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Multinucleated spermatogonia in cryptorchid boys: A possible association with an increased risk of testicular malignancy later in life

Multinucleated spermatogonia in cryptorchid boys: A possible association with an increased risk of testicular malignancy later in life
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  APMIS 111: 25–31, 2003 Copyright  C  APMIS 2003Printed in Denmark . All rights reserved ISSN 0903-4641 Multinucleatedspermatogoniaincryptorchidboys:Apossibleassociationwithanincreasedriskoftesticularmalignancylaterinlife? DINA CORTES, 1,2 JØRGEN THORUP 1 and JAKOB VISFELDT 3 The Departments of   1 Pediatric Surgery and  3 Pathology, Rigshospitalet, and the Department of   2 Pediatrics,Glostrup Hospital, University of Copenhagen, DenmarkCortes D, Thorup J, Visfeldt J. Multinucleated spermatogonia in cryptorchid boys. A possible associ-ation with an increased risk of testicular malignancy later in life. APMIS 2003;111:25–31.At birth, undescended testes contain germ cells, but after 1 year of life, a reduced number of germcells is generally found. Microlithiasis and carcinoma-in-situ-testis occur in cryptorchid boys. Multi-nucleated germ cells, including at least 3 nuclei in the cell, exist in impaired spermatogenesis and inthe senescent testis.  Aim of the study.  We investigated whether multinucleated spermatogonia werepresent in undescended testes of cryptorchid boys, and if such a pattern is associated with specialclinical features.  Results.  Multinucleated spermatogonia occurred in 13/168 (8%) of 163 consecutivecryptorchid boys, who underwent surgery for cryptorchidism with simultaneous testicular biopsyshowing seminiferous tubules. The patients with multinucleated spermatogonia more often exhibiteda normal germ cell number (Fisher’s exact test, p  0.0005), and were younger at surgery (MannWhitney, p  0.005) than the rest of the patients. Before surgery, 3 patients underwent treatment withErythropoietin because of renal failure. An intra-abdominal testis underwent clipping and division of the spermatic vessels, and a biopsy at final surgery 7 months later, exhibited multinucleated spermato-gonia. In 1 case the undescended testicular position, a fixed retraction, was acquired after surgery foran inguinal hernia. Multinucleated spermatogonia were found in cases of carcinoma-in situ-testis in2 cryptorchid boys. No case of multinucleated germ cells appeared in our normal material.  Conclusion. Multinucleated spermatogonia are a further abnormality present in cryptorchidism. The cryptorchidboys with multinucleated spermatogonia in general exhibited rather many germ cells. This featuremay be associated with an increased risk of testicular malignancy later in life, and we propose acareful follow up regime in these cases including ultrasound examination and a testicular biopsy incases of symptoms or clinical findings.Key words: Multinucleated spermatogonia; germ cells; cryptorchidism; carcinoma-in-situ-testis; mi-crolithiasis.Dina Cortes, Department of Pediatrics, Glostrup Hospital, University of Copenhagen, Ndr Ringvej,DK-2600 Glostrup, Denmark. e-mail: DICO / At birth, undescended testes contain germ cells.However, a reduced number of germ cells pertubular cross section (S/T) may be found in in-tra-abdominal and intra-canalicular testes innewborn boys (1). After 1 year of life, onlyabout 10% of undescended testes exhibit a nor-mal number of germ cells. From 18 months of age, a lack of germ cells has been reported (1,252). After that age, a progressive number of biop-sies from undescended testes at surgery. Theabsence of germ cells at orchidopexy is associ-ated with subsequent infertility (2, 3).Microlithiasis (4–6) and carcinoma in situ tes-tis have been reported in cryptorchid boys (2, 3,5). Examination for testicular neoplasm is rec-ommended in cases of intra-abdominal testis,  CORTES  et al  . Fig. 1.  