• Clinical and Experimental Reproductive Medicine
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• 제32권1호
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• pp.17-26
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• 2005

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.

• 한국발생생물학회지:발생과생식
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• 제17권3호
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• pp.269-274
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• 2013

This study was performed to evaluate the effects of fibroblast co-culture on in vitro maturation (IVM) of prepubertal mouse preantral follicles. The intact preantral follicles were obtained from the ovaries of 12-14 day old mice and these were cultured individually in ${\alpha}$-minimal essential medium (${\alpha}$-MEM) supplemented with 5% fetal bovine serum (FBS), $100mIU/m{\ell}$ recombinant follicle stimulating hormone (rFSH), 1% insulin-transferrin-selenium, $100{\mu}g/ml$ penicillin and $50{\mu}g/m{\ell}$ streptomycin as base medium for 12 days. A total of 200 follicles were cultured in base medium co-cultured with mouse embryonic fibroblast (MEF) (MEF group) (n=100) or only base medium as control group (n=100). Survival rate of follicles on day 12 of culture were significantly higher in the MEF group of 90.0%, compared with 77.0% of the control group (p=0.021). Follicle diameters on day 6 and 8 of the culture period were significantly larger in the MEF group than those in the control group (p=0.021, p=0.007, respectively). Estradiol levels in culture media on day 4, 6, 8, 10 and 12 of the culture period were significantly higher in the MEF group (p=0.043, p=0.021, p=0.006, p<0.001 and p=0.008, retrospectively). Our data suggest that MEF cell co-culture on IVM of mouse preantral follicle increases survival rate and promotes follicular growth and steroid production.

• Clinical and Experimental Reproductive Medicine
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• 제30권2호
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• pp.111-118
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• 2003

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 Methods: 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 $1.0{\mu}M$ of vinblastine (20.3%), $5.0{\mu}M$ of nocodazole (28.1%) and $1.0{\mu}M$ colcemid (55.6%), respectively. The highest MA rate (91.2%) in the mouse embryos was obtained by a mixture of vinblastine ($1.0{\mu}M$) and nocodazole ($1.0{\mu}M$). 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.

• 한국발생생물학회지:발생과생식
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• 제18권3호
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• pp.145-152
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• 2014

This study was performed to investigate the effect of of transdermal testosterone gel (TTG) on controlled ovarian stimulation (COS) and IVF outcomes and ovarian morphology according to pretreatment duration in poor responders. A total of 120 women were recruited for this pilot study. They were randomized into control, 2 weeks, 3 weeks or 4 weeks TTG treatment groups. For three TTG treatment groups, 12.5 mg TTG was applied daily for 2 weeks, 3 weeks or 4 weeks in preceding period of study stimulation cycle. After 3 weeks of TTG pretreatment, significant increase of antral follicle count (AFC) and significant decreases of mean follicular diameter (MFD) and resistance index (RI) value of ovarian stromal artery were observed (p=0.026, p<0.001, p<0.01, respectively). The total dose of rhFSH administered for COS significantly decreased after 3 and 4 weeks TTG treatment both compared with control group (p<0.001, p<0.001). The numbers of oocytes retrieved and mature oocytes were significanty higher in 3 and 4 weeks TTG treatment groups than control group (p<0.001, p<0.001 in the number of oocytes retrieved; p<0.001, p<0.001 in the number of mature oocytes). The clinical pregnancy rate and live birth rate were increased only in 4 weeks TTG treatment group compared with control group (p=0.030 and p=0.042, respectively). These data demonstrated that TTG pretreatment for 3 to 4 weeks increases AFC and ovarian stromal blood flow, thereby potentially improving the ovarian response to COS and IVF outcome in poor responders undergoing IVF/ICSI.

• Clinical and Experimental Reproductive Medicine
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• 제23권1호
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• pp.61-66
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• 1996

Vasectomy has become a popular method for male sterilization and this, in tum, has been followed by an increase in the number of patients requiring vasectomy reversal. Recently, many authors have reported a high success rate of vasovasostomy using microsurgical techniques. However, a significant discrepancy exist between the anatomical patency rate and pregnancy rate despite improvements in surgical techniques. Number of 420 patients who underwent vasovasostomy by a modified one layer reanastomosis from January 1986 to December 1994 were reviewed. Of the total, Complete follow up were possible in 115 patients. Of the 115 patients, 74 patients were treated by macroscopic reanastomosis, and microscopic technique were applied in 41 patients. Duration of vasal obstruction, gross apperance of vasal fluid, operative method, presence or absence of sperm and sperm granuloma, and results of postoperative semen analysis were analyzed as factors influencing the pregnancy rate. Success rates for patency and for pregnancy were 81% and, 42% respectively. Rate of pregnancy were increased if there were shorter periods of obstruction(<10years), bilateral observation of watery vasal fluid, presence of sperm bilaterally, bilateral presence of sperm granuloma at the vasectomy site, and normal results on postoperative semenalysis. With these results, we can conclude that all factors mentioned may affect the success rate of pregnancy following vasovasostomy.

