- Email: [email protected]
The Relation between Adult Dark Spermatogonia and Other Parameters of Fertility Potential in Cryptorchid Testes
The Relation between Adult Dark Spermatogonia and Other Parameters of Fertility Potential in Cryptorchid Testes Jorgen Thorup,* Kolja Kvist, Erik Clasen-Linde, Bodil Laub Petersen and Dina Cortes From the Departments of Pediatric Surgery (JT, KK) and Pathology (ECL, BLP), Rigshospitalet, Faculty of Health Science, University of Copenhagen (JT, BLP, DC) and Section of Endocrinology, Department of Pediatrics, Hvidovre Hospital (DC), Copenhagen, Denmark
Abbreviations and Acronyms Ad ¼ adult dark Ad/T ¼ Ad spermatogonia per transverse tubule FSH ¼ follicle-stimulating hormone LH ¼ luteinizing hormone PLAP ¼ placental-like alkaline phosphatase S/T ¼ germ cells per transverse tubule Accepted for publication January 17, 2013. Study received Copenhagen ethics committee approval. Supported by the Beckgaard Foundation. * Correspondence: Rigshospitalet, Copenhagen, Denmark.
Purpose: The fertility potential of boys with cryptorchidism may be related to the number of adult dark spermatogonia per tubular transverse section in testicular biopsies taken at orchiopexy. Placental-like alkaline phosphatase positive gonocytes in testes within year 1 of life indicate preserved ability for germ cell transformation. We related these parameters to the total number of tubular germ cells and other factors associated with fertility potential. Materials and Methods: The study comprised 89 boys 0.7 to 3 years old (median age 1.8) who underwent bilateral testicular biopsy at bilateral orchiopexy and provided blood samples for gonadotropins and inhibin B. Results: Of 76 boys with adult dark spermatogonia 44 (58%) had a normal mean number of spermatogonia per tubular transverse section compared to 2 of 13 (15%) without adult dark spermatogonia (p <0.05). In the 30 boys with good fertility potential, including a normal mean number of tubular germ cells, and normal gonadotropins and inhibin B, the mean number of adult dark tubular germ cells was 0.081 vs 0.031 in the 38 with low fertility potential, including impaired tubular germ cells and/or low inhibin B but no reactive increase in gonadotropins (p <0.05). In the 21 patients with increased gonadotropins the mean number of adult dark spermatogonia per tubular transverse section was 0.063. Of the 20 boys with normal mean adult dark spermatogonia per tubular transverse section 12 (60%) had good fertility potential, including a normal mean number of tubular germ cells, normal gonadotropins and normal inhibin B, compared to only 18 of 69 (26%) with an impaired mean number of adult dark spermatogonia per tubular transverse section (p <0.05). Of 46 boys with a normal mean number of tubular germ cells 26 (57%) had placental-like alkaline phosphatase positive cells compared to 14 of 43 (33%) with a decreased mean number of tubular germ cells (p <0.05). Conclusions: The number of placental-like alkaline phosphatase positive gonocytes and adult dark spermatogonia per tubular transverse section are important parameters related to the fertility potential of boys with cryptorchid testes. Key Words: testis; cryptorchidism; infertility, male; inhibin B; gonadotropins
THE number of S/T in testicular biopsies is the parameter most often used to predict later fertility in adulthood in boys with cryptorchidism.
1566
j
www.jurology.com
Recently, by combining the results of serum gonadotropin and serum inhibin B blood samples, and determining the S/T in boys with bilateral
0022-5347/13/1904-1566/0 THE JOURNAL OF UROLOGY® © 2013 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION AND RESEARCH, INC.
http://dx.doi.org/10.1016/j.juro.2013.01.058 Vol. 190, 1566-1571, October 2013 Printed in U.S.A.
ADULT DARK SPERMATOGONIA AND FERTILITY POTENTIAL IN CRYPTORCHID TESTES
cryptorchidism it was possible to identify those with a good prognosis at low risk for infertility, those with possible congenital abnormalities related to testicular descent at intermediate risk for infertility and those with insufficient gonadotropin stimulation as the primary pathogenic factor of cryptorchidism at related high risk for infertility.1 At birth patients with cryptorchidism harbor germ cells in the testes but in decreased number as a group compared to the normal population. From about age 15 months germ cells may be lacking.2 The greater the age at surgery for cryptorchidism, the higher the risk of finding no germ cells in testicular biopsy at surgery, which predicts infertility in adulthood.2,3 Probably induced by minipuberty, the neonatal gonocyte transforms into a type A spermatogonium during the first 12 months of age, a step that is now postulated to be crucial for subsequent fertility since stem cells for spermatogenesis are created in this structure.3,4 Impaired transformation of neonatal gonocytes into type A spermatogonia during the first 12 months of life age with subsequent germ cell apoptosis may be a pathogenic factor for infertility. Several groups identified a number of antigens that are highly expressed in the cells of intratubular germ cell neoplasia and gonocytes but not in normal germ cells of adult testes.5 PLAP is a tissue specific alkaline phosphatase with unknown biological function that is expressed in classical seminoma, primordial germ cells, early neonatal gonocytes and fetal spermatogonia.5,6 PLAP is one of the most commonly used markers for intratubular germ cell neoplasia and cancer in testicular biopsies.5 However, in the testes of normal boys PLAP positive germ cells are present within year 1 of life.6 PLAP positive gonocytes in normal testes within year 1 of life may indicate preserved ability for germ cell transformation and good fertility potential, as does the transformation of germ cells into a type Ad spermatogonium in cryptorchid testes within the first 12 months. However, it is uncertain how these parameters relate to total S/T and other factors associated with the fertility potential of boys with cryptorchidism, such as hormone profiles. We hypothesized that the presence and number of PLAP positive germ cells and type A spermatogonia in undescended testes during early infancy would correlate with our mentioned recent classification of boys with bilateral cryptorchidism into 3 groups at low, intermediate and high risk for infertility.
MATERIALS AND METHODS We prospectively included in the study 89 otherwise healthy, bilaterally cryptorchid boys 7 months to 3 years
1567
old (median age 1.8 years) who were seen consecutively by a single surgeon from 2008 to 2011. All had blood samples taken on the day of surgery and bilateral testicular biopsy was performed in all at orchiopexy. We excluded from analysis patients with chromosomal abnormalities, associated anomalies or previous inguinal surgery. None received hormonal therapy. Patients were divided into groups depending on whether the S/T number was normal or decreased, whether serum FSH was normal or increased and whether preoperative serum inhibin B was normal or decreased. Criteria for classifying patients based on 75% of the current material were described previously.1 In group 1 at intermediate risk for infertility all patients had preoperative serum FSH 1.3 IU/L or greater. In group 2 at high risk for infertility all patients had normal FSH, low S/T and/or low inhibin B. In group 3 at low risk for infertility all patients had normal S/T, normal FSH and normal inhibin B.
