No abstract available.

No abstract available.
Epidermolysis Bullosa ; Muscular Dystrophy, Duchenne ; Ornithine Carbamoyltransferase ; Polymerase Chain Reaction* ; Preimplantation Diagnosis*

OBJECTIVES: Klinefelter syndrome is the most common genetic cause of male infertility and presents with 47, XXY mainly or 46, XX/47, XXY mosaicism. It is characterized by hypogonadism and azoospermia due to testicular failure, however, sporadic cases of natural pregnancies have been reported. With the development of testicular sperm extraction (TESE) and intracytoplasmic sperm injection (ICSI), sperm can be retrieved successfully and ART is applied in these patients for pregnancy. It has been suggested that the risk of chromosome aneuploidy for both sex chromosome and autosome is increased in the sperms from 47, XXY germ cells. Considering the risk for chromosomal aneuploidy in the offspring, preimplantation genetic diagnosis (PGD) could be applied as a safe and more effective treatment option in Klinefelter syndrome. The aim of this study is to assess the outcome of PGD cycles by using FISH for sex chromosome and autosome in patients with Klinefelter syndrome. MATERIALS AND METHODS: From Jan. 2001 to Dec. 2003, PGD was attempted in 8 cases of Klinefelter syndrome but TESE was failed to retrieve sperm in the 3 cases, therefore PGD was performed in 8 cycles of 5 cases (four 47, XXY and one 46, XY/47, XXY mosaicism). In one case, ejaculated sperm was used and in 4 cases, TESE sperm was used for ICSI. After fertilization, blastomere biopsy was performed in 6~10 cell stage embryo and the chromosome aneuploidy was diagnosed by using FISH with CEP probes for chromosome X, Y and 17 or 18. RESULTS: A total of 127 oocytes were retrieved and ICSI was performed in 113 mature oocytes. The fertilization rate was 65.3+/-6.0% (mean+/-SEM) and 76 embryos were obtained. Blastomere biopsy was performed in 61 developing embryos and FISH analysis was successful in 95.1% of the biopsied blastomeres (58/61). The rate of balanced embryos for chromosome X, Y and 17 or 18 was 39.7+/-6.9%. The rate of aneuploidy for sex chromosome (X and Y) was 45.9+/-5.3% and 43.2+/-5.8% for chromosome 17 or 18, respectively. Embryo transfer was performed in all 8 cycles and mean number of transferred embryos was 2.5+/-0.5. In 2 cases, clinical pregnancies were obtained and normal 46, XX and 46, XY karyotypes were confirmed by amniocentesis, respectively. Healthy male and female babies were delivered uneventfully at term. CONCLUSION: The patients with Klinefelter syndrome can benefit from ART with TESE and ICSI. Considering the risk of aneuploidy for both sex chromosome and autosome in the sperms and embryos of Klinefelter syndrome, PGD could be offered as safe and more effective treatment option.
Amniocentesis ; Aneuploidy ; Azoospermia ; Biopsy ; Blastomeres ; Chromosomes, Human, Pair 17 ; Embryo Transfer ; Embryonic Structures ; Female ; Fertilization ; Germ Cells ; Humans ; Hypogonadism ; Infertility, Male ; Karyotype ; Klinefelter Syndrome* ; Male ; Mosaicism ; Oocytes ; Pregnancy ; Preimplantation Diagnosis* ; Prostaglandins D ; Sex Chromosomes ; Sperm Injections, Intracytoplasmic ; Spermatozoa

No abstract available.
Humans* ; Pregnancy* ; Sperm Injections, Intracytoplasmic* ; Spermatozoa*

