Cryopreservation is an option for the preservation of pre- or post-pubertal female or male fertility. This technique not only is beneficial for human clinical applications, but also plays a crucial role in the breeding of livestock and endangered species. Unfortunately, frozen germ cells, including oocytes, sperm, embryos, and spermatogonial stem cells, are subject to cryoinjury. As a result, various cryoprotective agents and freezing techniques have been developed to mitigate this damage. Despite extensive research aimed at reducing apoptotic cell death during freezing, a low survival rate and impaired cell function are still observed after freeze-thawing. In recent decades, several cell death pathways other than apoptosis have been identified. However, the relationship between these pathways and cryoinjury is not yet fully understood, although necroptosis and autophagy appear to be linked to cryoinjury. Therefore, gaining a deeper understanding of the molecular mechanisms of cryoinjury could aid in the development of new strategies to enhance the effectiveness of the freezing of reproductive tissues. In this review, we focus on the pathways through which cryoinjury leads to cell death and propose novel approaches to enhance freezing efficacy based on signaling molecules.

OBJECTIVE: To evaluate the effects of the reactive oxygen species (ROS) generated with a xanthine(X) and xanthine oxidase (XO) system on sperm function, the change of sperm characteristics, lipid peroxidation, and DNA fragmentation in bovine spermatozoa. MATERIALS AND METHODS: ROS were produced using a combination of 100 micrometer X and 50 mU/ml XO. The ROS scavengers: superoxide dismutase (SOD)(200mu/ml) and catalase (500mu/ml) were also tested. Spermatozoa were incubated for 2 hours in BWW medium with a combination of X-XO supplemented with or without ROS scavengers at 37degrees C under 5% CO2 incubator. Sperm movement characteristics by CASA (computer-aided sperm analysis), HOST (hypoosmotic swelling test), Ca-ionophore induced acrosome reaction, malondialdehyde formation for the analysis of lipid peroxidation, the percentage of DNA fragmentation using the method of TdT-mediated nick end labelling (TUNEL) by flow cytometry were determined after 2 hours incubation. RESULTS: The action of ROS on bovine spermatozoa resulted in a decreased in capacity for sperm motility, Ca-ionophore induced acrosome reaction and membrane integrity, an increased in malondialdehyde formation and the percentage of sperm with DNA fragmentation. In the effects of antioxidant, catalase completely alleviated the toxic effects induced by the ROS in terms of sperm function and characteristics, however SOD exhibited no capacity to reduce the toxic effects. CONCLUSION: The ROS can induce significant damages to sperm functions and characteristics. The useful ROS scavengers can minimized the defects of sperm function and various damages of spermatozoa.
Acrosome Reaction ; Catalase ; DNA Fragmentation* ; DNA* ; Flow Cytometry ; Incubators ; Lipid Peroxidation* ; Malondialdehyde ; Membranes ; Reactive Oxygen Species* ; Sperm Motility ; Spermatozoa* ; Superoxide Dismutase ; Xanthine Oxidase

In spite of much progress in in vitro fertilization and embryo transfer(IVF-ET) program,the pregnancy rate remains at 20~30%, and the endometrial implantation rate per embryotransferred at 10%. Although IVF-ET is widely applied in the treatment of coupleswith male factor infertility, it may fail in many infertile couples with normal semen parameters,and certain couples cannot be accepted for standard IVF-ET due to unfertilization orextremely low fertilization rate of oocytes. Recently, several procedures of microassistedfertilization(MAF) using micromanipulation have been introduced, and pregnancies and birthshave been obtained after intracytoplasmic sperm injection(ICSI).This clinical study was performed to develop and establish ICSI as an effective procedureof MAF in infertile couples who could not be accepted for standard IVF-ET becauseof extremely impaired semen characteristics(Group A) and because of failure in fertilizationof extremely low fertilization rate of oocytes with the conventional fertilization technique inthe previous IVF-ET cycles(Group B). From March, 1995 to December, 1996, a total of 114cycles of IVF-ET with ICSI in 65 infertile patients were included in study group, and theoutcomes of ICSI were analyzed according to fertilization rate, cumulative embryo score(CES), and pregnancy rate.In Group A, 34 patients were evaluated with semen score such as number of totalmotile sperms, and then divided into 4 groups accordingly. In 62 ICSI cycles, the numberof oocytes retrieved after controlled ovarian hyperstimulation(COH) was 12.4+/-6.8, and thenumber of oocytes optimal for ICSI procedure was 8.8+/-5.5. The fertilization rate of 65.7+/-23.6% could be obtained after ICSI. The number of embryos transferred was 4.4+/-2.2 withthe mean CES of 50.5+/-34.3 in ICSI cycles. The overall pregnancy rate was 24.2%(15/62)per cycle and 44.1%(15/34) per patient. There were no significant differences in the pregnancyrates among 4 groups. Although more mature oocytes were retrieved, the fertilizationrate was significantly lower in Group A-1 compared with Group A-IV. However, semenscore did not clearly affect the outcomes of ICSI in couples with severe male factor infertility.In Group B, the number of oocytes retrieved after COH was 10.5+/-6.1 in 49 previouscycles, and 10.8+/-5.7 in 52 ICSI cycles. In ICSI cycles, the number of oocytes optimal forICSI procedure was 8.5+/-5.1 with the fertilization rate of 72.4+/-22.5%. The number ofembryos transferred was 1.4+/-2.4 in previous cycles, and 4.7+/-1.8 with the mean CES of 50.4+/-29.9 in ICSI cycles. In ICSI cycles, the overall pregnancy rate was 30.8%(16/52) percycles and 51.6%(16/31) per patients.In conclusion, MAF of human oocytes with ICSI is a promising fertilization method forIVF-ET patients, especially with few spermatozoa for the conventional methods of in vitroinsemination and with the past history of failure in fertilization or low fertilization rate inthe previous cycles, and ICSI using micromanipulation procedures applied to human oocyteswill provide a range of novel techniques which may dramatically improve the pregnancy ratein IVF-ET program and contribute much to the effective management of infertile couples.
Embryonic Structures ; Family Characteristics ; Fertilization in Vitro ; Fertilization* ; Humans* ; Infertility ; Male ; Micromanipulation ; Oocytes* ; Pregnancy ; Pregnancy Rate ; Semen ; Sperm Injections, Intracytoplasmic* ; Spermatozoa