Hormonal Assays Blood samples were obtained by venipuncture between 8:00 and 11:00 a.m. Serum samples were separated from the clot by 10-minute centrifugation at 2,000 gravity. Serum was stored at e80C until analysis. Serum inhibin B was measured using an inhibin B enzymelinked immunosorbent assay kit (Serotec, Oxford, United Kingdom) with a research kit, as recommended by the manufacturer. The lower detection limit was 5 pg/ml and measurements were made in duplicate. A similar inhibin B kit was predominantly used in previous studies of inhibin B in normal boys and those with cryptorchidism.7 Normal serum inhibin B reference levels were defined as described by Andersson et al (fig. 1).8 LH and FSH were measured by sandwich electrochemiluminescence immunoassay. The lowest measured
Figure 1. Serum inhibin B by age at surgery in patients with cryptorchidism and PLAP positive (red squares) vs negative (blue diamonds) germ cells in testicular biopsies. Normal reference serum inhibin B levels were considered those described by Andersson et al.8 Solid curves indicate lower and upper normal ranges. Dashed curve indicates median.
1568
ADULT DARK SPERMATOGONIA AND FERTILITY POTENTIAL IN CRYPTORCHID TESTES
RESULTS
Figure 2. Histological images of testis show normal number of germ cells per tubular transverse section at periphery and more centrally in seminiferous tubules but fetal spermatogonia, neonatal gonocytes or adult spermatogonia could not be characterized with certainty.15 A, 1-year-old boy with PLAP positive stained germ cells. B, 15-month-old boy with PLAP negative stained germ cells.
FSH was 0.05 IU/L (normal median 0.5, range 0.1 to 1.4). The lowest measured LH was also 0.05 IU/L (normal median 0.1, range 0.05 to 0.3). FSH 1.3 IU/L or greater was considered a high serum value for this age group.
Testicular Biopsies All tissue specimens were fixed in Stieve solution and embedded in paraffin. Sections (4 mm) were stained with hematoxylin and eosin, CD99/MIC-2 and PLAP. In blinded fashion one of us (DC) measured the total S/T number, including gonocytes and Ad spermatogonia, from at least 100 tubular transverse sections. For each patient the mean Ad/T and S/T were found. Mean S/T was considered normal when the value was at least 1.0 at birth, 0.65 at age 6 months and 0.38 at ages 1 to 4 years based on our previously published normal material.9e11 The number of Ad/T per patient was stratified as normaldgreater than 0.1 or decreasedd0.1 or less based on the previous criteria of Hadziselimovic et al.12,13
Statistical Analysis and Ethics Nonparametric statistics were used for analysis. The chi-square, Fisher exact, Kruskal-Wallis and MannWhitney tests were used to asses statistical significance with 2-sided p <0.05 considered significant. The study was done according to the Helsinki II declaration and informed consent was obtained from the parents of patients. The study received approval from the Copenhagen ethics committee.
In our study 52% of patients overall (46 of 89) had normal mean S/T and 22% (20 of 89) had normal mean Ad/T. Also, in 45% of the boys (40 of 89) germ cells stained positive for PLAP (figs. 1 and 2). Groups 1, 2 and 3 included 21, 38 and 30 patients, respectively (table 1). Mean S/T and Ad/T were significantly different in the 3 groups and lowest in group 2 (table 1). Table 2 lists further group 2 details. Median age in patients with normal mean Ad/T was 2.0 years (range 0.7 to 2.9), equivalent to the age of those with a decreased mean Ad/T (1.8, range 0.8 to 3.0, Mann-Whitney test p ¼ 0.27). Of boys with normal mean S/T 43% (20 of 46) had normal Ad/T compared to 0% (0 of 43) with decreased mean S/T (Fisher exact test p <0.001, tables 1 to 3). Only 32% of group 2 patients had PLAP positive staining of germ cells, significantly lower than in groups 1 and 3 pooled together (55% or 28 of 51, Fisher exact test p <0.05). Of the 46 boys with normal mean S/T 57% (26) had PLAP positive cells compared to 33% (14 of 43) with decreased mean S/T (Fisher exact test p <0.05). Of the 20 boys with normal mean Ad/T 60% (12) had PLAP positive cells compared to 41% (28 of 69) with decreased mean Ad/T (Fisher exact test p ¼ 0.1358). Median age in patients with PLAP positive germ cells was 1.5 years (range 0.7 to 2.9). Thus, they were younger than patients with PLAP negative germ cells (median age 2.2 years, range 0.8 to 3.0, Mann-Whitney test p <0.005). Of boys younger than 1 year 92% (11 of 12), 1 to younger than 2 years 41% (19 of 46) and 2 to 3 years old 32% (10 of 31) had PLAP positive germ cells. Figure 1 shows serum inhibin B levels in patients with PLAP positive and negative germ cells.
