Application Of Fluorescence In Situ Hybridization In Assisted Reproductive Medicine

It has been 20 years since the first success of test tube baby of preimplantation genetic diagnosis(PGD) in 1990.Preimplantation genetic diagnosis(PGD) is introduced as a method to analyze embryo hereditary substance before implantation, and to identify which embryo is normal and suitable to transfer.Thus it prevents genetic disorders.Compared with prenatal diagnosis,PGD can select unaffected embryos to transfer,thus avoiding selective and repeated abortion and moral conflict resulted from abortion.As a result,PGD was received more and more emphasis.On the other hand,the rapid progress in the field of in vitro fertilization-embryo transfer lays solid basement for the development of PGD.Although many novel technologies have applied in this special field for single cell diagnosis such as comparative genetic hybridization(CGH),array-CGH and microarray,there was no other alternate methods which can replace two basic methods for PGD:Fluorescence in situ hybridization(FISH) and polymerase chain reaction(PCR),single-cell PCR has special limitations:amplification efficiency,contamination,and Allele drop-out.FISH has advantages over PCR especially in the study of mosaicism and chromosome number and structural abnormality.So at present FISH is an important and effective method in PGD. Unfortunately,there are many difficulties encountered in clinical FISH-PGD. Firstly,the diagnosis accuracy of FISH depends on the fixation of blastomere in PGD. The ideal unicellular preparation should maintain the integrity of the chromosomal DNA,while removing the cytoplasm completely,and should be easy to learn and perform.At present,there are three different fixation methods for blastomere (Methanol/glacial acetic acid;Tween-20/HCl;Tween-20/HCl+ Methanol/glacial acetic acid),and each of them was adopted by many different PGD centers.However, the situation such as masking nucleus,residual cytoplasm,even cell loss often meet in the procedure of single cell fixation.Moreover,the results of fixation and signal rates of these methods have been inconsistent,and the fixation method is still in need of improvement.Secondly,there are high rate of aneuploidy in pre-implantation embryo which can lead clinical misdiagnosis and low clinical pregnancy rate.There are different standpoints about the embryo aneuploidy risk of chromosome balanced translocation carriers.And the effect of chromosome position on single diagnosis and embryo developmental potentiality remains unclear and become research hotspots. Thirdly,the prognostic factor and genetic counseling about chromosome aberration also become hot spots in the research of PGD.According to the difficulties and hotspots in this special field,the study consists of five parts as follows:PartⅠ:To simplify the procedure based on the Tween 20-HCL and acetic acid/methanol method,and compared it with the other standard methods to determine the suitability for FISH-PGD.PartⅡ:To determine the aneuploidy in pre-implantation embryos of balanced chromosome translocation couples.And to analyze the distribution of chromosome position in nucleus.PartⅢ:To determine whether the proportion of normal sperm is predictive of the proportion of normal embryos from couples in which the males are chromosomal disorder carriers by FISH.PartⅣ:To apply the modified unicellular fixation to clinical FISH-PGD.Till now,16 PGD cycles performed in our center including 9 cycles of Robertsonian translocation carrers,2 cycles for reciprocal translocation carriers,2 cycles for 47,XXY,1 cycles for AZFc deletion,and 2 cycles for aneuploidy screening for chromosome 21.And to retrospectively analyze the effect of Robertsonian translocation on ovarian responsiveness in controlled ovarian hyperstimulation,et al.PartⅤ:To perform FISH on uncultured amniocyte with domestic probes(Beijing Golden probe company).PartⅠThe effect of modified unicellular spreading method on the nucleus area and FISH signalObjectiveTo modify the unicellular fixation method based on Tween-20/HCl+ Methanol/glacial acetic acid and to analyzed the effect of modified unicellular spreading method on the nucleus area and FISH signal.MethodsThe embryos for this study were donated for research by patients treated with IVF/ICSI in our Reproductive Medical Unit.Written consent was obtained from all the patients.Only arrested cleavage-stage human embryos were used in this study.Isolated blastomeres with visible nuclei were randomly divided into 3 groups and fixed by 3 different methods(Methanol/glacial acetic acid;Tween-20/HCl+ Methanol/glacial acetic acid,modified Tween-20/HCl+ Methanol/glacial acetic acid),then perform FISH on the spreading nuclei with 5 probes.To calculate the fixed nuclei area by the special FISH software Imstar2.1 and the overlap signal rate and splitted signal rate for each group.Results1.The Methanol/glacial acetic acid method resulted in 86.73%fixation,which was significantly low than other two methods.2.The mean fixed nucleus area for Methanol/glacial acetic acid method was 55.3 um~3,which significantly larger than that of the other two methods. 