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Pre- and postoperative status of gonadotropins (FSH and LH) and inhibin-B in relation to testicular histopathology at orchiopexy in infant boys with unilateral undescended testes
Journal of Pediatric Urology (2015) 11, 25.e1e25.e5
Pre- and postoperative status of gonadotropins (FSH and LH) and inhibin-B in relation to testicular histopathology at orchiopexy in infant boys with unilateral undescended testes a
The Department of Pediatric Surgery, Rigshospitalet, Copenhagen, Denmark
b
Faculty of Health Science, University of Copenhagen, Denmark
c The Department of Pathology, Rigshospitalet, Copenhagen, Denmark
d
Section of Endocrinology, Department of Pediatrics, Hvidovre Hospital, Copenhagen, Denmark Correspondence to: J. Thorup, 4272 Department of Paediatric Surgery, Rigshospitalet, 9 Blegdamsvej, DK-2100 Copenhagen, Denmark, Tel.: þ45 3545 4868; fax: þ45 3545 3888
[email protected], jorgenthor [email protected], joergen. [email protected] (J. Thorup) Keywords Testis; Cryptorchidism; Orchiopexy; Inhibin-B; Germ cells; FSH LH
Jorgen Thorup a,b, Erik Clasen-Linde c, Sebastian Cortes Thorup a, Dina Cortes b,d Summary Purpose In recent publications of boys with cryptorchidism, gonadotropins are higher and serum inhibin-B lower than normal. To some extent, serum values of inhibin-B reflect the state of germinative epithelium in cryptorchid testes. The aim of the present study was to evaluate the impact of unilateral orchiopexy on levels of gonadotropins and inhibin-B and correlate the hormone findings to the histopathology of the unilateral undescended testis. Methods 50 boys (mean age: 1 year and 2 months) operated for unilateral cryptorchidism had blood samples for serum luteinizing hormone (LH), folliclestimulating hormone (FSH) and inhibin-B taken preoperatively and 3 months to 2 years postoperatively. Testicular biopsies were performed at orchiopexy. The total germ cell number per
transverse tubule and presence of adult dark spermatogonia were estimated. Results Preoperatively, 8 patients had impaired inhibin-B levels. In contrast with the expected normal physiological decline, 16 patients had an absolute rise in serum inhibin-B level postoperatively. The 4 patients who normalized the preoperative impaired serum level of inhibin-B all had low germ cell number in the testis at time of operation. Nine patients had preoperatively elevated serum levels of both LH 0.5e2.3 IU/l (mean: 1.0 IU/l) and FSH 1.3e2.2 IU/l (mean: 1.6 IU/l). Eight of these patients normalized both LH and FSH serum values postoperatively. None of the 4 boys with impaired inhibin-B level that did not normalize after surgery had elevated gonadotropins. Conclusion Orchiopexy in unilateral cryptorchidism had a clear impact on the serum level of inhibin-B and gonadotropins of 32% and 16%, respectively.
Received 29 December 2013 Accepted 12 August 2014 Available online 16 September 2014
http://dx.doi.org/10.1016/j.jpurol.2014.08.007 1477-5131/ª 2014 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.
25.e2 In boys with cryptorchidism the total number of germ cells per transverse tubule (S/T) in testicular biopsies has been the parameter most often used to predict later fertility in adulthood. However, recently the presence or not of Adult dark Spermatogonia (Ad-S) in seminiferous tubules also has been shown to be of great importance when estimating the fertility potential [1,2]. By combining the results of blood samples of serum gonadotropins and serum inhibin-B and determination of S/T from boys with bilateral cryptorchidism, we identified cases with a good fertility prognosis and a low risk of infertility, cases with possible congenital abnormalities related to the descent of the testes (intermediate risk of infertility) and cases with insufficient gonadotropin stimulation as the primary pathogenic factor in cryptorchidism and related high risk of infertility [3]. However, compared with bilateral cryptorchidism, the boys with the unilateral undescended testis present other clinical entities and there are various etiologies of unilateral cryptorchidism. In unilateral cryptorchidism the risk of infertility in adulthood is not as dramatically increased as described in bilateral cases. Lee et al. [4] found that 10% (95% CL 5e15%) of men operated in childhood for unilateral cryptorchidism did not achieve paternity in 1 year compared with 5% in a control group. This figure is in accordance with the findings of Cortes et al. [5] that 9% (95% CL 3e19%) were suspected to be infertile according to semen and hormonal analysis. In a review of 14 studies of men who had unilateral orchiopexy in childhood, 70% of 747 men had least 20 million sperm cells/ml in the ejaculate, which according to the WHO was the lower normal range at the time of the study [6]. That means that there is an effect on fertility among some patients after unilateral orchiopexy, because only 49% of men with persistent unilateral cryptorchidism in adulthood had least 20 million sperm cells/ml in the ejaculate [6]. So in some unilateral cases there are also fertility problems. This is in accordance with histopathological studies showing that in a proportion of cases with unilateral cryptorchidism there is evidence of a bilateral disease [1]. In patients with no germ cells in the biopsy of the unilateral undescended testis at the time of surgery, 33% ended up being infertile according to semen and hormonal analysis, even though the contralateral testis was descended [5]. In recent materials on boys with cryptorchidism, gonadotropins are higher and serum inhibin-B lower than normal [7]. To some extent serum values of inhibin-B reflect the state of germinative epithelium in cryptorchid testes [3]. In selected boys with bilateral cryptorchidism, we found a significant hormonal response to orchiopexy when analyzing pre- and postoperative serum values of gonadotropins and inhibin-B. However, in unilateral cryptorchidism the interpretation of hormone levels before and after surgery has been difficult with no strict conclusion [8]. The aim of the present study was to evaluate the impact of unilateral orchiopexy on serum gonadotropin and inhibin-B levels, and to correlate the hormone findings with the histopathology of the unilateral undescended testis. It is our hypothesis that in unilateral cryptorchidism there is also a hormonal response to orchiopexy when analyzing pre- and postoperative serum values of gonadotropins and inhibin-B.