DISCUSSION To our knowledge we report the first evaluation of the fertility potential of bilaterally cryptorchid male infants and boys performed by estimating the presence of Ad spermatogonia and PLAP positive
Table 1. Hormonal profiles and germ cell parameters in 3 groups of boys with bilateral cryptorchidism
No. pts (%) Median yrs age at orchiopexy (range) Mean S/T (range) Median IU/L FSH (range) Median IU/L LH (range) Median pg/ml inhibin B (range) Mean Ad/T (range) No. normal Ad/T (%) No. pos PLAP staining (%) Median gm birth wt (range)
Group 1
Group 2
Group 3
p Value
21 (24) 1.8 (0.7-2.7) 0.28 (0.04-1.57) 1.6 (1.3-3.0) 0.6 (0.1-3.2) 116 (44-246) 0.06 (0-0.16) 4 (19) 11 (52) 3,330 (1,600-4,700)
38 (43) 1.9 (0.8-3.0) 0.23 (0.06-0.87) 0.7 (0.3-1.2) 0.2 (0.1-1.1) 118 (27-281) 0.03 (0-0.15) 4 (11) 12 (32) 3,460 (1,676-4,610)
30 (34) 1.8 (0.8-2.9) 0.65 (0.38-2.66) 0.7 (0.2-1.2) 0.2 (0.1-0.7) 134 (85-263) 0.08 (0-0.21) 12 (40) 17 (57) 3,335 (2,000-5,300)
0.668 (Kruskal-Wallis) <0.0001 (Kruskal-Wallis) Not applicable Not applicable 0.040 (Kruskal-Wallis) <0.0001 (Kruskal-Wallis) 0.013 (chi-square) 0.087 (chi-square) 0.919 (Kruskal-Wallis)
ADULT DARK SPERMATOGONIA AND FERTILITY POTENTIAL IN CRYPTORCHID TESTES
1569
Table 2. Group 2 subdivision into 3 subgroups based on normal or decreased S/T and inhibin B
No. pts Median yrs age at orchiopexy (range) Mean S/T (range) Median pg/ml inhibin B (range) Median IU/L FSH (range) Median IU/L LH (range) Mean Ad/T (range) No. normal Ad/T (%) No. pos PLAP staining (%)
Subgroup 1*
Subgroup 2†
Subgroup 3‡
25 1.9 (0.8-3.0) 0.21 (0.06-0.60) 134 (55-281) 0.7 (0.3-1.2) 0.2 (0.1-1.1) 0.03 (0-0.07) 0 8 (32)
7 2.4 (0.9-2.7) 0.54 (0.39-0.87) 51 (38-70) 0.6 (0.4-1.1) 0.1 (0.1-0.4) 0.11 (0.03-0.15) 4 (57) 3 (43)
6 1.9 (1.5-2.3) 0.18 (0.08-0.28) 71 (27-94) 0.7 (0.3-0.9) 0.1 (0.1-0.7) 0.01 (0-0.05) 0 1 (17)
* Normal inhibin B and low S/T. † Low inhibin B and normal S/T. ‡ Low inhibin B and S/T.
germ cells in testicular biopsies and relating these parameters to the patient S/T and hormonal profile. PLAP definitely stains neonatal gonocytes and fetal spermatogonia.5,6,14 The fact that 45% of our patients with a median age of 1.8 years had PLAP positive germ cells shows that these cells surely persist for a longer period in cryptorchid than in normal infant testes.6 The age dependence of this finding was also clearly shown in the current study. However, although germ cell transformation was delayed compared to that in normal males, we hypothesized that PLAP positive gonocytes in the testes within the first years of life would indicate preserved ability for germ cell transformation and good fertility potential.15 PLAP positive gonocytes gradually decrease in number during the first years of life, as seen in this study. Of boys with normal mean S/T a significantly higher percent (57%) had PLAP positive cells compared to 33% with decreased mean S/T. No patient with a decreased mean S/T had any Ad spermatogonia, which may be a prerequisite for normal fertility potential.3,4,12 Thus, when PLAP positive gonocytes do not transform into spermatogonia as they should but increased apoptosis occurs instead, the end result would be an impaired total germ cell number, no Ad spermatogonia and few if any PLAP positive gonocytes left in
the biopsy. This was also indirectly confirmed by the fact that 100% of the patients with low inhibin B (less than 50 pg/ml) lacked PLAP positive germ cells vs 51% with inhibin B greater than 50 pg/ml. Inhibin B is produced by Sertoli cells and the serum level partly reflects the histological state of the seminiferous tubules (fig. 1).1,7 The results of the new germ cell parameters estimated in this study fit into our previous classification of 3 groups of boys with cryptorchidism (table 1).1 Notably, the 3 groups have an almost identical age distribution. Group 3 encompasses a milder variance of the cryptorchidism spectrum with a good prognosis for fertility. According to the definition, all patients have normal gonadotropins, inhibin B and S/T.1 This group includes also the most patients with normal Ad/T and PLAP positive germ cells, indicating preserved ability for further germ cell transformation when the testes have reached a scrotal position. In group 2 there was no gonadotropin response to the impaired histological state of the seminiferous tubules, as reflected directly by the mean S/T count or indirectly by serum inhibin B. The reason could be transient prepubertal hypothalamus-pituitarygonadal hypofunction.3,16 Impaired transformation of neonatal gonocytes into type A spermatogonia
Table 3. Group 1 subdivision into 4 subgroups based on normal or decreased S/T and inhibin B Results
Subgroup 1*
Subgroup 2†
Subgroup 3‡
Subgroup 4§
No. pts Median yrs age at orchiopexy (range) Mean S/T (range) Median pg/ml inhibin B (range) Median IU/L FSH (range) Median IU/L LH (range) Mean Ad/T (range) No. normal Ad/T (%) No. pos PLAP staining (%)
9 1.8 (1.1-2.7) 0.21 (0.04-0.33) 120 (89-246) 1.5 (1.3-2.3) 0.5 (0.1-1.5) 0.02 (0.01-0.08) 0 4 (44)
3 1.5 (0.9-1.8) 0.84 (0.59-1.57) 53 (51-95) 1.6 (1.4-3.0) 0.6 (0.4-1.1) 0.12 (0-0.17) 2 (67) 3 (100)
3 1.6 (0.9-2.1) 0.24 (0.07-0.25) 81 (44-119) 1.6 (1.5-2.3) 0.6 (0.5-1.3) 0.01 (0-0.01) 0 1 (33)
6 2.1 (0.7-2.3) 0.64 (0.48-1.36) 141 (85-230) 1.7 (1.3-2.5) 1.5 (0.3-3.2) 0.09 (0.06-0.15)jj 2 (33)jj 4 (67)jj
* Normal inhibin B and low S/T. † Low inhibin B and normal S/T. ‡ Low inhibin B and S/T. § Normal inhibin B and S/T. jj Two patients 0.7 and 2 years old with normal mean Ad spermatogonia/T and positive PLAP had FSH 1.6 and 2.3 IU/L, respectively, and so by definition were in group 1, although younger patient might be considered for borderline group 3 with all normal parameters.
1570
ADULT DARK SPERMATOGONIA AND FERTILITY POTENTIAL IN CRYPTORCHID TESTES
during the first 12 months of life and subsequent germ cell apoptosis may be pathogenic factors. This transformation may be impaired if stimulating gonadotropins are insufficient. When the testis is undescended, this leads to further germ cell deterioration. The hypothesis fits well with the findings of the lowest percent of boys with normal Ad/T and PLAP positive germ cells in group 2, indicating gonocyte apoptosis before sufficient transformation into type Ad spermatogonia due to insufficient gonadotropin stimulation and a nonscrotal position of the testes. Group 1 matches the hypothesis of primary testicular dysfunction. Undescended testis may be part of a congenital malformation and reactive high gonadotropins are noted. The testes in this group may not be equally damaged at surgery, as also reflected by the prevalence of boys with normal Ad/T and PLAP positive cells between that of the other 2 groups (tables 1 and 3). If not already damaged, these testes may benefit from early orchiopexy in respect of fertility potential.1 Most followup studies of fertility in adulthood related to histology of the testes at orchiopexy in childhood have only evaluated the total germ cell number when classifying the histopathological status of the testis.2,17 The results of the study by Hadziselimovic and Hoecht3 clearly support our findings that estimating the number of Ad spermatogonia per tubular transverse section is probably an important parameter for predicting fertility potential. Hadziselimovic and Herzog matched histological findings of the testes in 31 patients who underwent orchiopexy before age 2 years with the total number of sperm in adulthood.4 When Ad spermatogonia were present, 94% of the men (17 of 18) had a total sperm count of 40 million or greater per ejaculate. In contrast, despite successful early surgery, if Ad spermatogonia were absent, only 8% of patients (1 of 13) had normal spermiograms. Thus, the transformation of gonocytes into Ad spermatogonia is crucial for male fertility.
