Clinical analysis of preimplantation genetic diagnosis with HLA matching for beta-thalassemia / 中华妇产科杂志

Objective To investigate the efficacy and feasibility of preimplantation genetic diagnosis (PGD) with human leukocyte antigen (HLA) matching for beta-thalassemia. Methods A total of 43 referred beta-thalassemia couples, with at least on child in need of hematopoietic stem cell transplantation (HSCT), underwent PGD for HLA matching at the First Affiliated Hospital of Sun Yat-sen University from 2010 to 2015. PGD cycles of these couples were retrospectively analyzed, and 15 infants born from PGD-HLA were followed up. Results A total of 84 oocyte retrieval cycles were performed, providing 14±7 oocytes per cycle. Fifty nine embryos biopsied cycles were done, including 24 cleavage stage and 35 blastocyst stage biopsy cycles. In cleavage stage, 259 embryos were biopsied, 93.4% (242/259) of them with conclusive molecular diagnosis, and the percentage of unaffected embryos (normo-homozygote and heterozygote) was 71.4%(185/259). The percentage of HLA matched unaffected embryos was 9.3%(24/259). In blastocyst stage, 306 embryos were biopsied, while 93.8% (287/306) of them were conclusive, and the percentage of unaffected embryos was 70.6% (216/306). The percentage of HLA matched unaffected embryos in blastocyst stage biopsy was 14.4%(44/306), which was higher than in cleavage stage biopsy (P<0.05). Forty three female carriers underwent 48 embryo transfer cycles including 3 fresh and 45 frozen-thawed embryo transfer cycles. Three fresh embryo transfer cycles were done after cleavage stage biopsy, resulted in a birth of healthy twins born at 36 weeks′gestation. All the embryos were frozen after blastocyst biopsied. Totally, 54 frozen-thawed embryos that were transferred in 45 frozen-thawed embryo transfer cycles included 25 embryo from cleavage stage biopsy and 29 embryo from blastocyst stage biopsy, and 42 of them were HLA matched. Clinical pregnancy rate and implantation rate per cycle in frozen-thawed embryo transfer were 38%(17/45) and 37%(20/54) respectively. A total of 15 infants were born, 2 were from a fresh embryo transfer cycle, and 13 were from frozen-thawed embryo transfer cycles. Results of prenatal diagnosis from delivered cases were matched to that of PGD. Four sick children have been cured by HSCT from these HLA matched born siblings. Conclusion PGD with HLA matching is an established method for conceiving a child who may donate hematopoietic stem cells to save an ill sibling.

The management of extracorporeal circulation in modified total arch replacement combined with stented elephant trunk / 中国医师进修杂志

Objective To study the clinical experience of extracorporeal circulation management in modified total arch replacement combined with stented elephant trunk.Methods Fifty-eight patients with Stanford A aortic dissection underwent modified total arch replacement combined with stented elephant trunk.With the modified surgical technique,the technology of extracorporeal circulation was also modified with bilateral antegrade cerebral perfusion,the management of the temperature and blood protection.Results All patients were operated successfully.The operative time was 248-485 (396 ± 67) min,extracorporeal circulation time was 175-260 (181 ± 33) min,cross clamp time was 64-104 (85 ± 12) min,stop circulation time was 22-48 (32 ± 5) min,and selective cerebral perfusion time was 26-54 (39 ± 7) min.The ventilator assisted breathing time was 5.0-35.5 (23.0 ± 4.5) h,and ICU monitoring time was 24-140 (88 ± 12) h.Postoperative complications included transient neurologic deficit in 3 cases (5.2％,3/58),renal dysfunction in 5 case (8.6％,5/58),and pulmonary infection in 4 cases (6.9％,4/58).Conclusion To modified total arch replacement combined with stented elephant trunk,the modified management of extracorporeal circulation with bilateral antegrade cerebral perfusion,the management of temperature and blood protection,has a low prevalence of morbidity and mortality.

Preimplantation genetic diagnosis of infantile malignant osteopetrosis in a Chinese family / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore the application of preimplantation genetic diagnosis (PGD) for infantile malignant osteopetrosis (IMO).</p><p><b>METHODS</b>For a family affected with IMO, PGD was provided using combined parental mutation detection and haplotype constructions with microsatellite markers spanning the TCIRG1 gene. Prenatal diagnosis was performed on the chorionic villus and amniocentesis samples by direct sequencing.</p><p><b>RESULTS</b>Prenatal diagnosis showed that the fetus by the third pregnancy has carried the parental mutations [c.242delC (p.Pro81Argfs*85) and c.1114C>T (p.Gln372*)], and the pregnancy was terminated. PGD was subsequently performed through mutations detection and haplotype analyses following whole genome amplification (WGA) of each of 13 cells. The results showed that 6 of the 13 embryos were unaffected, 3 were carriers and 4 were affected. Well developed unaffected/carrier embryos were selected and transferred into the uterus. A single pregnancy was confirmed. Subsequently pre- and post-natal diagnoses have confirmed development of a healthy child.</p><p><b>CONCLUSION</b>The study demonstrated the advantage of PGD over prenatal diagnosis when natural pregnancies have repeatedly produced IMO children/fetuses.</p>