J. Thorup et al.
Material and methods Patients We included 50 infant boys aged 7 months to 2.5 years (mean: 1 year and 2 months) operated because of unilateral cryptorchidism. All had blood samples prospectively scheduled to be taken immediately prior to surgery and 1 year postoperatively. At surgery the positions of the undescended testes were classified clinically. At the time of orchiopexy all had unilateral testicular biopsies performed. For clinical practical reasons postoperative blood samples were taken 3 months to 2 years (mean: 12 months) after surgery. We excluded patients with intra-abdominal testes, chromosomal abnormalities, associated anomalies and previous inguinal surgery or a non-satisfactory result of surgery regarding position and size of the testis. None had hormonal therapy.
Hormonal assays Blood samples were obtained by venipuncture between 8 am and 11 am. Serum samples were separated from the clot by 10 min centrifugation at 2000 g. Serum was stored at 80 C until analysis. Serum inhibin-B levels were measured using a commercially available inhibin-B ELISA kit (Serotec Ltd., UK) with research kit as recommended by the manufactory instructions. The lower detection limit was 5 pg/ml, and the measurements were made in duplicate. Normal reference serum levels of inhibin-B were defined as described by Andersson et al. [9]. Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were measured by sandwich electrochemiluminescence-immunoassay (ECLIA) singly, with commercially available reagents from Roche (catalog no. 11732234122 for LH and catalog no. 11775863122 for FSH). The lowest value of FSH and LH to be measured was 0.05 IU/l. The intra- and interassay coefficients of variation were less than 7% for both. Normal reference serum levels of gonadotropins were defined as described by Andersson et al. [9].
Testicular biopsies Tissue specimens were fixed in Stieve’s solution, embedded in paraffin, and 4-mm sections were stained with hematoxylineeosin and CD99 (MIC-2). In blinded fashion the number of spermatogonia and gonocytes per tubular transverse section was measured from at least 100 tubular transverse sections (S/T) [5,10]. For every patient the S/T was found. The S/T was considered normal when the value was at least 1.0 at birth, 0.65 at six month and 0.38 in boys aged 1e4 years [5,10] (Fig. 1). A-dark Spermatogonia (Ad-S) was noted if present in the tubular transverse sections (AdS/T 0.01).
Statistical analyses Chi-square and Student-t tests were used to assess statistical significance, and two-sited p values less than 0.05 were considered significant.
Pre- and postoperative status of gonadotropins
25.e3
Figure 1 Spermatogonia and gonocytes per tubular transverse section measured from at least 100 tubular transverse sections (S/ T) in 50 unilateral cryptorchid boys. S/T for every patient is related to the age of the boy at surgery. The 8 cases with preoperative low serum levels of inhibin-B are marked with stars, the 12 cases with serum inhibin-B within normal range and an absolute increase of serum values postoperatively are marked with squares, the rest with small diamonds. The line shows normal lower S/T range based on our previously published normal material [5,10].
Ethics The study was conducted according to the Helsinki II declaration, and informed consent was obtained from the parents of the patients (ethical committee Copenhagen file number KF-01299830).
Results None of the included patients were lost to follow-up. The per-operative positions of the testis were classified as placed in the inguinal canal in 15 cases, placed at the superficial inguinal ring in 32 cases and suprascrotal in 3 cases. The germ cell number per tubular cross section (S/T) in relation to age of surgery is seen in Fig. 1. In 58% (29/50) of the cases S/T was impaired. Six of these cases had also serum inhibin-B below normal range. A total of 8 cases (16%) had serum inhibin-B below the normal range before surgery. Four of these, all with impaired S/T, normalized their serum inhibin-B level postoperatively. After surgery 88% (44/50) had normal serum levels of inhibin-B (Fig. 2A and B). Five (10%) of our 50 patients had a serum level of inhibin-B above the normal median (50 percentile) preoperatively compared with 20 (40%) postoperatively (Fig. 2A and B) (chi-square test: p < 0.05). Among the 42 patients with normal serum inhibin-B at surgery, 12 patients had an absolute rise in serum inhibin-B level from preoperative mean 124 41 pg/ml to postoperative mean 147 39 pg/ml (Fig. 2A). The rest of those 42 patients, except for 2 cases, just followed the normal
percentile decline of serum inhibin-B level physiologically seen in this age group (Fig. 2A and B). Fourteen of these 28 testes had normal S/T. There was a trend that S/T (0.37 0.42) in the testicular biopsies of those patients who increased the serum inhibin-B level after surgery was lower than S/T (0.64 0.64) in the testicular biopsies of those patients who did not have an increase in serum inhibin-B postoperatively (Student-t test: p Z 0.09). Overall, 16 of the 50 patients had a rise in inhibin-B after orchiopexy. Nine patients (18%) had preoperatively elevated serum levels of both luteinizing hormone (LH) 0.5e2.3 IU/l (mean: 1.0 IU/l) and follicle-stimulating hormone (FSH) 1.3e2.2 IU/ l (mean: 1.6 IU/l). Eight of these patients normalized both LH and FSH serum values postoperatively. The last patient persisted with elevated serum level of FSH (2.2 IU/l). Additionally, 2 boys had normal serum FSH but elevated serum levels of LH (0.6 and 0.8 IU/l). Both normalized LH levels postoperatively. Furthermore, 4 boys had normal serum LH but elevated serum levels of FSH (1.4e2.0 IU/l (mean: 1.6 IU/l)). Two of these boys had unchanged values postoperatively (1.4 and 1.5 IU/l). The boys with elevated gonadotropins were slightly older (9 monthse2.5 years old (mean: 1 year and 4 months)) than those with normal preoperative values (7 monthse1 year and 9 months old (mean: 1 year and 1 month)). None of the 4 boys with impaired inhibin-B level before as well as after surgery had elevated gonadotropins. Seventeen of 21 testes (81%) with normal S/T had presence of A-dark spermatogonia compared with 10 of 29 testes (35%) with impaired S/T (chi-square test: p < 0.05).