3.As for the overlap signal rate and splitted signal rate,there was no statistical significance between the three groups.ConclusionThe modified Tween-20/HCl+ Methanol/glacial acetic acid method do not increase the overlap signal rate and the splitted signal rate,but shorten the during of fixation. For the modified blastomere fixation method,it is easier to locate the nucleus of a blastomere and to learn which is critical for PGD.Considering all these points,the modified method of blastomere fixation for PGD is acceptable.PartⅡThe study on the aneuploidy screening of 11 cycles ofPGD for balanced translocation carriers and the correlation of chromosome position and aneuploidy of preimplantation embryosObjectiveTo perform aneuploidy screening of 11 cycles of PGD for balanced translocation carriers and analyze the correlation of chromosome position and aneuploidy of preimplantation embryos.Methods1.The procedure for PGD was the same as the SOP of our center.2.The second round FISH was performed on the well-fixed nuclei for aneuploidy screening of chromosome 13,18,21,X and Y.3.The relative distance(RD) of the signal was calculated with Imstar2.1 software. The RD=SC/R.Results1.139 well-fixed nuclei were selected for re-analysis,and 130 nuclei got the integrated FISH signals.937 FISH signals were analysized,including 304 signals from euploid nuclei and the others from aneuploid nuclei.2.The euploidy rate was 71.4%(30/42) from balanced embryos,while the euploidy rate was 9.1%(8/88) from unbalanced embryos,and there was significant difference between them(x~2 =53.4,P＜0.05)。Complex aneuploidy was the manifest phenomenon for nuclei from unbalanced embryos.The aneuploidy rate for good-quality embryo was 60%(56/92).3.304 FISH signals from 38 euploid nuclei were calculated which include 21 male nuclei and 21 female nuclei.There was no significant difference between them, howener,signal 13,21,X were prone to locate in the center of nucleus(RD＜0.5).while signal 18,Y trend to distribute in the periphery of nucleus(RD＞0.5).4.The distribution of FISH signal for aneupioid nuclei remains significant difference.signal 13,18,21,Y were prone to locate in the center of nucleus(RD＞0.5),especial for chromosome 18.however it is different for chromosome X.5.the signal distribution of euploidy was statistically significant different from that of aneploid nuclei.The signal location for euploid centralized the center of the fixed nucleus,however the signal distribution for aneuploidy centralized the periphery of the nucleus.Conclusion1.The euploidy rate for balance embryo was higher than that of the unbalance embryo.2.There was no significant difference between the embryos form different grades. The embryos to transfer may be the aneuploid embryos which lead to pregnancy failure.The aneuploidy rate for good-quality was 60%,so it is suggestion that PGS was performed during the procedure of PGD for balanced embryos.3.There is no significant difference for the distribution of chromosome position from euploid nuclei.4.The signal distribution for aneuploidy centralized the periphery of the nucleus, especial for chromosome 18.however,it is different for chromosome X. PartⅢThe Importance of Sperm Fluorescence in situ Hybridization in Preimplantation Genetic Diagnosis for Male Chromosome AbnormalityObjectiveTo determine whether the proportion of normal sperm is predictive of the proportion of normal embryos from couples in which the males are chromosomal disorders.Methods1.Sperm fluorescence in situ hybridization(FISH) and preimplantation genetic diagnosis were done for 9 couples of male chromosomal abnormality including 9 cycles of Robertsonian translocation carriers,2 cycles for reciprocal translocation carriers,1 cycles for Klinefelter's syndrome(47,XXY).The partners have normal karyotype.Informative consent was obtained from all the patients.2.The procedure for PGD was the same as the SOP of our center.3.Sperm pre-treatment:After semen washing,the specimen was fixed with Methanol/glacial acetic acid(3:1),and to dilute the sperm concentration to 20×10~6/ml, then drop on the slide to air dry.The sperm was decondensized by rinsing in 1M NaOH for 5min.FISH was applied to the specimen immediately.4.The program for FISH was the same as the PartⅠ.Results1.The rate of normal/balanced sperm of carriers for Robertsonian translocation, reciprocal translocation and Klinefelter's syndrome was 85.7%,30.4%and 68.8% respectively.2.The rate of normal/balanced embryo of carriers for Robertsonian translocation, reciprocal translocation and Klinefelter's syndrome was 28.97%,6.25%and 33.3% respectively.3.A correlation was found between the percentage of normal embryos and the percentage of normal sperms(R=0.75,P=0.02).Conclusion1.The rate of normal/balanced sperm of carriers for Robertsonian translocation