Objective: The safety of IVM program using PMSG+hCG of high dosage (10 iu/mL each) has been inquired. Furthermore, hCG priming before oocyte retrieval of unstimulated PCOS patient had no stimulatory effect on the developmental competence of oocytes matured after supplementation of PMSG+hCG. This study was conducted to evaluate whether different hormonal supplementations to IVM medium affect the developmental competence of immature oocytes retrieved from hCG primed, unstimulated PCOS patients. Materials and Methods: Oocytes were retrieved from patients with subcutaneous administration of 10,000 iu hCG at 36 h before oocyte aspiration. Retrieved oocytes were cultured for IVM in 20% (v/v) FBS-containing TCM-199 supplemented with rFSH (0.075 iu/mL) + hCG (0.5 iu/mL) + E2 (1 μg/mL), FSH-HP (0.075 iu/mL) + hCG + E2 or PMSG+hCG. At 24–48 h after IVM, matured oocytes were fertilized by ICSI of our standard protocol. The number of oocytes matured, fertilized and cleaved after IVM with different media was monitored and statistical analysis in each parameter was performed by ANOVA and the least square method in PROC-GLM of SAS program. Results: From 9 patients with hCG administration, a total of 116 germinal vesicle stage oocytes with morphologically normal were provided for this study. Among 3 supplementations, there were no significant difference in the proportion of oocytes matured (51.4–53.7%), fertilized (34.2–43.9%), formed two (male and female) pronuclei (26.8–32.4%) and cleaved (23.7–32.4%). Conclusion: The results of this study demonstrated that PMSG+hCG supplementation had a similar supporting action on the development of immature oocytes compared with rFSH or FSH-HP + hCG + E2 supplementation to IVM medium. Accordingly, a choice of PMSG+hCG as IVM medium supplements was not detrimental to maturation, fertilization and cleavage of oocytes and IVM medium including PMSG+hCG can be used for the maturation of oocytes retrieved from unstimulated PCOS patients with hCG-priming. ∗ Supported by a grant from the Interdisciplinary Research Program of the KOSEF (1999-2-205-002-5).

Objective: Preimplantation genetic diagnosis (PGD) for embryo sexing in couples with X-linked diseases, chromosomal abnormalities as well as those screened with single gene disorders were actually offered as an alternative to prenatal diagnosis in our institute. We present in this report the clinical results including pregnancy rate associated with the PGD program conducted from June 1994 to December 1999. Design: Eight-cell embryos generated during ICSI followed by IVF cycle were biopsied to obtain one blastomere for genetic analysis using either polymerase chain reaction (PCR) or fluorescence in situ hybridization (FISH). Materials and Methods: Fifty PGD cycles were performed in 36 patients presenting either with a carrier(s) or family history of single gene disorders (e.g., Duchenne's muscular dystrophy (DMD), Fragile X syndrome, Spinal muscular atrophy) or as a carrier of chromosomal abnormalities (e.g., translocation, mosaic Turner's syndrome) and those for sex determination (e.g., Incontinentia pigmenti, Hemophilia, Pelizaeus-Merzbacher syndrome). Embryos were biopsied at 8-cell stage to obtain one blastomere in most patients or two blastomeres for linkage PCR of non-deleted DMD and Fragile X syndrome. In terms of diagnostic methods, PCR was performed in 10 cycles from 7 patients with single gene disorders while FISH was performed in 40 cycles from 29 patients with sex determinations for X-linked disorders and chromosomal abnormalities. Embryo was transferred on day 4 or 5. All affected embryos reconfirmed the original diagnosis. Results: Five hundred forty one embryos were biopsied (10.7 embryos per cycle). The efficiency of PCR and FISH were 89.8% (87/98) and 88.3% (391/443), respectively. Embryo transfer was performed in (48/50) cycles. Among these cycles, clinical pregnancy rate were 25.7% (9/35) per embryo transfer and 18% (9/50) per cycle. Twenty three percent (8/36) of pregnancy rate was noticed in the 36 patient couples and four have delivered normal babies. There was no twin fetus in this PGD program. Two pregnancies ended with a spontaneous abortion. There was no difference in the efficiency of the diagnostic methods of PCR and FISH. In all cases, prenatal amniocentesis has been indicated as a confirmation of the original preimplantation diagnosis. Screening test on mosaics from the affected remnant embryos have been analyzed.

