIPT Physical examination was done in two-handed technique. Ultrasound of the testes was performed, and length, volume, and echogenicity of the parenchyma were assessed.

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Inguinal orchidofuniculolysis was done in a standardized technique, orchiopexy was performed according to Shoemaker (scrotal dartos pouch). A patent processus vaginalis and abnormalities such as dissociation of testis and epididymis were documented. A testicular biopsy was taken from the affected side and divided. In boys with bilateral AC, only unilateral specimens were taken, either from the smaller testis, or the one in higher position. One section was put in formalin for histological analysis, the second section was kept in Karnovsky solution for assessment with the resin semithin sections examination. The first specimen was divided again, one half underwent hematoxylin and eosin (HE) staining, the other was incubated with immunohistochemical primary antibodies for anti-PLAP (placental alkaline phosphatase) and anti-OCT-4 (octamer-binding transcription factor 4). PLAP and OCT4 served as markers for atypical germ cells.

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For the resin semithin section examination, the second sample was embedded in Epon resin and polymerized. The Epon block was sectioned in semi-thin sections (0.7µm). After drying the sections were double-stained with toluidine blue and fuchsine, and additionally triplestained according to Laczko for glycogen and TIN detection. The number of tubules (T) containing germ cells (GC), and the number of tubules with atrophic sertoli cells, and tubule morphology were assessed, as well as the amount of germ cells and adult dark spermatogonia (Ad-S). Glycogen storage as a parameter for a disturbance of local metabolism and potential TIN formation as well as the Leydig score were assessed [13]. (Figure 1) All results were analyzed using SPSS Statistics 23, the appropriate statistical test was then selected: the two sample t-test or the Mann-Whitney rank sum test were used to compare the distributions of continuous variables between groups, depending on whether the data were normally distributed or not.

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Results

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This study was reviewed and approved by the Ethical Committee of the University Medical Center of Regensburg (reference no. 13-101-0142). Written informed consent was obtained from all parents.

Twenty-one boys, mean age 90 months (range 39-153 months) were included in our study (Figure 2). Nineteen/21 boys were prepubertal. Four boys (19%) were born prematurely, however, in 3 gestational age was 37 weeks. All 3 boys had a family history of UDT. Mean birth-weight was 3132 g, range 1900 g to 4100 g. Two (9.5%) of the maturely born boys were small for gestational age (SGA). Family history was positive for UDT in 9/21boys (43%), in 4/9 paternally. AC occurred unilaterally in 14 (66%) (10 right-sided, 4 left-sided) and bilaterally in 7 boys (33%). The time at which AC was diagnosed was reported by 16/21 parents to be between 2 to 48 months prior to presentation in our hospital, however, most parents were unable to provide an exact time point. Five parents were entirely unaware when the AC occurred. 3

ACCEPTED MANUSCRIPT Ultrasound of the testes showed age-appropriate testicular volume in most boys. 2/21 had testes smaller than the average, 4 showed a size difference compared to the contralateral testis. Three of the 4 had a unilateral AC. Echogenicity was normal in all boys, but 2 (9.5%) showed testicular microlithiasis (TM). Intraoperatively a patent processus vaginalis was found in 12 boys (57%) and dissociation of testis and epididymis in one boy.

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The HE stains showed no indication of atypical cells or fibrosis. Immunohistological staining for PLAP and OCT 4 was negative in all specimens. Resin semithin section studies showed normal morphology of the tubules in all specimens. Leydig score was 3 (normal) in 19 boys, and 2.7 and 2.3 (reduced) respectively in 2 boys.

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Assessment of tubules containing germ cells as well as germ cell number per tubule was notably reduced in all age groups, as judged by normal reference values. There was a statistically significant difference in germ cell number per tubule, comparing the samples of boys above and below 9 years of age (p = 0.04). The findings are summarized in table 2, asterixes indicate statistically significant differences. There was no correlation between histological findings and the presence or absence of a patent processus vaginalis.

Discussion

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Although AC has nowadays been widely accepted as a relevant clinical entity, risk factors for the occurrence of ascension have not yet been established, and the potential risk for testicular damage is still subject to debate. However, there is growing evidence that the potential harm is as serious as in cUDT [4, 9, 12].