25.e4
J. Thorup et al.
Figure 2 The serum levels of inhibin-B for every patient before and after surgery are connected with lines. The normal 50% (median) percentile and normal upper and lower range reference serum levels of inhibin-B in relation to age as described by Andersson et al. [9] are marked with thin lines. (A) The 16 cases with absolute increment of serum inhibin-B level after surgery. (B) The 34 cases with serum inhibin-B level following the normal physiological percentile decline of serum inhibin-B level seen in this age group.
Discussion We found that orchiopexy in unilateral cryptorchidism had some positive impact on the serum level of inhibin-B, because overall 16 of the 50 patients had a rise in inhibin-B after orchiopexy in contrast with the normal physiological decline expected in this age group. This is in agreement with Irkilata et al. [11] who found an increment of serum inhibin-B in 22 of 27 boys, whereas 2 boys only had bilateral undescended testes, the rest were unilateral cases. In the study by Irkilata et al. [11], there was a failure of postoperative increase in inhibin-B in 2 out of 3 cases correlated with reduced germ cell number. However, all these boys were much older (5, 10 and 11 years old) than in our study. It is well known that in older boys with cryptorchidism, irreversible damage of testicular epithelium (Sertoli cells only) is more prominent [5,6]. Furthermore, 2 out of these 3 cases actually had bilateral cryptorchidism. In the study by Kollin et al. [8] of pre- and postoperative inhibin-B serum values in 58 and 62 unilateral 1-year-old cryptorchid boys, the inhibin-B serum level seemed higher in the postoperative group (168 88.4 pg/ml) compared with the preoperative group (160 66.5 pg/ml). Inhibin-B is produced by Sertoli cells, and the serum level partly reflects the histological state of seminiferous tubules. Interestingly, we found a high proportion of testes with impaired germ cell number among those boys who had an absolute increment of inhibin-B serum values. So the serum inhibin-B increment may actually reflect an improvement of the histological state of seminiferous tubules of the undescended testis among some after surgery. Our findings are indirectly supported by other studies [8,12]. One study showed that the serum level of inhibin-B correlated positively with testicular volume in newborn boys [12]. This was confirmed by Kollin et al. [8], who found that boys with unilateral cryptorchid testes with high levels of inhibin-B at 6 months and 4 years also had bigger testes at 4 years than boys with low levels of inhibin-B. Furthermore, a good correlation was found between the number of germ and Sertoli cells and testicular volume at time of operation with biopsy [8]. Interestingly, only 10% (5/ 50) of our patients had a serum level of inhibin-B above the
normal median (50 percentile) preoperatively compared with 40% (20/50) postoperatively (Fig. 2A and B). So there was a trend towards a more normal distribution of serum levels of inhibin-B postoperatively. Previously we found a positive correlation between germ cell number in cryptorchid testes operated in childhood and testicular volume of the same testes at follow-up in adulthood [13]. Based on these studies [8,12,13] and our present findings, we found good evidence that, generally in unilateral cryptorchidism, an increment of the serum level of inhibin-B postoperatively may indicate testicular growth as a result of improvement of the histological state of seminiferous tubules. The patients just following the normal physiological percentile decline of serum inhibin-B level seen in this age group seemed to have better S/T when operated, so therefore testicular growth as a result of further improvement of the histological state of seminiferous tubules probably could not be expected to the same degree. It is also important to acknowledge that there is more than a single etiology of unilateral cryptorchidism. In a previous study of fertility parameters among adult men operated in childhood for unilateral cryptorchidism, we found that 9% (6/65) were probably infertile based on evaluation of sperm samples [14]. All these patients had very low S/T when operated in childhood, but only 1 of these 6 infertile patients had increased serum FSH level in adulthood. Kraft et al. [15] recently related childhood undescended testis histology with adult hormone levels and semen analysis. Based on bilateral testicular biopsies taken in childhood at orchiopexy in unilateral cryptorchidism, they also found 9% (8/91) in a high-risk infertility group. At follow-up of these patients, the median sperm density was 2.3 million/ml similar to 3.9 million/ml in the comparable group of patients in our aforementioned study [14]. In the present study, only 3 patients had increased serum level of FSH postoperatively; the other 10 patients with increased serum level of FSH preoperatively had normalized serum values postoperatively. There is a biological decrease of FSH serum levels seen between 6 months and 2 years, which should be taken into consideration and a lower postoperative serum level of FSH would be expected [9]. However, the decrease seen in most patients of this group is