OBJECTIVE: To investigate the positive or negative effect of delaying embryo transfer(ET) one day in IVF-ET. METHODS: From May to July, 1997, a total of 64 patients was emolled in this prospective randomized case-controlled study. When the timing of oocyte retrieval was decided, random allocation of patients was made to one of the two groups: day 2 transfer or day 3 transfer. In day 3 transfer group, embryos were cultured in M3 media(Medi-Cult) for further 24 hours. RESULTS: There were no significant differences in age of patients, infertility factor, basal serum FSH level, and serum E2 level on hCG day between two poups, but number of previous IVF-ET cycles was significantly higher in day 3 transfer group(p 0.042). Number of oocytes retrieved, fertilization rate, cleavage rate, and number of embryos transferred had no significant difference, but cumulative embryo score(CES) was significantly higher in day 3 transfer group(p 0.0001). Clinical pregnancy and implantation rates were bigher in day 3 transfer group, but without significance(34.4% vs. 21.9%; 8.7% vs, 5.4%). There were also no significant differences in spontaneous miscarriage and multiple pregnancy rates. Especially in patients over 35 years of age, clinical pregnancy and implantation rates were more higher in day 3 transfer group, but without significance(41.7% vs, 8.3%; 8.5% vs. 1.6%). CONCLUSION: Considering the higher number of previous cycles in day 3 transfer group, it is at least likely that delaying ET one day may be clinically beneficial in IVF-ET, especially in patients with old age or repeated failure of previous cycles.
Abortion, Spontaneous ; Case-Control Studies ; Embryo Transfer* ; Embryonic Structures* ; Female ; Fertilization ; Fertilization in Vitro* ; Humans ; Infertility ; Oocyte Retrieval ; Oocytes ; Pregnancy ; Pregnancy Outcome ; Pregnancy, Multiple ; Prospective Studies ; Random Allocation

OBJECTIVE: To investigate the intluence of previous tuberculous epididymitis in infertile males with obstructive azoospermia on the outcome of sperm retrieval and intracytoplasmic sperm injection(ICSI) in IVF-ET propam. METHODS: Retrospective analysis was paformed in 26 patients with obstructive azoospermia undergoing sperm retrieval and ICSI at Seoul National University Hospital from January, 1996 to August, 1997; 12 cycles in 5 patients with tuberculous obstructive azoospermia(Group A), and 40 cycles in 21 patients with non-tuberculous obstructive azoospermia(Group B). RESULTS: There was no significant difference in the clinical pregnancy rate(PR) per fresh transfer between Groups A and B(20.0%[2/10] vs. 26.8%(11/41)). The rates of embryo implantation and clinical miscarriage were also comparable in both groups(6.3% vs. 11.1%, 50.0% vs. 9.1%). This tendencies were also similar after including five cryopreserved-thawed transfer cycles. CONCLUSION: Embryo quality and pregnancy outcome in sperm retrieval and ICSI were comparable in both the tuberculous and non-tuberculous obstructive azoospermia patients. Our results suggest that previous history of tuberculous epididymitis in patients with obstructive azoospermia does not affect the outcome of sperm retrieval and ICSI.
Abortion, Spontaneous ; Azoospermia* ; Embryo Implantation ; Embryonic Structures ; Epididymitis* ; Female ; Humans ; Male ; Pregnancy ; Pregnancy Outcome ; Retrospective Studies ; Seoul ; Sperm Injections, Intracytoplasmic* ; Sperm Retrieval* ; Spermatozoa*