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In our study, 19 % of the boys with AC were born prematurely, which is above the average rate of prematurity in Germany (9%) [14]. With regard to prematurity, it is well-known that it implies a risk for cUDT, but no data is available for AC [15, 16]. Also, compared to the normal population, in our study almost twice as many boys born at term were SGA, which is an independent risk factor for cUDT [17]. Therefore prematurity and SGA may pose the same risk for AC as for cUDT. In our study 9/21 (43%) boys had a positive family history, in 4/9 paternally, in the remaining boys, brothers or uncles were affected. In cUDT, a positive family history is known as a risk factor. Elert et al. found almost 23% with cUDT have a positive family history versus 7.5% in the control group, 4.6-fold higher, if the father is affected [18]. In our study we found an even higher number of relatives with UDT, suggesting that a positive family history is a strong predictor for AC. To our knowledge this is the first time risk factors for AC have been established. Some studies suggest that retractile testes pose a risk for development of AC [9,16]. In our study we were not able to obtain individual patient records in order to answer this question. In our study, the time at which AC was noticed was reported by 16/21 parents to be between 2 to 48 months prior to presentation in our hospital, and to determine the actual occurrence 4

ACCEPTED MANUSCRIPT of AC was not possible. Five parents were even entirely unaware when the ascension of the testis occurred. This is a common problem when working with AC patients. Caused by increasing intervals of routine check-ups in later childhood and less surveillance by the parents, years without noticing and diagnosing the AC may pass. The fact that in our study up to 2 years after occurrence went by, before the patient was referred for assessment, may reflect the issue that the existence and importance of AC is often still unknown to parents and primary care physicians [19].

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Ultrasound of the testes showed age-appropriate testicular volume in most boys. Two/21 had smaller than average testes, and 4 showed – despite regular testicular volume - a size difference compared to the contralateral normally descended testis [20]. Van Brakel et al. examined adult patients with a history of unilateral cUDT and AC [10]. They found that the affected testes in AC often have a smaller than average testicular volume, and a lower testicular volume than the contralateral normally descended testis. In 2 boys (9.5%) TM was found. In descended testes in prepubertal boys the incidence of microlithiasis is only 4.2% [21]. Van Brakel described an incidence of 16% of TM in AC [10]. Whether or not this poses an increased risk for development of testicular cancer is yet unknown, however, counseling of the parents is advisable. It is of note that ultrasound examination, although providing interesting information, does not influence the therapeutical pathway and may therefore not be of critical value in the assessment of this condition. In our health care system, however, it is part of any routine examination of the testicles and performed by pediatric urologists or surgeons themselves in their office, but we do not suggest that it provides information that influences the decision-making process.

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Intraoperatively variable degrees of a patent processus vaginalis were found in 12 boys (57%), of those, 10 boys had a small or nearly obliterated processus vaginalis. This is in line with findings of other studies, who reported that the processus vaginalis was nearly closed in most patients with AC [4]. However, others describe a significantly higher percentage of patent processus vaginalis (28 to 78%), compared to our findings [4, 8, 22, 23, 24]. This may be due to the lack of a clear definition of what constitutes a patent processus vaginalis, as well as the differences in age at surgery.

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Only one of our patients (5%) showed an epididymal anomaly. This is in contrast to the findings of van Brakel et al., who found epididymal anomalies in 11% of his patients with AC. The clinical relevance of this finding has not yet been investigated [24]. The results of the conventional and the immunohistochemical stains showed, according to other authors, no indication of malignancy or atypical cells, but significant alteration in the resin semithin section examination (see below) [4, 25]. This is in contrast to the findings of Suskind et al., who found that fibrosis correlates with a decreased amount of germ cells [25]. We found a significant reduction of tubules containing germ cells as well as germ cell number per tubule in all age groups, however, it was significantly more pronounced in boys older than 9 years. This is in accordance with Tasian et al., who found the degree of germ cell depletion to be dependent on age at surgery in cUDT [26]. This suggests a similar pathophysiology of testicular damage, although the duration of testicular malposition was obviously much shorter in AC. Also, the amount of Ad-spermatogonia was markedly reduced in both age groups, again more pronounced in the older boys. This is surprising, considering the hypothesis of Hadziselimovic, who postulated that the main cause for testicular alterations is the incomplete course of the so called “mini-puberty” in early infancy, which 5

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does not apply to AC [27]. In general, the hormonal axis as cause for the alterations in UDT has lately been put into question. In a basic science study on androgen receptor knockout mice, Li et al. found that other, nonandrogenic factors may be responsible to postnatal germ cell development [28]. Nonetheless, to our knowledge, Rusnack et al. were the first to investigate histological changes in AC, although they did not assess risk factors for AC [4]. They found changes very similar to ours, showing a reduced amount of germ cells in tubules in boys with AC compared to congenital UDT, and also considered the findings a relevant risk factor for infertility. Cortes et al. also confirmed in another histological study that fertility potential is impaired in boys comparable to our patient group, however, they found no predictors for testicular cancer [29].

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Whether patients with AC benefit from immediate orchiopexy remains controversial. Van der Plas et al. showed that awaiting spontaneous descent, which occurred in their study in more than 50% of the cases, seems not to impair fertility potential further in cases of unilateral AC, compared to patients who underwent immediate orchiopexy [12].However, in cases of bilateral AC, early surgical intervention was beneficial with regard to fertility parameters. Bearing in mind that in unilateral UDT paternity rates have not been found to be reduced [30], it will be hard to prove a beneficial effect in cases of unilateral AC without histological follow-up. Nevertheless, one can extrapolate from the findings of van de Plas et al that early surgery does have a positive influence on the respective testicle in patients with AC.

