Rat in vitro spermatogenesis promoted by chemical supplementations and oxygen-tension control.

In vitro spermatogenesis (IVS) using air-liquid interphase organ culture method is possible with mouse testis tissues. The same method, however, has been hardly applicable to animals other than mice, only producing no or limited progression of spermatogenesis. In the present study, we challenged IVS of rats with modifications of culture medium, by supplementing chemical substances, including hormones, antioxidants, and lysophospholipids. In addition, reducing oxygen tension by placing tissues in an incubator of lower oxygen concentration and/or applying silicone cover ceiling on top of the tissue were effective for improving the spermatogenic efficiency. Through these modifications of the culture condition, rat spermatogenesis up to round spermatids was maintained over 70 days in the cultured tissue. Present results demonstrated a significant progress in rat IVS, revealing conditions commonly favorable for mice and rats as well as finding rat-specific optimizations. This is an important step towards successful IVS in many animal species, including humans.

Genetic disorders and male infertility.

BACKGROUND: At present, one out of six couples is infertile, and in 50% of cases, infertility is attributed to male infertility factors. Genetic abnormalities are found in 10%-20% of patients showing severe spermatogenesis disorders, including non-obstructive azoospermia. METHODS: Literatures covering the relationship between male infertility and genetic disorders or chromosomal abnormalities were studied and summarized. MAIN FINDINGS RESULTS: Genetic disorders, including Klinefelter syndrome, balanced reciprocal translocation, Robertsonian translocation, structural abnormalities in Y chromosome, XX male, azoospermic factor (AZF) deletions, and congenital bilateral absence of vas deferens were summarized and discussed from a practical point of view. Among them, understanding on AZF deletions significantly changed owing to advanced elucidation of their pathogenesis. Due to its technical progress, AZF deletion test can reveal their delicate variations and predict the condition of spermatogenesis. Thirty-nine candidate genes possibly responsible for azoospermia have been identified in the last 10 years owing to the advances in genome sequencing technologies. CONCLUSION: Genetic testing for chromosomes and AZF deletions should be examined in cases of severe oligozoospermia and azoospermia. Genetic counseling should be offered before and after genetic testing.

Antioxidant vitamins and lysophospholipids are critical for inducing mouse spermatogenesis under organ culture conditions.

In vitro mouse spermatogenesis using a classical organ culture method became possible by supplementing basal culture medium with only the product of bovine serum albumin purified by chromatography (AlbuMAX), which indicated that AlbuMAX contained every chemical factor necessary for mouse spermatogenesis. However, since the identity of these factors was unclear, improvements in culture media and our understanding of the nutritional and signal substances required for spermatogenesis were hindered. In the present study, chemically defined media (CDM) without AlbuMAX was used to evaluate each supplementary factor and their combinations for the induction of spermatogenesis. Similar to in vivo conditions, retinoic acid, triiodothyronine (T3 ), and testosterone (T) were needed. Based on differences in spermatogenic competence between AlbuMAX, fetal bovine serum, and adult bovine serum, we identified α-tocopherol, which strongly promoted spermatogenesis when combined with ascorbic acid and glutathione. Differences were also observed in the abilities of lipids extracted from AlbuMAX using two different methods to induce spermatogenesis. This led to the identification of lysophospholipids, particularly lysophosphatidylcholine, lysophosphatidic acid, and lysophosphatidylserine, as important molecules for spermatogenesis. New CDM formulated based on these results induced and promoted spermatogenesis as efficiently as AlbuMAX-containing medium. In vitro spermatogenesis with CDM may provide a unique experimental system for research on spermatogenesis that cannot be performed in in vivo experiments.

Early and late paternal effects of reactive oxygen species in semen on embryo development after intracytoplasmic sperm injection.