The number of spermatogonia and gonocytes per tubular transverse section, S/T, in 163 cryptorchidboys. In 13 patients multinucleated spermatogonia were demonstrated. abnormal external genitalia or in cases of aknown abnormal karyotype (3).In multinucleated germ cells at least 3 nucleishare a common cytoplasm. All developmentalstages of germ cells, viz. spermatogonia, sper-matocytes and spermatids, may be affected. Inmultinucleated spermatogonia the nuclei maybe of the same type, either type A pale or typeA dark, or a mixture of both types (7).In the human testis, multinucleated germ cellshave been observed in men with disturbed sper-matogenesis, for example following treatmentwith antiandrogens, cystostatic agents or X-rays(8), and in the regressively altered senescentseminiferous epithelium (9, 10). In rats, multi-nucleated spermatids have been reported undervarious experimental conditions, including ex-posure to drugs or chemicals such as cimetidine,a contraceptive agent (1-(2,4-dichlorobenzyl)-indazol-3-carbohydrazide), and herbicides (11).Furthermore, transgenic mice such as those de-ficient in aromase (cyp 19) gene, BAX or hor-mone-sensitive lipase, exhibited multinucleatedspermatids (11). The presence of multinucleatedgerm cells has been taken as an early sign of testicular atrophy or involution (9, 10), as wellas a result of testicular degeneration associated26with infertility (11). We report the occurrence of multinucleated germ cells in cryptorchid boys.MATERIALS AND METHODS The study includes 163 consecutive cryptorchid boyswho in the period from April 1997 to April 2002underwent surgery for cryptorchidism with simul-taneous testicular biopsy, showing seminiferous tu-bules. The case reports included the age of patientsat surgery, the testicular location and the informationof previous hormonal treatment before surgery. Ex-cluded was a boy with fallopian tubes and a uterus.All tissue specimens were fixed in Stieve’s solution,embedded in paraffin, and 4 m m-sections were stainedwith haematoxylin-eosin and van Gieson’s staining.The majority of sections furthermore underwentstaining with Anti-MIC-2, as previously reported(12). In short, Anti-MIC-2 is an immunohistochem-ical method with DAKO antibody to the MIC2 geneproduct (MIC2, 12 E7, code no. M306) in order toobtain a ‘‘negative reaction’’ of germ cells, con-trasting with the stained Sertoli cells.In blinded fashion, the number of spermatogoniaand gonocytes per tubular cross-section (S/T) wasmeasured from at least 100 tubular cross sections, aspreviously described (1, 2). Gonocytes are locatedcentrally in the seminiferous tubules, are round orovoid, have relatively little cytoplasm, and relatively  MULTINUCLEATED SPERMATOGONIA – BOYS Fig. 2.  A. Biopsy from a bilateral cryptorchid boy, 1.8 years of age at surgery, showing multinucleated spermato-gonia and microlithiasis. Treatment with Erythropoietin was given before surgery because of renal failure. HE ¿ 90. B. Biopsy from a unilateral cryptorchid boy, 2.5 years of age at surgery with multinucleated spermatogon-ia. The testis was located intra-abdominally, and 7 months before the testis underwent clipping and division of the spermatic vessels, as part of Fowler-Stephens procedure. Anti-MIC 2 ¿ 180. C. Biopsy from a unilateralcryptorchid boy, 1.3 years of age at surgery, showing multinucleated spermatogonia. Anti-MIC 2 ¿ 180. D.Biopsy from an undescended testis that had been cultured for 21 days showing multinucleated spermatogonia(arrow). At surgery a normal number of germ cells, but no multinuclear spermatogonia were found. Anti-MIC2 ¿ 180.large bright nuclei with a fine chromatin pattern and1 to 2 nucleoli. In contrast, spermatogonia are larger,flattened, and located on the basement membrane.Spermatogonia have a rather abundant pale cyto-plasm and their nuclei are large and round or ovoidwith fine granular chromatin and 1 to 2 nucleoli, orthey are smaller and round with dark granularchromatin (1, 7). We investigated if multinucleargerm cells were present. In cases of multinuclear germcells the affected cells were classified, and it was cal-culated how often the cells appeared as multi-nucleated. For example, if 250 tubular cross sectionswere counted, 200 spermatogonia and gonocytes weredemonstrated, 5 spermatogonia were multinucleated,and no further germ cells were identified, then the S/T was 200/250 Ω 0.8, and multinucleated spermato-gonia were found in 5/200 ¿ 100% Ω 2.5% of the germcells. 