Pre- and postoperative status of gonadotropins more prominent than would have been expected according to changes in age, and the boys with elevated gonadotropins were actually slightly older than those with normal preoperative values. So there is good evidence that early surgery with placing the testes in the scrotum had a beneficial effect on gonadotropin levels, even in those boys with unilateral cryptorchidism. In the report by Kraft et al. [15], more than half of the patients with severe GC/T (germ cells per tubule) histopathology ended up without increased serum level of gonadotropins. In our present material, none of the 4 boys with impaired inhibin-B level before as well as after surgery had elevated gonadotropins. This can be explained by the presence of so-called prepubertal transient hypothalamusepituitaryegonadal hypofunction [1,16]. That means that it is likely that at least 8% of unilateral cryptorchid cases may have a prepubertal transient hypothalamusepituitaryegonadal hypofunction [1,16]. The presence of A-dark spermatogonia in tubular transverse sections in the present study was seen in a significantly higher proportion in testes with normal S/T compared with those with impaired S/T. This is in accordance with our findings in bilateral cases [2]. Impaired transformation of the neonatal gonocytes into type A-dark spermatogonia during the first 12 months of age and subsequent apoptosis of germ cells, may be a pathogenic factor for decreased fertility potential. The transformation of the neonatal gonocytes into type A-dark spermatogonia may be impaired if gonadotropins are insufficient, and when the testis is undescended this leads to further germ cell deterioration. So, if there is an underlying endocrinopathy causing inadequate maturation of the testis, merely putting the testis into the scrotum will not correct that endocrinopathy [17]. A potential weakness of the study relates to the modest sample size of the material and the 1 year and 9 months range of patient age at inclusion in a period of life during which significant normal biological changes in hormone levels and germ cell numbers occur. In fact, fluctuations in inhibin-B and FSH levels were common in boys followed for the first 2 years of life in the longitudinal series reported by Andersson et al. [9]. There is no control group to show what would have happened without orchiopexy. However, it is only speculative to suggest increase of serum inhibin-B levels in such a large group of cryptorchid patients without surgery, when serum inhibin-B levels of normal boys decline in this age group. When taking findings of previous studies into consideration, we find our data add important information to otherwise sparse knowledge on the subject. In conclusion, we found that orchiopexy in unilateral cryptorchidism had a positive impact on the serum levels of inhibin-B and gonadotropins. The serum inhibin-B increment may actually reflect an improvement of the histological state of seminiferous tubules of the undescended testis after surgery.
Conflict of interest None.
25.e5
Funding None.
References [1] Hadziselimovic F, Hoecht B. Testicular histology related to fertility outcome and postpubertal hormone status in cryptorchidism. Klin Pa ¨diatr 2008;220:302. [2] Thorup J, Kvist K, Clasen-Linde E, Petersen BL, Cortes D. The relation between adult-dark spermatogonia and other parameters of fertility potential in cryptorchid testes. J Urol 2013;190:1566. [3] Thorup J, Petersen BL, Kvist K, Cortes D. Bilateral undescended testes classified according to preoperative and postoperative status of gonadotropins and inhibin B in relation to testicular histopathology at bilateral orchiopexy in infant boys. J Urol 2012;188:1436. [4] Lee PA, O’Leary LA, Songer NJ. Paternity after unilateral cryptorchidism: a controlled study. Pediatrics 1996;98:676. [5] Cortes D, Thorup J, Visfeldt J. Cryptorchidism: aspects of fertility and neoplasms. Horm Res 2001;55:21. [6] Cortes D. Cryptorchidism: aspects of pathogenesis, histology and treatment. Scand J Urol Nephrol 1998;32(Suppl 196): 1e54. [7] Suomi AM, Main KM, Kaleva M, Schmidt IM, Chellakooty M, Virtanen HE, et al. Hormonal changes in 3-month-old cryptorchid boys. J Clin Endocrinol Metab 2006;91:953e8. [8] Kollin C, Stukenborg JB, Nurmio M, Sundqvist E, Gustafsson T, So ¨der O, et al. Boys with undescended testes: endocrine, volumetric and morphometric studies on testicular function before and after orchidopexy at nine months or three years of age. J Clin Endocrinol Metab 2012;97:4588. [9] Andersson AM, Toppari J, Haavisto AM, Petersen JH, Simell T, Simell O, et al. Longitudinal reproductive hormone profiles in infants: peak of inhibin B levels in infant boys exceeds levels in adult men. J Clin Endocrinol Metab 1998;83:675. [10] Cortes D, Thorup J, Beck BL. Quantitative histology of germ cells in the undescended testes of human fetuses, neonates and infants. J Urol 1995;154:1188. [11] Irkilata HC, Yildirim I, Onguru O, Aydur E, Musabak U, Dayanc M. The influence of orchiopexy on serum inhibin B level: relationship with histology. J Urol 2004;172:2402. [12] Main KM, Toppari J, Suomi AM, Kaleva M, Chellakooty M, Schmidt IM, et al. Larger testes and higher inhibin B levels in Finnish than in Danish newborn boys. J Clin Endocrinol Metab 2006;91:2732. [13] Cortes D, Thorup J. Histology of testicular biopsies taken at operation for bilateral maldescended testes in relation to fertility in adulthood. BJU 1991;68:285. [14] Cortes D, Thorup J, Lindenberg S, Visfeldt J. Infertility despite surgery for cryptorchidism in childhood can be classified by patients with normal or elevated follicle-stimulating hormone and identified at orchiopexy. BJU Int 2003;91:670. [15] Kraft KH, Canning DA, Snyder 3rd HM, Kolon TF. Undescended testis histology correlation with adult hormone levels and semen analysis. J Urol 2012;188(4 Suppl.):1429. [16] Hadziselimovic F, Herzog B. Hodendystopie. In: Thu ¨roff JW, Schulte-Wissermann, editors. Kinderurologie in Klinik und Praxis. 2 ed., 484. Stuttgart-New York: Thieme; 2000. [17] Thorup J, McLachlan R, Cortes D, Nation TR, Balic A, Southwell BR, et al. What is new in cryptorchidism and hypospadias e a critical review on the testicular dysgenesis hypothesis. J Pediatr Surg 2010;45:2074.