BACKGROUND: Clonidine depress the surge of sympathetic system outflow via central alpha 2 adrenergic effect. Still on a debate is the receptor relevant to analgesic effect of clonidine. METHODS: Intrathecal catheter(PE-10, 10 cm in length) was inserted via the atlanto-occipital membrane and the tip of intrathecal catheter was allowed to reach at the lumbar area. At the fifth day after catheter insertion, all experimental animals were ramdomly divided to two groups. Clonidine (5 microgram) in clonidine group and morphine (45 microgram) in morphine group was administered into subarachnoid space 20 minutes before tail-clamping test. Heart rate and blood pressure changes were recorded during the experimental period. Then naloxone was given intravenously 5 minutes after the first tail-clamping test. In 2 minutes after that, the second tail clamping was done. RESULTS: Results were as follows. First, comparing the highest blood pressure changes before and after administration of naloxone, the elevation of blood pressure was significant after administration of naloxone in morphine group(p<0.05), but not in clonidine group. Second, comparing the change of heart rate, in morphine group there was significant elevation of heart rate before and after administration of naloxone. And comparing the highest elevation of heart rate, morphine group showed significant difference before and after naloxone administration(p<0.05), but not in clonidine group. CONCLUSIONS: From above results, we assumed that the analgesic effect of clonidine was not related to the opiate receptor.
Adrenergic Agents ; Animals ; Blood Pressure ; Catheters ; Clonidine* ; Constriction ; Heart Rate ; Membranes ; Morphine ; Naloxone* ; Rats* ; Receptors, Opioid ; Subarachnoid Space

OBJECTIVE: Preimplantation genetic diagnosis (PGD) is reserved for couples with a risk of transmitting a serious and incurable disease, and hence avoids the undesirable therapeutic abortion. In this study, we evaluated the efficacy of PGD for Duchenne muscular dystrophy (DMD) cases by the fluorescent PCR with polymorphic linked markers and the conventional duplex-nested PCR methods. METHODS: Biopsy of one or two blastomeres was done from the embryos fertilized by ICSI on the third day after fertilization. We performed two cases of PGD-DMD by the duplex-nested PCR for the causative mutation loci and the SRY gene on Y chromosome. The triplex fluorescent PCR for the mutation loci, the SRY gene and the polymorphic microsatellite marker on X chromosome was applied for two cases of PGD-DMD. RESULTS: By the duplex-nested PCR, successful diagnosis rate was 95.5% (21/22), but we could not discriminate the female embryos whether normal or carrier in this X-linked recessive disease. However, the triplex fluorescent PCR method showed 100% (27/27) of successful diagnosis rate, and all female embryos (n=17) were distinguished normal (n=10) from carrier (n=7) embryos. Unaffected and normal embryos were transferred into mother's uterus after diagnosis. A healthy normal male was achieved after PGD with the duplex-nested PCR method and a twin, a male and a female, were delivered with triplex fluorescent PCR method. The normality of dystrophin gene was confirmed by amniocentesis and postnatal genetic analysis in all offsprings. CONCLUSION: The fluorescent PCR with polymorphic marker might be useful in improving the specificity and reliability of PGD for single gene disorders.
Abortion, Therapeutic ; Amniocentesis ; Biopsy ; Blastomeres ; Diagnosis ; Dystrophin ; Embryonic Structures ; Family Characteristics ; Female ; Fertilization ; Genes, sry ; Humans ; Male ; Microsatellite Repeats ; Muscular Dystrophy, Duchenne* ; Polymerase Chain Reaction* ; Pregnancy ; Preimplantation Diagnosis* ; Prostaglandins D ; Sensitivity and Specificity ; Sperm Injections, Intracytoplasmic ; Twins ; Uterus ; X Chromosome ; Y Chromosome

