Germ cells positive for PLAP and c-Kit in 11-16 year old normal boys with ongoing spermatogenesis.

INTRODUCTION: Positive staining of testicular germ cells for PLAP and c-Kit beyond infancy may be associated with the presence of GCNIS (Germ Cell Neoplasia In Situ). We recently reported our findings of positive staining of normal, infantile germ cells for PLAP, and c-Kit up to 2 years of age, contrary to previous studies. The present study aims to elucidate whether otherwise normal testes of boys undergoing puberty express PLAP, c-Kit, Oct3/4, or D2-40. MATERIALS AND METHODS: Biopsies were taken from 31 boys (11.5-16.5 years of age, mean and median of 13.5 years), who underwent surgery either for torsion of the testis (15) or a history suspicious of intermittent torsion of the testis (16). 21 were biopsied on both sides, making a total of 52 biopsies. Four testes were necrotic. The biopsies were fixed in Stieve's medium, cut into 2 µm sections, and mounted on coated slides. One slide was processed for H-E, and the others incubated with primary antibody for PLAP, c-Kit, D2-40, and Oct3/4. RESULTS: 87% of the boys stained positive for both PLAP and c-Kit. None were positive for either D2-40 or Oct3/4. None had any histological features characteristic of GCNIS. Only two boys showed no signs of having initiated spermatogenesis. Those positive for PLAP were likewise for c-Kit, and vice versa, except 2; one boy, 13 years, was positive for PLAP, but negative for c-KIT, another, 16 years, was negative for PLAP and positive for c-Kit. Three boys stained positive for PLAP and c-Kit on the right side, and negative on the left. One boy was negative for c-Kit on the right side, positive on the left, and positive for PLAP bilaterally. CONCLUSION: Positive staining of testicular germ cells for PLAP and c-Kit seems to be a normal finding in boys not having completed puberty. Rather than indicating pre-malignant transformation, the positivity is indicative of an ongoing maturational process of the germ cells.

Postnatal germ cell development in cryptorchid boys.

Cryptorchidism is associated with infertility in adulthood. Early orchiopexy is suggested to reduce the risk. Information is lacking on the potential link between infant germ cell maturation and the risk of future infertility. The objective of the study was to evaluate age-related germ cell development in cryptorchidism. Immunostaining for markers of germ cell development (octamer-binding transcription factor 3/4 [OCT3/4], placental alkaline phosphatase [PLAP], KIT proto-oncogene [C-KIT], podoplanin [D2-40], Lin-28 homolog A [LIN28], and G antigen 7 [GAGE-7]) was performed in testicular biopsies from 40 cryptorchid boys aged 4-35 months. Germ cell numbers and distributions were evaluated in cross sections of seminiferous tubules, with and without immunostaining. OCT3/4, D2-40, and LIN28 were generally expressed in the early stages of germ cell development, as shown by positive expression in germ cells in the central region of seminiferous tubules. In contrast, PLAP and GAGE-7 were expressed in both central and peripheral parts of the tubules in the early stages of development and expressed mainly in a peripheral position with advancing age. Germ cell maturation was delayed in this study population as compared with that observed in our previous study on germ cell markers in a healthy population. The number of GAGE-7-positive germ cells per tubular cross section obtained by immunostaining was significantly higher than that obtained by standard hematoxylin and eosin staining. Double immunostaining revealed heterogeneity in germ cell development in cryptorchid testes. These results shed light on the pathophysiology of germ cell development in boys with cryptorchidism.

The Expression of Markers for Intratubular Germ Cell Neoplasia in Normal Infantile Testes.

BACKGROUND: Positive immunohistochemical expression of testicular cancer markers is often reported beyond 12 months of age in cryptorchid testes, which is assumed to indicate delayed maturation of the fetal germ cells, or neoplastic changes. These findings allowed for questions as to the extent of positive reaction in normal testes. The aim of the study was to clarify the expression of these markers in a normal material up to 2 years. METHODS: Testicular material from 69 boys aged 1-690 days, who died of causes with no association of testicular pathology. Histology sections were incubated with primary antibodies including anti-placental-like alkaline phosphatase (PLAP), anti-C-Kit, anti-D2-40, and anti-Oct3/4. The mean germ cell number per tubular transverse section (G/T) was calculated based on the G/T of both testes of every boy. RESULTS: The mean G/T declined through the 690 days. PLAP appeared stably expressed throughout the ages studied. The likelihood of a positive reaction for C-Kit waned with increasing age within the study period. Positive staining for D2-40 and Oct3/4 was demonstrated up to 6 and 9 months respectively. CONCLUSION: Up to 1 or 2 years of age, normal infantile testes contain germ cells positive for the immunohistochemical markers commonly utilized to aid in the detection of testicular cancer. This finding supports the concept of germ cells undergoing a continuous maturational process in a heterogeneous fashion, and that this process is not complete by 2 years of age.

