Clinical outcomes of microdissection testicular sperm extraction and intracytoplasmic sperm injection in Japanese men with Y chromosome microdeletions.

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 < .001). Thirty-three couples underwent ICSI using testicular spermatozoa retrieved by microTESE, and eight couples underwent ICSI using ejaculatory spermatozoa. The fertilization rate and clinical pregnancy rate per embryo transfer cycle were significantly higher in the ejaculatory group than that of the testicular group (66.4% vs 43.7%, P < .001, 53.3% vs 24.7%, P = .03, respectively). When compared with the control group, the fertilization rate was significantly lower in the testicular group with AZFc microdeletions (43.7% vs 53.6%, P < .001). CONCLUSIONS: Our study highlights that although microTESE in azoospermic men with AZFc microdeletions led to a higher SRR, ICSI outcomes of these men were worse than that of men without AZF deletions, even if testicular spermatozoa were retrieved.

A variant of Runx2 that differs from the bone isoform in its splicing is expressed in spermatogenic cells.

Background. Members of the Runx gene 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 the Runx2 gene is also expressed in testes. Methods. Murine testes from 1-, 2-, 3-, 4-, and 10-week-old male mice of the C57BL/6J strain and W∕W (v) strain 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 (Met(1) to Gly(15)) as an antigen, immunoblot analyses were performed to detect the predicted polypeptide of 106 aa residues with the initiating Met(1). 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. A Runx2 transcript was detected in testes and was specifically expressed in germ cells. Determination of the transcript structure indicated that the testicular Runx2 is 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 Met(1) in the deduced open reading frame of Runx2 is 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 of Runx2 that 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 single Runx2 gene 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.

Progressing management of non-obstructive azoospermia in the era of microdissection testicular sperm extraction.

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.

A birth of twins-one boy and one girl-from a single embryo transfer and a possible natural pregnancy.

PURPOSE: To describe a rare case of a birth of dizygotic twins with different-sex infants from a single embryo transfer. METHODS AND RESULTS: 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). CONCLUSIONS: A single embryo transfer could produce different-sex twins.

Fresh testicular sperm retrieved from men with spinal cord injury retains equal fecundity to that from men with obstructive azoospermia via intracytoplasmic sperm injection.

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.

Fresh motile testicular sperm retrieved from nonobstructive azoospermic patients has the same potential to achieve fertilization and pregnancy via ICSI as sperm retrieved from obstructive azoospermic patients.

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.

[Occult lumbar vertebral body metastasis of non-seminomatous germ cell tumor eradicated by radiation and salvage surgery 9 years after initial onset].

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.

Up-regulation of DNA-methyltransferase 3A expression is associated with hypomethylation of intron 25 in human testicular germ cell tumors.

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.

Incidental testicular cancers that subsequently developed in oligozoospermic and azoospermic patients: report of three cases.

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.

Seven pregnancies and deliveries from non-mosaic Klinefelter syndrome patients using fresh and frozen testicular sperm.

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.

Clinical features of testicular tumors in children.

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.

Risk factors in past histories and familial episodes related to development of testicular germ cell tumor.

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.

[Carcinoma in situ detected by contralateral testicular biopsy of 55 germ cell tumor patients].

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.

Gene expression profiling identifies a set of transcripts that are up-regulated inhuman testicular seminoma.

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, 12pll, 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.

Immunostaining of stage-specific embryonic antigen-4 in intratubular germ cell neoplasia unclassified and in testicular germ-cell tumors.

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.

Natural killer cells attack tumor cells expressing high levels of sialyl Lewis x oligosaccharides.

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 alpha1,3-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.

Clinical features of prostate cancer patients younger than 50 years: report of seven cases.

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 < 50 years. METHODS: Between 1988 and 2000, we studied seven cases of prostate cancer in men aged under 50 years. The clinicopathological results included: the first sign or symptom; prostate-specific antigen (PSA) at the time of diagnosis; existence of abnormal digital rectal examination (DRE); the differentiation of the cancer and Gleason score; and the outcome of treatment. RESULTS: Six cases were diagnosed as stage D2. One case was diagnosed as stage B2 and the patient underwent radical prostatectomy. None of the cases were detected by mass screening. The PSA at diagnosis was < 10 ng/mL in only one case and that patient underwent radical prostatectomy. Six cases were diagnosed pathologically as poorly differentiated adenocarcinoma. The only patient who survived more than 5 years underwent radical prostatectomy. CONCLUSION: Six of seven cases of prostate cancer were detected at advanced stage. Only one case was thought to be curable and this patient's cancer was detected by chance occult blood test. Because young prostate cancer patients are potential candidates for radical prostatectomy and the sensitivity of PSA might be higher in young men, high-risk groups could be screened by PSA.