Erik Clasen-Linde

Cryopreserved ovarian cortex from patients with leukemia in complete remission contains no apparent viable malignant cells

Some women suffering from leukemia require bone marrow transplantation to be cured. Bone marrow transplantation is associated with a high risk of sterility, and some patients are offered fertility preservation by cryopreservation of the ovarian cortex. Transplantation of the ovarian cortex to women cured of leukemia who became menopausal is currently not performed because of the risk of introducing the disease. In this study, individual pieces of ovarian cortex intended for reimplantation from 25 patients with leukemia were transplanted to each of 25 nude mice for 20 weeks. The ovarian cortex was examined before and after transplantation by histology and immunohistochemistry, and RT-quantitative PCR (in the 7 patients with a known marker). Seventeen patients had the ovarian cortex retrieved when they were in complete remission. Before transplantation, 4 of 7 pieces (2 from patients in complete remission) of ovarian cortex had a positive RT-quantitative PCR. After transplantation, none of the mice revealed any sign of disease, neither in the pieces of ovarian cortex transplanted nor in any of the murine organs evaluated. Thus, the ovaries from patients in complete remission do not appear to contain viable malignant cells contrasting ovarian tissue retrieved before treatment.

The Relation between Adult Dark Spermatogonia and Other Parameters of Fertility Potential in Cryptorchid Testes

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.

The Sertoli cell hormones inhibin-B and anti Müllerian hormone have different patterns of secretion in prepubertal cryptorchid boys

OBJECTIVES AND HYPOTHESES The Sertoli-cells produce inhibin-B and Anti-Müllerian-Hormone (AMH). Much is still unknown about these hormones in prepubertal cryptorchids. The Sertoli-cells are mandatory for germ cell development. The aim of the study was to investigate if there are differences in secretion pattern of Sertoli-cell hormones and their gonadotropin feed-back mechanisms. METHODS Included were 94 prepubertal cryptorchid boys 0.5-13.1years with measurements of serum-inhibin-B, Anti-Müllerian-Hormone (AMH), Luteinizing Hormone (LH) and Follicle Stimulation Hormone (FSH). The serum values were measured using commercially available kits. The hormonal values were related to age-matched normal values. Testicular biopsy was taken at orchiopexy. RESULTS Inhibin-B positively correlated to AMH for 1-13year-old patients (p<0.0001), but not for 0.5-1year-old patients (p=0.439). For 0.5-1year-old patients inhibin-B-values tended to decrease (p=0.055), in contrast to AMH-values (p=0.852). LH was elevated more often than FSH (p=0.014). FSH and LH were positively associated in patients both 0.5-1year (p=0.042) and 1-13years of age (p<0.0001). LH correlated positively to inhibin- B (p=0.001). In contrast, FSH did not correlate to inhibin-B or AMH (p=0.755 and p=0.528). The number of A-dark spermatogonia per tubular transverse section was positively correlated to inhibin-B serum level. CONCLUSION Our new finding of an association between LH and inhibin-B in infancy of cryptorchid boys may be essential for the transformation of gonocytes to A-dark spermatogonia. Previously, LH associated to inhibin-B was described in early puberty only. During the first year of life inhibin-B values decreased faster than AMH. The AMH-levels may just reflect the increased Sertoli cell number that occurs during the first 3months of life.

Serum inhibin B values in boys with unilateral vanished testis or unilateral cryptorchidism.