However, others found less clear results in a similar study but this could partly be explained by the modest patient sample size.18,19 Kraft et al reported a significant association between abnormal Ad/T and decreased sperm density.18 Increased FSH was noted in formerly unilateral cryptorchid cases with abnormal Ad/T. Normal Ad/T was associated with normal sperm density in 88% of unilateral (29 of 33) and 100% of bilateral (2 of 2) cases.19 Progressive additive deterioration caused by high testicular temperature in the cryptorchidism cases, as described by Mieusset et al,20 may be prevented if the testes are brought to the scrotum early. If PLAP positive gonocytes are still in place and adequate natural or supplementary gonadotropin stimulation is present, gonocyte transformation into Ad spermatogonia may still be possible, although with some delay.1 Estimating PLAP positive germ cells and evaluating the number of Ad/T are probably additional important parameters when patient selection is considered for supplementary gonadotropin treatment. The limitations of our study are related to the modest sample size, especially when divided into subgroups, as well as possible irregularity in immunohistochemical staining and subjectivity in Ad/T estimation. However, the blinded fashion of cell evaluation makes the data reliable for drawing conclusions.
CONCLUSIONS Estimating the number of PLAP positive germ cells in infant cryptorchid testes and evaluating the number of Ad/T are additional important parameters related to the fertility potential of boys with cryptorchidism. Within the first years of life, when orchiopexy is often performed, the normal range of the number of Ad/T and the presence of PLAP positive germ cells are highly age dependent and not yet fully elucidated. Further investigations are needed to determine specific prognostic criteria related to fertility potential.
REFERENCES 1. Thorup J, Petersen BL, Kvist K et al: 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. 2. Cortes D: Cryptorchidism: aspects of pathogenesis, histology and treatment. Scand J Urol Nephrol, suppl., 1998; 32: 1. 3. Hadziselimovic F and Hoecht B: Testicular histology related to fertility outcome and postpubertal hormone status in cryptorchidism. Klin P€adiatr 2008; 220: 302.
4. Hadziselimovic F and Herzog B: The importance of both an early orchidopexy and germ cell maturation for fertility. Lancet 2001; 358: 1156. 5. Rajpert-De Meyts E, Bartkova J, Samson M et al: The emerging phenotype of the testicular carcinoma in situ germ cell. APMIS 2003; 111: 267. 6. Jørgensen N, Giwercman A, M€uller J et al: Immunohistochemical markers of carcinoma in situ of the testis also expressed in normal infantile germ cells. Histopathology 1993; 22: 373. 7. Cortes D, Thorup J, Hogdall E et al: The relation of germ cells per tubule in testes, serum inhibin
B and FSH in cryptorchid boys. Pediatr Surg Int 2007; 23: 163. 8. Andersson AM, Toppari J, Haavisto AM 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. 9. Cortes D, Thorup J and Visfeldt J: Cryptorchidism: aspects of fertility and neoplasms. Horm Research 2001; 55: 21. 10. Cortes D, Thorup J and Beck BL: Quantitative histology of germ cells in the undescended
ADULT DARK SPERMATOGONIA AND FERTILITY POTENTIAL IN CRYPTORCHID TESTES
testes of human fetuses, neonates and infants. J Urol 1995; 154: 1188. 11. Thorup J, Cortes D and Nielsen OH: Clinical and histopathologie evaluation of operated maldescended testes after luteinizing hormone-releasing hormone treatment. Pediatr Surg Int 1993; 8: 419. 12. Hadziselimovic F, Zivkovic D, Bica DT et al: The importance of mini-puberty for fertility in cryptorchidism. J Urol 2005; 174: 1536. 13. Hadziselimovic F, Emmonsa LR and Buser MW: A diminished postnatal surge of Ad spermatogonia in cryptorchid infants is additional evidence for hypogonadotropic hypogonadism. Swiss Med Wkly 2004; 134: 381.
14. Jørgensen N, Rajpert-De Meyts E, Graem N et al: Expression of immunohistochemical markers for testicular carcinoma in situ by normal human fetal germ cells. Lab Invest 1995; 72: 223. 15. Hutson JM, Li R, Southwell BR et al: Germ cell development in the postnatal testis: the key to prevent malignancy in cryptorchidism? Front Endocrinol 2013; 3: 176. 16. Hadziselimovic F and Herzog B: Hodendystopie. In: Kinderurologie in Klinik und Praxis, 2nd ed. Edited by JW Th€uroff and H SchulteWissermann. New York: Thieme 2000; pp 484e500. 17. Rusnack SL, Wu HY, Huff DS et al: Testis histopathology in boys with cryptorchidism correlates
1571
with future fertility potential. J Urol 2003; 169: 659. 18. Kraft KH, Canning DA, Snyder HM et al: Undescended testis histology correlation with adult hormone levels and semen analysis. J Urol 2012; 188: 1429. 19. Cortes D: Editorial comment: Undescended testis histology correlation with adult hormone levels and semen analysis. J Urol 2012; 188: 1435. 20. Mieusset R, Fonda PJ, Vaysse P et al: Increase in testicular temperature in case of cryptorchidism in boys. Fertil Steril 1993; 59: 1319.
