| Objective:To apply fluorescence in-situ hybridization(FISH) - preimplantation genetic diagnosis(PGD) to the couples with chromosomal structural abnormalities after confirming the combination of labeled probes for each couple who will accept PGD and determining the frequency of normal/balanced gametes by FISH analysis.Method:(1) Sixty-eight infertile outpatients who desired to accept PGD were confirmed having chromosome abnormality by G-banding.According to the carrier's karyotype, appropriate fluorescence labeled probes were used to hybridize with peripheral blood lymphocyte.In case of inaccurate G- banding karyotype or partial probe signals,we change probe combination or adjust probe concentration before performing FISH on peripheral blood lymphocyte again.(2) Multi-color FISH was performed on sperm samples in five inv(Y)(p11.1;q11.2) carriers,four inv(1) carriers and one t(Y;10)(q12;q24) cartier.(3)FISH-PGD was performed in 2 cases of pericentric inversion carriers,14 cases of Robertsonian translocations,10 cases of reciprocal translocations,1 case of Y/autosome translocation. Results:(1) FISH was performed in 68 carriers on peripheral blood lymphocyte,and in 32 cases,probe concentration were adjusted due to partial probe signals.After alteration, the average hybridization efficiency and specificity for the 68 mixed probes were 96%-100%and 100%respectively.Probe combination was changed because of wrong diagnosis with G-banding in three carriers.FISH-PGD was unable to be performed in two carriers.(2) The frequency of spermatozoa with abnormal sex chromosome numbers and recombinant Y chromosome was not statistically different between the inv(Y) patients and controls.The frequencies of non-recombinant gametes and recombinant sperm were 83.8%-98.7%and 0-17.3%in four inv(1) carriers.The frequencies of normal and balanced translocation segregations were 28.2%and 20.3% respectively in t(Y;10)(q12;q24) carrier.(3)There were 63 couples that could be predicted non-balanced embryos by FISH.After consent was obtained from patients,31 PGD cycles were performed for 27 couples.Total 223 embryos from 27 couples were biopsied 209 embryos(93.7%) were successfully diagnosed by FISH.Among them there were no embryos that could be transferred in 5 PGD cycles for 5 couples because of no normal embryos.Forty-eight normal/balanced embryos were transferred into the uterus of 22 patients totally in 26 PGD cycles,the transferred embryo of every PGD cycle was 1-3.The transferred embryos resulted in 14 pregnancies,with a clinical pregnancy rate of 53.8%(14/26),an implantation rate of 29.2%(14/48).Up to now,3 boys and 5 girls were born,3 patients are in ongoing pregnancies and 5 patients underwent abortion.Conelutions:(1)The preliminary experiment of peripheral blood lymphocytes before FISH-PGD for the couples with chromosomal structural abnormalities is valuable and inevitable to confirm break point,select probe and establish optimal experimental condition. (2)Sperm-FISH studies are useful for genetic counseling and predict the risk of transmission of unbalanced embryo of PGD.PGD was not necessary for inv(Y)(p11.1;q11.2) and inv(1)(p11;q12) carriers.(3)Chromosomally abnormal embryos can be effectively distinguished by FISH-PGD in pericentric inversion;Robertsonian translocation;reciprocal translocation and Y/autosome translocation.Therefore, FISH-PGD is an efficient treatment for these patients.(4)Twenty-seven couples with high genetic risk accepted PGD and resulted in 14 pregnancies.Three boys and 5 girls were born in this research.(5)The FISH-PGD was only efficient for different forms of segregation during the meiosis division with chromosome translocation or inversion,so the genetic information was partially.The PGD technique should be improved for patients with structural chromosomal abnormalities. |
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