Clinical investigation to compare aCGH and FISH in preimplantationgenetic diagnosis of chromosome translocation carriers / 中华妇产科杂志

Objective To investigate the clinical use of array comparative genomic hybridization (aCGH) with fluorescence in situ hybridization (FISH) in preimplantion genetic diagnosis (PGD)for reciprocal and Robertsonian translocation carriers.Methods From Jan.2012 to Jun.2013,a total of 220 PGD cycles from 151 reciprocal translocation and 62 Robertsonian translocation carrier couples,including 33 cycles for reciprocal translocation carriers and 22 cycles for Robertsonian translocation carriers performed using array CGH,and 119 cycles for reciprocal translocation carriers and 46 cycles for Robertsonian translocation carriers performed using FISH were retrospectively studied.The rate of accurate diagnosis was compared between two methods.Results Normal and/or balance rates of the two translocated chromosomes detected by aCGH for both reciprocal and Robertsonian translocation carriers were 38.20％ (123/322) and 67.20％ (127/189),significantly higher than 15.39％ (195/1 267) and 30.75％ (202/657) by FISH (all P ＜0.05).Abnormal rates of the two translocated chromosomes detected by aCGH for both reciprocal and Robertsonian translocation carriers were 59.32％ (191/322) and 30.69％ (58/189),significantly lower than 83.03％ (1 052/1 267) and 67.43％ (443/657) by FISH (all P ＜ 0.05).And the rate of aneu ploidy in non-translocated chromosome from reciprocal translocation embryos was 20.19％ (65/322),which was significantly lower than 38.62％ (13/189) from Robertsonian translocation embryos (P ＜ 0.01).Conclusions Normal and/or balance rates of the two translocated chromosomes detected by array CGH were significantly higher than FISH.And the rate of aneuploidy in non-translocated chromosomes from reciprocal translocation embryos was significantly lower than that from Robertsonian translocation embryos.

A preliminary study on the application of array comparative genomic hybridization for preimplantaion genetic diagnosis / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To assess the value of array comparative genomic hybridization (array CGH) technique for preimplantation genetic diagnosis (PGD).</p><p><b>METHODS</b>Array CGH was performed on three types of cells, which included 3-5 cells isolated from B2/C38/A1 embryonic stem cell lines, single cells isolated from two discarded normal fertilized embryos, and 10 blastocysts biopsied from 5 couples undergoing PGD for chromosomal translocations. For the 10 blastocysts, 8 were abnormal embryos, 1 appeared to be normal but showed arrested development, and 1 embryo was without any fluorescence signals. 24sure V3 or 24sure+ array chips were applied for CGH analysis. The results were analyzed with a BlueFuse Multi software.</p><p><b>RESULTS</b>(1) The results of cells from B2/C3/A1 embryo stem cells by array CGH were consistent with karyotyping analysis. (2) For the 6 single cell samples from two discarded embryos, 2 blastomeres from one embryo were diagnosed as with aneuploidy and a normal karyotype, respectively. Two out of 4 blastomeres biopsied from another embryo were normal, whilst the remaining two were diagnosed with aneuploidies of -22 and +13. Repeated detection with 24sure+ array was consistent with the 24sure V3 result. (3) Ten cell masses from 10 embryos in PGD cycles were successfully analyzed with array CGH, among which four were confirmed with fluorescence in situ hybridization (FISH) on day 3. In two of them, array CGH confirmed FISH diagnosis. For the remaining two, additional aneuploidies for chromosomes not tested by FISH were discovered by array CGH. Another embryo diagnosed as no signal by FISH was found to have trisomy 13 by array CGH. The remaining 5 embryos also showed discordant results by FISH and array CGH. One embryo from a Robertsonian translocation carrier was found to have monosomy 13 by FISH but trisomy 14 and additional aneuploidies by both 24sure V3 and 24sure+ chips. One embryo with many fragments and arrested development by D5 showed discordant results by FISH and array CGH. However, the FISH and array CGH results for other two embryos from this reciprocal translocation carrier were consistent. Three embryos with inconsistent results by FISH and array CGH had a chromosomal translocation involving q11 region.</p><p><b>CONCLUSION</b>Array CGH is useful for PGD applications for its capability to detect structural chromosomal abnormalities through screening of aneuploidies. However, the 24sure V3 array may not suit detection of translocations with breakpoints close to the q11 region of chromosomes.</p>