EDITORIAL COMMENT Testicular biopsies have implicated a decreased germ cell count as the histological basis of subfertility in patients with undescended testes. The authors convincingly report that the presence and number of PLAP positive germ cells should be considered additional histological parameters when assessing fertility potential in bilateral cryptorchidism cases. Including adult hormone levels, semen analysis or paternity data in this cohort would further clarify the clinical relevance of these findings, particularly since associations between germ cell histology and these variables suggest that testicular biopsy at bilateral orchiopexy may help predict future fertility
(reference 18 in article). Nonetheless, these results underscore the value of reviewing testis histology in patients with bilateral cryptorchidism when counseling families about fertility potential. Further investigation is warranted to determine whether testicular biopsy at bilateral orchiopexy should be routine clinical practice for detecting boys at risk for future infertility. Kate H. Kraft Department of Urology University of Michigan Health System Ann Arbor, Michigan
Abbreviations and Acronyms Ad ¼ adult dark Ad/T ¼ Ad spermatogonia per transverse tubule FSH ¼ follicle-stimulating hormone LH ¼ luteinizing hormone PLAP ¼ placental-like alkaline phosphatase S/T ¼ germ cells per transverse tubule Accepted for publication January 17, 2013. Study received Copenhagen ethics committee approval. Supported by the Beckgaard Foundation. * Correspondence: Rigshospitalet, Copenhagen, Denmark.
Purpose: The fertility potential of boys with cryptorchidism may be related to the number of adult dark spermatogonia per tubular transverse section in testicular biopsies taken at orchiopexy. Placental-like alkaline phosphatase positive gonocytes in testes within year 1 of life indicate preserved ability for germ cell transformation. We related these parameters to the total number of tubular germ cells and other factors associated with fertility potential. Materials and Methods: The study comprised 89 boys 0.7 to 3 years old (median age 1.8) who underwent bilateral testicular biopsy at bilateral orchiopexy and provided blood samples for gonadotropins and inhibin B. Results: Of 76 boys with adult dark spermatogonia 44 (58%) had a normal mean number of spermatogonia per tubular transverse section compared to 2 of 13 (15%) without adult dark spermatogonia (p <0.05). In the 30 boys with good fertility potential, including a normal mean number of tubular germ cells, and normal gonadotropins and inhibin B, the mean number of adult dark tubular germ cells was 0.081 vs 0.031 in the 38 with low fertility potential, including impaired tubular germ cells and/or low inhibin B but no reactive increase in gonadotropins (p <0.05). In the 21 patients with increased gonadotropins the mean number of adult dark spermatogonia per tubular transverse section was 0.063. Of the 20 boys with normal mean adult dark spermatogonia per tubular transverse section 12 (60%) had good fertility potential, including a normal mean number of tubular germ cells, normal gonadotropins and normal inhibin B, compared to only 18 of 69 (26%) with an impaired mean number of adult dark spermatogonia per tubular transverse section (p <0.05). Of 46 boys with a normal mean number of tubular germ cells 26 (57%) had placental-like alkaline phosphatase positive cells compared to 14 of 43 (33%) with a decreased mean number of tubular germ cells (p <0.05). Conclusions: The number of placental-like alkaline phosphatase positive gonocytes and adult dark spermatogonia per tubular transverse section are important parameters related to the fertility potential of boys with cryptorchid testes. Key Words: testis; cryptorchidism; infertility, male; inhibin B; gonadotropins
THE number of S/T in testicular biopsies is the parameter most often used to predict later fertility in adulthood in boys with cryptorchidism.
1566
j
www.jurology.com
Recently, by combining the results of serum gonadotropin and serum inhibin B blood samples, and determining the S/T in boys with bilateral
0022-5347/13/1904-1566/0 THE JOURNAL OF UROLOGY® © 2013 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION AND RESEARCH, INC.
http://dx.doi.org/10.1016/j.juro.2013.01.058 Vol. 190, 1566-1571, October 2013 Printed in U.S.A.
ADULT DARK SPERMATOGONIA AND FERTILITY POTENTIAL IN CRYPTORCHID TESTES
cryptorchidism it was possible to identify those with a good prognosis at low risk for infertility, those with possible congenital abnormalities related to testicular descent at intermediate risk for infertility and those with insufficient gonadotropin stimulation as the primary pathogenic factor of cryptorchidism at related high risk for infertility.1 At birth patients with cryptorchidism harbor germ cells in the testes but in decreased number as a group compared to the normal population. From about age 15 months germ cells may be lacking.2 The greater the age at surgery for cryptorchidism, the higher the risk of finding no germ cells in testicular biopsy at surgery, which predicts infertility in adulthood.2,3 Probably induced by minipuberty, the neonatal gonocyte transforms into a type A spermatogonium during the first 12 months of age, a step that is now postulated to be crucial for subsequent fertility since stem cells for spermatogenesis are created in this structure.3,4 Impaired transformation of neonatal gonocytes into type A spermatogonia during the first 12 months of life age with subsequent germ cell apoptosis may be a pathogenic factor for infertility. Several groups identified a number of antigens that are highly expressed in the cells of intratubular germ cell neoplasia and gonocytes but not in normal germ cells of adult testes.5 PLAP is a tissue specific alkaline phosphatase with unknown biological function that is expressed in classical seminoma, primordial germ cells, early neonatal gonocytes and fetal spermatogonia.5,6 PLAP is one of the most commonly used markers for intratubular germ cell neoplasia and cancer in testicular biopsies.5 However, in the testes of normal boys PLAP positive germ cells are present within year 1 of life.6 PLAP positive gonocytes in normal testes within year 1 of life may indicate preserved ability for germ cell transformation and good fertility potential, as does the transformation of germ cells into a type Ad spermatogonium in cryptorchid testes within the first 12 months. However, it is uncertain how these parameters relate to total S/T and other factors associated with the fertility potential of boys with cryptorchidism, such as hormone profiles. We hypothesized that the presence and number of PLAP positive germ cells and type A spermatogonia in undescended testes during early infancy would correlate with our mentioned recent classification of boys with bilateral cryptorchidism into 3 groups at low, intermediate and high risk for infertility.
MATERIALS AND METHODS We prospectively included in the study 89 otherwise healthy, bilaterally cryptorchid boys 7 months to 3 years
1567
old (median age 1.8 years) who were seen consecutively by a single surgeon from 2008 to 2011. All had blood samples taken on the day of surgery and bilateral testicular biopsy was performed in all at orchiopexy. We excluded from analysis patients with chromosomal abnormalities, associated anomalies or previous inguinal surgery. None received hormonal therapy. Patients were divided into groups depending on whether the S/T number was normal or decreased, whether serum FSH was normal or increased and whether preoperative serum inhibin B was normal or decreased. Criteria for classifying patients based on 75% of the current material were described previously.1 In group 1 at intermediate risk for infertility all patients had preoperative serum FSH 1.3 IU/L or greater. In group 2 at high risk for infertility all patients had normal FSH, low S/T and/or low inhibin B. In group 3 at low risk for infertility all patients had normal S/T, normal FSH and normal inhibin B.