• Clinical and Experimental Reproductive Medicine
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• 제19권2호
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• pp.163-168
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• 1992

This study was carried out to set up the ovum bank for ovum donation and to determine the best freezing method for human immature oocytes. Human immature follicular oocytes were cryopreserved by slow freezing and rapid thawing method. Immature follicular oocytes were treated by propanediol(PROH) solution by 2 and 4 step method in protocols A & B, respectively. In protocol C, immature oocytes were exposed to sucrose prior to treatment of PROH by 4 step method. We compared survival rate, maturation rate, and fertilization rate of immature oocytes among three protocols. Results were as follows. 1. Oocytes treated by the protocol C showed the highest survival rate( 70.3 %) and maturation rate(34.6%) after thawing. 2. Survival rate of oocytes treated by the protocol C was significantly higher than that of the protocol B after thawing(p<0.05). In conclusion, treatment of oocytes with sucrose prior to expose PROH was the best freezing method. Sucrose may have reduced the toxic effect of cryoprotectant to oocytes. We failed to induce fertilization of oocytes, which were treated by any protocols, by conventional insemination method, but obtained 28.8% fertilization rate by using partial zona dissection(PZD) method. This result suggests that micromanipulation(PZD) of the thawed oocytes before insemination will improve the fertilization rate.

• Clinical and Experimental Reproductive Medicine
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• 제18권1호
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• pp.55-61
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• 1991

In order to increase the pregnancy rate by means of cryopreservation of the excess oocytes in IVF-ET program, the survival rate of the frozen-thawed oocytes of mouse was examined according to the stages of maturation, cryoprotectants and their treatment. The results were summarized as follows. First, during the continuous treatment with cryoprotectant media, the survival rate of oocytes was higher in DMSO than in PROH, and higher at low temperature($4^{\circ}C$) than at room temperature($25^{\circ}C$). Second, as regard with the maturation of immature(GV-intact) oocytes after treatment with cryoprotectant media, the rate of maturation in DMSO-treated group(52%) was higher than in PROH-treated group(35%). Third, according to the treatment of cryoprotectant media, the survival rate of frozen-thawed oocytes in DMSO-treated group (45%) was higher than in PROH-treated group(29%), and that of oocytes in DMSO 4-step treated group was higher than any other groups. Finally, in the post-thaw oocytes frozen at various stage of maturation, the survival rate of immature oocytes with GV was the highest in all groups. These results suggest that in the cryopreservation of mouse oocytes, DMSO was better than PROH as cryoprotectant, in treatment of cryprotectant the multi-step treatment was better than single-step, and the post-thaw survival rate of oocytes was closely related to the maturity of oocytes. It is assumed that the highest survival rate of mouse oocytes with GV is due to the stability of the structures in nucleus and intracelluar organelles, and of physiological function.

• Clinical and Experimental Reproductive Medicine
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• 제21권3호
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• pp.247-252
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• 1994

Intracytoplasmic sperm injection(ICSI) was known as effective method in treatments of couples who unable to be helped by conventional in vitro fertilization. In 78 treatment cycles of 78 infertile couples using ICSI performed at our infertility clinic between May and August 1994 were analyzed. These patients were classified two groups, andrological factor(AF) and non-andrological factor(non-AF) group. The AF group, which had abnormal sperm physiology, included oligozoospermia, asthenozoospermia, oligoasthenoteratozoospermia(OATS) and microsurgical epididymal sperm aspiration(MESA) patients. The non-AF group, which had abnormal oocyte physiology, included abnormal zona pellucida, poor quality of oocyte and immune factor infertile patients. A single spermatozoon was injected into the ooplasm of 776 metaphase II oocytes. The fertilization rate was 44.6%(346/776) and 319 embryos were transferred. After 73 embryo transfers(93.6% of treatment cycles) 23 pregnancies were estabilshed, i. e. pregnancy rate of 29.4% per started cycle and 31.5% per embryo transfer. Fertilization rate of AF and non-AF group was 46.2% and 35.8%, pregnancy rate was 34.5%(20/58) and 20.0%(3/15), respectively. In order to increase the pregnancy rate, assisted hatching(AHA) has done after lCSl in 47 treatment cycles. Pregnancy rate of ICSI with AHA and without AHA group was 34. 0% (16/47) and 26.9%(7/26), respectively. ICSI was more effective in andrological factor infertility and the pregnancy rate was increased by ICSI with AHA procedure.

