The Investigation Of Sperm Fluorescence In Situ Hybridization And Preimplantation Screening On Aneuploidy

Aneuploidy phenomenon is common in preimplanted embryos, which results in unsatisfactory outcome in therapy of assisted reproductive technology (ART). At present, the IVF/ICSI technique is mainly based on pronuclear and morphology scoring to choose the good quality embryos, but it can not rule out aneuploidy embryos.Preimplantation genetic diagnosis for aneuploidy screening (PGS), means to biopsy blastomeres in vitro fertilization (IVF/ICSI) process, and to applicate Fluorescence In Situ Hybridization (FISH) to screen out the embryos with normal number of chromosome. These can improve the success rate of IVF/ICSI, reduce the abortion rate, and reduce the rate of trisomy risk in neonatus. But there were a few reports about such research by now in China. And most of them only choosed chromosome X, Y and21probes for FISH to diagnoze embryos’ sex and trisome. But at least eight probes are needed for testing to improve implantation rate and reducing the abortion rate.In addition, it can provide basis for assess offspring’s genetic risk to analyze the aneuploidy rate in semen. There were few reports about aneuploidy rates in sperms of patients with mosaic Klinefelter syndrome. And there was no report about wether the sperms’aneuploidy rate will reduce after optimization.The purpose of this study is to establish FISH technique based on11different chromosome probes (except for8chromosome probes, increasing1,7,17probes) to test blastomeres, to analyze the sperms’aneuploidy rate of patients with mosaic Klinefelter syndrome and compare sperm aneuploidy rate of XXY/XY patients and XYY patients by optimization, density gradient centrifugation and swim-up. The study is divided into three parts as follows..Part1. FISH detection of peripheral blood samples from patients with aneuploidyObjective:To establish the FISH techniques for the detection of11chromosomes (1,7,13,15,16,17,18,21,22, X and Y chromosome) in three groups, and compare the results of G-banding test and FISH.Methods:To culture peripheral blood lymphocyte of normal male and fix11probes are divided into three groups:PB probes (including13,16,18,21,22chromosome probe), mixed probes1(including X, Y and chromosome17probe) and mixed probes2(including chromosome1,7,15probes) and then to apply FISH hybridization. If the signal is not ideal, then adjust the probe concentration or experimental conditions. To Culture and fix lymphocytes from three patients with Klinefelter syndrome mosaicism and then analyze using three-color FISH (chromosome17, X, Y), and compare the results with G banding karyotype.Results:The cell signal rate of PB probes was98.7%(512/519). The rate of mixed probes1was99%(502/507). And the rate of mixed probes2was98.6%(490/497). Comparing the G banding karyotype results of three XY/XXY patient’s with FISH results, there is no significant difference (P=0.868,0.755,0.644). But in FISH detection the small proportion of47, XYY cell line was found in patient1.Part2. FISH detection on semen samples of aneuploidy patientsObjective:To investigate the sperm aneuploidy rate of mosaic Klinefelter syndrome patients (XY/XXY) and super male syndrome (XYY), and assess offspring genetic risk. To compare sperm aneuploidy rate of XXY/XY patients and XYY patients by optimization, density gradient centrifugation and swim-up, so that to provide a basis for selection of semen processing method for the treatment of IVF/ICSI.Methods:Semen from cases including3patients with Klinefelter syndrome mosaicism,2patients with XYY and1control, were divided into3parts after liquefaction, and treated by centrifugal concentration, density gradient centrifugation and swim-up methods respectively. Then fixed and detected by three-color FISH hybridization (chromosome17, X, Y). Each patient’s result was compared with that of control. The results of XY/XXY patients treated by three methods were compared each other.Results:Compared with the control cases, X:Y of three cases of XXY/XY patients showed no significant difference (P>0.05). Sperm chromosome abnormality rate increased. XY sperm proportion increased. But patient1 showed no significant difference (P>0.05). Patient2and patient3showed statistical difference (P<0.05). Compared with the control cases, X:Y of two cases with XYY showed no significant difference (P>0.05). Sperm chromosome abnormality rate, YY sperm proportion and XY sperm proportion increased. Patient4and patient5showed statistical difference (P<0.05).Part3. The applification of FISH in preimplantation genetic diagnosis for aneuploidy screeningObjective:To establish the FISH technique for the detection of11chromosomes (1,7,13,15,16,17,18,21,22, X, Y chromosome) in three rounds to test blastomeres, and to investigate the mosaic phenomenon in D3embryos.Methods:44D3fresh embryos abandoned during IVF/ICSI treatment, and16frozen embryos donated were collected. Digesting zona with Pronase, extractig single blastomeres from32embryos abandoned fresh embryos and12donated frozen embryos, and extracting2blastomeres from the remaining16embryos. To Fix blastomeres using0.2%Tween-20/0.01N HC1+methanol/acetic acid (3:1), and detect by three rounds of FISH hybridization using11chromosome probes.Results:A total of112blastomeres were collected.108nuclears were fixed successfully. The fixed rate was96.4%.103nuclear had complete FISH signal, and the hybridization rate was95.4%. The abnormality rates were29.5%,54.5%and65.9%respectively using5,8,11probes for tesing. The normal rate was34.1%in44embryos which analyzed single blastomere. The normal rate of frozen embryo donated was41.7%. The normal rate in16fresh abandoned embryos was31.3%.7embryos were mosaic among16embryos which analyze single blastomeres, and the mosaic rate was43.8%.Conclusion1. The sperm of mosaic XXY patients and XYY patients are mostly aneuploidy. And X sperm are equal to Y sperm. Aneuploid rate increased slightly, mainly in XY sperm.2. Density gradient centrifugation and swim-up methods can not reduce the aneuploid rate in patients with XXY/XY and XYY.3. Establish the FISH technique for the detection of11chromosomes (1,7,13,15,16,17,18,21,22, X, Y chromosome) in three rounds to test blastomeres.4. In the diagnosis of PGS, increasing the number of probes can improve the detection rate of abnormal embryos.5. The good quality embryos according to morphological standard still have high rate of chromosome aneuploidy.6. Mosaic phenomenon in D3embryos is common, and mainly is diploid mosaic.