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Purpose We investigated the clinical results of Japanese men with Y chromosome microdeletions. Methods This study retrospectively examined 2163 azoospermic or severe oligozoospermic patients. We investigated the frequency of azoospermia factor (AZF) deletions and sperm retrieval rate (SRR) by microTESE in patients with these deletions, then analyzed the ICSI outcomes. Results Azoospermia factor deletions were found in 201 patients. SRR was significantly higher than that of the control group (74.0% vs 20.4%, P

Background.Members of theRunxgene family encode transcription factors that bind to DNA in a sequence-specific manner. Among the three Runx proteins, Runx2 comprises 607 amino acid (aa) residues, is expressed in bone, and plays crucial roles in osteoblast differentiation and bone development. We examined whether theRunx2gene is also expressed in testes.Methods.Murine testes from 1-, 2-, 3-, 4-, and 10-week-old male mice of the C57BL/6J strain andW∕Wvstrain were used throughout the study. Northern Blot Analyses were performed using extracts form the murine testes. Sequencing of cDNA clones and 5′-rapid amplification of cDNA ends were performed to determine the full length of the transcripts, which revealed that the testicular Runx2 comprises 106 aa residues coding novel protein. Generating an antiserum using the amino-terminal 15 aa of Runx2 (Met1to Gly15) as an antigen, immunoblot analyses were performed to detect the predicted polypeptide of 106 aa residues with the initiating Met1. With the affinity-purified anti-Runx2 antibody, immunohistochemical analyses were performed to elucidate the localization of the protein. Furthermore, bioinformatic analyses were performed to predict the function of the protein.Results.ARunx2transcript was detected in testes and was specifically expressed in germ cells. Determination of the transcript structure indicated that the testicularRunx2is a splice isoform. The predicted testicular Runx2 polypeptide is composed of only 106 aa residues, lacks a Runt domain, and appears to be a basic protein with a predominantly alpha-helical conformation. Immunoblot analyses with an anti-Runx2 antibody revealed that Met1in the deduced open reading frame ofRunx2is used as the initiation codon to express an 11 kDa protein. Furthermore, immunohistochemical analyses revealed that the Runx2 polypeptide was located in the nuclei, and was detected in spermatocytes at the stages of late pachytene, diplotene and second meiotic cells as well as in round spermatids. Bioinformatic analyses suggested that the testicular Runx2 is a histone-like protein.Discussion.A variant ofRunx2that differs from the bone isoform in its splicing is expressed in pachytene spermatocytes and round spermatids in testes, and encodes a histone-like, nuclear protein of 106 aa residues. Considering its nuclear localization and differentiation stage-dependent expression, Runx2 may function as a chromatin-remodeling factor during spermatogenesis. We thus conclude that a singleRunx2gene can encode two different types of nuclear proteins, a previously defined transcription factor in bone and cartilage and a short testicular variant that lacks a Runt domain.

From a developmental point of view, human testicular germ cell tumor (TGCT) can be traced back to the primordial germ cells in the embryo, which, upon transformation, become either seminoma or non-seminoma. Thus, TGCT provides a useful model system for the study of gene regulation involved in oncogenesis as well as development. In this study, we focused and analyzed the expression and epigenetic alteration of DNA-methyltransferase (DNMT) genes in TGCT tissues. The examined genes included DNMT1, DNMT3A and DNMT3B that function to maintain or generate a methylation status of genomic DNA. Using semi-quantitative reverse transcription-polymerase chain reaction, we found that the expression of DNMT3A, but not DNMT1 or DNMT3B, was up-regulated markedly in TGCT specimens compared to non-tumor testicular tissues. To explore mechanisms involved in the up-regulation of DNMT3A, we examined the methylation status of CpGs in the gene. The distal and proximal promoter regions of DNMT3A were non-methylated in both TGCT and non-tumor tissues. In contrast, non-tumor testicular tissues exhibited a mixture of methylated and non-methylated CpGs in intron 25 of DNMT3A, whereas most CpGs in intron 25 were demethylated in TGCT specimens. This difference in the degree of methylation was confirmed by Southern blot analysis, in which an EcoRI site in intron 25 could be digested only when the CpG was non-methylated. Thus, epigenetic alteration of intron 25 toward de-methylation is associated with increased expression of DNMT3A in TGCT. The intron 25 may represent a differentially-methylated region in DNMT3A that is modulated during development and/or tumorigenesis of germ cells.