Pre- and postoperative status of gonadotropins (FSH and LH) and inhibin-B in relation to testicular histopathology at orchiopexy in infant boys with unilateral undescended testes a
The Department of Pediatric Surgery, Rigshospitalet, Copenhagen, Denmark
b
Faculty of Health Science, University of Copenhagen, Denmark
c The Department of Pathology, Rigshospitalet, Copenhagen, Denmark
d
Section of Endocrinology, Department of Pediatrics, Hvidovre Hospital, Copenhagen, Denmark Correspondence to: J. Thorup, 4272 Department of Paediatric Surgery, Rigshospitalet, 9 Blegdamsvej, DK-2100 Copenhagen, Denmark, Tel.: þ45 3545 4868; fax: þ45 3545 3888
[email protected], jorgenthor [email protected], joergen. [email protected] (J. Thorup) Keywords Testis; Cryptorchidism; Orchiopexy; Inhibin-B; Germ cells; FSH LH
Jorgen Thorup a,b, Erik Clasen-Linde c, Sebastian Cortes Thorup a, Dina Cortes b,d Summary Purpose In recent publications of boys with cryptorchidism, gonadotropins are higher and serum inhibin-B lower than normal. To some extent, serum values of inhibin-B reflect the state of germinative epithelium in cryptorchid testes. The aim of the present study was to evaluate the impact of unilateral orchiopexy on levels of gonadotropins and inhibin-B and correlate the hormone findings to the histopathology of the unilateral undescended testis. Methods 50 boys (mean age: 1 year and 2 months) operated for unilateral cryptorchidism had blood samples for serum luteinizing hormone (LH), folliclestimulating hormone (FSH) and inhibin-B taken preoperatively and 3 months to 2 years postoperatively. Testicular biopsies were performed at orchiopexy. The total germ cell number per
transverse tubule and presence of adult dark spermatogonia were estimated. Results Preoperatively, 8 patients had impaired inhibin-B levels. In contrast with the expected normal physiological decline, 16 patients had an absolute rise in serum inhibin-B level postoperatively. The 4 patients who normalized the preoperative impaired serum level of inhibin-B all had low germ cell number in the testis at time of operation. Nine patients had preoperatively elevated serum levels of both LH 0.5e2.3 IU/l (mean: 1.0 IU/l) and FSH 1.3e2.2 IU/l (mean: 1.6 IU/l). Eight of these patients normalized both LH and FSH serum values postoperatively. None of the 4 boys with impaired inhibin-B level that did not normalize after surgery had elevated gonadotropins. Conclusion Orchiopexy in unilateral cryptorchidism had a clear impact on the serum level of inhibin-B and gonadotropins of 32% and 16%, respectively.
Received 29 December 2013 Accepted 12 August 2014 Available online 16 September 2014
http://dx.doi.org/10.1016/j.jpurol.2014.08.007 1477-5131/ª 2014 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.
25.e2 In boys with cryptorchidism the total number of germ cells per transverse tubule (S/T) in testicular biopsies has been the parameter most often used to predict later fertility in adulthood. However, recently the presence or not of Adult dark Spermatogonia (Ad-S) in seminiferous tubules also has been shown to be of great importance when estimating the fertility potential [1,2]. By combining the results of blood samples of serum gonadotropins and serum inhibin-B and determination of S/T from boys with bilateral cryptorchidism, we identified cases with a good fertility prognosis and a low risk of infertility, cases with possible congenital abnormalities related to the descent of the testes (intermediate risk of infertility) and cases with insufficient gonadotropin stimulation as the primary pathogenic factor in cryptorchidism and related high risk of infertility [3]. However, compared with bilateral cryptorchidism, the boys with the unilateral undescended testis present other clinical entities and there are various etiologies of unilateral cryptorchidism. In unilateral cryptorchidism the risk of infertility in adulthood is not as dramatically increased as described in bilateral cases. Lee et al. [4] found that 10% (95% CL 5e15%) of men operated in childhood for unilateral cryptorchidism did not achieve paternity in 1 year compared with 5% in a control group. This figure is in accordance with the findings of Cortes et al. [5] that 9% (95% CL 3e19%) were suspected to be infertile according to semen and hormonal analysis. In a review of 14 studies of men who had unilateral orchiopexy in childhood, 70% of 747 men had least 20 million sperm cells/ml in the ejaculate, which according to the WHO was the lower normal range at the time of the study [6]. That means that there is an effect on fertility among some patients after unilateral orchiopexy, because only 49% of men with persistent unilateral cryptorchidism in adulthood had least 20 million sperm cells/ml in the ejaculate [6]. So in some unilateral cases there are also fertility problems. This is in accordance with histopathological studies showing that in a proportion of cases with unilateral cryptorchidism there is evidence of a bilateral disease [1]. In patients with no germ cells in the biopsy of the unilateral undescended testis at the time of surgery, 33% ended up being infertile according to semen and hormonal analysis, even though the contralateral testis was descended [5]. In recent materials on boys with cryptorchidism, gonadotropins are higher and serum inhibin-B lower than normal [7]. To some extent serum values of inhibin-B reflect the state of germinative epithelium in cryptorchid testes [3]. In selected boys with bilateral cryptorchidism, we found a significant hormonal response to orchiopexy when analyzing pre- and postoperative serum values of gonadotropins and inhibin-B. However, in unilateral cryptorchidism the interpretation of hormone levels before and after surgery has been difficult with no strict conclusion [8]. The aim of the present study was to evaluate the impact of unilateral orchiopexy on serum gonadotropin and inhibin-B levels, and to correlate the hormone findings with the histopathology of the unilateral undescended testis. It is our hypothesis that in unilateral cryptorchidism there is also a hormonal response to orchiopexy when analyzing pre- and postoperative serum values of gonadotropins and inhibin-B.