OBJECTIVES: This study was performed to evaluate the laboratory system for successful PGD using fluorescence in situ hybridization (FISH) and the clinical outcome of PGD cycles in five years experiences. METHODS: A total of 181 PGD-FISH cycles of 106 couples were performed, and diagnosed chromosome normality in the preimplantation embryos. The laboratory and clinical data were classified by the following optimization steps, and statistically analyzed. Phase I: Blastomere biopsy with two kinds of pipettes, removal of cytoplasmic proteins without treatment of pepsin and culture of biopsied embryos with single medium; Phase II: Blatomere biopsy with single pipette, removal of cytoplasmic proteins with pepsin and culture of biopsied embryos with single medium; Phase III: Blastomere biopsy with single pipette, removal of cytoplasmic proteins with pepsin and culture of biopsied embryos with sequential media. RESULTS: A total of 3,209 oocytes were collected, and 83.8% (2,212/2,640) of fertilization rate was obtained by ICSI procedure. The successful blastomere biopsies were accomplished in 98.6% (2,043/2,071) of embryos, and the successful diagnosis rate of FISH was 94.7% (1,935/ 2,043) of blastomeres from overall data. Embryo transfers with normal embryos were conducted in 93.9% (170/181) of started cycles. There was no difference in the successful rate of biopsy and diagnosis among Phase I, II and III. However, the pregnancy rate per embryo transfer of Phase III (38.8%, 26/67) was significantly (p<0.05) higher than those of Phase I (13.9%, 5/36) and Phase II (14.9%, 10/67). CONCLUSIONS: The laboratory optimization and experience for the PGD with FISH procedure can increase the pregnancy rate to 38.8% in the human IVF-ET program. Our facility of PGD with FISH provides the great possibility to get a normal pregnancy for the concerned couples by chromosomal aberrations.
Biopsy ; Blastocyst ; Blastomeres ; Chromosome Aberrations ; Cytoplasm ; Diagnosis ; Embryo Transfer ; Embryonic Structures ; Family Characteristics ; Fertilization ; Fluorescence ; Humans ; In Situ Hybridization ; Oocytes ; Pepsin A ; Pregnancy Rate* ; Pregnancy* ; Preimplantation Diagnosis* ; Prostaglandins D ; Sperm Injections, Intracytoplasmic

OBJECTIVES: The development of an useful method for obtaining metaphase chromosomes from a biopsied blastomere would allow differentiation between embryos with balanced and normal chromosome complements in the preimplantation genetic diagnosis for chromosomal translocations. This study was performed to evaluate the effects of microtubule depolymerizing agents (MTDAs) on the blastomeres of mouse and human preimplantation embryos, and to establish an effective method for obtaining metaphase chromosomes of biopsied blastomeres in human early embryos. MATERIALS AND MEHTODS: Early embryos (2-4 cell stage) from superovulated mice (ICR strain) were collected and treated with single or mixture MTDAs, such as vinblastine, nocodazole and colcemid. After the treatment of MTDAs for 16 hours, the metaphase aquisition (MA) rates were evaluated by the observation of chromosome status with bis-benzimide or DAPI staining. The optimal condition from the above experiment was applied to human embryos, which were developed from abnormal fertilization (3-pronuclei). Fluorescence in-situ hybridization (FISH) with whole chromosome probes was conducted on the human metaphase chromosomes by the MTDAs. RESULTS: In mouse embryos, the effective concentrations of each MTDAs for obtaining metaphase chromosomes were 1.0 micrometer of vinblastine (20.3%), 5.0 micrometer of nocodazole (28.1%) and 1.0 micrometer colcemid (55.6%), respectively. The highest MA rate (91.2%) in the mouse embryos was obtained by a mixture of vinblastine (1.0 micrometer) and nocodazole (1.0 micrometer). In the human embryos, the metaphase chromosomes of blastomeres were obtained in 44 of 113 blastomeres (38.9%) by treatment of the mixture of vinblastine and nocodazole. FISH signals of the metaphase chromosomes were successfully observed in human individual blastomeres. CONCLUSIONS: The treatment of a mixture MTDAs for obtaining metaphase chromosomes was an efficient method, and the MA rate was above 90% in the mouse embryos. However, only a relatively small proportions of the blastomeres yielded metaphase chromosomes by the MTDAs in the human embryos. The inconsistent effects of MTDAs may be related to the variation of different species and the poor developmental potency of abnormally fertilized human embryos. We should develop more reliable and efficient methods for obtaining the metaphase chromosomes in the biopsied blastomeres of human preimplantation embryos.
Animals ; Blastocyst ; Blastomeres* ; Complement System Proteins ; Demecolcine ; Embryonic Structures* ; Fertilization ; Fluorescence ; Humans* ; Metaphase* ; Mice* ; Microtubules* ; Nocodazole ; Preimplantation Diagnosis ; Translocation, Genetic ; Vinblastine