Purpose: The aim of this study was to investigate the feasibility of using frozen-thawed testicular sperm as well as the timing of testicular sperm extraction (TESE) in patients with non-mosaic Klinefelter syndrome. Methods: Intracytoplasmic sperm injection (ICSI) was performed in six of 17 (35%) patients whose sperm was recovered by TESE. Multiple biopsies of both testes were performed on the day of oocyte retrieval in all but one of the six patients. Results: Seven pregnancies and deliveries were achieved in five couples, and one couple was unsuccessful. Five pregnancies were achieved using fresh motile sperm, and two were achieved using frozen-thawed sperm. Sperm cryopreservation was not possible in one of the five couples because of the small number of recovered sperm, and possible in four other couples for subsequent ICSI. One woman whose husband had TESE performed prior to ovarian stimulation did not become pregnant. This may be due to the attainment of only a few immotile sperm following the frozen-thawed procedure. Conclusion: The outcome of ICSI using fresh or frozen-thawed testicular sperm in patients with non-mosaic Klinefelter syndrome was identical; however, TESE should be performed on the day of oocyte retrieval until such time as a procedure with a higher sperm yield from TESE is available. Moreover, an improved recovery procedure after cryopreservation-thawing of a single spermatozoon must be developed.

Aim: Testicular tumors are not common pediatric solid tumors, especially in Asian children. There have been few reviews of cases in Japan to date. We present the clinical features of 14 pediatric testicular tumor patients. Methods: Clinical features of 14 testicular tumor patients, such as chief complaints, age at diagnosis, pathology, stages, treatments and prognosis, were examined from medical records. Two patients had their semen tested at adolescence. Results: Of the 14 prepubescent patients, 12 (85.7%) patients were diagnosed before 3 years of age. Ten cases (71.4%) were diagnosed as yolk sac tumors, three (21.4%) as mature teratomas and one case as an epidermoid cyst. Nine cases (90.0%) among the 10 cases of yolk sac tumor were diagnosed as stage I and one case was stage IV. One stage I yolk sac tumor patient developed lung metastasis later. Eventually, two yolk sac tumor patients died, despite chemotherapy. While all the cases with a diagnosis before 2 years of age survived, 67% (2/3) of cases with a diagnosis after the age of 2 died of tumors. Semen analysis in two patients showed normospermia. Conclusion: In the present study, the most common testicular tumors were yolk sac tumors and the patients diagnosed before 2 years of age showed favorable results. Age could be a relapse risk factor in yolk sac tumors. Guidelines for handling testicular tumors in children is not yet well established in Japan. An organized system seems necessary to gather and accumulate the results of the cases in Japan in order to develop better guidelines for treatment.

Background: A retrospective study was conducted to examine the host factors of 240 testicular germ cell tumor patients. This study was performed to address a new theory proposed by Skakkebaek called testicular dysgenesis syndrome which claims that cryptorchism, hypospadias, poor semen quality and testicular germ cell tumors are symptoms of an underlying testicular dysgenesis in uterus. Methods: The past health histories and familial episodes of 240 testicular germ cell tumor patients were examined. The past health histories included cryptorchism, hypospadias, infertility, atrophic testis and inguinal hernia. Results: Of the 240 patients, 13 (5.4%) had a history of cryptorchism or orchidopexy. Two (0.8%) showed existence of hypospadias or had experienced urethroplasty. Among 129 married couples, 104 (80.6%) couples were fertile. Three (1.3%) patients developed testicular tumors after they were diagnosed as infertile or came to the hospital with the complaints of infertility. Four (1.7%) had contralateral atrophic testis. 19 (7.9%) had experienced inguinal herniorrhaphy before age 15. Three (1.3%) had testicular germ cell tumor patients among their family or relatives. Conclusions: The testicular germ cell tumor patients showed a considerable incidence of complications such as cryptorchism, hypospadias and incomplete closure of processus vaginalis. Cryptorchism, perinatal factors and familial factors could be risks for developing testicular germ cell tumors.