Although reactive oxygen species in semen are associated with unfavorable results with respect to assisted reproductive technology, their effects based on the detailed stages of embryo development are unclear. We investigated the relationship between reactive oxygen species in semen and the oocyte fertilization rate, cleavage rate, and blastulation rate of intracytoplasmic sperm injections. This retrospective study enrolled 77 couples who underwent intracytoplasmic sperm injection and analyzed 887 eggs from 141 cycles of intracytoplasmic sperm injection. The reactive oxygen species level in semen was compared between the fertilized and nonfertilized groups, between the good-cleavage-embryo and non-developed-embryo groups, and between the good-quality-blastocyst and poor-quality-blastocyst groups. The cut-off level of reactive oxygen species was calculated to predict good-cleavage-embryo and good-quality-blastocyst development. The fertilization rate was 65.4%, and the mean reactive oxygen species levels were not significantly different between the fertilized and nonfertilized groups. The reactive oxygen species level was significantly higher in the non-developed-embryo group than in the good-cleavage-embryo group (P = 0.0026) and was significantly lower in the good-quality-blastocyst group than in the poor-quality-embryo group (P = 0.015). Cleavage embryos and blastocysts were divided into high- and low-reactive-oxygen-species groups using a cut-off value of 6601 and 4926 relative light units, as calculated from the receiver operating characteristic curve. The rates of good-cleavage embryos and good-quality blastocysts were lower in the high-reactive-oxygen-species group than in the low-reactive-oxygen-species group, which were both statistically significant. To conclude, reactive oxygen species in semen is considered to have an adverse effect on both the early and late stages of embryo development in intracytoplasmic sperm injection.Abbreviations: GnRH, gonadotropin-releasing hormone; ICSI, intracytoplasmic sperm injection; IVF, in vitro fertilization; LPO, lipid peroxidation; NADPH, nicotinamide adenine dinucleotide phosphate; RLU, relative light units; ROC, receiver operating characteristic; ROS, reactive oxygen species.

In vitro spermatogenesis in two-dimensionally spread mouse testis tissues.

PURPOSE: Mouse in vitro spermatogenesis is possible with classical organ culture methods, by placing the testis tissue at the interphase between culture medium and air. In this condition, however, a tissue piece tends to round up to be compact, whose central region suffers from shortage of nutrients and oxygen. In this study, the authors improved the culture condition by spreading each tissue thin and flat, by which they were able to get better access to the oxygen and nutrients. METHODS: Immature mouse testis tissues placed on agarose gel block were forced to spread flat by covering with a polydimethylsiloxane (PDMS) ceiling chip (PC chip). They were then cultured for weeks and evaluated by the transgene expression of Acr-Gfp, which reflects the progression of spermatogenesis. RESULTS: Testis tissues covered with PC chip initiated and maintained spermatogenesis in its wider region than those without PC chip covering. Flow cytometric analysis demonstrated that the PC method yielded more numerous meiotic germ cells than those without PC. Immunohistochemical examination confirmed the authentic histological figure of spermatogenesis from spermatogonia up to round or elongating spermatids. CONCLUSIONS: The PC chip method is simple and effective to improve the efficiency of in vitro spermatogenesis in the organ culture system.

Successful onco-testicular sperm extraction from a testicular cancer patient with a single testis and azoospermia.

Onco-testicular sperm extraction is used to preserve fertility in patients with bilateral testicular tumors and azoospermia. We report the case of a testicular tumor in the solitary testis of a patient who had previously undergone successful contralateral orchiectomy and whose sperm was preserved by onco-testicular sperm extraction. A 35-year-old patient presented with swelling of his right scrotum that had lasted for 1 month. His medical history included a contralateral orchiectomy during childhood. Ultrasonography revealed a mosaic echoic area in his scrotum, suggesting a testicular tumor. The lesion was palpated within the normal testicular tissue along its edge and semen analysis showed azoospermia. Radical inguinal orchiectomy and onco-testicular sperm extraction were performed simultaneously. Motile spermatozoa were extracted from normal seminiferous tubules under microscopy and were frozen. Eventual intracytoplasmic sperm injection using the frozen spermatozoa is planned. Onco-testicular sperm extraction is an important fertility preservation method in patients with bilateral testicular tumors or a history of a previous contralateral orchiectomy.

An infertile patient with Y chromosome b1/b3 deletion presenting with congenital bilateral absence of the vas deferens with normal spermatogenesis.