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.

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.

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.

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.

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.

PURPOSE: In recent series of boys with cryptorchidism gonadotropin levels have been higher and serum inhibin B levels have been lower than normal. To some extent the serum values of inhibin B reflect the state of germinative epithelium in cryptorchid testes. We evaluated whether blood samples of gonadotropins and inhibin B as well as histopathology could be used to classify undescended testes. MATERIALS AND METHODS: A total of 69 boys (median age 2 years) who underwent surgery for bilateral cryptorchidism had blood samples taken preoperatively and 3 months to 2.1 years postoperatively. Testicular biopsies were performed bilaterally at orchiopexy. The average germ cell number per tubular transverse tubule was measured. RESULTS: Group 1 included 17 patients with increased follicle-stimulating hormone levels. Serum follicle-stimulating hormone and luteinizing hormone decreased significantly after surgery. In 77% of patients (13 of 17) follicle-stimulating hormone levels were normalized. Of these boys 35% (6 of 17) had a low postoperative serum inhibin B. Group 2 consisted of 27 patients with a decreased germ cell number and/or low preoperative inhibin B, but not increased serum follicle-stimulating hormone or luteinizing hormone. There were no significant postoperative changes in follicle-stimulating hormone and luteinizing hormone. Of these boys 22% (6 of 27) had a low serum inhibin B postoperatively. In group 3 there were 25 patients with a normal germ cell number, normal preoperative serum inhibin B and normal gonadotropins. There were no significant changes in luteinizing hormone and follicle-stimulating hormone postoperatively. Only 1 boy in this group had a low postoperative serum inhibin B. CONCLUSIONS: Patients with increased gonadotropin levels may have testicular dysgenesis and some may benefit from early surgery. Patients with normal gonadotropin levels and a decreased germ cell number have transient hypothalamus-pituitary-gonadal hypofunction and a poor fertility prognosis. These patients may benefit from gonadotropin treatment after orchiopexy. Patients with normal gonadotropins, inhibin B and germ cell number have a good fertility prognosis after surgery.

Bilateral vanished testes diagnosed with a single blood sample showing very high gonadotropins (follicle-stimulating hormone and luteinizing hormone) and very low inhibin B.

OBJECTIVE: In boys with cryptorchidism median serum values of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are higher and median serum values of inhibin B lower than in normal controls. Serum values of inhibin B reflect the state of germinative epithelium in cryptorchid testes. The aim of the study was to evaluate whether a simple blood sample of gonadotropins and inhibin B could diagnose bilateral vanished testes. MATERIAL AND METHODS: Group I included five boys (4 months to 6 years and 3 months old) with bilateral vanished testes at laparoscopy. Group II included 82 boys with bilateral cryptorchidism younger than 7 years of age at surgery for bilateral cryptorchidism (median age 1 year and 9 months). RESULTS: The serum levels of hormones for the patients with vanished testes were: inhibin B 5?18 pg/ml, FSH 41-191 IU/l and LH 3.9?56 IU/l. The patients all had karyotype 46,xy. The serum levels of hormones from group II were: inhibin B median 122 (range 20?404) pg/ml, FSH median 0.8 (range 0.2?3.5) IU/l and LH median 0.2 (range 0.1-3.2) IU/l. The serum levels of inhibin B, FSH and LH from the boys with vanished testes were significantly different from the serum levels of the boys with bilateral cryptorchidism (p = 0.0026, p < 0.0001 and p < 0.0001, respectively). CONCLUSIONS: The serum values of gonadotropins and inhibin B from boys with bilateral vanished testes were significantly different from those of bilateral cryptorchid boys, indicating no germinative epithelium, no Sertoli cells and compensatory high gonadotropins. If such abnormal serum values are obtained from boys with bilateral non-palpable testes, tubular tissue is not present and surgery can be avoided.