OBJECTIVES: The development of an useful method for obtaining metaphase chromosomes from a biopsied blastomere would allow differentiation between embryos with balanced and normal chromosome complements in the preimplantation genetic diagnosis for chromosomal translocations. This study was performed to evaluate the effects of microtubule depolymerizing agents (MTDAs) on the blastomeres of mouse and human preimplantation embryos, and to establish an effective method for obtaining metaphase chromosomes of biopsied blastomeres in human early embryos. MATERIALS AND MEHTODS: Early embryos (2-4 cell stage) from superovulated mice (ICR strain) were collected and treated with single or mixture MTDAs, such as vinblastine, nocodazole and colcemid. After the treatment of MTDAs for 16 hours, the metaphase aquisition (MA) rates were evaluated by the observation of chromosome status with bis-benzimide or DAPI staining. The optimal condition from the above experiment was applied to human embryos, which were developed from abnormal fertilization (3-pronuclei). Fluorescence in-situ hybridization (FISH) with whole chromosome probes was conducted on the human metaphase chromosomes by the MTDAs. RESULTS: In mouse embryos, the effective concentrations of each MTDAs for obtaining metaphase chromosomes were 1.0 micrometer of vinblastine (20.3%), 5.0 micrometer of nocodazole (28.1%) and 1.0 micrometer colcemid (55.6%), respectively. The highest MA rate (91.2%) in the mouse embryos was obtained by a mixture of vinblastine (1.0 micrometer) and nocodazole (1.0 micrometer). In the human embryos, the metaphase chromosomes of blastomeres were obtained in 44 of 113 blastomeres (38.9%) by treatment of the mixture of vinblastine and nocodazole. FISH signals of the metaphase chromosomes were successfully observed in human individual blastomeres. CONCLUSIONS: The treatment of a mixture MTDAs for obtaining metaphase chromosomes was an efficient method, and the MA rate was above 90% in the mouse embryos. However, only a relatively small proportions of the blastomeres yielded metaphase chromosomes by the MTDAs in the human embryos. The inconsistent effects of MTDAs may be related to the variation of different species and the poor developmental potency of abnormally fertilized human embryos. We should develop more reliable and efficient methods for obtaining the metaphase chromosomes in the biopsied blastomeres of human preimplantation embryos.
Animals ; Blastocyst ; Blastomeres* ; Complement System Proteins ; Demecolcine ; Embryonic Structures* ; Fertilization ; Fluorescence ; Humans* ; Metaphase* ; Mice* ; Microtubules* ; Nocodazole ; Preimplantation Diagnosis ; Translocation, Genetic ; Vinblastine

OBJECTIVE: To determine whether the serum beta-human chorionic gonadotropin (hCG) profile following preimplantation genetic diagnosis (PGD) is lower than that of intracytoplasmic sperm injection (ICSI) cycles. METHODS: A total of 129 PGD cycles and 1,161 age-matched ICSI cycles, which resulted in pregnancy (serum beta-hCG> or =5 mIU/mL) on post-ovulation day (POD) 12 were included. We compared the mean serum beta-hCG levels on POD 12, 14, 21, and 28, doubling time of serum hCG, and created a cut-off value for predicting a singleton pregnancy in each group. RESULTS: The mean serum beta-hCG concentration of the PGD group was significantly lower than that of the control group on POD 12, 14, and 21. The doubling time of serum beta-hCG at each time interval showed no significant difference. The cut-off-value of serum beta-hCG for predicting a single viable pregnancy was 32.5 mIU/mL on POD 12 and 113.5 mIU/mL on POD 14 for the PGD group, which was lower than that for the control group. CONCLUSION: Blastomere biopsy may decrease the beta-hCG-producing activity of the trophoblasts, especially in early pregnancy. Setting a lower cut-off value of serum beta-hCG for predicting pregnancy outcomes in PGD may be needed.
Biopsy ; Blastomeres ; Chorionic Gonadotropin ; Female ; Humans ; Pregnancy ; Pregnancy Outcome ; Preimplantation Diagnosis ; Prostaglandins D ; Sperm Injections, Intracytoplasmic ; Trophoblasts