Clinical analysis of 100 preimplantation genetic diagnosis cycles / 中华妇产科杂志

Objective To investigate influence of chromosomal translocations on early embryo development and to evaluate the efficacy and feasibility of preimplantation genetic diagnosis (PGD)techniques through clinical analysis on PGD cycles. Methods Embryo development, efficacy of PGD and clinical outcome of 100 cycles were studied retrospectively, including 23 cycles with Robertsonian translocations, 19 cycles with reciprocal translocations, and 58 cycles for α-Thalassaemia. Results Among 354 embryos biopsied by PGD for translocations, 321 (90. 7% ) presented fluorescence in situ hybridization (FISH) results. The rate of normal/balanced embryos in the Robertsonian translocation was 38. 3% (64/167),which was significantly higher than 20. 8% (32/154) in the reciprocal translocation group. Amplification was achieved in 443 blastomeres from 537 embryos in Thalassaemia group, which given to an amplification efficiency rate of 82. 5% ( 443/537 ). Totally, 140 normal homozygous, 112 heterozygotes and 155 affected homozygous embryos were identified, while 36 embryos had uncertain result. The successful diagnostic rate was 75.8% (407/537). After 3 days in the translocation groups, the rate of normal and/or balanced translocations in biopsed embryos with ≥7 cells was 34. 4% (77/224), which was significantly higher than 19. 6% ( 19/97 ) of biopsed embryos with ＜ 7 cells. After 4 days, the compaction rate in normal/balanced embryos was 59.4% ( 57/96 ), which was significantly higher than 34. 2% ( 77/225 ) in imbalanced embryos significantly. Seventy-five embryos transferred in 37 cycles with translocations group led to clinical pregnancy rate of 27.0% (10/37), and 170 embryos transferred in 58 cycles with Thalassaemia got a clinical pregnancy rate of 43. 1% ( 25/58 ) . Conclusions PGD can provide management efficiently for both chromosome translocations and Thalassaemia. Translocations might have slightly negative impact on embryo development before implantation.

Genetic analysis of chorionic villi specimen in spontaneous abortion using various methods / 中华妇产科杂志

Objective To explore the efficacy of multiplex ligation-dependent probe amplification (MLPA)combined with fluorescence in situ hybridization(FISH)and comparative genomie hybridization (CGH)combined with FISH in genetic analysis of chorionic villi specimen(CVS)of spontaneous abortion.Methods CGH+FISH and MLPA+FISH were used for genetic analysis of 29 CVS from spontaneous abortion and 6 normal CVS from selective abortion,in the mean time,those results were compared with conventional eytogenetic karyotyping.Results The report time were 40 hours in MLPA+FISH and 120 houm in CGH+FISH.The mean time of chorionic villi culture was(240±72)hours.The successful rate of specimen analysis were 97%(34/35)in CGH,100%(35/35)in MLPA,100%(35/35)in FISH and 91%(32/35)in conventional cytogenetic karyotyping.Apart from 1 case failed in CGH analysis,the results from MLPA+FISH were almost similar to that from CGH+FISH,however,that 1 specimen failed in CGH were detected successfully by MLPA+FISH.The discrepancy rate were 13%(4/31)in CGH+FISH and 12%(4/32)in MLPA+FISH respectively when compared with conventional cytogenetic analysis.Conclusions MLPA+FISH analysis present shorter detecting time and achieve 100%tale of successful report.This combined method was an important adjuvant approach to conventional cytogenetic karyotyping in CVS from spontaneous abortion.

Efficiency comparison between two preimplantation genetic diagnostic methods for chromosomal translocation carriers / 中华妇产科杂志

Objective To compare the diagnostic efficiency between blastomere preimplantation genetic diagnosis (PGD) and polar body PGD for chromosomal translocation carriers. Methods Group A had 8 cycles using whole painting probes for the first polar body diagnosis, while group B had 29 cycles using two subtelomeric probes and one centromeric probe for the blastomere diagnosis. Results The fertilization rate of group A was significantly lower than group B [66. 1% (72/109) vs 85.2% (304/357) , P < 0.05]. There was no significant difference in the successful biopsy rate between two groups. However, group A had a significantly higher loss rate during fixation and higher no signal rate after fluorescence in situ hybridization [ FISH, 9. 6% (12/104) vs 1.6% (4/252), 11.2% (10/89) vs 3.0% (7/233) ]. Totally, the diagnostic efficiency in group A (72. 5% ,79/109 ) was significantly lower than that in group B( 89. 8%, 230/256, P < 0. 05 ). Although both the clinical pregnancy rate( 3/7 ) and implantation rate( 22. 2% ,4/18 ) of group A were higher, the differences were not statistically significant ( P > 0.05 ). Conclusion Both methods can be used efficiently in the PGD for chromosomal translocation carriers. Blastomere PGD has a higher diagnostic rate.