and Klinefelter's syndrome are higher than that of reciprocal translocation carriers2.Since the correlation was established between the percentage of normal embryos and the percentage of normal sperms,it is advisable to recommend for being routinely incorporated into the genetic screening offered prior to preimplantation genetic diagnosis.PartⅣThe clinical analysis of 16 cycles of Preimplantation genetic diagnosisObjectiveTo analyze 16 PGD cycles which were performed in our reproductive center including 9 cycles of Robertsonian translocation carrers,2 cycles for reciprocal translocation carriers,2 cycles for Klinefelter's syndrome(47,XXY),1 cycles for AZFc deletion,and 2 cycles for aneuploidy screening for chromosome 21 from January,2006 to March,2009.MethodsTo perform FISH in clinical PGD.All treatment cycles consisted of controlled ovarian hyperstimulation(COH),oocyte retrieval,ICSI and culture in Quinn's-1026 media after fertilization.We observed the development of embryos on day3 after fertilization,then biopsied after laser-assisted ZP drilling.The standard for biopsy 1-2 blastomeres is the normally fertilized embryo which is above 6-8 cells,less than 20% fragment.The isolated blastomeres were fixed,then hybridized with corresponding probes;the embryos were cultured in Quinn's-1029 media.The normal embryos were transferred according to the diagnosis of FISH and the quality on D 4-5.Results1.We harvested 310 oocytes totally from 16 cycles.The number of MII oocyte was 282.The rate of normal fertilization,cleavage and good embryo were 89.7% (253/282),92.9%(235/253) and 73.0%(165/226) respectively.180 embryos were biopsied successfully in the 195 embryos which were suitable for biopsy,and the fixation rate was 94.4%(170/180).149 embryos were got the FISH result and the rate of FISH diagnosis rate was 87.6%(149/170).2.4 couples had pregnancy in 16 PGD cycles.They were retrieved as the first success of pregnancy of PGD for Robertsonian translocation and Klinefelter's syndrome in Henan province.3 In this study,we retrospectively reviewed 9 cycles controlled ovarian hyperstimulation in PGD for Robertsonian translocations including 4 cycles for female carriers(study group) and 5 cycles for male carriers(controlled group).In the two groups,we analysized the data including age,body mass index(BMI),basal FSH/LH/E2 level,number of down-regulation days,number of days of Gn stimulation,total doses of gonadotropin,E2 level at hCG,number of oocytes retrieved, 2PN fertilization rate,2PN cleavage rate,number of ETs.There were no significant difference in age,body mass index(BMI),basal FSH/LH/E2 level,number of down-regulation days,number of days of Gn stimulation,2PN fertilization rate,2PN cleavage rate,number of ETs between two groups.However,total doses of gonadotropin of study group was higher than that of controlled group(2493.75±555.79 vs 1600.00±173.20,P＜0.05).E2 level at hCG and number of retrieved oocytes of study group was lower than that of controlled group(4309.18±1687.26 vs 7711.00±380.76,17.50±4.51 vs 34.67±2.89,P＜0.05).Conclusion1.Modified modified Tween-20/HCl+ Methanol/glacial acetic acid method applied in clinical PGD.2.Four couples had pregnancy in 16 PGD cycles.This enabled them realize their desire of having healthy offsprings,and laid solid foundation for further research and clinical application of PGD.3.Robertsonian translocation carriers have high rate chaotic embryos. 4.Chromosome Robertsonian translocation maybe is one of the risk factors of poor ovarian responsiveness during controlled ovarian hyperstimulation.PartⅤThe clinical application of Fluorescence in situ Hybridization for uncultured amniocyte in detection of prenatal genetic disordersObjectiveTo detect the prenatal genetic abnormality with amniocyte FISH.Methods1.To performed FISH and karytyping of amniocyte for the cases needed to prenatal genetic diagnosis.2.Standard amniocyte karytyping was adopted and performed in Genetic Research Institute of Henan renmin hospital.3.The protocol FISH for uncultured amniocyte was strictly performed with the instruction of the probe manufacturer(Beijing Golden Probe company).Results1.Totally,100 cases were detected by amniocyte karytyping and FISH which consists of 55 male fetuses and 45 female fetuses.The age range was 25-40 years and the range of gestational week was 17-25 weeks.2.The results of abnormal amniocyte karytypes consists of 1 case of 21-trisomy. The chromosomal variation includes 1 case of inversion(9),1 case of 46 XX,1qh+, 1 case of 46 XY,Yqh+.3.FISH results were got less than 24hrs.Only one case was abnormal which indicate 47,XX,+21.Conclusion1.The result of uncultured amniocyte FISH is feasible and reliable.The diagnosis accuracy of FISH for amniocyte chromosome aneuploidy is 100%. 2.Considered the advantages of uncultured amniocyte FISH such as quickness and reliability,the technology has wonderful prospective application in the prenatal diagnosis,even with the low cost using domestic probes.