OBJECTIVE: This study was designed to investigate the interrelationship and clinical usefulness of sperm morphology by strict criteria (SM), acrosome reaction following ionophore challenge test (ARIC) and sperm penetration assay (SPA) using zona-free hamster ova as prognostic factors in in vitro fertilization. MATERIALS AND METHODS: Semen samples were provided by 83 patients undergoing IVF. We first evaluated the differences between normal fertilization group and poor fertilization group on three andrologic tests. Secondly, we analyzed the relationship between the three andrologic tests and in vitro fertilization on IVF settings. Finally, we evaluated the effectiveness of the three andrologic tests as the prognostic indicators for fertilizing ability. RESULTS: The fertilization rate of all men in the poor fertilization group was less than 30%; but there was no evidence that this poor fertilization was due to oocyte defects. The results of three andrologic tests were significatly higher in normal fertilization group. Fertilization rate (%) in vitro was highly correlated (p<0.001) with % normal sperm by SM, ARIC value (%), and SPA result. By using Receiver-Operator-Characteristic curve (ROC), we evaluated the effectiveness of these three tests. The sensitivity and specificity of SM, ARIC test and SPA in predicting fertilization potential in IVF setting were 76% and 75%, 84% and 90%, and 76% and 95%, respectively. CONCLUSION: Our data suggest that the three andrologic tests can be reliable tools as prognostic factors of sperm fertilizing ability. Among these test, ARIC test and SPA gave more accurate information on fertilizing capacity. ARIC test was shown to have a predictive value for fertilizing ability comparable to that of SPA that appears to be a simple and cost-effective addition to current andrology laboratory. Combined application of these three tests may give more information on predicting sperm fertilizing capacity.
Acrosome Reaction* ; Acrosome* ; Andrology ; Animals ; Cricetinae* ; Diagnosis* ; Fertilization ; Fertilization in Vitro* ; Humans ; Infertility, Male* ; Male ; Male* ; Oocytes ; Ovum* ; Semen ; Sensitivity and Specificity ; Sperm-Ovum Interactions* ; Spermatozoa*

OBJECTIVE: The purpose of this study was to determine whether intracytoplasmic sperm injection(ICSI) could overcome the defects of oocytes in IVF-ET patients with previous fertilization failure by conventional fertilization technique. Design: Retrospective study Materials and METHODS: A total of 119 ICSI cycles in 57 IVF-ET patients performed from May, 1995 to December, 1997 was enrolled. Subjects were divided into two groups: FR group included 66 ICSI cycles in 35 patients with normal sperm who underwent ICSI due to past history of failed or poor fertilization in the previous IVF-ET cycles, and OAT group included 53 ICSI cycles in 22 patients with severe oligoasthenoterato- zoospermia(OAT) which was defined as sperm concentration < 20 million/ml, mo#dlity < 30% and normal morphology < 4% by strict morphologic criteria. The outcomes of ICSI were analyzed and compared in both groups. RESULTS: The age of female patients, basal serum FSH level, and the numbers of oocytes retrieved and metaphase II oocytes were all comparable in both groups. The fertilization rate after ICSI was similar in both groups(68.7+/-25.3% vs. 67.7+/-24.5%), as were the cleavage rate of normally fertilized oocytes(93.1+/-21.4% vs. 89.3+/-21.6%), the number of embryos transferred(4,00+/-1.98 vs. 4.64+/-2.10), and cumulative embryo score(CES) indicating the quality of embryos(47.3+/-33.2 vs. 54.1+/-33.2). The implantation rate(4.3+/-10.5% vs. 3.8+/-11.0%) and the clinical pregnancy rate per cycle(15.2% vs. 13.2%) were also comparable in both groups. CONCLUSIONS: Although it has been shown that there is a higher risk of chromosomal abnormalities in oocytes from IVF-ET patients with pevious failed or poor fertilization, higher implantation and clinical pregnancy rates wer#e not observed in patients with OAT following ICSL Therefore, the functional defect of sperm such as loss of capacitation, defect of aaasome reaction, and abnormality of nucleus decondensation should be also considered in patients with previous failed or poor fertilization.
Avena ; Chromosome Aberrations ; Embryonic Structures ; Female ; Fertilization* ; Humans ; Male* ; Metaphase ; Oocytes ; Pregnancy Rate ; Retrospective Studies ; Sperm Injections, Intracytoplasmic ; Spermatozoa*