Quality of Life and Influence Factors in Patients with Stroke / 中国康复理论与实践

@#Objective To explore the quality of life(QOL) of stroke patients and the influence factors.Methods The QOL of stroke patients was measured with Shot Form 36 Health Survey Questionnaire(SF-36) and questionnaire survey before discharge and six months after discharge;the patients' state of life was analyzed with the ordinal Logistic regression method to find out influence factors.Results The QOL of the stroke patients decreased obviously with an average sphere score between 38.18～67.57.Sphere scores increased during the six months of post-discharge,but only those of physical functioning(PF),role limitations due to physical health(RP),bodily pain(BP),general health perceptions(GH) and vitality(VT) rose significantly.Some common factors that would affect physical and mental health of the patients included: marriage state,economic conditions,medical care assurance,health care,psychological obstacles,rehabilitation and viability.Besides,age,the features of pathological changes and the complication had significant effect on the physical health of the patients,while children and aphasia affected mental health.Conclusion Early and canonical rehabilitation physical care,language and psychological training and so on,as well as family caring and nursing all are contributive to improve the QOL of the stroke patients.

Quality of Life and Influence Factors in Patients with Stroke / 中国康复理论与实践

@#Objective To explore the quality of life(QOL) of stroke patients and the influence factors.Methods The QOL of stroke patients was measured with Shot Form 36 Health Survey Questionnaire(SF-36) and questionnaire survey before discharge and six months after discharge;the patients' state of life was analyzed with the ordinal Logistic regression method to find out influence factors.Results The QOL of the stroke patients decreased obviously with an average sphere score between 38.18～67.57.Sphere scores increased during the six months of post-discharge,but only those of physical functioning(PF),role limitations due to physical health(RP),bodily pain(BP),general health perceptions(GH) and vitality(VT) rose significantly.Some common factors that would affect physical and mental health of the patients included: marriage state,economic conditions,medical care assurance,health care,psychological obstacles,rehabilitation and viability.Besides,age,the features of pathological changes and the complication had significant effect on the physical health of the patients,while children and aphasia affected mental health.Conclusion Early and canonical rehabilitation physical care,language and psychological training and so on,as well as family caring and nursing all are contributive to improve the QOL of the stroke patients.

Study on the mutation of human short tandem repeats at three loci / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To understand the mutational patterns and mechanism of short tandem repeats(STRs).</p><p><b>METHODS</b>The DNA samples of 19 parent-child pairs with mutations in three loci (FGA, D12S391, and D11S554) were genotyped by silver staining on STR. Alleles to be sequenced were excised from gels, reamplified by PCR, and purified. Sequencing was performed by use of cycle sequencing.</p><p><b>RESULTS</b>There were 18 out of 19 pedigrees in which the 'new' alleles gained or lost a single repeat (8 gains, 7 losses, and 3 being indistinguishable). Only one pedigree lost two repeats. In the 19 pedigrees, there were 13 pedigrees whose 'new' alleles came from fathers, 3 from mothers, 3 from either father or mother. The ratio was 4 1 between fathers and mothers. The mutation of three STR loci occurred in the long, uninterrupted tetranucleotide repeat regions ('CTTT' in FGA, 'AGAT' in D12S391, and 'AAAG' in D11S554).</p><p><b>CONCLUSION</b>Single- step mutations accounted for 95% of STR mutation events in these three loci: FGA, D12S391, and D11S554. The rest were double step mutations. There was no insertion or deletion of an incomplete repeat in any of the pedigrees. The mutation was mainly caused by fathers. The long, uninterrupted tetranucleotide repeats in these three loci might be susceptible to mutation.</p>

The evaluation of PowerPlex~?6 used in paternity testing / 中国法医学杂志

Objective To evaluate the power of the PowerPlex?6 system for paternity testing. Method 633 cases of paternity testing were studied. After the megaplex STR amplification, the PCR products were genotyped by using the ABI377 DNA Sequencer. Results Among the 879 unrelated individuals, 197 alleles and 739 different genotypes were observed. The cumulated discriminating power was 1x 10-30. And the cumulative chance of exclusion was 0.999999999999987. 95 out of 633 cases were excluded. The RCP of all unexcluded cases were ≥0.9990. Gene mutations were found in 19 cases. Conclusion The Po-werPlex?16 system is powerful and reliable for paternity testing.