Objective: The motility of sperm obtained from testicular tissue is generally very low. Sometimes they showed no motility. In our previous studies, we have achieved better results by culturing testicular sperm in vitro with Vero cells for a long period (up to 1 week) compared with simple culturing or no culture. However, the results of long term in vitro sperm culturing are unpredictable and give us additional laboratory work. Therefore, we tried to induce testicular sperm motility acquiring by using Pentoxyphylline, known as a good sperm motility stimulator, in a short term culture period and also tried to cryopreserve the motile sperm for the next ICSI cycles.Design: Testicular sperm were cultured in vitro with and without Pentoxyphylline before and after sperm cryopreservation and motility was evaluated to compare short term culture with Pentoxyphylline and long term culturing method.Materials and Methods: Testicular tissues were excised from seven patients with spermatogenesis problems or CAVD just before ICSI or coming ICSI cycles. Excised testicular tissues were minced into small pieces by blade and recovered sperm were cultured in Ham's F-10 medium supplemented 10% FBS for 3 days or Pentoxyphylline (3.6 mM) was added in the same culture medium for short term (1 hr) culture. For culture, 37°C and 5% carbon dioxide atmosphere of incubator was used. Just after sperm retrieval, 1 hr and 3 days of culture, the sperm motility was evaluated in both groups. At least 100 sperm were counted for motility scoring. After 1 hr of culture with Pentoxyphylline or 3 days of culture, sperm cryopreservation was performed by vapour freezing method.Results: Total sperm solution volume was adjusted to 4 ml and mean sperm concentration was 0.3 × 106 ± 0.2/ml. Initial sperm motility was 12.0 ± 13.8% and the motility increased to 25.7 ± 17.3% (simple medium) and 62.4 ± 21.8% (Pentoxyphylline) after 1 hr of culture. After 3 days, the motility was changed 41.9 ± 19.0% (simple medium) and 44.3 ± 16.2% (Pentoxyphylline). In the Pentoxyphylline treated group, the sperm were cryopreserved and thawed and the motility fell greatly to 6.3 ± 6.1%. Long term cultured sperm were reserved for actual ICSI cycles. Pentoxyphylline treatment shortens the culture time to attain motility of testicular sperm in vitro. Only 1 hr of culture was resulted to get sperm motility over 60%.Conclusion: Results of the present study suggest that Pentoxyphylline treatment is a useful method and should be substituted as the long term culture method. Objective: The motility of sperm obtained from testicular tissue is generally very low. Sometimes they showed no motility. In our previous studies, we have achieved better results by culturing testicular sperm in vitro with Vero cells for a long period (up to 1 week) compared with simple culturing or no culture. However, the results of long term in vitro sperm culturing are unpredictable and give us additional laboratory work. Therefore, we tried to induce testicular sperm motility acquiring by using Pentoxyphylline, known as a good sperm motility stimulator, in a short term culture period and also tried to cryopreserve the motile sperm for the next ICSI cycles. Design: Testicular sperm were cultured in vitro with and without Pentoxyphylline before and after sperm cryopreservation and motility was evaluated to compare short term culture with Pentoxyphylline and long term culturing method. Materials and Methods: Testicular tissues were excised from seven patients with spermatogenesis problems or CAVD just before ICSI or coming ICSI cycles. Excised testicular tissues were minced into small pieces by blade and recovered sperm were cultured in Ham's F-10 medium supplemented 10% FBS for 3 days or Pentoxyphylline (3.6 mM) was added in the same culture medium for short term (1 hr) culture. For culture, 37°C and 5% carbon dioxide atmosphere of incubator was used. Just after sperm retrieval, 1 hr and 3 days of culture, the sperm motility was evaluated in both groups. At least 100 sperm were counted for motility scoring. After 1 hr of culture with Pentoxyphylline or 3 days of culture, sperm cryopreservation was performed by vapour freezing method. Results: Total sperm solution volume was adjusted to 4 ml and mean sperm concentration was 0.3 × 106 ± 0.2/ml. Initial sperm motility was 12.0 ± 13.8% and the motility increased to 25.7 ± 17.3% (simple medium) and 62.4 ± 21.8% (Pentoxyphylline) after 1 hr of culture. After 3 days, the motility was changed 41.9 ± 19.0% (simple medium) and 44.3 ± 16.2% (Pentoxyphylline). In the Pentoxyphylline treated group, the sperm were cryopreserved and thawed and the motility fell greatly to 6.3 ± 6.1%. Long term cultured sperm were reserved for actual ICSI cycles. Pentoxyphylline treatment shortens the culture time to attain motility of testicular sperm in vitro. Only 1 hr of culture was resulted to get sperm motility over 60%. Conclusion: Results of the present study suggest that Pentoxyphylline treatment is a useful method and should be substituted as the long term culture method.

Objective: To describe methods of sperm retrieval for intracytoplasmic sperm injection (ICSI) in patients with male factor infertility and to review the clinical results using sperm from the different sources.Design: The literature on sperm-obtaining methods and ICSI was reviewed. Studies related to this topic were identified through MEDLINE.Result(s): This review describes the evolution of sperm retrieval methods. Sperm can be obtained by microepididymal sperm aspiration (MESA), percutaneous sperm aspiration (PESA), and testicular sperm extraction (TESE), from patients with congenital absence of the vas deferens or acquired vas obstruction. When ICSI is performed with ejaculated, epididymal, or testicular sperm, good fertilization and pregnancy rates are achieved without significant differences among the various sperm sources. The original percutaneous sperm aspiration method has been modified slightly and yields successful results.Conclusion(s): Viable pregnancies can be achieved with ICSI by using not only ejaculated sperm, but also epididymal and testicular sperm. Microepididymal sperm aspiration, percutaneous sperm aspiration, modified percutaneous sperm aspiration, and testicular sperm extraction can be considered standard procedures to treat male factor infertility. (C) 1997 by American Society for Reproductive Medicine.