J. Thorup et al.
Material and methods Patients We included 50 infant boys aged 7 months to 2.5 years (mean: 1 year and 2 months) operated because of unilateral cryptorchidism. All had blood samples prospectively scheduled to be taken immediately prior to surgery and 1 year postoperatively. At surgery the positions of the undescended testes were classified clinically. At the time of orchiopexy all had unilateral testicular biopsies performed. For clinical practical reasons postoperative blood samples were taken 3 months to 2 years (mean: 12 months) after surgery. We excluded patients with intra-abdominal testes, chromosomal abnormalities, associated anomalies and previous inguinal surgery or a non-satisfactory result of surgery regarding position and size of the testis. None had hormonal therapy.
Hormonal assays Blood samples were obtained by venipuncture between 8 am and 11 am. Serum samples were separated from the clot by 10 min centrifugation at 2000 g. Serum was stored at 80 C until analysis. Serum inhibin-B levels were measured using a commercially available inhibin-B ELISA kit (Serotec Ltd., UK) with research kit as recommended by the manufactory instructions. The lower detection limit was 5 pg/ml, and the measurements were made in duplicate. Normal reference serum levels of inhibin-B were defined as described by Andersson et al. [9]. Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were measured by sandwich electrochemiluminescence-immunoassay (ECLIA) singly, with commercially available reagents from Roche (catalog no. 11732234122 for LH and catalog no. 11775863122 for FSH). The lowest value of FSH and LH to be measured was 0.05 IU/l. The intra- and interassay coefficients of variation were less than 7% for both. Normal reference serum levels of gonadotropins were defined as described by Andersson et al. [9].
Testicular biopsies Tissue specimens were fixed in Stieve’s solution, embedded in paraffin, and 4-mm sections were stained with hematoxylineeosin and CD99 (MIC-2). In blinded fashion the number of spermatogonia and gonocytes per tubular transverse section was measured from at least 100 tubular transverse sections (S/T) [5,10]. For every patient the S/T was found. The S/T was considered normal when the value was at least 1.0 at birth, 0.65 at six month and 0.38 in boys aged 1e4 years [5,10] (Fig. 1). A-dark Spermatogonia (Ad-S) was noted if present in the tubular transverse sections (AdS/T 0.01).
Statistical analyses Chi-square and Student-t tests were used to assess statistical significance, and two-sited p values less than 0.05 were considered significant.
Pre- and postoperative status of gonadotropins
25.e3
Figure 1 Spermatogonia and gonocytes per tubular transverse section measured from at least 100 tubular transverse sections (S/ T) in 50 unilateral cryptorchid boys. S/T for every patient is related to the age of the boy at surgery. The 8 cases with preoperative low serum levels of inhibin-B are marked with stars, the 12 cases with serum inhibin-B within normal range and an absolute increase of serum values postoperatively are marked with squares, the rest with small diamonds. The line shows normal lower S/T range based on our previously published normal material [5,10].
Ethics The study was conducted according to the Helsinki II declaration, and informed consent was obtained from the parents of the patients (ethical committee Copenhagen file number KF-01299830).
Results None of the included patients were lost to follow-up. The per-operative positions of the testis were classified as placed in the inguinal canal in 15 cases, placed at the superficial inguinal ring in 32 cases and suprascrotal in 3 cases. The germ cell number per tubular cross section (S/T) in relation to age of surgery is seen in Fig. 1. In 58% (29/50) of the cases S/T was impaired. Six of these cases had also serum inhibin-B below normal range. A total of 8 cases (16%) had serum inhibin-B below the normal range before surgery. Four of these, all with impaired S/T, normalized their serum inhibin-B level postoperatively. After surgery 88% (44/50) had normal serum levels of inhibin-B (Fig. 2A and B). Five (10%) of our 50 patients had a serum level of inhibin-B above the normal median (50 percentile) preoperatively compared with 20 (40%) postoperatively (Fig. 2A and B) (chi-square test: p < 0.05). Among the 42 patients with normal serum inhibin-B at surgery, 12 patients had an absolute rise in serum inhibin-B level from preoperative mean 124 41 pg/ml to postoperative mean 147 39 pg/ml (Fig. 2A). The rest of those 42 patients, except for 2 cases, just followed the normal
percentile decline of serum inhibin-B level physiologically seen in this age group (Fig. 2A and B). Fourteen of these 28 testes had normal S/T. There was a trend that S/T (0.37 0.42) in the testicular biopsies of those patients who increased the serum inhibin-B level after surgery was lower than S/T (0.64 0.64) in the testicular biopsies of those patients who did not have an increase in serum inhibin-B postoperatively (Student-t test: p Z 0.09). Overall, 16 of the 50 patients had a rise in inhibin-B after orchiopexy. Nine patients (18%) had preoperatively elevated serum levels of both luteinizing hormone (LH) 0.5e2.3 IU/l (mean: 1.0 IU/l) and follicle-stimulating hormone (FSH) 1.3e2.2 IU/ l (mean: 1.6 IU/l). Eight of these patients normalized both LH and FSH serum values postoperatively. The last patient persisted with elevated serum level of FSH (2.2 IU/l). Additionally, 2 boys had normal serum FSH but elevated serum levels of LH (0.6 and 0.8 IU/l). Both normalized LH levels postoperatively. Furthermore, 4 boys had normal serum LH but elevated serum levels of FSH (1.4e2.0 IU/l (mean: 1.6 IU/l)). Two of these boys had unchanged values postoperatively (1.4 and 1.5 IU/l). The boys with elevated gonadotropins were slightly older (9 monthse2.5 years old (mean: 1 year and 4 months)) than those with normal preoperative values (7 monthse1 year and 9 months old (mean: 1 year and 1 month)). None of the 4 boys with impaired inhibin-B level before as well as after surgery had elevated gonadotropins. Seventeen of 21 testes (81%) with normal S/T had presence of A-dark spermatogonia compared with 10 of 29 testes (35%) with impaired S/T (chi-square test: p < 0.05).