PURPOSE: We performed contra-lateral testicular biopsies in 55 testicular tumor patients when high orchiectomy was performed. In these cases, two cases developed invasive testicular tumor later although the biopsies had not revealed testicular CIS. Then we re-examined the sensitivity of biopsies and judged if our results are contradictory against Skakkebaek's theory. PATIENTS AND METHODS: The paraffin blocks of two cases who later developed testicular tumor were sliced again and re-examined by H/E staining and immunostaining with PLAP antibody (clone No. 8A9). The other 53 H/E samples were re-examined and the result of the contra-lateral testis was re-searched in the case that CIS was detected in the specimen. RESULTS: CIS was detected in one of the two cases who later developed contra-lateral testicular tumor and another case among the other 53 cases. We could not reveal the result of the testis of case No. 3 because of the patient's disappearance. CIS existed 3.6% (2/55) and two cases were found to have been false negative. CONCLUSION: It is important for both urologists and pathologists to know well about testicular CIS and to perform biopsy according to Skakkebaek's guidance for raising the sensitivity to detect testicular CIS.

Seminoma constitutes one subtype of human testicular germ cell tumors and is uniformly composed of cells that are morphologically similar to the primordial germ cells and/or the cells in the carcinoma in situ. We performed a genome-wide exploration of the genes that are specifically up-regulated in seminoma by oligonucleotide-based microarray analysis. This revealed 106 genes that are significantly and consistently up-regulated in the seminomas compared to the adjacent normal tissues of the testes. The microarray data were validated by semi-quantitative RT-PCR analysis. Of the 106 genes, 42 mapped to a small number of specific chromosomal regions, namely, 1q21, 2p23, 6p21-22, 7p14-15, 12p11, 12p13, 12q13-14 and 22q12-13. This list of up-regulated genes may be useful in identifying the causative oncogene(s) and/or the origin of seminoma. Furthermore, immunohistochemical analysis revealed that the seminoma cells specifically expressed the six gene products that were selected randomly from the list. These proteins include CCND2 and DNMT3A and may be useful as molecular pathological markers of seminoma.

Previously, it was absolutely impossible for azoospermic men to reproduce except in some obstructive azoospermic cases for whom reconstruction of the seminal pathway was successful. However, nowadays, intracytoplasmic sperm injection and microdissection testicular sperm extraction have brought about chances of biological paternity in some non-obstructive azoospermic men. It is almost 15 years since the first trials of testicular sperm retrieval using surgical microscopy for non-obstructive azoospermia were reported. In this manuscript, the progress and outcomes of these novel techniques since then are reviewed, the controversial points are discussed and the latest research to achieve pregnancies in tough non-obstructive azoospermic couples are introduced. Not only the bright side of the renovations, but the underlying concerns are also discussed.

The chromosomal inversion (16)(p13q22), which is associated with the M4-eosinophilia subtype of human acute myeloid leukemia, causes the fusion of two distinct genes. The polypeptide encoded by the chimeric gene, PEBP2p/CBFp-SMMHC, retains the ability to interact with, and dominantly interfere with the function of proteins possessing the Runt homology domain. The Runt protein homologs constitute the DNA binding subunit of the PEBP2/CBF transcription factor. We examined the subcellular localization of PEBP2beta/CBFbeta-SMMHC, as well as that of Runt protein homologs in leukemic cells carrying inversion 16 by immunoblot analysis. A significant amount of the PEBPbeta/CBFbeta-SMMHC protein was recovered from the nuclear fraction along with the Runt protein homologs. Furthermore, some of both polypeptides was retained in the DNA pellet that represents the material remaining after extraction of nuclear fraction with high salt. These observations suggest that the so-called dominant interfering effect of PEBPbeta/CBFbeta-SMMHC on PEBP2/CBF occurs inside the nucleus. In addition, we could detect PEBP2beta/CBFbeta-SMMHC in the cytoplasmic membrane fraction as well. The function of this membrane-located PEBP2beta/CBFbeta-SMMHC, if any, appears to be unrelated to that of Runt protein homologs.

