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Objective: To explore the significance of karyotype analysis in screening sperm donors., Methods: From January 1, 2004 to December 31, 2008, a total of 2537 potential sperm donors passed our preliminary screening, and all were routinely karyo-typed via peripheral blood. Follow-ups were conducted on the pregnancy outcome and congenital malformation after artificial insemination with the sperm from the qualified donors., Results: Among the 2537 qualified sperm donors, 2362 were of the normal karyotype 46, XY and 135 showed polymorphism. Abnormal karyotype was found in 6 cases, and controversial abnormal karyotype in 34., Conclusion: Karyotype analysis can reduce the risk of chromosomal disease in neonates from artificial insemination, and genetic counseling for abnormal karyotype sperm donors may help them solve their future reproductive problems.

Objective: To determine the karyotype of a patient with Prader-Willi-like syndrome features., Methods: Chromosomal high resolution banding was carried out to analyze the karyotype of the patient, and methylation-specific PCR was used to analyze the imprinting region of chromosome 15. Subtelomeric region was screened by multiplex ligation-dependent probe amplification (MLPA), and fluorescent in situ hybridization (FISH) and real-time quantitative PCR were further performed to identify the deleted region., Results: No abnormality was discovered by high resolution karyotype analysis and methylation-specific PCR studies. MLPA analysis showed that the patient had a deletion of 1p subtelomeric area, which was confirmed by FISH analysis. The deleted region was shown within a 4.2 Mb in the distal 1p by 3 BAC FISH probes of 1p36 combined with real-time PCR technique. Family pedigree investigation showed the chromosome abnormality was de novo. Therefore, partial monosomy 1p36 was likely responsible for the mental retardation of the patient., Conclusion: Molecular cytogenetic techniques should be performed to those patients with Prader-Willi-like syndrome features, to determine their karyotypes.

Objective: To establish a single-cell whole genome amplification (WGA) technique, in combination with comparative genomic hybridization (CGH), for analyzing chromosomal copy number changes, and to explore its clinical application in preimplantation genetic diagnosis (PGD)., Methods: Twelve single-cell samples with known karyotypes, including 5 chorionic villus samples, 4 human embryonic stem cell (hESC) samples and 3 peripheral lymphocyte samples, and 4 single blastomere samples carrying chromosomal abnormalities detected by PGD, were collected for whole genome amplification by combining primer extension preamplification (PEP) with degenerate oligonucleotide primed-PCR (DOP-PCR) amplification. The amplified products labeled by red fluorescence were mixed with control DNA labeled by green fluorescence, and then the mixture was analyzed by CGH. As a comparison, 10 single cell samples were amplified by DOP-PCR only and then CGH analysis was performed., Results: The amplification using PEP-DOP-PCR was more stable than traditional DOP-PCR. The products of PEP-DOP-PCR range from 100 bp to 1000 bp, with the mean size being about 400 bp. The CGH results were consistent with analyses by other methods. However, only 6 out of 10 single cell samples were successfully amplified by DOP-PCR, and CGH analysis showed a high background and 2 samples showed inconsistent results from other methods., Conclusion: PEP-DOP-PCR can effectively amplify the whole genome DNA of single cell. Combined with CGH, this WGA method can successfully detect single-cell chromosomal copy number changes, while DOP-PCR was easy to fail to amplify and amplify inhomogeneously, and CGH analysis using this PCR product usually showed high background. These results suggest that PEP-DOP-CGH is a promising method for preimplantation genetic diagnosis.

Objective: To analyze the karyotypes of 11 cases of Turner syndrome with marker chromosome, and study the phenotypic effects resulting from the abnormal karyotype., Methods: Eleven Turner syndrome patients had a mosaic karyotype and carried a marker chromosome, and 6 marker chromosomes were ring chromosomes. Their karyotypes were showed as mos. 45, X/46, X, + mar or mos. 45, X/46, X, + r. Fluorescence in situ hybridization (FISH) technique with X/Y centromere probes was performed to determine the origin of the marker chromosome. Reverse chromosome painting technique was used to identify the breakpoints of two largest markers. Phenotype effects with different chromosome breakpoints were compared., Results: All the 11 marker chromosomes were ring X chromosomes. The breakpoints of the r(X) were involved in Xp22, Xq22, Xq24 and Xq26, etc., Conclusions: The marker chromosomes in Turner syndrome mainly originate from X chromosome and form ring chromosome X. Each r(X) in our patients was mosaic, indicating it was originated from mitosis error during early embryo development. To analyze the origin of the marker chromosome and the breakpoint of r(X) will provide guidance for the therapy and prognosis of the Turner syndrome patient.