• 한국환경보건학회지
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• 제33권4호
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• pp.306-316
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• 2007

This study have a object to found out the effects of oriental herb medicine, Antaeeum, to dams of rats and their offsprings. The Antaeeum was savaged to female Sprague-Dawley rats at a dose of 5 mg/kg/day for 3 weeks during gestation periods. Dams of rat were sacrificed at 20th day of gestation, and were observed major internal and reproductive organs. Approximately live fetuses in the 20th days of gestation were selected randomly and examined with stereo microscopes. Others offsprings were fixed with 95% ethanol for skeletal examinations. The fixed fetuses were stained with alcian blue and alizarin red S to observe skeletal variations or malformations. Maternal body weight of Antaeeum treated dams have a tendency of increasing compared with control dams. There were no significant difference in internal and reproductive organs of weight or findings. The spleenic organ relative weight of treated dams were decreased compared with the control significaltly (p<0.05). There were no significant changes between two groups in blood chemistry and hematological values. There were no significant changes in number of corpus luteum, implantation, live fetuses and implantation rate, delivery rate, late resorption rate and sex ratio. But in the Antaeeum treated group showed lower early resorption rate than that of the control dams. Fetal body weight and number of fetus a dam at Antaeeum treated group were higher than that of control group. The fetuses of dams treated with Antaeeum didn't induced external malformations. Vertebral and sternal variations were observed in Antaeeum group, but compared with the control, those variations were not significant. The ossification numbers of rib, cervical, thoracic, and lumber were normal. Fetuses treated with Antaeeum to the dams showed no significant difference in the number of caudal vertebra (P>0.01). From these results, it can be concluded that Antaeeum showed no toxicity effects on maternal side especially on body weight, early resorption rate, and number of live fetuses. Also there were no significant changes on maternal organ weights except spleen, hematological data, reproductive organs. Although skeletal variations were examined at vertebra and sternum, this Antaeeum could not induced significant choses in bone malformation.

• Clinical and Experimental Reproductive Medicine
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• 제35권2호
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• pp.99-110
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• 2008

목 적: 착상전 유전진단은 환아를 출산할 가능성이 높은 부부들에게 정상아의 출산 기회를 제공해주는 보조생식술의 하나로서 널리 시행되고 있다. 골형성부전증은 상염색체 우성 유전질환으로서 뼈와 피부를 구성하는 결합조직의 이상으로 뼈가 잘 부러지는 특징을 가지고 있으며, 질환의 95% 이상은 COL1A1 또는 COL1A2 유전자의 이상으로 발병한다고 알려져 있다. 본 논문에서는 골형성부전증 환자 2 가계를 대상으로 5 주기의 착상전 유전진단을 시행하여 임신에 성공한 사례에 대해 보고하고자 한다. 연구방법: 착상전 유전진단 수행 전에 환자의 단일 림프구에서 증폭성공률과 allele drop-out (ADO) rate을 확인하기 위하여 임상전 검사를 수행하였다. 각각 원인 돌연변이로 확인된 COL1A1 유전자 c.2452G>A와 c.3226G>A를 가지고 있는 골형성부전증 2 가계를 대상으로 5 주기의 착상전 유전진단을 시행하였다. 생검한 할구로부터 원인 돌연변이를 진단하기 위하여 nested PCR 후 HaeIII 제한효소를 이용한 restriction fragment length polymorphism (RFLP) 분석방법과 direct sequencing 방법을 이용하여 진단을 실시하였다. 결 과: Genomic DNA를 이용하여 COL1A1 유전자 검사를 해 본 결과 각 가계의 원인 돌연변이는 c.2452G>A와 c.3226G>A임을 재확인하였으며, 단일세포를 이용한 임상전 검사 결과 첫 번째 사례의 경우 94.2%의 증폭성공률과 22.5%의 ADO rate을 나타내었고, 두 번째 사례의 경우 98.1%의 증폭성공률과 1.9%의 ADO rate를 나타내었다. 착상전 유전진단 결과 첫 번째 사례의 경우, 3 주기의 착상전 유전진단에서 총 34개의 배아 중 31개의 배아에서 진단에 성공하여 91.2% (31/34)의 진단성공률을 나타내었고, 총 19개의 정상 배아 중 8개의 배아 (2.7개/배아이식)를 이식하였다. 세 번째 주기에서 임신에 성공하였고 제왕절개로 건강한 아이를 분만하였다. 두 번째 사례의 경우 2 주기의 착상전 유전진단을 시행하였으며, 총 19개의 배아 모두 진단에 성공하여 100.0% (19/19)의 진단성공률을 나타내었고, 총 11개의 정상 배아 중 4개 (2개/배아이식)의 배아를 이식하였다. 두 번째 착상전 유전진단에서 임신에 성공하였고, 건강한 아이를 분만하였다. 결 론: 착상전 유전진단을 통한 골형성부전증 환자 2 가계에서의 성공적인 임신과 출산은 국내에서 처음으로 보고되는 임상 결과로, 본원의 착상전 유전진단방법은 효과적이고 확실한 방법으로써 앞으로도 더 많은 단일 유전자 이상의 유전질환을 가진 환자들에게 적용하게 될 것이며, 이는 이와 유사한 유전질환을 가지고 있거나 유전질환의 이환 가능성이 있는 부부들에게 정상아의 임신과 출산의 기회를 더욱 많이 제공할 수 있을 것이다.