Hormonal Assays Blood samples were obtained by venipuncture between 8:00 and 11:00 a.m. Serum samples were separated from the clot by 10-minute centrifugation at 2,000 gravity. Serum was stored at e80C until analysis. Serum inhibin B was measured using an inhibin B enzymelinked immunosorbent assay kit (Serotec, Oxford, United Kingdom) with a research kit, as recommended by the manufacturer. The lower detection limit was 5 pg/ml and measurements were made in duplicate. A similar inhibin B kit was predominantly used in previous studies of inhibin B in normal boys and those with cryptorchidism.7 Normal serum inhibin B reference levels were defined as described by Andersson et al (fig. 1).8 LH and FSH were measured by sandwich electrochemiluminescence immunoassay. The lowest measured
Figure 1. Serum inhibin B by age at surgery in patients with cryptorchidism and PLAP positive (red squares) vs negative (blue diamonds) germ cells in testicular biopsies. Normal reference serum inhibin B levels were considered those described by Andersson et al.8 Solid curves indicate lower and upper normal ranges. Dashed curve indicates median.
1568
ADULT DARK SPERMATOGONIA AND FERTILITY POTENTIAL IN CRYPTORCHID TESTES
RESULTS
Figure 2. Histological images of testis show normal number of germ cells per tubular transverse section at periphery and more centrally in seminiferous tubules but fetal spermatogonia, neonatal gonocytes or adult spermatogonia could not be characterized with certainty.15 A, 1-year-old boy with PLAP positive stained germ cells. B, 15-month-old boy with PLAP negative stained germ cells.
FSH was 0.05 IU/L (normal median 0.5, range 0.1 to 1.4). The lowest measured LH was also 0.05 IU/L (normal median 0.1, range 0.05 to 0.3). FSH 1.3 IU/L or greater was considered a high serum value for this age group.
Testicular Biopsies All tissue specimens were fixed in Stieve solution and embedded in paraffin. Sections (4 mm) were stained with hematoxylin and eosin, CD99/MIC-2 and PLAP. In blinded fashion one of us (DC) measured the total S/T number, including gonocytes and Ad spermatogonia, from at least 100 tubular transverse sections. For each patient the mean Ad/T and S/T were found. Mean S/T was considered normal when the value was at least 1.0 at birth, 0.65 at age 6 months and 0.38 at ages 1 to 4 years based on our previously published normal material.9e11 The number of Ad/T per patient was stratified as normaldgreater than 0.1 or decreasedd0.1 or less based on the previous criteria of Hadziselimovic et al.12,13
Statistical Analysis and Ethics Nonparametric statistics were used for analysis. The chi-square, Fisher exact, Kruskal-Wallis and MannWhitney tests were used to asses statistical significance with 2-sided p <0.05 considered significant. The study was done according to the Helsinki II declaration and informed consent was obtained from the parents of patients. The study received approval from the Copenhagen ethics committee.
In our study 52% of patients overall (46 of 89) had normal mean S/T and 22% (20 of 89) had normal mean Ad/T. Also, in 45% of the boys (40 of 89) germ cells stained positive for PLAP (figs. 1 and 2). Groups 1, 2 and 3 included 21, 38 and 30 patients, respectively (table 1). Mean S/T and Ad/T were significantly different in the 3 groups and lowest in group 2 (table 1). Table 2 lists further group 2 details. Median age in patients with normal mean Ad/T was 2.0 years (range 0.7 to 2.9), equivalent to the age of those with a decreased mean Ad/T (1.8, range 0.8 to 3.0, Mann-Whitney test p ¼ 0.27). Of boys with normal mean S/T 43% (20 of 46) had normal Ad/T compared to 0% (0 of 43) with decreased mean S/T (Fisher exact test p <0.001, tables 1 to 3). Only 32% of group 2 patients had PLAP positive staining of germ cells, significantly lower than in groups 1 and 3 pooled together (55% or 28 of 51, Fisher exact test p <0.05). Of the 46 boys with normal mean S/T 57% (26) had PLAP positive cells compared to 33% (14 of 43) with decreased mean S/T (Fisher exact test p <0.05). Of the 20 boys with normal mean Ad/T 60% (12) had PLAP positive cells compared to 41% (28 of 69) with decreased mean Ad/T (Fisher exact test p ¼ 0.1358). Median age in patients with PLAP positive germ cells was 1.5 years (range 0.7 to 2.9). Thus, they were younger than patients with PLAP negative germ cells (median age 2.2 years, range 0.8 to 3.0, Mann-Whitney test p <0.005). Of boys younger than 1 year 92% (11 of 12), 1 to younger than 2 years 41% (19 of 46) and 2 to 3 years old 32% (10 of 31) had PLAP positive germ cells. Figure 1 shows serum inhibin B levels in patients with PLAP positive and negative germ cells.
DISCUSSION To our knowledge we report the first evaluation of the fertility potential of bilaterally cryptorchid male infants and boys performed by estimating the presence of Ad spermatogonia and PLAP positive
Table 1. Hormonal profiles and germ cell parameters in 3 groups of boys with bilateral cryptorchidism
No. pts (%) Median yrs age at orchiopexy (range) Mean S/T (range) Median IU/L FSH (range) Median IU/L LH (range) Median pg/ml inhibin B (range) Mean Ad/T (range) No. normal Ad/T (%) No. pos PLAP staining (%) Median gm birth wt (range)
Group 1
Group 2
Group 3
p Value
21 (24) 1.8 (0.7-2.7) 0.28 (0.04-1.57) 1.6 (1.3-3.0) 0.6 (0.1-3.2) 116 (44-246) 0.06 (0-0.16) 4 (19) 11 (52) 3,330 (1,600-4,700)
38 (43) 1.9 (0.8-3.0) 0.23 (0.06-0.87) 0.7 (0.3-1.2) 0.2 (0.1-1.1) 118 (27-281) 0.03 (0-0.15) 4 (11) 12 (32) 3,460 (1,676-4,610)
30 (34) 1.8 (0.8-2.9) 0.65 (0.38-2.66) 0.7 (0.2-1.2) 0.2 (0.1-0.7) 134 (85-263) 0.08 (0-0.21) 12 (40) 17 (57) 3,335 (2,000-5,300)
0.668 (Kruskal-Wallis) <0.0001 (Kruskal-Wallis) Not applicable Not applicable 0.040 (Kruskal-Wallis) <0.0001 (Kruskal-Wallis) 0.013 (chi-square) 0.087 (chi-square) 0.919 (Kruskal-Wallis)
ADULT DARK SPERMATOGONIA AND FERTILITY POTENTIAL IN CRYPTORCHID TESTES
1569
Table 2. Group 2 subdivision into 3 subgroups based on normal or decreased S/T and inhibin B
No. pts Median yrs age at orchiopexy (range) Mean S/T (range) Median pg/ml inhibin B (range) Median IU/L FSH (range) Median IU/L LH (range) Mean Ad/T (range) No. normal Ad/T (%) No. pos PLAP staining (%)
Subgroup 1*
Subgroup 2†
Subgroup 3‡
25 1.9 (0.8-3.0) 0.21 (0.06-0.60) 134 (55-281) 0.7 (0.3-1.2) 0.2 (0.1-1.1) 0.03 (0-0.07) 0 8 (32)
7 2.4 (0.9-2.7) 0.54 (0.39-0.87) 51 (38-70) 0.6 (0.4-1.1) 0.1 (0.1-0.4) 0.11 (0.03-0.15) 4 (57) 3 (43)
6 1.9 (1.5-2.3) 0.18 (0.08-0.28) 71 (27-94) 0.7 (0.3-0.9) 0.1 (0.1-0.7) 0.01 (0-0.05) 0 1 (17)
* Normal inhibin B and low S/T. † Low inhibin B and normal S/T. ‡ Low inhibin B and S/T.