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Our results show more severe alteration, the older the patient. It has to be assumed that the longer the respective testis remains in the ascended position, the more pronounced the histological changes are. Unfortunately we were frequently not able to define exactly for how long the testis was in ascended position prior to diagnosis. The ascended testis was mostly diagnosed during the in our health care system mandatory routine pediatric checkup examinations, which are done according to a defined schedule (see table 1). The interval increases during childhood, therefore the time point of ascension can only be estimated, which becomes even more difficult when the diagnosis was made after a long interval between two checkup examinations. Although one can assume that in the older boys the testicle could have ascended years prior to diagnosis, thereby explaining the more pronounced alterations, this may be speculative. Moreover, although there is still scarce data whether AC has the same harmful potential in testicular cancer development like cUDT, it has been well established that in case of UDT early orchiopexy generally lowers the risk for the occurrence of testicular cancer [31]. Therefore we believe that boys with AC, regardless if uni- or bilateral, should undergo early orchiopexy to reduce the risk of further damage to the testicular parenchyma. There are limitations in our study. Besides the small patient number, the lack of histological control samples, either from the contralateral or normal testis, is a common weakness of studies like this one, however, in order to ensure the integrity of the unaffected testis, no control biopsies were obtained. The issue of not being able to assess the exact time of onset of AC has already been addressed above. Conclusion In our patient population, prematurity, SGA and a positive family history of UDT appear to be predictors for AC. Histological work-up revealed extensive testicular alterations, which were comparable to those described by others in cUDT. The fact that the patients had 6

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presumably gone through mini-puberty during infancy suggests a pathophysiological pathway other than not going through that particular developmental phase. The alterations in testicles with AC were more severe in boys older than 9 years, compared to younger children. Whether or not this is caused by a possibly longer duration of inguinal position is unclear, but this finding suggests that routine checks of testicular position throughout childhood are needed and that there is a cause for continued efforts in educating and altering parents and primary care physicians regarding AC. Current data supports the notion of surgical correction once the diagnosis is made.

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Conflict of interest None

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ACCEPTED MANUSCRIPT References [1] Kolon TF, Herndon CDA, Baker LA, et al. Evaluation and Treatment of Cryptorchidism: AUA Guideline. J Urol 2014; 192: 337-345. [2] Mechlin CW, Kogan BA. What lessons can be learned from testicular histology in undescended testes? Trans Androl Urol 2014; 3(4): 365-369.

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[3] Villmsen AL, Zachau-Christiansen B. Spontaneous alterations in position of the testes. Arch Dis Childh 1966; 41:198-200. [4] Rusnack SL, Wu HY, Huff DS et al. The ascending testis and the testis undescended since birth share the same histopathology. J Urol 2002; 168: 2590-2591.

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[5] Hack WWM, Meijer RW, van der Voort-Doedens LM et al. Previous testicular position in boys referred for an undescenden testis: further explanation of the late orchidopexy enigma? BJU 2003; 92: 293-296.

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[6] Fenton EJM, Woodward AA, Hudson IL, Marschner I. The ascending testis. Pediatr Surg Int 1990; 5: 6-9. [7] Hutson JM, Thorup J. Evaluation and management of the infant with cryptorchidism. Curr Opin Pediatr 2015; 27: 520-524. [8] Meij-de Vries A, Hack WWM, Heif HA, Meijer RW. Perioperative surgical findings in congenital and acquired undescended testis. J Pediatr Surg 2010; 45: 1874-1881.

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[9] Hutson JM, Southwell BR, Li R et al. The regulation of testicular descent and the effects of cryptorchidism. Endocr Rev 2013; 34 (5): 725-752. [10] van Brakel J, de Muinck Keizer-Schrama SMPF, van Casteren NJ et al. Scrotal ultrasound findings in previously congenital and acquired unilateral undescended testes and their contralateral normally descended testis. Andrology 2015; 3: 888-894.

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[11] Dadfar MR. Orchidopexy for retractile testes in infertile men – a prospective clinical study. Urol J 2007; 4: 164-168.