27 Previously reported normal material served as ref-erence for the normal number of spermatogonia andgonocytes per tubular cross-section (S/T), and for oc-currence of multinucleated germ cells. The normalmaterial has previously been published, and includedthe testes of 46 boys and adolescents who were 0– 17.9 years old when they died suddenly and unexpect-edly, or from meningitis or pneumonia less than 6days in duration. The lowest normal S/T value was0.69 in 0–0.9 year-old boys, 0.38 in 1–3.9 year-oldboys, 0.65 in 4–9.9 year-old boys, 1.8 in 10–13.9 year-old boys and 4.9 in 14–18 year-old boys (1, 2).We compared the data of the patients with multi-nucleated germ cells to the rest of the patients toevaluate if multinucleated germ cells were associatedwith: a normal number of germ cells, bilateral crypt-orchidism, an intra-abdominal testicular position,unsuccessful hormonal treatment with human chori-  CORTES  et al  . Fig 3.  Biopsy from a bilateral cryptorchid boy 10.9years old at surgery showing multinucleated sperm-atogonia and carcinoma-in-situ testis. HE ¿ 180.onic gonadotropin (hCG) or Gonadotropin Re-leasing Hormone (Gn-RH) before surgery for crypt-orchidism, or another medical treatment beforesurgery for cryptorchidism (Fisher’s Exact test). Fur-thermore, we calculated if the patients with multi-nucleated germ cells had a lower age at surgery thanthe rest of the patients (Mann-Whitney test). RESULTSWe demonstrated multinucleated germ cells in13 of 163 cryptorchid boys (8%), Figs. 1–2. Thepatients with multinucleated spermatogoniahad unilateral cryptorchidism in 10 cases, andbilateral in 3 cases. The testes were located atthe external inguinal ring in 14 cases, in theinguinal canal in 1 case, and 1 testis was intra-abdominally placed. In all cases the multi-nucleated germ cells were spermatogonia, whichwere the highest differentiated germ cell presentin these tubules. The multinucleated spermato-gonia were seen in median 6% of the germ cells(range 2.4–16%).The patients with multinucleated germ cellsexhibited a normal number of spermatogoniaand gonocytes per tubular cross-section (S/T)with a higher frequency than in the group of patients without multinucleated spermatogoniagerm cells (Fisher’s Exact test, p  0.00005). Thepatients with multinucleated germ cells wereaged from 0.5 to 7.9 years, median 1.5 years, atsurgery for cryptorchidism, and were youngerat surgery than the rest of the patients (Mann-Whitney test, p  0.005), Table 1.Treatment with Erythropoietin had been28given to 4 patients because of renal function im-pairment. Multinucleated spermatogonia ap-peared at surgery in 3 of these patients, therebymaking the frequency of previous treatmentwith Erythropoietin go higher in the cryptor-chids with multinucleated spermatogonia thanin the rest of the patients (Fisher’s Exact test,p  0.005). The number of multinucleatedspermatogonia in the patients treated with Er-ythropoietin was; 2.8%, 3.0% and 6.6% of thegerm cells, which was equivalent to the numberof multinucleated spermatogonia in the otheraffected patients (Mann Whitney test, p Ω 0.573). The 4th patient who had undergone pre-vious treatment with Erythropoietin totallylacked germ cells.In the group of patients with multinucleatedspermatogonia one patient had the testis intra-abdominally located, and in one case the undes-cended state of the testis was acquired aftersurgery for an inguinal hernia. The latter casewas 7.9 years old at surgery, and the oldest pa-tient with multinucleated spermatogonia.In no other ways did the patients with multi-nucleated germ cells differ from the rest of thepatients, Table 1.No case of multinucleated germ cells ap-peared in our normal material.DISCUSSIONMultinucleated spermatogonia occurred in 8%of the cryptorchid boys. These patients ex-hibited a normal number of germ cells in 77%of cases, which is a higher frequency than isnormal in material of undescended testes (1, 2).Multinucleated spermatogonia occured aftertreatment with Erythropoietin because of renalfailure (13). Erythropoietin is a growth factorwhich is involved in the regulation of testicularfunction like epidermal growth factor, insulin-like growth factor I, transforming growth fac-tor-beta, fibroblast growth factor and insulin(14). These growth factors influence the Leydigcell steroidogenesis directly by binding to speci-fic plasma membrane receptors (14). Human re-combinant erythropoietin treatment improvessexual function in end-stage renal failure pa-tients. In healthy young men injection of humanrecombinant erythropoietin increased the tes-tosterone level in the spermatic veins, but not  MULTINUCLEATED SPERMATOGONIA – BOYS TABLE 1.  