germ cells in testicular biopsies and relating these parameters to the patient S/T and hormonal profile. PLAP definitely stains neonatal gonocytes and fetal spermatogonia.5,6,14 The fact that 45% of our patients with a median age of 1.8 years had PLAP positive germ cells shows that these cells surely persist for a longer period in cryptorchid than in normal infant testes.6 The age dependence of this finding was also clearly shown in the current study. However, although germ cell transformation was delayed compared to that in normal males, we hypothesized that PLAP positive gonocytes in the testes within the first years of life would indicate preserved ability for germ cell transformation and good fertility potential.15 PLAP positive gonocytes gradually decrease in number during the first years of life, as seen in this study. Of boys with normal mean S/T a significantly higher percent (57%) had PLAP positive cells compared to 33% with decreased mean S/T. No patient with a decreased mean S/T had any Ad spermatogonia, which may be a prerequisite for normal fertility potential.3,4,12 Thus, when PLAP positive gonocytes do not transform into spermatogonia as they should but increased apoptosis occurs instead, the end result would be an impaired total germ cell number, no Ad spermatogonia and few if any PLAP positive gonocytes left in
the biopsy. This was also indirectly confirmed by the fact that 100% of the patients with low inhibin B (less than 50 pg/ml) lacked PLAP positive germ cells vs 51% with inhibin B greater than 50 pg/ml. Inhibin B is produced by Sertoli cells and the serum level partly reflects the histological state of the seminiferous tubules (fig. 1).1,7 The results of the new germ cell parameters estimated in this study fit into our previous classification of 3 groups of boys with cryptorchidism (table 1).1 Notably, the 3 groups have an almost identical age distribution. Group 3 encompasses a milder variance of the cryptorchidism spectrum with a good prognosis for fertility. According to the definition, all patients have normal gonadotropins, inhibin B and S/T.1 This group includes also the most patients with normal Ad/T and PLAP positive germ cells, indicating preserved ability for further germ cell transformation when the testes have reached a scrotal position. In group 2 there was no gonadotropin response to the impaired histological state of the seminiferous tubules, as reflected directly by the mean S/T count or indirectly by serum inhibin B. The reason could be transient prepubertal hypothalamus-pituitarygonadal hypofunction.3,16 Impaired transformation of neonatal gonocytes into type A spermatogonia
Table 3. Group 1 subdivision into 4 subgroups based on normal or decreased S/T and inhibin B Results
Subgroup 1*
Subgroup 2†
Subgroup 3‡
Subgroup 4§
No. pts Median yrs age at orchiopexy (range) Mean S/T (range) Median pg/ml inhibin B (range) Median IU/L FSH (range) Median IU/L LH (range) Mean Ad/T (range) No. normal Ad/T (%) No. pos PLAP staining (%)
9 1.8 (1.1-2.7) 0.21 (0.04-0.33) 120 (89-246) 1.5 (1.3-2.3) 0.5 (0.1-1.5) 0.02 (0.01-0.08) 0 4 (44)
3 1.5 (0.9-1.8) 0.84 (0.59-1.57) 53 (51-95) 1.6 (1.4-3.0) 0.6 (0.4-1.1) 0.12 (0-0.17) 2 (67) 3 (100)
3 1.6 (0.9-2.1) 0.24 (0.07-0.25) 81 (44-119) 1.6 (1.5-2.3) 0.6 (0.5-1.3) 0.01 (0-0.01) 0 1 (33)
6 2.1 (0.7-2.3) 0.64 (0.48-1.36) 141 (85-230) 1.7 (1.3-2.5) 1.5 (0.3-3.2) 0.09 (0.06-0.15)jj 2 (33)jj 4 (67)jj
* Normal inhibin B and low S/T. † Low inhibin B and normal S/T. ‡ Low inhibin B and S/T. § Normal inhibin B and S/T. jj Two patients 0.7 and 2 years old with normal mean Ad spermatogonia/T and positive PLAP had FSH 1.6 and 2.3 IU/L, respectively, and so by definition were in group 1, although younger patient might be considered for borderline group 3 with all normal parameters.
1570
ADULT DARK SPERMATOGONIA AND FERTILITY POTENTIAL IN CRYPTORCHID TESTES
during the first 12 months of life and subsequent germ cell apoptosis may be pathogenic factors. This transformation may be impaired if stimulating gonadotropins are insufficient. When the testis is undescended, this leads to further germ cell deterioration. The hypothesis fits well with the findings of the lowest percent of boys with normal Ad/T and PLAP positive germ cells in group 2, indicating gonocyte apoptosis before sufficient transformation into type Ad spermatogonia due to insufficient gonadotropin stimulation and a nonscrotal position of the testes. Group 1 matches the hypothesis of primary testicular dysfunction. Undescended testis may be part of a congenital malformation and reactive high gonadotropins are noted. The testes in this group may not be equally damaged at surgery, as also reflected by the prevalence of boys with normal Ad/T and PLAP positive cells between that of the other 2 groups (tables 1 and 3). If not already damaged, these testes may benefit from early orchiopexy in respect of fertility potential.1 Most followup studies of fertility in adulthood related to histology of the testes at orchiopexy in childhood have only evaluated the total germ cell number when classifying the histopathological status of the testis.2,17 The results of the study by Hadziselimovic and Hoecht3 clearly support our findings that estimating the number of Ad spermatogonia per tubular transverse section is probably an important parameter for predicting fertility potential. Hadziselimovic and Herzog matched histological findings of the testes in 31 patients who underwent orchiopexy before age 2 years with the total number of sperm in adulthood.4 When Ad spermatogonia were present, 94% of the men (17 of 18) had a total sperm count of 40 million or greater per ejaculate. In contrast, despite successful early surgery, if Ad spermatogonia were absent, only 8% of patients (1 of 13) had normal spermiograms. Thus, the transformation of gonocytes into Ad spermatogonia is crucial for male fertility.