Objective: To characterize a supernumerary marker chromosome (SMC) by comparative genomic hybridization (CGH), fluorescence in situ hybridization (FISH) and traditional cytogenetic techniques, and to explore the clinical application of these techniques in delineating de novo marker chromosomes., Methods: A mental retardation patient received chromosome test by ordinary G banding. CGH and FISH techniques were used to analyze the origin of the de novo SMC, and N banding technique and C banding techniques were used to analyze the SMC structure. The phenotypic effects of the SMC were analyzed after the karyotype was determined., Results: By G banding technique, the patient was showed to have a mosaic karyotype with SMC: mos.47, XX, +mar [31]/48, XX, +2mar[29]. CGH analysis showed a gain of 15q11 --> q14, and the result was confirmed by FISH with chromosome 15 painting probe. The further FISH analysis showed the SMC had two signals with UBE3A probe for detecting Prader-willi syndrome/Angelman syndrome (PWS/AS). N banding and C banding analysis showed the SMC had a double satellite and double centromere, respectively. Combined with the above results, the karyotype of the patient was: mos.47, XX, +der (15) (pter --> q14::q14 --> pter) [31]/48, XX, +2der (15) (pter --> q14::q14 --> pter) [29]. ish der(15)(WCP15+, UBE3A++, PML-)., Conclusion: CGH is a valuable method to detect imbalanced chromosomal rearrangement. Combined with FISH and the traditional cytogenetic technique, it provides a valuable technique platform for characterizing the structure of the de novo SMC, and a basis for exploring the relation between karyotype and phenotype, prognosis and recurrent risk.

Premature ovarian failure (POF) is a complicated and heterogeneous disease. In majority of cases the underlying cause is not identified. Among the known causes, genetic aberration plays very important role. POF not only causes infertility, also adds the risk of osteoporosis and coronary heart disease because of the low level estrogen. The major therapy measures in present include hormone replacement therapy and infertility treatment with donated oocytes, but the effect is not ideal. To identify the genes of POF is the basis for treating and preventing this disease. A large number of candidate genes of POF are found on X chromosome and autosomes. The present paper reviewed the advance in mutation analysis on the candidate genes of POF, which is aimed to provide a basis to explore its molecular mechanism.

In order to assess the value of applying human chromosomal telomeric band painting probes (TBPs) in genetic diagnosis, we used three human telomeric band painting probes in fluorescence in situ hybridization (FISH) to analyze two cases who had a history of habitual abortion and were suspected to carrying a minute translocation. The probes were prepared by microdissection, and the covered regions were 11q23.3-->qter12q24.1-->qter and 22q13.1-->qter, respectively. According to the FISH results, both the cases were cryptic minute translocation carriers. The involved chromosomes were chromosome 11 and 12 in case 1, and chromosome 11 and 22 in case 2. Combined with chromosome G banding, their breakpoints were 11q23.3, 12q24.1, and 22q13.1, respectively. The results showed that the telomeric band painting probes can be used as a tool to determine minute chromosomal abnormality and the precise breakpoints.

Objective: To search the forming cause and the correlation between the clinical phenotype and chromosome band by the cytogenetic analysis on a case of ring chromosome 21 syndrome., Methods: Identification and location of 21 ring chromosome were performed with the G-banding, C-banding, N-banding, high-resolution banding and fluorescence in situ hybridization (FISH) techniques., Results: It was found that the karyotypes of the patient's parents are normal. The patient's karyotype is 46,XY, r(21)[91]/46,XY,r(21;21)(p11q22.3;p11q22.3) [5]/45,XY,-21[4]., Conclusion: The clinical phenotype of ring chromosome 21 syndrome is related to the deletion of distal segment of 21q, and the abnormal sexual development of male is related with the deletion of 21q22.3.

Aim: To examine the sensitivity and accuracy of real-time polymerase chain reaction (PCR) for the quantification of hepatitis B virus (HBV) DNA in semen., Methods: Hepatitis B viral DNA was isolated from HBV carriers' semen and sera using phenol extraction method and QIAamp DNA blood mini kit (Qiagen, Germany). HBV DNA was detected by conventional PCR and quantified by TaqMan technology-based real-time PCR (quantitative polymerase chain reaction (qPCR)). The detection threshold was 200 copies of HBV DNA for conventional PCR and 10 copies of HBV DNA for real time PCR per reaction., Results: Both methods of phenol extraction and QIAamp DNA blood mini kit were suitable for isolating HBV DNA from semen. The value of the detection thresholds was 500 copies of HBV DNA per mL in the semen. The viral loads were 7.5X10(7) and 1.67X10(7) copies of HBV DNA per mL in two HBV infected patients' sera, while 2.14X10(5) and 3.02X10(5) copies of HBV DNA per mL in the semen., Conclusion: Real-time PCR is a more sensitive and accurate method to detect and quantify HBV DNA in the semen.

