근이영양증에 대한 착상전 유전진단에서 Duplex-nested PCR과 Fluorescent PCR 방법의 효용성

이형송,최혜원,임천규,박소연,김진영,궁미경,전진현,강인수,Lee, Hyoung-Song,Choi, Hye Won,Lim, Chun Kyu,Park, So Yeon,Kim, Jin Young,Koong, Mi Kyoung,Jun, Jin Hyun,Kang, Inn Soo 대한생식의학회 2005 Clinical and Experimental Reproductive Medicine Vol.32 No.1

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.

한국 남성 불임환자에서 Protamine 1과 2 유전자의 Single Nucleotide Polymorphism에 관한 연구

이형송,최혜원,박용석,서주태,궁미경,전진현,Lee, Hyoung-Song,Choi, Hye Won,Park, Yong-Seog,Seo, Ju Tae,Koong, Mi Kyoung,Jun, Jin Hyun 대한생식의학회 2005 Clinical and Experimental Reproductive Medicine Vol.32 No.3

Objective: Although several genetic factors have been associated with defects in human spermatogenesis, the unambiguous causative genes have not been elucidated. The male infertility by haploinsufficiency of PRM1 or PRM2 has been reported in mouse model. The aim of this study was to identify the single nucleotide polymorphisms (SNPs) of PRM1 and PRM2, related to the genotype of Korean infertile men. Methods: Genomic DNAs were extracted from peripheral bloods of infertile men with oligozoospermia or azoospermia, and analyzed using polymerase chain reaction (PCR) and direct sequencing. We carried out the direct sequencing analysis of amplified fragments in PRM1 (557 nucleotides from -42 to 515) and PRM2 (599 nucleotides from 49 to 648) genes, respectively. Results: Three SNPs of coding region in the PRM1 gene was found in the analysis of 130 infertile men. However, the SNPs at a133g (aa 96.9%, ag 3.1% and gg 0.0%), c160a (cc 99.2%, ca 0.8% and aa 0.0%) and c321a (cc 56.9%, ca 35.4% and aa 7.7%) coded the same amino acids, in terms of silence phenotypes. On the other hand, as results of the PRM2 gene sequencing in 164 infertile men, only two SNPs, g398c (gg 62.2%, gc 31.1% and ga 6.7%) and a473c (aa 63.4%, ac 29.9% and cc 6.7%), were identified in the intron of the PRM2 gene. Conclusions: There was no mutation and significant SNPs on PRM1 and PRM2 gene in Korean infertile men. These results suggest that the PRM1 and PRM2 genes are highly conserved and essential for normal fertility of men.

Y 염색체 미세결실과 정자형성장애의 연관성에 대한 연구

이형송,최혜원,박용석,궁미경,강인수,윤종민,이유식,서주태,전진현,Lee, Hyoung-Song,Choi, Hye-Won,Park, Yong-Seog,Koong, Mi-Kyoung,Kang, Inn-Soo,Yun, Jong-Min,Lee, You-Sik,Seo, Ju-Tae,Jun, Jin-Hyun 대한생식의학회 2002 Clinical and Experimental Reproductive Medicine Vol.29 No.4

Objective s: To estimate the frequency of Y chromosome microdeletions in the Korean population of infertile men and to evaluate the relationship between microdeletion on the Y chromosome and clinical phenotypes of infertile men with idiopathic azoospermia and oligozoospermia. Materials and Methods: Genomic DNA was extracted from blood samples collected from 330 infertile men attending the Infertility Clinic at Samsung Cheil Hospital, Korea. Six sequence tagged sites (STSs) spanning the azoospermia factor (AZF) regions of the Y chromosome were amplified by polymerase chain reactions (PCRs). Results: Microdeletions on Y chromosome were detected in 35 (10.6%) of the 330 infertile men. Most of the microdeletions (91.4%) involved AZFb or AZFc. The high incidence of microdeletions were found in AZFc region (57.1%), but the low in AZFa (8.6%) and AZFb (5.7%). Larger microdeletions involving two or three AZF regions were detected in 28.6% of cases. All patients (6 patients) with deletion of AZFa region showed no germ cell phenotypes, Sertoli cell only syndrome or Leydig cell hyperplasia in histopathologic examinations. Conclusion: Microdeletions on the Y chromosome, especially, at AZFc/DAZ regions may be the major cause of azoospermia and severe oligozoospermia. We suggest that idiopathic infertile men have genetic counselling and microdeletion analysis on the Y chromosome before IVF-ET and ART program.