We report the case of a 46-year-old Chinese male patient who visited our clinic complaining of infertility. Semen analysis revealed azoospermia, and azoospermia factor c region partial deletion (b1/b3) was detected using Y chromosome microdeletion analysis. Testicular sperm extraction was performed after genetic counseling. The bilateral ductus deferens and a portion of the epididymis were absent, whereas the remaining epididymis was expanded. Motile intratesticular spermatozoa were successfully extracted from the seminiferous tubule. On histopathology, nearly complete spermatogenesis was confirmed in almost every seminiferous tubule. To our knowledge, this is the first case report of b1/b3 deletion with a congenital bilateral absence of the vas deferens and almost normal spermatogenesis.

A monolayer microfluidic device supporting mouse spermatogenesis with improved visibility.

In our previous study, we produced a microfluidic device (MFD) which successfully maintained spermatogenesis for over 6 months in mouse testis tissues loaded in the device. In the present study, we developed a new MFD, a monolayer device (ML-D) with a barrier structure consisting of pillars and slits, which is simpler in design and easier to make. This ML-D was also effective for inducing mouse spermatogenesis and maintained it for a longer period than the conventional culture method. In addition, we devised a way of introducing sample tissue into the device during its production, just before bonding the upper layer of polydimethylsiloxane (PDMS) and bottom glass slide. The tissue can obtain nutrients horizontally from the medium running beside it and oxygen vertically from above through PDMS. In addition, the glass slide set at the bottom improved the visibility of the sample tissue with an inverted microscope. When we took photos of cultured tissue of the Acr-Gfp transgenic mouse testis in ML-D sequentially every day, morphological changes of the acrosome during spermiogenesis were successfully recorded. The ML-D is simple in design and useful for culturing testis tissue for inducing and maintaining spermatogenesis with clearer visibility. Due to the new method of sample loading, tissues other than testis should also be applicable.

In vitro mouse spermatogenesis with an organ culture method in chemically defined medium.

We previously reported the successful induction and completion of mouse spermatogenesis by culturing neonatal testis tissues. The culture medium consisted of α-minimum essential medium (α-MEM), supplemented with Knockout serum replacement (KSR) or AlbuMAX, neither of which were defined chemically. In this study, we formulated a chemically defined medium (CDM) that can induce mouse spermatogenesis under organ culture conditions. It was found that bovine serum albumin (BSA) purified through three different procedures had different effects on spermatogenesis. We also confirmed that retinoic acid (RA) played crucial roles in the onset of spermatogonial differentiation and meiotic initiation. The added lipids exhibited weak promoting effects on spermatogenesis. Lastly, luteinizing hormone (LH), follicle stimulating hormone (FSH), triiodothyronine (T3), and testosterone (T) combined together promoted spermatogenesis until round spermatid production. The CDM, however, was not able to produce elongated spermatids. It was also unable to induce spermatogenesis from the very early neonatal period, before 2 days postpartum, leaving certain factors necessary for spermatogenic induction in mice unidentified. Nonetheless, the present study provided important basic information on testis organ culture and spermatogenesis in vitro.

Reactive oxygen species measured in the unprocessed semen samples of 715 infertile patients.

Purpose: To determine whether reactive oxygen species (ROS) in semen samples could be measured with the Monolight™ 3010 Luminometer. Methods: Using the Monolight™ 3010 Luminometer, the ROS was measured in the unprocessed semen samples of infertile male patients, as well as the luminescence of 190 semen samples. The samples were classified as "luminescence-detectable" (n = 89) and "luminescence-undetectable" (n = 101). Thereafter, the luminescence of the semen samples that had been obtained from the 715 infertile patients was measured and compared by using Sperm Motility Analyzing System measurements. Moreover, in order to investigate the ROS measurement consistency, the chemiluminescence values of 84 samples were measured concurrently by using the Monolight™ 3010 Luminometer and the 1251 Luminometer™. Results: The semen volume, sperm motility, and progressive motility of the samples were significantly higher in the luminescence-undetectable samples. The sperm motility, straight-line velocity, curvilinear velocity, mean amplitude head displacement, beat cross frequency, and progressive motility showed an inverse correlation with the logarithmic-transformed luminescence level in the luminescence-detected samples. The integrated chemiluminescence levels in the 84 samples were correlated. Conclusion: The substance that was measured in the unprocessed semen with the Monolight™ 3010 Luminometer and stimulated chemiluminescence is ROS.

Pumpless microfluidic system driven by hydrostatic pressure induces and maintains mouse spermatogenesis in vitro.