PURPOSE Boys with cryptorchidism have overall increased gonadotropin and decreased serum inhibin B levels compared to normal. Serum inhibin B levels, produced by Sertoli cells, may reflect the state of germinative epithelium in cryptorchid testes. We evaluated whether serum inhibin B levels differed between boys with unilateral vanished testis and those with unilateral cryptorchidism. MATERIALS AND METHODS Blood samples from 297 boys 1.5 to 5 years old were included, of whom 222 had unilateral cryptorchidism, 29 had unilateral vanished testis and 46 had undergone unilateral orchiopexy 1 year previously. Serum inhibin B levels were measured using a commercially available ELISA kit and were compared to normal range. RESULTS Serum inhibin B levels in boys with unilateral vanished testis were not different from those with unilateral cryptorchidism. Serum inhibin B values were above the normal median in 43% of boys previously operated on for unilateral cryptorchidism, compared to 17% at surgery (p = 0.0003). The percentage of patients with inhibin B levels below normal range was 14% in those with unilateral vanished testis, 23% in those with unilateral cryptorchidism and 11% in those who had undergone orchiopexy 1 year previously for unilateral cryptorchidism. The percentage of boys with inhibin B levels above normal median was 24% in those with unilateral vanished testis, 17% in those with unilateral cryptorchidism and 43% in those who had undergone orchiopexy. However, in boys with a vanished testis the frequency of serum inhibin B above normal median was only 5% before age 1.5 years, after which the rate was 67% (p = 0.0022). CONCLUSIONS Our findings may reflect the development of contralateral testicular hypertrophy in boys with unilateral vanished testis. The initial low inhibin B values may be explained by impaired total number of Sertoli cells. Serum inhibin B values also indicated that in 6-month to 5-year-old boys with cryptorchidism orchiopexy was beneficial for the germinative epithelium.

Adult immunohistochemical markers fail to detect intratubular germ cell neoplasia in prepubertal boys with cryptorchidism.

PURPOSE Intratubular germ cell neoplasia is a precursor to testicular germ cell cancer. The condition is characterized by large germ cells with large nuclei with a hyperchromatic, coarse chromatin pattern, large prominent nucleoli and abundant pale cytoplasm. In prepubertal boys these cells are located centrally and peripherally mixed with normal cells in the seminiferous tubules. We evaluated the impact of adult intratubular germ cell neoplasia marking immunohistochemistry in screening for intratubular germ cell neoplasia in boys with cryptorchidism. MATERIALS AND METHODS Histology sections of 236 testicular biopsies were retrieved from 170 boys 1 month to 15 years old operated on for cryptorchidism (excluding disorders of sex development). Specimens were incubated with primary antibodies, including anti-placental-like alkaline phosphatase, anti-Oct3/4, anti-C-kit and anti-D2-40 receptor. RESULTS A 1-year, 1-month-old boy had intratubular germ cell neoplasia and all positive markers. The prevalence of placental-like alkaline phosphatase positive staining of germ cells in testicular biopsies was 98% in boys younger than 1 year, 82% in those 1 to less than 2 years old, 74% in those 2 to less than 3 years old and 60% in those 3 to 15 years. Similarly the prevalence of C-kit positive staining was 71% in boys younger than 1 year, 49% in those 1 to less than 2 years, 16% in those 2 to less than 3 years and 34% in those 3 to 15 years. Placental-like alkaline phosphatase negative germ cells did not express any of the other described antigens. In none of the 116 testes from boys older than 1 year and 7 months were any Oct3/4 or D2-40 positive germ cells identified. Up to that age 33% and 8% of biopsies were Oct3/4 and D2-40 positive, respectively. CONCLUSIONS Adult intratubular germ cell neoplasia/cancer immunohistochemical markers cannot be used alone for intratubular germ cell neoplasia screening in male infants with cryptorchidism because positive immunohistochemistry is commonly seen within this age group, when most orchiopexies are performed. It is generally not plausible that intratubular germ cell neoplasia originates during fetal development in patients with cryptorchidism.

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

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), follicle-stimulating 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 0.5-2.3 IU/l (mean: 1.0 IU/l) and FSH 1.3-2.2 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.