Objective: To explore the relationship between chromosome anomaly and spontaneous abortion, and to provide useful information for genetic counseling and prenatal diagnosis in reproductive clinic., Methods: A total of 1 780 patients who had a history of spontaneous abortion before 24 weeks of gestation were enrolled. The lymphocyte culture and harvest were performed according to standard methods. Karyotypes were analyzed by G-banding in all cases and C- banding in some cases in addition., Results: Altogether 57 abnormal karyotypes were found and the overall incidence of chromosomal abnormalities was 3.20% (women 3.32%; men 2.12%). Among them 23 cases were the balanced translocation; 14 cases were the Robertsonian translocation, 3 cases were the complex chromosomal rearrangement, and the other 17 cases were the other abnormalities. In women with 1, 2, 3 or more spontaneous abortion, the incidence of chromosomal abnormalities was 1.7%, 2.3%, and 5.8%, respectively., Conclusion: Translocations are the major abnormal karyotpes associated with spontaneous abortions. The chance of finding chromosomal aberration increases with the number of abortions. Chromosomal abnormalities are more common in women with 3 or more spontaneous abortions.

Objective: To determine a complex chromosomal rearrangement by advanced molecular cytogenetic techniques and analyze its clinical effect., Methods: A complex chromosomal rearrangement (CCR) involved in chromosomes 5, 16 and 20 in a 29-year-old male carrier was determined by chromosomal microdissection and multicolor fluorescence in situ hybridization (M-FISH), and family degree investigation was further performed., Results: The karyotype of the case was a complex chromosomal translocation among chromosomes 5, 20 and 16, and accompanied with a band of chromosome 20 inserted into chromosome 5. His mother and sister both had the same abnormal karyotype by familial investigation., Conclusion: The combined use of M-FISH and chromosome microdissection is a powerful tool to determine CCR. The complex chromosomal rearrangement could be transmitted stably in the family, but still the carriers could give birth to a healthy baby by chance.

Objective: To evaluate the feasibility of using fluorescence in situ hybridization(FISH) for the detection of a few common chromosome aneuploidies on interphase nuclei of uncultured amniotic fluid cells., Methods: Amniotic fluid samples were taken from 55 women at 16-32 weeks of pregnancy; interphase FISH was performed for diagnosing Down syndrome and aneuploidies of other four chromosomes 13, 18, X and Y. Then the karyotypes from standard cytogenetic analysis after percutaneous umbilical blood sampling(PUBS) were compared to the FISH results., Results: Each of the 55 uncultured amniotic fluid samples tested with FISH was enumerated 200 nuclei. Fifty-three samples were normal. Two samples were found to have trisomy 21(one is a case of standard trisomy 21 with three signals in all 200 nuclei, the other is a mosaic trisomy 21)., Conclusion: Interphase FISH analysis of uncultured amniotic fluid cells is a rapid, accurate and very sensitive method. It could be used in the prenatal cytogenetic laboratory.

Objective: To search for the possible relation between tortilcollis and partial chromosome 13q trisomy., Methods: Fluorescence in situ hybridization (FISH) technique combined with chromosome banding was performed to determine the karyotype of two patients with typical clinical features of partial 13q trisomy syndrome, then their manifestations were compared with those of the literatures published previously., Results: The two cases were partial trisomy of 13q14--> ter with a different second derivative chromosome, in spite of this difference, both of them had tortilcollis., Conclusion: It is suggested that a potential site for tortilcollis may locate on the long arm of chromosome 13. With reference to a report previously published, the more precise candidate related region may be 13q32--> qter.

In order to identify those easily overlooked minute chromosomal structural abnormality on the chromosomal regions, and to provide a valuable guidance for pregnancy, fluorescence in situ hybridization (FISH) technique by whole chromosome 7 painting probe, Xq probe and subterminal probe of 7q36-->qter was performed to analyze two cases. Case 1 had a history of recurrence spontaneous abotion and with an uncertain minute translocation on the chromosomal terminal regions. Case 2 was a premature ovarian failure patient with a balanced translocation between chromosome X and chromosome 7 by G banding. The results showed that case 1 was a cryptic minute translocation on the chromosomal terminal regions, and the breakpoint of case 2 was accurately determined, that is, the breakpoint was not on 7q36 but on 7qter. Therefore FISH technique with whole chromosome painting probe and subterminal probe could be used to diagnose the minute chromosomal structural abnormality on the chromosomal regions. It could be used widely in the clinical genetics and was an effective tool for genetic counseling and reproductive guidance.