Three-dimensional aggregation and organ culture methods are critical for recreating in vivo cellular phenomena outside the body. Previously, we used the conventional gas liquid interphase organ culture method to induce complete mouse spermatogenesis. After incorporating microfluidic systems, we achieved a significant increase in efficiency and duration of spermatogenesis. One of the major drawbacks preventing the popularization of microfluidics, however, is the use of a power-pump to generate medium flow. In this study, we produced a pumpless microfluidic device using hydrostatic pressure and a resistance circuit to facilitate slow, longer lasting medium flow. During three months of culture, results in induction and maintenance of spermatogenesis showed no difference between pumpless and pump-driven devices. Correspondingly, the spermatogonial population was favorably maintained in the pumpless device compared to the conventional method. These results show the advantage of using microfluidic systems for organ culture experiments. Our pumpless device could be applied to a variety of other tissues and organs, and may revolutionize organ culture methods as a whole.

Inverse correlation between reactive oxygen species in unwashed semen and sperm motion parameters as measured by a computer-assisted semen analyzer.

This study investigated the correlation between sperm motion parameters obtained by a computer-assisted semen analyzer and levels of reactive oxygen species in unwashed semen. In total, 847 patients, except for azoospermic patients were investigated. At the time of each patient's first consultation, semen parameters were measured using SMAS™ or CellSoft 3000™, and production of reactive oxygen species was measured using a computer-driven LKB Wallac Luminometer 1251 Analyzer. The patients were divided into two groups: reactive oxygen species - positive and negative. The semen parameters within each group were measured using one of the two computer-assisted semen analyzer systems and then compared. Correlations between reactive oxygen species levels and sperm motion parameters in semen from the reactive oxygen species - positive group were also investigated. Reactive oxygen species were detected in semen samples of 282 cases (33.3%). Sperm concentration (P < 0.01; P < 0.01), motility (P < 0.01; P < 0.05), and progressive motility (P < 0.01; P < 0.01) were markedly lower in the reactive oxygen species - positive group than in the reactive oxygen species - negative group. Among the sperm motion parameters in the reactive oxygen species - positive group, sperm concentration (P < 0.01; P < 0.01), motility (P < 0.05; P < 0.01), mALH (P < 0.05; P < 0.01), and progressive motility (P < 0.05; P < 0.01) also showed inverse correlations with the logarithmic transformed reactive oxygen species levels. Therefore, this study demonstrated that excessive reactive oxygen species in semen damage sperm concentration, motility, and other sperm motion parameters.

Long-term ex vivo maintenance of testis tissues producing fertile sperm in a microfluidic device.

In contrast to cell cultures, particularly to cell lines, tissues or organs removed from the body cannot be maintained for long in any culture conditions. Although it is apparent that in vivo regional homeostasis is facilitated by the microvascular system, mimicking such a system ex vivo is difficult and has not been proved effective. Using the culture system of mouse spermatogenesis, we addressed this issue and devised a simple microfluidic device in which a porous membrane separates a tissue from the flowing medium, conceptually imitating the in vivo relationship between the microvascular flow and surrounding tissue. Testis tissues cultured in this device successfully maintained spermatogenesis for 6 months. The produced sperm were functional to generate healthy offspring with micro-insemination. In addition, the tissue kept producing testosterone and responded to stimulation by luteinizing hormone. These data suggest that the microfluidic device successfully created in vivo-like conditions, in which testis tissue maintained its physiologic functions and homeostasis. The present model of the device, therefore, would provide a valuable foundation of future improvement of culture conditions for various tissues and organs, and revolutionize the organ culture method as a whole.

Clinical application of neutrophil CD64 quantification for differential diagnosis of acute scrotum.

The management of acute scrotum can be challenging, especially in infants or patients with a neurological or neurodevelopmental disorder in whom presentation, diagnosis and definitive management tends to be delayed. This leads to poor outcomes, such as loss of the affected testis. Here we present two cases of testicular torsion in patients with neurodevelopmental disorders, and a further two cases of epidydimo-orchitis in whom measurement of CD64 expression on neutrophils was helpful for differential diagnosis. These data suggest that the levels of expression of CD64 by neutrophils, known as a marker of infection, could also be useful for differentiating between testicular torsion and infection in acute scrotum.