Postnatal Germ Cell Development in the Cryptorchid Testis: The Key to Explain Why Early Surgery Decreases the Risk of Malignancy

Purpose Cryptorchidism is a risk factor for testicular malignancy and surgical treatment lowers this risk. This study aimed to investigate the germ cell behavior in prepubertal cryptorchid testes using immunohistochemical markers for germ cell malignancy to understand how early orchiopexy may possibly prevent cancer developing. Materials and Methods Histology sections from 1,521 consecutive testicular biopsies from 1,134 boys aged 1 month to 16.5 years operated for cryptorchidism were incubated with antibodies including antiplacental‐like alkaline phosphatase (PLAP), anti‐Oct3/4, anti‐C‐kit, and anti‐D2‐40. Results Oct3/4 and D2‐40‐positive germ cells are found throughout the first 2 years of life, with declining frequency thereafter. After 2 years, they should have disappeared and may indicate neoplasia. PLAP‐positive cells were seen in 57 to 82% and C‐kit‐positive cells in 5 to 21% of cryptorchid testes between 4 and 13 years. Not until puberty did PLAP and C‐kit‐positive undifferentiated spermatogonial stem cells vanish. Only 0.3% of the present material had obvious prepubertal intratubular germ cell neoplasia (ITGCN) and they all had syndromic cryptorchidism. An additional three boys (0.3%) older than 2 years had weak Oct3/4 expression in undescended testes, but all cases were D2‐40 negative. Conclusion Prepubertal ITGCN was rare and mostly seen in syndromic cryptorchidism. In nonsyndromic cryptorchidism PLAP‐positive undifferentiated spermatogonial stem cells persisted in a significant proportion of nontreated undescended testes and they will be especially sensitive to long‐lasting abnormally high temperature that may be the single most important cause facilitating the accumulation of mutations during cell replication and the development of ITGCN to be prevented by orchiopexy.

The value of positive Oct3/4 and D2-40 immunohistochemical expression in prediction of germ cell neoplasia in prepubertal boys with cryptorchidism

Abstract Objective: Intratubular germ cell neoplasia (ITGCN) is a precursor to testicular germ cell cancer. Adult germ cell cancer immunohistochemical markers fail to detect ITGCN in prepubertal boys with congenital cryptorchidism, because positive immunohistochemistry is commonly seen below 18 months old, where most orchiopexies are performed. The aim of the study was to evaluate the ability of Oct3/4 and D2-40 immunohistochemical markers to detect ITGCN in boys older than 2 years with cryptorchidism. Materials and methods: Histological sections from 309 testicular biopsies from 234 boys aged 1 month to 14 years, 6 months operated on for cryptorchidism were incubated with primary antibodies including anti-placental-like alkaline phosphatase, anti-Oct3/4, anti-C-kit and anti-D2-40 receptor. Results: One 3-year, 8-month-old boy with 45X/46XY disorder of sexual development had ITGCN and all positive markers. Besides this case, none of the 192 testes except one from boys older than 2 years had any Oct3/4- or D2-40-positive germ cells identified. The germ cells of the right testis from a 3-year, 7-month-old boy had weak Oct3/4 expression but were D2-40 negative. The prevalences of Oct3/4- and D2-40-positive staining of germ cells in testicular biopsies were, for each age group: < 6 months, 100% and 50%; 6 months to < 1 year, 65% and 16%; 1 to < 2 years, 15% and 3%; and 2 years to < 14 years, 6 months, 2% and 1%, respectively. Conclusion: Oct3/4 and D2-40 immunohistochemical markers may be beneficial in detecting ITGCN in boys older than 2 years with cryptorchidism. Even when immunohistochemistry is applied, prepubertal ITGCN is so rarely demonstrated in cryptorchid testes that it is not plausible that ITGCN generally originates during fetal development in cryptorchidism.