Congenital Adrenal Hyperplasia 환자에서 21 - hydroxylase ( CYP21 ) 유전자 돌연변이의 분자 진단에 관한 연구

이형송(Hyoung Song Lee),천강우(Kang Woo Cheon),박용석(Yong Seok Park),한인권(In Kwon Han),강인수(Inn Soo Kang) 대한산부인과학회 2001 Obstetrics & Gynecology Science Vol.44 No.6

N/A Objective: Congenital adrenal hyperplasia (CAH) is an autosomal recessive diseav which is most often caused by a deficiency in steroid 21-hydroxylase (21-0H), a microsomal enzyme encoded by the CYP21 gene. Although several CAH causing mutations have been identified in the CYP21 gene of patients with 21-OH deficiency, genotyping of the 21-OH locus is quite complex because of the high frequency of gene conversion and the presence of multiple mutations on single CAH alleles. This study was aimed to analyze the complete characterization of the CYP21 gene coding region in a Korean CAH patient and to conform the PCR-based single strand conformation polymorphism (SSCP) and heteroduplex analysis as a diagnostic tool. Methods: We used a highly sensitive, non-radioactive method allowing PCR-based single strand c<formation polymorphism (SSCP) analysis. This method was applied to the characterization of all the exons and intron-exon junctions of the CYP21 gene in one patients affected by the salt wasting form and 4 normal controls. Results: In all samples showing SSCP abnormal band pattems, sequence analysis showed the presence of sequence variants. In particular, one mutation (I172N) which is already known to cause the disease and 3 silent mutations were detected. Conclusion: PCR-based single strand conformation polymorphism (SSCP) and heteroduplex analysis should be useful for the clinical application as a diagnostic tool for the detection of 21-hydroxylase gene mutations.

단일 유전자 질환에 대한 착상전 유전진단

이형송(Hyoung-Song Lee),김민지(Min Jee Kim),강인수(Inn Soo Kang) 대한의학유전학회 2009 대한의학유전학회지 Vol.6 No.2

착상전 유전진단은 유전질환이 이환될 가능성이 있는 부부들을 대상으로 산전진단을 통한 임신중절의 위험성 없이 정상적인 아이를 가질 수 있게 도와주는 보조생식술의 한 방법으로 확립되었다 . 단일 할구를 대상으로 하는 분자생물학 및 분자생물학적 기술의 발전은 착상전 유전진단의 정확성을 높은 수준에 이르게 하였고 whole genome amplification 방법을 이용함으로써 단일세포로부터 여러 가지 다양한 진단을 동시에 수행 가능케 하였으며 단일 유전자 질환에 대한 착상전 유전진단에서의 오진을 감소시킬 수 있었다. 따라서 PCR을 이용한 단일 유전자 질환에 대한 착상전 유전진단의 적용가능 유전질환은 더욱 확대될 것이며 건강한 아이의 출산을 원하는 더 많은 부부들에게 기회를 제공해 줄 것이다. 본 종설에서는 현재 단일유전자 질환에 대한 착상전 유전진단을 시행하는 대부분의 센터에서 시행하고 있는 생검 방법과 multiplex PCR, PCR 후 진단 방법, 그리고 multiple displacement amplification 등의 분자생물학적 방법과 단일 세포 분석에서의 문제점 등을 포함한 단일 유전자 질환에 대한 착상전 유전진단 전반에 관하여 논의할 것이다. Preimplantation genetic diagnosis (PGD) has become an assisted reproductive technique for couples who are at risk that enables them to have unaffected baby without facing the risk of pregnancy termination after invasive prenatal diagnosis. The molecular biology and technology for single-cell genetics has reached an extremely high level of accuracy, and has enabled the possibility of performing multiple diagnoses on one cell using whole genome amplification. These technological advances have contributed to the avoidance of misdiagnosis in PGD for single gene disorders. Polymerase chain reaction (PCR)-based PGD will lead to a significant increase in the number of disorders diagnosed and will find more widespread use, benefiting many more couples who are at risk of transmitting an inherited disease to their baby. In this review, we will focus on the molecular biological techniques that are currently in use in the most advanced centers for PGD for single gene disorders, including biopsy procedure, multiplex PCR and post-PCR diagnostic methods, and multiple displacement amplification (MDA) and the problems in the single cell genetic analysis.