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[12] van der Plas EM, van Brakel J, Meij-de Vries A et al. Acquired undescended testes and fertility potential: is orchiopexy at diagnosis better than awaiting spontaneous descent? Andrology 2015; 3:677-684. [13] Johnson SG. Testicular biopsy score count – a method for registration of spermatogenesis in human testes: normal values and results in 335 hypogonadal males. Hormons 1970; 1: 2-25. [14] AQUA - Institut für angewandte Qualitätsförderung und Forschung im Gesundheitswesen GmbH. Bundesauswertun zum Verfahrensjahr 2010 16/1 – Geburtshilfe. [15] Berkowitz GS, Lapinski RH. Risk factors for cryptorchidism: a nested case-control study. Paediatr Perinat Epidemiol 1996; 10 (1): 39-51. [16] Sijstermans K, Hack WWM, Meijer RW, van der Voort-Doedens LM. The frequency of undescended testis from birth to adulthood: a review. Int J Androl. 2008; 31(1): 1-11.

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ACCEPTED MANUSCRIPT [17] Wollmann HA. Zu klein bei Geburt (SGA). Monatsschrift Kinderheilkunde 2004; 152: 528-535. [18] Elert A, Jahn K, Heidenreich A, Hofmann R. The familial undescended testis. Klein Padiatr 2003; 215 (1): 40-45.

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[19] Hensel KO, Caspers T, Jenke AC et al. Operative management of cryptorchidism: guidelines and reality – a 10-year observational analysis of 3587 cases. BMC Pediatrics 2015; 15: 116-124. [20] Goede J, Hack WW, Sijstermans K et al: Normative values for testicular volume measured by ultrasonography in a normal population from infancy to adolescence. Horm Res Paediatr 2011; 76:56-64.

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[21] Goede J, Hack WW, van der Voort-Doedens LM et al. Prevalence of testicular microlithiasis in asymptomatic males 0-19 years old. J Urol 2009; 182: 1516-1520.

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[22] Donell SC, Rickwood AM, Jee LD et al. Congenital testicular maldescent: significance of the complete hernia sac. Br J Urol 1995; 75: 702-703. [23] Guven A, Kogan BA. Undescended testis in older boys: further evidence that ascending testes are common. J Pediatr Surg 2008; 43: 1700-1704. [24] van Brakel J, Dohle R, de Muinck Keizer-Schrama SMPF. Different surgical findings in congenital and acquired undescended testes. BJU International 2012; 110: E387-E391.

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[25] Suskind A, Hayner-Buchan A, Feustel PJ, Kogan BA. Fibrosis correlates with detailed histological analysis of human undescended testes. BJU International 2008; 101: 1441-1445. [26] Tasian GE, Hittelman AB, Kim GE et al. Age at orchiopexy and testis palpability predict germ and Leydig cell loss: clinical predictors of adverse histological features of cryptorchidism. J Urol 2009; 182: 704-709.

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[27] Hadziselimovic F, Herzog B. Importance of early postnatal germ cell maturation for fertility of cryptorchid male. Horm Res 2001; 55: 6-10.

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[28] Li R, Vannitamby A, Meijer J et al. Postnatal germ cell development during mini-puberty in the mouse does not require androgen receptor: implications for managing cryptorchidism. J Urol 2015; 193: 1361-1367. [29] Cortes D, Thorup JM, Visfeldt J. Cryptorchidism: aspect of fertility and neoplasms. Horm Res 2001; 55: 21-27. [30] Lee PA, Coughlin MT. The single testis: paternity after presentation as unilateral cryptorchidism. J Urol. 2002; 168: 1680-1683. [31] Pettersson A, Richiardi L, Nordenskjold A et al. Age at surgery for undescended testis and risk of testicular cancer. N Engl J Med 2007; 356: 1835-1841.

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Number of patients

Figure 2:

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Figure 1: Yellow arrows indicate Adult dark-spermatogonia Green arrows indicate Glycogen storage

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Age of patients in years

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ACCEPTED MANUSCRIPT Table 1: Time period of checkups

#1

Directly after birth

#2

3rd-10th day of life

#3

4th-5th week of life

#4

3rd-4th month of life

#5

6th-7th month of life

#6

10th-12th month of life

#7

21st-24th month of life

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Routine checkup

#7a

34th-36th month of life 46th-48th month of life

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#8 #9

60th-64th month of life

#10

12th-14th year of life

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Table 2 < 9 years

> 9 years

(n=10)

(n=11)

median

Reference value [7,25]

median

Reference value [7,25]

43%

100%

29%*

100%

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n= 21

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N° of tubules w/ germ cells N° of tubules w/ atrophic sertoli cells

13.5%

Germ cells per tubule

1.06

>4

0.60*

>4

Ad-spermatogonia per tubule

0.02

0.1-0.2

0.01

0.1-0.2

17.5%

Asteriks indicates statistically significant difference from < 9 years (p < 0.05)

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ACCEPTED MANUSCRIPT Key: Figure 1: Representative Resin semi-thin section of a testicular biopsy (63-fold magnification) Figure 2: Number of patients with AC by age

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Table 1: Timing of routine pediatric checkups

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Table 2: Histological findings from testicular biopsies sorted by age groups (younger and older than 9 years).

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