The diagnostic impact of testicular biopsies for intratubular germ cell neoplasia in cryptorchid boys and the subsequent risk of testicular cancer in men with prepubertal surgery for syndromic or non-syndromic cryptorchidism

INTRODUCTION Cryptorchidism is a risk factor for testicular cancer in adult life. It remains unclear how prepubertal surgery for cryptorchidism impacts later development of adult testicular cancer. The aim of study was to investigate tools to identify the cryptorchid boys who later develop testicular cancer. METHODS The study cohort consisted of 1403 men operated prepubertally/pubertally for undescended testis between 1971 and 2003. At surgery testicular biopsies were taken from the cryptorchid testes. The boys were followed for occurrence of testicular cancer. The testicular cancer risk was compared to the risk in the Danish Population. Testicular biopsies from the boys who developed testicular cancer during follow-up underwent histological examination with specific diagnostic immunohistochemical markers for germ cell neoplasia. RESULTS The cohort was followed for 33,627 person years at risk. We identified 16 cases with testicular cancer in adulthood. The standardized incidence ratio was 2.66 (95% CI: 1.52-4.32). At time of primary surgery in prepubertal/pubertal age Intratubular Germ Cell Neoplasia (ITGCN) was diagnosed in 5 cases and the boys were unilaterally orchiectomized. At follow-up new immunohistochemical staining indicated ITGCN in two of the 16 cancer cases at reevaluation of the original biopsies from time of prepubertal/pubertal surgery. One had syndromic cryptorchid and developed seminoma, and another showed nonsyndromic cryptorchidism and developed embryonic teratocarcinoma. Totally, ITGCN was diagnosed in 0.5% (7/1403) of prepubertal cryptorchid boys, whereof 57% (4/7) in syndromic-cryptorchidism. DISCUSSION ITGCN is predominantly observed prepubertally in boys with syndromic-cryptorchidism. In nonsyndromic cryptorchidism testicular cancer develops postpubertally, generally not based on dormant germ cells of ITGCN caused by an early fetal maldevelopment. LEVELS OF EVIDENCE LEVEL I.

Positive Oct -3/4 and D2–40 Immunohistochemical Expression in Germ Cells and Suspected Histology Pattern of Intratubular Germ Cell Neoplasia in Boys with Cryptorchidism Vanish after the Age of 2 Years

Introduction Intratubular germ cell neoplasia (ITGCN) is a precursor to testicular germ cell cancer. Adult germ cell cancer immunohistochemical markers may fail to detect ITGCN in prepubertal boys with congenital cryptorchidism, because positive immunohistochemistry is commonly seen in boys younger than the age of 2 years, where most orchiopexies are performed. The aim of the study was to evaluate the diagnostic challenge to differentiate between a histological pattern of ITGCN and a histological pattern with some atypical germ cells and all positive cancer immunohistochemical markers, but no increased risk of malignancy. Materials and Methods Histology sections from 373 testicular biopsies from 289 boys aged 1 month to 2 years operated for cryptorchidism were incubated with primary antibodies including anti‐placental‐like‐alkaline phosphatase, antiOct‐3/4, anti‐C‐kit, anti‐D2‐40, and in case of repeat biopsy with anti‐stem cell factor (SCF) receptor. Results The prevalence of Oct‐3/4 and D2‐40‐positive staining of germ cells in testicular biopsies were in age groups less than 6 months, 100% and 50%; 6‐12 months, 60% and 17%; and 1‐2 years, 12% and 4%. A 1 year, 1‐month‐old boy with Prader‐Willi syndrome treated with growth hormone had ITGCN in both cryptorchid testes. In another three bilateral nonsyndromic cases, 8 months, 8 months and 1‐year‐old, a histological pattern in accordance with ITGCN was found. These three boys had a repeat biopsy from both testes performed at the age of 3 years, 4 months, 3.5 years, and 3 years, 10months, respectively. In all cases, the Oct‐3/4 and D2‐40 positive germ cells turned negative and the histological pattern normalized completely. The primary biopsies had SCF negative germ cells. Conclusion This study is valuable in identifying the age‐related change in Oct‐3/4 or D2‐40 immunopositive germ cells in seminiferous tubules. An ITGCN‐like histological pattern in nonsyndromic cryptorchidism will vanish after the age of 3 years. Even when immunohistochemistry is applied, prepubertal ITGCN is so rarely demonstrated in cryptorchid testes, that it is not plausible that ITGCN generally originates during fetal development in cryptorchidism.