삼핵산 반복서열 질환인 헌팅톤병, 척수소뇌성 운동실조증, X-염색체 취약 증후군의 착상전 유전진단 방법에 대한 연구

김민지,이형송,임천규,조재원,김진영,궁미경,송인옥,강인수,전진현,Kim, Min-Jee,Lee, Hyoung-Song,Lim, Chun-Kyu,Cho, Jae-Won,Kim, Jin-Young,Koong, Mi-Kyoung,Son, In-Ok,Kang, Inn-Soo,Jun, Jin-Hyon 대한생식의학회 2007 Clinical and Experimental Reproductive Medicine Vol.34 No.3

목 적: 본 연구에서는 삼핵산 반복서열 확장에 의해 발병하는 헌팅톤병, 척수소뇌성 운동실조증과 X-염색체 취약 증후군 등에 대한 착상전 유전진단을 시행하기 위한 전임상 검사에서 진단 방법을 최적화하는 과정을 통해 얻은 결과들에 대해 기술하고자 한다. 연구방법: 단일 림프구를 이용한 임상전 검사에서는 서로 다른 allele를 갖고 있는 환자의 단일 세포를 사용하였으며, 헌팅톤병과 척수소뇌성 운동실조증에서는 fluorescent semi-nested PCR 시행 후 fragment analysis를 수행하였다. X-염색체 취약 증후군의 경우 multiple displacement amplification (MDA) 방법을 이용한 whole genome amplification에서 얻어진 MDA 산물로 fluorescent PCR을 시도하였다. 결 과: 헌팅톤병의 경우 단일 림프구 시료 모두에서 CAG repeats 증폭에 성공하여 100.0%의 증폭성공률과 14.0% allele drop-out (ADO) rate를, 척수소뇌성 운동실조증의 경우 94.7%의 증폭성공률과 5.6%의 ADO rate을 나타내었다. X-염색체 취약 증후군의 경우 fluorescent semi-nested PCR 방법만으로는 단일 림프구 시료에서 CGG repeats이 증폭되지 않았지만, MDA 산물을 이용한 fluorescent PCR 결과 84.2%의 증폭성공률과 31.3%의 ADO rate을 얻을 수 있었다. 결 론: 본 연구를 통해 헌팅톤병과 척수소뇌성 운동실조증의 착상전 유전진단에는 fluorescent semi-nested PCR 방법의 적용이 가능함을 확인하였으며, X-염색체 취약 증후군의 경우에는 MDA를 이용한 fluorescent PCR 방법을 사용해야 함을 알 수 있었다. 유전자의 변이에 대한 분석이 쉽지 않은 단일 유전자 이상에 대한 착상전 유전진단의 경우 다양한 유전자 분석 방법을 이용한 단일 세포에서의 진단 방법의 최적화 연구가 필수적으로 선행되어야 할 것으로 사료된다. Objectives: Many neurological diseases are known to be caused by expansion of trinucleotide repeats (TNRs). It is hard to diagnose the alteration of TNRs with single cell level for preimplantation genetic diagnosis (PGD). In this study, we describe methods optimized for PGD of TNRs related diseases such as Huntington's disease (HD), spinocerebellar ataxia 3 (SCA3) and fragile X syndrome (FXS). Methods: We performed the preclinical assays with heterozygous patient's lymphocytes by single cell PCR strategy. Fluorescent semi-nested PCR and fragment analysis using automatic genetic analyzer were applied for HD and SCA 3. Whole genome amplification with multiple displacement amplification (MDA) method and fluorescent PCR were carried out for FXS. Amplification and allele drop-out (ADO) rate were evaluated in each case. Results: The fluorescent semi-nested PCR of single lymphocyte showed 100.0% of amplification and 14.0% of ADO rate in HD, and 94.7% of amplification and 5.6% of ADO rate in SCA3, respectively. We could not detect the PCR product of CGG repeats in FXS using the fluorescent semi-nested PCR alone. After applying the MDA method in FXS, 84.2% of amplification and 31.3% of ADO rate were achieved. Conclusions: Fluorescent semi-nested PCR is a reliable method for PGD of HD and SCA3. The advanced MDA method overcomes the problem of amplification failure in CGG repeats of FXS case. Optimization of methods for single cell analysis could improve the sensitivity and reliability of PGD for complicated single gene disorders of TNRs.

