Effect of feeder free poly(lactide-co-glycolide) scaffolds on morphology, proliferation, and pluripotency of mouse embryonic stem cells.

The aim of the study has been to evaluate the morphology, proliferation, and pluripotency maintenance of mouse embryonic stem cells (mESCs) cultivated on poly(lactic-co-glycolic acid) scaffolds. The scaffolds were hydrolyzed with NaOH (treated) and nonhydrolyzed (untreated). Morphological and mechanical characterization of the scaffolds was performed. mESC were evaluated for cell viability, cytotoxicity, expression of pluripotency markers, colony morphology, and overall distribution. The treatment generated a reduction in the hydrophobic characteristics of the scaffolds, leading to a higher wettability compared to the untreated group. The viability, cytotoxicity, number of colonies, and the thickness of the cell layer presented similar results between the scaffold groups. The viability test showed that it was possible to cultivate the mESCs on the scaffolds. The cytotoxicity analysis showed that the PLGA scaffolds were not harmful for the cells. The cells maintained the expression of the pluripotency markers Oct4 and Sox2. The number of colonies and the thickness of the cell layer on the scaffold showed that they were not able to colonize the entire volume of the scaffolds. The area occupied by the mESCs was the same between the treated and untreated groups after 14 days in culture. It is possible to conclude that both conditions are equally suitable for maintaining mESC culture. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 424-432, 2017.

Evaluation of the effectiveness of semen processing techniques to remove bovine viral diarrhea virus from experimentally contaminated semen samples.

The aim of this study was to evaluate the capacity of three semen processing techniques, Percoll gradient centrifugation, Swim-up and a combination of Swim-up and Percoll gradient centrifugation, to reduce the viral load of bovine viral diarrhea virus (BVDV) in experimentally infected semen samples. The evaluation was performed using two approaches: first, searching for the presence of virus in the processed samples (via virus titration and RT-PCR) and second, ascertaining the possible interference on in vitro embryo production. The sperm count and DNA integrity (Comet assay) of the processed samples were analyzed (Experiment 1). The amount of virus in the processed samples was determined by titration in cell culture (Experiment 2). The samples processed by Swim up/Percoll gradient centrifugation were utilized for in vitro embryo production, and the embryos produced were tested for BVDV by RT-PCR (Experiment 3). Sperm concentration, Comet assay and embryo production were analyzed by chi-squared tests (P<0.05). There was a significant difference between sperm separation techniques when the sperm count and Comet assay were analyzed. The sperm count obtained from the Swim up/Percoll gradient centrifugation group was lower than that obtained in either of the two other groups (Swim up and Percoll gradient centrifugation), and the Comet assay showed that the combination of the two semen processing techniques (Swim up/Percoll gradient) produced a 1.1% prevalence of Comet level 2, which was not observed in the other groups. The BVDV titer (10(6.68)TCID(50)/mL) added to experimentally infected semen samples decreased after Percoll gradient centrifugation to 10(2.3)-10(1)TCID(50)/mL; for the Swim up group, the titer range was 10(3.3)-10(1.87)TCID(50)/mL, and in the Swim up/Percoll gradient centrifugation group, BVDV was undetectable. The decreases in titer varied from 99.9% in the Swim up-processed group to 100% in the Swim up/Percoll gradient centrifugation group. In vitro embryo production displayed similar blastocyst development rates among all groups, and RT-PCR was negative for the produced embryos. The data showed that the combination of Swim up/Percoll gradient centrifugation promoted the elimination of BVDV from the semen samples without damaging spermatozoa cells and also allowed successful in vitro embryo production free of BVDV. Hence, the risk of BVDV contamination is negligible for the embryo recipient.

Testicular histopathological diagnosis as a predictive factor for retrieving spermatozoa for ICSI in non-obstructive azoospermic patients.

OBJECTIVE: Histological testicular pattern has a predictive role in the possibility of finding spermatozoa for ICSI in cases of non-obstructive azoospermia because some individuals could show residual spermatogenic sites in the testis. The aim of this study was to evaluate the sperm retrieval rate in each of the histopathological groups (hypospermatogenesis--Hypo, spermatogenic maturation arrest--MA, Sertoli cell only--SCO and testicular hyalinization) in patients assisted in our clinic. MATERIALS AND METHODS: Retrospective study from March 1997 to October 2002. We analyzed 14 patients with mean age of 34.3 +/- 0.7, with non-obstructive azoospermia. All patients were submitted to previous diagnostic biopsy (Bx) elsewhere and came to our institution for treatment. After an average of 12 months (8-20), they were submitted to a new Bx procedure to retrieve sperm. RESULTS: Previous diagnostic Bx showed the following histopathological results: 5 patients with MA, 4 with Hypo and 5 SCO. In the following Bx (for sperm retrieval) spermatozoa was found in 33% of the procedures in patients with MA, 50% in patients with Hypo and 40% of the procedures in patients with SCO. CONCLUSION: Previous diagnostic Bx can help in patient counseling concerning the result of sperm retrieval.