Objective To examine the outcomes of intracytoplasmic sperm injection (ICSI) with testicular sperm retrieved from men with spinal cord injury. Design Retrospective study. Setting Private hospital-based infertility research laboratory. Patient(s) Twenty-two couples of whom one partner was a man with spinal cord injury (SCI). Intervention(s) Reviewing the outcomes of testicular sperm extraction (TESE)–ICSI. Main Outcome Measure(s) Testicular sperm retrieval rate, fertilization rate, pregnancy rate, comparison with patients with obstructive azoospermia. Result(s) Testicular sperm were retrieved from 19 of 22 (86%) patients with SCI. Intracytoplasmic sperm injection resulted in a fertilization rate of 236 of 364 (65%). Of 19 couples, 14 couples achieved 18 pregnancies, and 22 infants (14 singleton and 4 twin) were born. (Pregnancy per couple was 74% and that per ICSI was 54%). There was no significant difference in pregnancy rate at the first ICSI between SCI couples and obstructive azoospermia couples (68% SCI, 68% obstructive azoospermia). However, pregnancy rate per fresh testicular sperm–ICSI was significantly higher than that per frozen-thawed sperm–ICSI in SCI couples (64% SCI fresh, 25% SCI frozen-thawed) although no significant difference was seen in obstructive azoospermia couples (76% obstructive azoospermia fresh, 63% obstructive azoospermia frozen-thawed). There was no significant difference in pregnancy rate between fresh ET cycle and frozen-thawed ET cycle in SCI couples. Conclusion(s) Testicular sperm in men with SCI may possess disadvantages in freezing and thawing compared with that in men with obstructive azoospermia. Fresh testicular sperm–ICSI may offer optimum outcome for SCI couples desirous of pregnancy.

Objective To compare the fertilization and pregnancy rates using fresh testicular sperm between nonobstructive azoospermic (NOA) patients and obstructive azoospermic (OA) patients. Design We evaluated sperm quality of testicular sperm retrieved by microdissection testicular sperm extraction (MD-TESE) in NOA patients and compared the fertilization rate and pregnancy rate via intracytoplasmic sperm injection (ICSI) between NOA and OA patients. Setting Private hospital–based infertility research laboratory. Patient(s) A total of 58 couples in which the husband was diagnosed with azoospermia. Intervention(s) Analytic examination of the outcomes and description of the NOA cases achieving pregnancies; oocyte retrieval and testicular sperm retrieval were performed simultaneously. Main Outcome Measure(s) Comparison of fertilization rate and pregnancy rate at the first ICSI attempt. Result(s) Testicular sperm were retrieved from 17 of 40 NOA patients and 18 of 18 OA patients. Motile sperm were successfully retrieved from 16 of the 17 NOA patients. There was no significant difference in fertilization rate and pregnancy rate between OA and NOA cases. Of the 17 NOA patients, nine pregnancies were achieved using fresh motile testicular sperm. Conclusion(s) Fresh motile testicular sperm retrieved from NOA patients may have the same potential to achieve fertilization and pregnancy as sperm retrieved from OA patients. The MD-TESE technique might contribute to the high retrieval rate of fresh motile testicular sperm even in NOA patients.

In this report we describe a case of late relapse non-seminomatous germ cell tumor eradicated after 9 years of initial onset. A 20-year-old man complaining of recent aches, vomiting and headaches was diagnosed with right testicular tumor with solitary brain and bilateral lung metastases. At presentation, human chorionic gonadotropin (HCG) was elevated to 22,000 mIU/ml, and alpha-fetoprotein to 79 ng/ml. A right high orchiectomy was performed, followed by a right occipital osteoplastic craniotomy due to the presence of left hemiplesia and anisocoria prior to chemotherapy. Pathologically, the tumors were embryonal carcinoma and yolk sac tumor. The patient received 5 cycles of cisplatin-based PEP chemotherapy (cisplatin, etoposide and peplomycin) after which all the tumor markers fell to within the normal range. The remaining right lung tumor was removed surgically and the remnant lesion was found to be scar tissue. Four years after initial therapy, elevated serum HCG levels were detected. The tumor metastasis showed only HCG elevation responsive to chemotherapy each time followed by relapse and undetectable with all kinds of imaging examinations for 5 years. Finally when the tumor became chemorefractory, conventional computed tomography scan on bone window detected the occult tumor in L4 corporal body. After radiation therapy the tumor was removed by total spondylectomy and there was no viable tumor cells in the specimen pathologically. HCG fell to within normal range according to its half life period after the operation and there is no relapse of HCG after 18 months follow up. CT bone window photography may be sometimes useful to detect occult bone metastasis and salvage surgery combined with radiation therapy may be worth trying in patients with chemorefractory non-seminomatous germ cell tumors.