Clinical data of 13 cryptorchid patients with multinucleated spermatogonia compared to 150 cryptor-chid patients without multinucleated spermatogonia Variable Patients with multi- Patients without multi- Statisticalnucleated spermatogonia nucleated spermatogonia analysisNo of patients 13 150Age at surgery (years)* 1.5 (0.5–7.9) 4.4 (0.25–14.9) p  0.005No of patients  2 years ¤  11/13 19/150 p  0.00005Normal S/T-value (ref. 1, 2) . 9/13 10/150 p  0.00005No germ cells . 0/13 34/150 p Ω 0.0836Intra-abdominal testis . 1/13 28/150 p Ω 0.5741Bilateral cryptorchidism . 3/13 35/150 p Ω 1.0000Unsuccesful hormonal 1/13 25/(150–8 § ) p Ω 0.6418treatment (hCG or Gn-RH) . EPO treatment . 3/13 1/150 p  0.005* Mean (range), Mann Whitney test. . Frequency, Fisher Exact test. ¤  At time of surgery for cryptorchidism. § In 8 cases it was unknown if the patient had undergone unsuccessful hormonal treatment before surgery forcryptorchidism. in the peripheral veins. On the other hand theinfusion had no effect on plasma LH and FSHlevels in peripheral or spermatic veins (15). Theinteraction between Leydig cell steroidogenesisand germ cell proliferation is not fully under-stood (2).Multinucleated spermatogonia also occurredafter a Fowler Stephens procedure with clippingand division of the spermatic vessels. Thatspecial patient was one out of 10 with repeattesticular biopsies after clipping and dividing of the spermatic vessels, and he was the one withthe highest number of germ cells in that series(16). Compromised artery supply has been as-sociated with multinucleated germ cells inelderly men and experimental in rats (17, 18).The features of such testes are a mixture of tu-bules with complete spermatogenesis and a re-duced number of spermatids, tubules withmultinucleated germ cells, spermatocyte andspermatid degeneration or spermatid sloughing,and sclerosed tubules (9).Furthermore, we found multinucleatedspermatogonia in the biopsy of a patient whodeveloped cryptorchidism after surgery foringuinal hernia. In such cases, the general pat-tern is a normal number of germ cells beforethe acquired fixed retraction out of the scrotum,but a decreased number of germ cells appearsafter 1 year of undescended position (1). Scrotaltesticular position is so important for the sper-matogenesis that non-surgical testicular fixation29close to the inguinal canal has been proposed asa contraceptive method (18).Previously, we have published material on tes-ticular biopsies that were taken at the time of surgery for cryptorchidism and had been cul-tured for up to 21 days in vitro (12). In thatmaterial we also noted the occurrence of multi-nucleated spermatogonia, Fig. 2D. These in vi-tro findings seen under unusual growth con-ditions are in agreement with the results of thepresent study. In general, the undescended testeswith multinucleated spermatogonia exhibited acombination of rather many spermatogonia andunusual growth conditions.However, we also found multinucleatedspermatogonia in 3 of 6 testes with carcinomain situ testis in 2 cryptorchid boys previouslydescribed, Fig. 3. These 3 testes were found intwo 10 year-old cryptorchid boys with bilateralcryptorchidism and abnormal external genitalia(3, 19, 20). Multinucleated spermatogonia havenot been described as tumour cells (7).Microlithiasis was associated with multi-nucleated spermatogonia in two of the patientsin the present material, Fig. 2A. One patienthad been treated with Erythropoietin beforesurgery for cryptorchidism, and the other onedeveloped crytorchidism after surgery for aninguinal hernia. Microlithiasis is associatedwith germ cell neoplasia including carcinoma insitu testis in adults and boys (4–6). In cases of microlithiasis in boys it has been recommended
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