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Figure 2 The serum levels of inhibin-B for every patient before and after surgery are connected with lines. The normal 50% (median) percentile and normal upper and lower range reference serum levels of inhibin-B in relation to age as described by Andersson et al. [9] are marked with thin lines. (A) The 16 cases with absolute increment of serum inhibin-B level after surgery. (B) The 34 cases with serum inhibin-B level following the normal physiological percentile decline of serum inhibin-B level seen in this age group.
Discussion We found that orchiopexy in unilateral cryptorchidism had some positive impact on the serum level of inhibin-B, because overall 16 of the 50 patients had a rise in inhibin-B after orchiopexy in contrast with the normal physiological decline expected in this age group. This is in agreement with Irkilata et al. [11] who found an increment of serum inhibin-B in 22 of 27 boys, whereas 2 boys only had bilateral undescended testes, the rest were unilateral cases. In the study by Irkilata et al. [11], there was a failure of postoperative increase in inhibin-B in 2 out of 3 cases correlated with reduced germ cell number. However, all these boys were much older (5, 10 and 11 years old) than in our study. It is well known that in older boys with cryptorchidism, irreversible damage of testicular epithelium (Sertoli cells only) is more prominent [5,6]. Furthermore, 2 out of these 3 cases actually had bilateral cryptorchidism. In the study by Kollin et al. [8] of pre- and postoperative inhibin-B serum values in 58 and 62 unilateral 1-year-old cryptorchid boys, the inhibin-B serum level seemed higher in the postoperative group (168 88.4 pg/ml) compared with the preoperative group (160 66.5 pg/ml). Inhibin-B is produced by Sertoli cells, and the serum level partly reflects the histological state of seminiferous tubules. Interestingly, we found a high proportion of testes with impaired germ cell number among those boys who had an absolute increment of inhibin-B serum values. So the serum inhibin-B increment may actually reflect an improvement of the histological state of seminiferous tubules of the undescended testis among some after surgery. Our findings are indirectly supported by other studies [8,12]. One study showed that the serum level of inhibin-B correlated positively with testicular volume in newborn boys [12]. This was confirmed by Kollin et al. [8], who found that boys with unilateral cryptorchid testes with high levels of inhibin-B at 6 months and 4 years also had bigger testes at 4 years than boys with low levels of inhibin-B. Furthermore, a good correlation was found between the number of germ and Sertoli cells and testicular volume at time of operation with biopsy [8]. Interestingly, only 10% (5/ 50) of our patients had a serum level of inhibin-B above the
normal median (50 percentile) preoperatively compared with 40% (20/50) postoperatively (Fig. 2A and B). So there was a trend towards a more normal distribution of serum levels of inhibin-B postoperatively. Previously we found a positive correlation between germ cell number in cryptorchid testes operated in childhood and testicular volume of the same testes at follow-up in adulthood [13]. Based on these studies [8,12,13] and our present findings, we found good evidence that, generally in unilateral cryptorchidism, an increment of the serum level of inhibin-B postoperatively may indicate testicular growth as a result of improvement of the histological state of seminiferous tubules. The patients just following the normal physiological percentile decline of serum inhibin-B level seen in this age group seemed to have better S/T when operated, so therefore testicular growth as a result of further improvement of the histological state of seminiferous tubules probably could not be expected to the same degree. It is also important to acknowledge that there is more than a single etiology of unilateral cryptorchidism. In a previous study of fertility parameters among adult men operated in childhood for unilateral cryptorchidism, we found that 9% (6/65) were probably infertile based on evaluation of sperm samples [14]. All these patients had very low S/T when operated in childhood, but only 1 of these 6 infertile patients had increased serum FSH level in adulthood. Kraft et al. [15] recently related childhood undescended testis histology with adult hormone levels and semen analysis. Based on bilateral testicular biopsies taken in childhood at orchiopexy in unilateral cryptorchidism, they also found 9% (8/91) in a high-risk infertility group. At follow-up of these patients, the median sperm density was 2.3 million/ml similar to 3.9 million/ml in the comparable group of patients in our aforementioned study [14]. In the present study, only 3 patients had increased serum level of FSH postoperatively; the other 10 patients with increased serum level of FSH preoperatively had normalized serum values postoperatively. There is a biological decrease of FSH serum levels seen between 6 months and 2 years, which should be taken into consideration and a lower postoperative serum level of FSH would be expected [9]. However, the decrease seen in most patients of this group is