[Case of azoospermia patient with a chromosomal abnormality considered a ring Y chromosome].

A 43-year-old man came to our clinic complaining of infertility and semen analysis showed azoospermia. Analysis of chromosomes showed a mosaic 45, XO/46, X, ＋mar1/46, X, ＋mar2 karyotype, and the marker chromosomes were considered to be two kinds of ring Y chromosomes. Y chromosome microdeletion analysis showed partial deletion of Azoospermic Factor (AZF) a, and complete deletion of AZFb and AZFc. The patient gave up having a child because these results indicated that no sperm would be collected even if Testicular Sperm Extraction (TESE) were performed.

[Unilateral adrenal tuberculosis: a case report].

Computed tomography (CT) performed for a 75-year-old man as a follow-up examination for deep vein thrombosis in October 2010 revealed a left adrenal mass (diameter, 8 mm). In December 2012, the adrenal mass increased to 28 mm in diameter, and he was referred to our department. Several blood examinations revealed that the adrenal mass was non-functioning, and only peripheral lesions were observed to be enhanced by using CT in the arterial phase. Malignancy was suspected due to the irregular shape and growth of the mass, and left adrenalectomy was performed in February 2013. The histopathological diagnosis was adrenal mycobacteriosis, and clinical diagnosis was adrenal tuberculosis. No other tuberculosis infection-related lesion was detected, and the patient was treated with multidrug antituberculous chemotherapy.

[A case of long-term survivor after combined modality therapy for brain metastasis of bladder carcinoma].

A 75-year-old man with advanced bladder cancer (cT4N1M0) received three courses of systemic chemotherapy with Methotrexate, Epirubicin and Nedaplatin (MEN). His metastatic lymph node completely disappeared. We performed total cystectomy. Three months after the surgery, he complained of neck pain and nausea. Brain magnetic resonance imaging (MRI) revealed a 3 cm tumor in his right cerebella and a 5 mm tumor in left parietal lobe. He underwent surgical resection of the right cerebellar tumor and a gamma knife therapy for the left parietal tumor. Pathological diagnosis was metastatic urothelial carcinoma. We performed three additional courses of chemotherapy of MEN. He has been well without local recurrence or distant metastasis for 18 months.

[Metastatic micropapillary variant of the urinary bladder: progression from low grade non-muscle invasive bladder carcinoma].

We report a case of metastatic micropapillary variant of the bladder that progressed from low grade non-muscle invasive bladder carcinoma. Lung, para-aortic and pelvic lymph nodes metastatic lesions were found in a 62-year-old woman, who had been followed due to non-muscle invasive bladder carcinoma. The bladder wall was found to be thick by computerized tomography (CT). She had had transurethral resection of bladder tumor (TURBT) at 60 and 61 years old, followed by intravesical therarubicin and bacille Calmette-Guérin therapy, respectively. Both TURBT specimens showed low grade, non-muscle invasive urothelial carcinoma. The thickened bladder wall was resected transurethrally and the pathological examination revealed that the recurrent tumor was entirely composed of micropapillary variant component. There must have been tiny lesions of a micro papillary variant component after the second TURBT. Several reports suggest that intravesical BCG therapy was ineffective for micropapillary variant. So the UC component was substituted for micropapillary component.

[Renal carcinoid tumor: a case report].

A right renal tumor was incidentally found in a 38-year-old woman by annual medical check up. She visited our hospital for further examination and treatment. She did not show typical symptoms of carcinoid. A computed tomography (CT) revealed a calcified solid tumor in the upper portion of the right kidney. The tumor was 6.0 cm in diameter and was not enhanced in either early or late phase. There was no evidence of extrarenal invasion or distant metastasis. Based on a clinical diagnosis of stage 1 renal cell carcinoma, laparoscopic nephrectomy was performed. The pathological diagnosis was renal carcinoid tumor. The tumor had trabecular and ribbon-like structures with a thin fibrovascular stroma. Immunohistochemicaliy, the tumor cells stained positive for chromogranin A, synaptophisin and CD56. The cell proliferation rate was estimated to be under 1% with Ki67 staining. To find the primary lesion, we performed upper and lower gastric endoscopy and chest computed tomography, but could not find any/other carcinoid tumors. At 1-year follow up, she had no evidence of local recurrence or metastasis.