골형성부전증의 착상전 유전진단을 위한 분자유전학적 방법의 조건 확립과 적용

김민지,이형송,최혜원,임천규,조재원,김진영,송인옥,강인수,Kim, Min-Jee,Lee, Hyoung-Song,Choi, Hye-Won,Lim, Chun-Kyu,Cho, Jae-Won,Kim, Jin-Young,Song, In-Ok,Kang, Inn-Soo 대한생식의학회 2008 Clinical and Experimental Reproductive Medicine Vol.35 No.2

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. Objectives: Preimplantation genetic diagnosis (PGD) has become an assisted reproductive technique for couples carrying genetic conditions that may affect their offspring. Osteogenesis imperfecta (OI) is an autosomal dominant disorder of connective tissue characterized by bone fragility and low bone mass. At least 95% of cases are caused by dominant mutations in the COL1A1 or COL1A2. In this study, we report on our experience clinical outcomes with 5 PGD cycles for OI in two couples. Methods: Before clinical PGD, we assessed the amplification rate and allele drop-out (ADO) rate of alkaline lysis and nested PCR protocol using heterozygous patient's single lymphocytes in the pre-clinical diagnostic tests for OI. We performed 5 cycles of PGD for OI by nested PCR for the causative mutation loci, COL1A1 c.2452G>A and c.3226G>A, in case 1 and case 2, respectively. The PCR products were analyzed by agarose gel electrophoresis, restriction fragment length polymorphism (RFLP) analysis with HaeIII restriction enzyme in the case 1 and direct DNA sequencing. Results: We confirmed the causative mutation loci, COL1A1 c.2452G>A in case 1 and c.3226G>A in case 2. In the pre-clinical tests, the amplification rate was 94.2% and ADO rate was 22.5% in case 1, while 98.1% and 1.9% in case 2, respectively. In case 1, a total of 34 embryos were analyzed and 31 embryos (91.2%) were successfully diagnosed in 3 PGD cycles. Eight out of 19 embryos diagnosed as unaffected embryos were transferred in all 3 cycles, and in the third cycle, pregnancy was achieved and a healthy baby was delivered without any complications in July, 2005. In case 2, all 19 embryos (100.0%) were successfully diagnosed and 4 out of 11 unaffected embryos were transferred in 2 cycles. Pregnancy was achieved in the second cycle and the healthy baby was delivered in March, 2008. The causative locus was confirmed as a normal by amniocentesis and postnatal diagnosis. Conclusions: To our knowledge, these two cases are the first successful PGD for OI in Korea. Our experience provides a further demonstration that PGD is a reliable and effective clinical techniques and a useful option for many couples with a high risk of transmitting a genetic disease.

한국 남성 불임환자에서 Y 염색체상의 AZF Gene에 대한 분석 및 DAZ Gene의 발현 양상

이호준,이형송,송견지,변혜경,서주태,김종현,이유식,Lee, Ho-Joon,Lee, Hyoung-Song,Song, Gyun-Jee,Byun, Hye-Kyung,Seo, Ju-Tae,Kim, Jong-Hyun,Lee, You-Sik 대한생식의학회 1997 Clinical and Experimental Reproductive Medicine Vol.24 No.1

Cytogenetic observations of loss of the distal portion of the Y chromosome long arm were found to be associated with disrupted spermatogenesis. The existence of a gene involved in the regulation of spermatogenesis, the azoospermia factor (AZF), was postulated. In this study, we screened the AZF region including DAZ and DAZH genes and observed the expression pattern of DAZ and DAZH transcript in infertile men with azoospermia and oligospermia by using a sequence-tagged site (STS)-based PCR method. PCR primers were synthesized for 11 STSs that span Yq interval 6, SRY, DAZ, and DAZH, functional DAZ homologue on chromosome 3. Microdeletions were detected in 4/32 (12.5%) azoospermic men and 1/11 (9%) severe oligospermic men. Only 2 of 5 patients had microdeletions of Yq that contained the DAZ gene, whereas the other 3 patients had deletions extending from intervals 5L-6F proximal to the DAZ gene on Yq. Testis biopsies of the azoospermic patients revealed a variety from Sertoli cell-only syndrome to testicular maturation arrest. Of 4 men with clinical data available, average testis size was R: 13.8 cc, L: 13.8 cc, serum T was $4.0{\pm}1.25$ ng/ml, LH was $3.63{\pm}1.90$ mIU/ml, and FSH was $8.85{\pm}5.13$ mIU/ml. These values did not differ significantly from the remainder of the patients tested. We could not observed the DAZ transcript in 2 patients, who have no mature spermatozoa. In 11.6% of patients microdeletions of the AZF could be detected. These deletions in the AZF region seem to be involved causing spermatogenic failure. But the frequency of microdeletions proximal to DAZ suggests that DAZ is not the only gene associated with spermatogenic failure.