Pre- and postoperative status of gonadotropins more prominent than would have been expected according to changes in age, and the boys with elevated gonadotropins were actually slightly older than those with normal preoperative values. So there is good evidence that early surgery with placing the testes in the scrotum had a beneficial effect on gonadotropin levels, even in those boys with unilateral cryptorchidism. In the report by Kraft et al. [15], more than half of the patients with severe GC/T (germ cells per tubule) histopathology ended up without increased serum level of gonadotropins. In our present material, none of the 4 boys with impaired inhibin-B level before as well as after surgery had elevated gonadotropins. This can be explained by the presence of so-called prepubertal transient hypothalamusepituitaryegonadal hypofunction [1,16]. That means that it is likely that at least 8% of unilateral cryptorchid cases may have a prepubertal transient hypothalamusepituitaryegonadal hypofunction [1,16]. The presence of A-dark spermatogonia in tubular transverse sections in the present study was seen in a significantly higher proportion in testes with normal S/T compared with those with impaired S/T. This is in accordance with our findings in bilateral cases [2]. Impaired transformation of the neonatal gonocytes into type A-dark spermatogonia during the first 12 months of age and subsequent apoptosis of germ cells, may be a pathogenic factor for decreased fertility potential. The transformation of the neonatal gonocytes into type A-dark spermatogonia may be impaired if gonadotropins are insufficient, and when the testis is undescended this leads to further germ cell deterioration. So, if there is an underlying endocrinopathy causing inadequate maturation of the testis, merely putting the testis into the scrotum will not correct that endocrinopathy [17]. A potential weakness of the study relates to the modest sample size of the material and the 1 year and 9 months range of patient age at inclusion in a period of life during which significant normal biological changes in hormone levels and germ cell numbers occur. In fact, fluctuations in inhibin-B and FSH levels were common in boys followed for the first 2 years of life in the longitudinal series reported by Andersson et al. [9]. There is no control group to show what would have happened without orchiopexy. However, it is only speculative to suggest increase of serum inhibin-B levels in such a large group of cryptorchid patients without surgery, when serum inhibin-B levels of normal boys decline in this age group. When taking findings of previous studies into consideration, we find our data add important information to otherwise sparse knowledge on the subject. In conclusion, we found that orchiopexy in unilateral cryptorchidism had a positive impact on the serum levels of inhibin-B and gonadotropins. The serum inhibin-B increment may actually reflect an improvement of the histological state of seminiferous tubules of the undescended testis after surgery.
Conflict of interest None.
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Funding None.
References [1] Hadziselimovic F, Hoecht B. Testicular histology related to fertility outcome and postpubertal hormone status in cryptorchidism. Klin Pa ¨diatr 2008;220:302. [2] Thorup J, Kvist K, Clasen-Linde E, Petersen BL, Cortes D. The relation between adult-dark spermatogonia and other parameters of fertility potential in cryptorchid testes. J Urol 2013;190:1566. [3] Thorup J, Petersen BL, Kvist K, Cortes D. Bilateral undescended testes classified according to preoperative and postoperative status of gonadotropins and inhibin B in relation to testicular histopathology at bilateral orchiopexy in infant boys. J Urol 2012;188:1436. [4] Lee PA, O’Leary LA, Songer NJ. Paternity after unilateral cryptorchidism: a controlled study. Pediatrics 1996;98:676. [5] Cortes D, Thorup J, Visfeldt J. Cryptorchidism: aspects of fertility and neoplasms. Horm Res 2001;55:21. [6] Cortes D. Cryptorchidism: aspects of pathogenesis, histology and treatment. Scand J Urol Nephrol 1998;32(Suppl 196): 1e54. [7] Suomi AM, Main KM, Kaleva M, Schmidt IM, Chellakooty M, Virtanen HE, et al. Hormonal changes in 3-month-old cryptorchid boys. J Clin Endocrinol Metab 2006;91:953e8. [8] Kollin C, Stukenborg JB, Nurmio M, Sundqvist E, Gustafsson T, So ¨der O, et al. Boys with undescended testes: endocrine, volumetric and morphometric studies on testicular function before and after orchidopexy at nine months or three years of age. J Clin Endocrinol Metab 2012;97:4588. [9] Andersson AM, Toppari J, Haavisto AM, Petersen JH, Simell T, Simell O, et al. Longitudinal reproductive hormone profiles in infants: peak of inhibin B levels in infant boys exceeds levels in adult men. J Clin Endocrinol Metab 1998;83:675. [10] Cortes D, Thorup J, Beck BL. Quantitative histology of germ cells in the undescended testes of human fetuses, neonates and infants. J Urol 1995;154:1188. [11] Irkilata HC, Yildirim I, Onguru O, Aydur E, Musabak U, Dayanc M. The influence of orchiopexy on serum inhibin B level: relationship with histology. J Urol 2004;172:2402. [12] Main KM, Toppari J, Suomi AM, Kaleva M, Chellakooty M, Schmidt IM, et al. Larger testes and higher inhibin B levels in Finnish than in Danish newborn boys. J Clin Endocrinol Metab 2006;91:2732. [13] Cortes D, Thorup J. Histology of testicular biopsies taken at operation for bilateral maldescended testes in relation to fertility in adulthood. BJU 1991;68:285. [14] Cortes D, Thorup J, Lindenberg S, Visfeldt J. Infertility despite surgery for cryptorchidism in childhood can be classified by patients with normal or elevated follicle-stimulating hormone and identified at orchiopexy. BJU Int 2003;91:670. [15] Kraft KH, Canning DA, Snyder 3rd HM, Kolon TF. Undescended testis histology correlation with adult hormone levels and semen analysis. J Urol 2012;188(4 Suppl.):1429. [16] Hadziselimovic F, Herzog B. Hodendystopie. In: Thu ¨roff JW, Schulte-Wissermann, editors. Kinderurologie in Klinik und Praxis. 2 ed., 484. Stuttgart-New York: Thieme; 2000. [17] Thorup J, McLachlan R, Cortes D, Nation TR, Balic A, Southwell BR, et al. What is new in cryptorchidism and hypospadias e a critical review on the testicular dysgenesis hypothesis. J Pediatr Surg 2010;45:2074.