No abstract available.
Chromosome Aberrations* ; Embryonic Structures* ; Humans ; Male* ; Preimplantation Diagnosis* ; Sperm Injections, Intracytoplasmic*

Fluorescence in situ hybridization (FISH) techniques allow the enumeration of chromosome abnormalities and from a great potential for many clinical applications. In order to produce quantitative and reproducible results, expensive tools such as a cooled CCD camera and a computer software are required. We have developed a Chromosome Image Processing System (Chips) using FISH that allows the detection and mapping of the genetic aberrations. The aim of our study, therefore, is to evaluate the capabilities of our original system using a black-and-white video camera. As a model system, three repetitive DNA probes (D18Zl, DXZI, and DYZ3) were hybridized to variety different clinical samples such as human metaphase spreads and interphase nuclei obtained from uncultured peripheral blood lymphocytes, uncultured amniocytes, and germ cells. The visualization of the FISH signals was performed using our system for image acquisition and pseudocoloring. FISH images were obtained by combining images from each of probes and DAPI counterstain captured separately. Using our original system, the aberrations of single or multiple chromosomes in a single hybridization experiment using chromosomes and interphase nuclei from a variety of cell types, including lymphocytes, amniocytes, sperm, and biopsied blastomeres, were enabled to evaluate. There were no differences in the image quality in accordance with FISH method, fluorochrome types, or different clinical samples. Always bright signals were detected using our system. Our system also yielded constant results. Our Chips would permit a level of performance of FISH analysis on metaphase chromosomes and interphase nuclei with unparalleled capabilities. Thus, it would be useful for clinical purposes.
Blastomeres ; Chromosome Aberrations ; DNA Probes ; Fluorescence* ; Germ Cells ; Humans ; In Situ Hybridization* ; Interphase ; Lymphocytes ; Metaphase ; Spermatozoa

Although the fertilization rate exceeds to 80~90% with much progress in vitro fertilizaton and embyo transfer(IVF-ET) program, the prgnancy rate rmains at 20~30%, and the endometrial implantaion rate per embryo transferred at 10~15%. Recently, many attempts have been made to improve embrynic implantion after IVF-ET including serveral procedures of assisted hatching(AH) using micromanipulation, and pregnacies and births have been obtained after AH. This clinical study was performed to develop and estabilish AH as an effective procedure to improve embryonic implantioan in IVF-ET patients who had previous repeated failure of standard IVF-ET more than 2 times(Group R), were more than 37 years old(Group A), or had high basal serun FSH levels more than 15 mIU/ml(Group F). From January, 1995 to Februry, 1996, 132 cycles of AH using partial zona dissection(PZD) were performed in 104 infertile patients, and the outcomes of AH were analyzed according to pregnancy rate. The number of oocytes retrieved after controlled ovarian hyperstimulation(COH) was 9.9+/-7.1 in 71 cycles of 54 patients who had previous repeated failure more than two times(Group I: Group R,R+A,R+F, and R+A+F), 8.4+/-5.9 in 62 cycles of 46 patients whose age was more than 37 years old(Group II : Groups A, R+A, A+F, and R+A+F), and 8.7+/-6.5 in 49 cycles of 47 patients who had high basal serum FSH levels more than 15 mIU/ml(Group III:Groups F,R+F, A+F, and R+A+F). The number of embroys transferred after AH was 4.7 +/-1.8 in Group I, 4.2 +/-1.9 in Group II, and 4.2+/-2.0 in Froup III. The mean cumulative embryo score(CES) was 56.8+/-30.0 in Group I, 52.6+/-30.6 in Group II, and 52.6+/-29.9 in Group III. There were no significant differences in the numbers of oocytes rerieved and embryos transferred, and CES among 3 groups. The overall clinical pregnancy rate was 14.4%(19/132) per cycle and 18.3%(19/104) per patient. THe clinical pregnancy rate per cycle and per patient was 12.7%(9/71) and 16.7%(9/54) in Group I, 4.8% (3/62) and 6.5%(3/46) in Group II, and 26.5%(13/49) and 27.7%(13/47) in Group III, and there was a significant difference between Group II and Group III. In conclusion, AH of human embryos using micromanipulation might be promising for IVF-ET patients, especially with the past history of repeated failure, old age, and high basal serum FSH level and AH will provide a range of novel techiques which may dramatically improve the implanatation and pregnancy rates in IVF-ET program and contribute much to effective management of infertile couples.
Embryo Transfer* ; Embryonic Structures* ; Family Characteristics ; Fertilization ; Herpes Zoster ; Humans ; Micromanipulation* ; Oocytes ; Parturition ; Pregnancy Rate