Objective To examine whether infertile men with poor semen count subsequently developed testicular cancers and to describe their clinical presentation. Design We reviewed 460 male patients with abnormal semen counts between 1989 and 2004. Setting University hospital. Patient(s) Infertile men who developed testicular cancers after assisted reproductive technologies (ART). Intervention(s) Description of patient characteristics: age at infertility, presentation, semen quality, and ART. Main Outcome Measure(s) The number of patients who subsequently developed testicular cancers and the period from ART to the development of clinical testicular cancers. Result(s) Of the 460 patients, 169 patients presented with mild oligozoospermia, 117 patients with severe oligozoospermia, and 174 patients with azoospermia. The follow-up periods were as follows: 1–192 months (median, 96.5 mo) for mild oligozoospermia, 1–156 months (median, 78.5 mo) for severe oligozoospermia, and 1–197 months (median, 99 mo) for azoospermia. We subsequently found three testicular cancers that had developed among severely oligozoospermic and azoospermic patients. The period from the claim of sterility to developing testicular cancers varied from 4 to 14 years (median, 9 y). Conclusion(s) These results indicate that severe semen abnormality may be a risk factor in developing testicular cancers. Self-examination of the testes could be used as an alternative or supplement to physical examination and testicular ultrasound as part of the infertility workup, even after ART.

Stage-specific embryonic antigen-4 (SSEA-4) is expressed in testicular germ-cell tumors (GCT) according to studies using thin-layer chromatography (TLC) immunostaining. To further understand the relationship between SSEA-4 and the histogenesis of testicular GCT, we examined the expression of SSEA-4 in 43 samples of testicular GCT either by immunohistochemical staining or by TLC immunostaining. Immunohistochemical staining detected SSEA-4 in spermatogonia from the non-tumor parts of the testis and in all of 8 samples of intratubular germ cell neoplasia unclassified (IGCNU). Immunohistochemical staining was SSEA-4 positive in all 9 seminomas, and in one yolk sac tumor and in 5 embryonal carcinomas among 9 non-seminomas. Immunostaining with TLC showed that SSEA-4 was retained in 15 of 16 seminomas and in 4 of 8 non-seminomas. These results demonstrate an antigenic link between spermatogonia, IGCNU, and testicular GCT. We suggest that SSEA-4 is associated with the histogenesis of testicular GCT.

Epithelial carcinoma and leukemia cells express sialyl Lewis x oligosaccharides as tumor-associated carbohydrate antigens. To determine the role of sialyl Lewis x oligosaccharides in tumor dissemination, human melanoma MeWo cells, which do not express sialyl Lewis x, were transfected with α1,3-fucosyltransferase III (FTIII), and cell lines expressing different amounts of sialyl Lewis x were isolated. When these cells were injected into the tail vein of nude mice, cells expressing moderate amounts of sialyl Lewis x (MeWo-FTIII⋅M) produced a significantly greater number of lung tumor foci than did parental MeWo cells. In contrast, cells expressing large amounts of sialyl Lewis x (MeWo-FTIII⋅H) produced few lung tumor foci in nude mice but were highly tumorigenic in beige mice, which have defective natural killer (NK) cells. In vitro assays demonstrated that MeWo-FTIII⋅H cells are much more sensitive to NK cell-mediated cytotoxicity than are MeWo-FTIII⋅M cells or parental MeWo cells and the susceptibility of MeWo-FTIII⋅H cells to NK cell-mediated cytolysis can be inhibited by preincubating MeWo-FTIII⋅H cells with anti-sialyl Lewis x antibody. Moreover, we discovered that NK cell-mediated cytolysis of MeWo-FTIII⋅H cells can be inhibited by the addition of an antibody against the NK cell receptor CD94 or sialyl Lewis x oligosaccharides. These results, combined with structural analysis of MeWo-FTIII⋅H cell carbohydrates, indicate that moderate amounts of sialyl Lewis x lead to tumor metastasis, whereas expression of high levels of sialyl Lewis x leads to an NK cell attack on tumor cells, demonstrating that expression of different amounts of sialyl Lewis x results in entirely different biological consequences.

Background: The incidence of prostate cancer increases with age and latent cancer is common in older men. But clinical prostate cancer is rare in men aged

To describe a rare case of a birth of dizygotic twins with different-sex infants from a single embryo transfer.A patient, who had her right ovary and tube removed, and her husband were treated with ICSI and a single embryo transfer. When a single fresh embryo was transferred on day 4, following oocyte retrieval using GnRH agonist-long protocol, two gestational sacs were recognized at 8 weeks of gestation. Healthy twins with a boy and a girl were delivered at 37 weeks 0 days of gestation by a cesarean section. The boy's weight was 2096g, and his height was 45.0 cm, while the girl's weight was 1988g, and her height was 41.5 cm. Peripheral lymphocyte chromosome analysis of the two infants showed normal karyotype, 46, XY (boy) and 46, XX (girl).A single embryo transfer could produce different-sex twins.