Testicular histopathological diagnosis as a predictive factor for retrieving spermatozoa for ICSI in non-obstructive azoospermic patients

OBJECTIVE: Histological testicular pattern has a predictive role in the possibility of finding spermatozoa for ICSI in cases of non-obstructive azoospermia because some individuals could show residual spermatogenic sites in the testis. The aim of this study was to evaluate the sperm retrieval rate in each of the histopathological groups (hypospermatogenesis-Hypo, spermatogenic maturation arrest-MA, Sertoli cell only-SCO and testicular hyalinization) in patients assisted in our clinic. MATERIALS AND METHODS: Retrospective study from March 1997 to October 2002. We analyzed 14 patients with mean age of 34.3 n 0.7, with non-obstructive azoospermia. All patients were submitted to previous diagnostic biopsy (Bx) elsewhere and came to our institution for treatment. After an average of 12 months (8 - 20), they were submitted to a new Bx procedure to retrieve sperm. RESULTS: Previous diagnostic Bx showed the following histopathological results: 5 patients with MA, 4 with Hypo and 5 SCO. In the following Bx (for sperm retrieval) spermatozoa was found in 33 percent of the procedures in patients with MA, 50 percent in patients with Hypo and 40 percent of the procedures in patients with SCO. CONCLUSION: Previous diagnostic Bx can help in patient counseling concerning the result of sperm retrieval.

Percutaneous epididymal sperm aspiration (PESA) in men with obstructive azoospermia

OBJECTIVES: Assessing the efficiency of repeated percutaneous epididymal sperm aspiration (PESA) in men with obstructive azoospermia, and also the possibility of cryopreservation of remaining material for future use in intracytoplasmic sperm injection (ICSI). METHOD: Retrospective study, in which 79 procedures of PESA were assessed in 58 patients (mean age = 45 years), whose partners had mean age of 34 years. Vasectomy was the most frequent cause of obstructive azoospermia (n = 46). RESULTS: Motile spermatozoa were obtained in 65 procedures (82 percent). PESA was twice repeated for 15 patients, 3 times for 5 patients, and 4 times for 1 patient. Spermatozoa were found in 13 (87 percent) patients in the second attempt, in 4 (80 percent) patients in the third attempt, and in the only patient that had accomplished 4 procedures. In 30 procedures (37 percent), we have obtained enough material for cryopreservation. In 12 among the 13 samples thawed (n = 13 patients), motile spermatozoa were found, and ICSI was accomplished. Four patients that did not use their samples requested the elimination of the material. Total rate of pregnancy per transference was 21/55 (38 percent). In 14 PESA procedures, it was not possible to find spermatozoa; in these cases, the patients opted for accomplishing the procedure of testicular sperm aspiration (TESA). CONCLUSION: PESA is an efficient and simple method of retrieving spermatozoa, allowing repeated procedures. Additionally, spermatozoa collected through PESA can be cryopreserved

Percutaneous epididymal sperm aspiration (PESA) in men with obstructive azoospermia.

OBJECTIVES: Assessing the efficiency of repeated percutaneous epididymal sperm aspiration (PESA) in men with obstructive azoospermia, and also the possibility of cryopreservation of remaining material for future use in intracytoplasmic sperm injection (ICSI). METHOD: Retrospective study, in which 79 procedures of PESA were assessed in 58 patients (mean age = 45 years), whose partners had mean age of 34 years. Vasectomy was the most frequent cause of obstructive azoospermia (n = 46). RESULTS: Motile spermatozoa were obtained in 65 procedures (82%). PESA was twice repeated for 15 patients, 3 times for 5 patients, and 4 times for 1 patient. Spermatozoa were found in 13 (87%) patients in the second attempt, in 4 (80%) patients in the third attempt, and in the only patient that had accomplished 4 procedures. In 30 procedures (37%), we have obtained enough material for cryopreservation. In 12 among the 13 samples thawed (n = 13 patients), motile spermatozoa were found, and ICSI was accomplished. Four patients that did not use their samples requested the elimination of the material. Total rate of pregnancy per transference was 21/55 (38%). In 14 PESA procedures, it was not possible to find spermatozoa; in these cases, the patients opted for accomplishing the procedure of testicular sperm aspiration (TESA). CONCLUSION: PESA is an efficient and simple method of retrieving spermatozoa, allowing repeated procedures. Additionally, spermatozoa collected through PESA can be cryopreserved.