However, others found less clear results in a similar study but this could partly be explained by the modest patient sample size.18,19 Kraft et al reported a significant association between abnormal Ad/T and decreased sperm density.18 Increased FSH was noted in formerly unilateral cryptorchid cases with abnormal Ad/T. Normal Ad/T was associated with normal sperm density in 88% of unilateral (29 of 33) and 100% of bilateral (2 of 2) cases.19 Progressive additive deterioration caused by high testicular temperature in the cryptorchidism cases, as described by Mieusset et al,20 may be prevented if the testes are brought to the scrotum early. If PLAP positive gonocytes are still in place and adequate natural or supplementary gonadotropin stimulation is present, gonocyte transformation into Ad spermatogonia may still be possible, although with some delay.1 Estimating PLAP positive germ cells and evaluating the number of Ad/T are probably additional important parameters when patient selection is considered for supplementary gonadotropin treatment. The limitations of our study are related to the modest sample size, especially when divided into subgroups, as well as possible irregularity in immunohistochemical staining and subjectivity in Ad/T estimation. However, the blinded fashion of cell evaluation makes the data reliable for drawing conclusions.
CONCLUSIONS Estimating the number of PLAP positive germ cells in infant cryptorchid testes and evaluating the number of Ad/T are additional important parameters related to the fertility potential of boys with cryptorchidism. Within the first years of life, when orchiopexy is often performed, the normal range of the number of Ad/T and the presence of PLAP positive germ cells are highly age dependent and not yet fully elucidated. Further investigations are needed to determine specific prognostic criteria related to fertility potential.
REFERENCES 1. Thorup J, Petersen BL, Kvist K et al: 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. 2. Cortes D: Cryptorchidism: aspects of pathogenesis, histology and treatment. Scand J Urol Nephrol, suppl., 1998; 32: 1. 3. Hadziselimovic F and Hoecht B: Testicular histology related to fertility outcome and postpubertal hormone status in cryptorchidism. Klin P€adiatr 2008; 220: 302.
4. Hadziselimovic F and Herzog B: The importance of both an early orchidopexy and germ cell maturation for fertility. Lancet 2001; 358: 1156. 5. Rajpert-De Meyts E, Bartkova J, Samson M et al: The emerging phenotype of the testicular carcinoma in situ germ cell. APMIS 2003; 111: 267. 6. Jørgensen N, Giwercman A, M€uller J et al: Immunohistochemical markers of carcinoma in situ of the testis also expressed in normal infantile germ cells. Histopathology 1993; 22: 373. 7. Cortes D, Thorup J, Hogdall E et al: The relation of germ cells per tubule in testes, serum inhibin
B and FSH in cryptorchid boys. Pediatr Surg Int 2007; 23: 163. 8. Andersson AM, Toppari J, Haavisto AM 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. 9. Cortes D, Thorup J and Visfeldt J: Cryptorchidism: aspects of fertility and neoplasms. Horm Research 2001; 55: 21. 10. Cortes D, Thorup J and Beck BL: Quantitative histology of germ cells in the undescended
ADULT DARK SPERMATOGONIA AND FERTILITY POTENTIAL IN CRYPTORCHID TESTES
testes of human fetuses, neonates and infants. J Urol 1995; 154: 1188. 11. Thorup J, Cortes D and Nielsen OH: Clinical and histopathologie evaluation of operated maldescended testes after luteinizing hormone-releasing hormone treatment. Pediatr Surg Int 1993; 8: 419. 12. Hadziselimovic F, Zivkovic D, Bica DT et al: The importance of mini-puberty for fertility in cryptorchidism. J Urol 2005; 174: 1536. 13. Hadziselimovic F, Emmonsa LR and Buser MW: A diminished postnatal surge of Ad spermatogonia in cryptorchid infants is additional evidence for hypogonadotropic hypogonadism. Swiss Med Wkly 2004; 134: 381.
14. Jørgensen N, Rajpert-De Meyts E, Graem N et al: Expression of immunohistochemical markers for testicular carcinoma in situ by normal human fetal germ cells. Lab Invest 1995; 72: 223. 15. Hutson JM, Li R, Southwell BR et al: Germ cell development in the postnatal testis: the key to prevent malignancy in cryptorchidism? Front Endocrinol 2013; 3: 176. 16. Hadziselimovic F and Herzog B: Hodendystopie. In: Kinderurologie in Klinik und Praxis, 2nd ed. Edited by JW Th€uroff and H SchulteWissermann. New York: Thieme 2000; pp 484e500. 17. Rusnack SL, Wu HY, Huff DS et al: Testis histopathology in boys with cryptorchidism correlates
1571
with future fertility potential. J Urol 2003; 169: 659. 18. Kraft KH, Canning DA, Snyder HM et al: Undescended testis histology correlation with adult hormone levels and semen analysis. J Urol 2012; 188: 1429. 19. Cortes D: Editorial comment: Undescended testis histology correlation with adult hormone levels and semen analysis. J Urol 2012; 188: 1435. 20. Mieusset R, Fonda PJ, Vaysse P et al: Increase in testicular temperature in case of cryptorchidism in boys. Fertil Steril 1993; 59: 1319.
EDITORIAL COMMENT Testicular biopsies have implicated a decreased germ cell count as the histological basis of subfertility in patients with undescended testes. The authors convincingly report that the presence and number of PLAP positive germ cells should be considered additional histological parameters when assessing fertility potential in bilateral cryptorchidism cases. Including adult hormone levels, semen analysis or paternity data in this cohort would further clarify the clinical relevance of these findings, particularly since associations between germ cell histology and these variables suggest that testicular biopsy at bilateral orchiopexy may help predict future fertility
(reference 18 in article). Nonetheless, these results underscore the value of reviewing testis histology in patients with bilateral cryptorchidism when counseling families about fertility potential. Further investigation is warranted to determine whether testicular biopsy at bilateral orchiopexy should be routine clinical practice for detecting boys at risk for future infertility. Kate H. Kraft Department of Urology University of Michigan Health System Ann Arbor, Michigan