고환조직 동결-융해 후 회수된 고환 정자에 대한 Hypo-osmotic Swelling (HOS) Test의 효과

박용석,이형송,송상진,김정욱,강인수,서주태,Park, Yong-Seog,Lee, Hyoung-Song,Song, Sang-Jin,Kim, Jeong-Wook,Kang, Inn-Soo,Seo, Ju-Tae 대한생식의학회 2000 Clinical and Experimental Reproductive Medicine Vol.27 No.3

Objectives: We have previous reported that thawed testicular sperm and sperm extracted from seminiferous tubule could achieved optimal fertilization and pregnancy in azoospermic patients. However, thawed testicular sperm did not show motility in many cases. Therefore we studied viability of immotile sperm extracted from frozen-thawed seminiferous tubule using hypo-osmotic swelling (HOS) test and eosin-Y test. Materials and Methods: After sperm extraction using for ICSI, the remained sections of seminiferous tubules were frozen with a computerized freezer. For thawing and preparation of testicular sperm, the seminiferous tubules were thawed by removing from $LN_2$ and letting them at room temperature for 10 min followed by %37^{\circ}C$ water bath for 10 min. The prepared samples were washed for free of preservation medium and sperm preparation method described previous. Sperm was suspended in 0.1 ml hypoosmotic solution. After 30 minutes, the type of distally coiled sperm were assessed. Results: In 44 cases of cryopreservation of seminiferous tubules in obstructive azoospennic patients, the fertilization rates with 2PN were 71.4% and pregnancy rates were 34.1%. The presence of motile spermatozoa on subsequent post-thaw testicular sperm remarked 15.1% and were increased to 77.3% just before ICSI. After sperm extracted from frozen-thawed seminiferous tubule, 3 hrs later in in vitro culture, the cases of presence of motile sperm, reaction of hypo-osmotic swelling test and viable sperm were 63.6% (28/44), 93.2% (41/44), and 77.3% (34/44), respectively. Conclusions: Just after post-thawed testicular sperm did not showed motility. Although motility was gained after in vitro culture, many cases showed non-motile sperm until optimal insemination time. However, HOS test showed positive reaction in non-motile sperm. Therefore, HOS test is an alternative method for the selection of viable sperm for ICSI.

폐쇄성 무정자증 환자의 신선고환조직 정자와 동결고환조직 정자의 운동성이 임신율에 미치는 영향

박용석,이형송,변혜경,염혜원,송상진,임천규,이유식,윤종민,서주태,송지홍,강인수,궁미경,Park, Yong-Seog,Lee, Hyoung-Song,Byun, Hye-Kyung,Youm, Hye-Won,Song, Sang-Jin,Lim, Chun-Kyu,Lee, You-Sik,Yun, Jong-Min,Seo, Ju-Tae,Song, Ji-Hong,Kang, I 대한생식의학회 2001 Clinical and Experimental Reproductive Medicine Vol.28 No.2

Objective: ICSI with testicular sperm could achieve optimal fertilization and pregnancy. This study was performed to observe the influence on fertilization and pregnancy of motility of fresh testicular sperm and sperm extracted from frozen-thawed seminiferous tubules in obstructive azoospermia. Materials and Methods: We analysed clinical outcome of ICSI using fresh testicular sperm and sperm extracted from thawed seminiferous tubules. The presence of motility were compared to determine the factor for optimal fertilization and pregnancy rates. Results: In 316 cases of TESE-ICSI in obstructive azoospermia, ICSI with fresh testicular sperm (fresh sperm group) were 163 cases and ICSI with sperm testicular sperm extracted from frozen-thawed seminiferous tubule (thawed sperm group) were 153 cases. The fertilization rates were 71.3% and pregnancy rates were 32.5% in fresh sperm group, in thawed sperm group, 65.1% and 33.3% respectively. The fertilization and pregnancy rates of motile and non-motile testicular sperm were 72.9% and 33.6%, 50.0% and 18.2%, respectively (p<0.05). The fertilization and pregnancy rates of motile and non-motile sperm extracted from the thawed seminiferous tubule were 67.8% and 34.7%, 55.1% and 28.1%, respectively (p<0.05). The comparative of the results of ICSI using motile fresh testicular sperm and motile sperm extracted from thawed seminiferous tubule, fertilization and pregnancy rates were not significantly different (72.9% and 33.6%, 67.8% and 34.7%, respectively). Conclusion: These results suggest that successful pregnancy in TESE-ICSI treatment is influenced by the motility of fresh testicular sperm and sperm extracted from thawed seminiferous tubule in obstructive azoospennic patients.