Exploration The Method Of Y Chromosome Microdeletion Detection By Multiple Displacement Amplification (MDA) And Multiple PCR

Objectives:The first part is to Explore the clinical significance of Y chromosome microdeletions, Through the analysis of idiopathic male infertility patients with Y chromosome microdeletions. For clinical diagnosis and treatment of male infertility and provide the theoretical basis of assisted reproductive technology.In the second part we retrospective analysis the embryo fertilization rate, cleavage rate, good-embryo rate, pregnancy rate and early abortion rate and other indicators of Y chromosome microdeletions the patients line ICSI-ET treatment. Preliminary discussion on the Y chromosome microdeletions would not affect the outcome of ICSI treatment.In the third part, we establish the method of a single cell AZF detected through multiple displacement amplification (MDA) combined with polymerase chain reaction (PCR) method.Provide a new method for the AZF detection in preimplantation embryos, provide the basis for a reasonable choice in patients with transplanted embryonic and more precise genetic counseling.Methods:In the first part,we collected122patients with azoospermia, severe oligoasthenozoospermia and oligozoospermia who undergoing reproductive treatment for the study. All patients according to the world health organization (WHO) recommended method and diagnostic criteria of diagnosis, the exclusion of endocrine disorders and vas deferens obstruction and other factors. Specimens from the patients with peripheral blood2mL ethylenediamine tetraacetate (EDTA) anticoagulated specimens for the detection of lead at-20℃. Thirty patients has normal fertility healthy male as control. DNA was extracted using salting-out method, Select the15sites for detection of Y chromosome microdeletions,included the AZFa region sY82、 sY84、sY86, the AZFb region sY124、sY127、sY128、sY133、sY134、sY143, AZFc area sY23、sY242、sY254、sY255, AZFd area sY145、sY152.These15bits composed of four sets of multiplex PCR system, respectively for the system Ⅰ:of SRY, sY254, sY143, sY242, sY255, System Ⅱ:SRY, sY84sY239, sY152system Ⅲ: SRY,sY86, sY127, sY145, sY124, system Ⅳ:SRY, SY134, SY82, SY128, SY133. Each system in the SRY gene as an internal control, both blank and normal fertility in male outside control.The second part, we collected5cases of Y chromosome microdeletions in patients6ICSI cycles and the same period of treatment no Y chromosome microdeletions in the serious oligoasthenozoospermia or azoospermia, a total of57cases,62ICSI cycles for study. All patients semen analysis according to WHO criteria, three times in a row semen analysis and precipitated by centrifugation no sperm is azoospermia, sperm concentration<5×106/ml is severe oligozoospermia. All patients were excluded from the abnormal karyotype and Y chromosome microdeletion detected. Observation and analysis between the two groups of patients with fertilization rate, cleavage rate, good quality embryo, embryo implantation rate, biochemical pregnancy, clinical pregnancy rate and early abortion rate.In the third part, Select86male infertility patients who with in vitro fertilization embryo transfer (IVF-ET) treatment in Tianjin certral obstetrics and gynecology hospital reproductive center for the study, Extracted from peripheral DNA and single cells, Using the MDA approach to amplification the whole genome from single/two lymphocytes,then use the DYZ1/DXZ1primers to line sex identification,the primer sequence are as follows: DYZ1:5’-TCCACTTTATTCCAGGCCTGTCC-3’,5’-TTGAATGGAATGGGAACG AATGG-3’DXZ1:5’-AATGTGCAAGTGGCTATTTAGCG-3’5’-TCCATCATAAGG AATGTTCAGCT-3’.Then detect AZF microdeletions,and then compare the AZF detection results between the peripheral blood DNA and single/two lymphocyte. The detection of single cell MDA-PCR-AZF method is feasible and accuracy, and analysis of the genetic relationship of Y chromosome microdeletion.Result:Part one:This study were collected122infertility patients,and12cases with microdeletions of AZF gene,total absence rate was9.8%(12/122). Azoospermia group, less serious weak azoospermia group and the oligozoospermia group of patients the missing rate was11.1%(5/45),10.9%(6/55),4.5%(1/22).The control group,30healthy men were not found AZF gene microdeletion.12patients with AZF microdeletions patients, nine patients with AZFc deletions, eight cases AZFd missing, three cases of AZFa, AZFb deletion is not found.There are three tapes deletion,eight cases areAZFc+d,the AZFa missing three cases,one cases AZFc.Azoospermia group and the less serious asthenospermia group patients with Y chromosome AZF microdeletion was significantly higher than the the oligozoospermia group(p<0.05). Y chromosome microdeletions have a significant impact on male infertility, is one of the important reasons for the cause of male infertility.Part two:Y chromosome microdeletions in patients fertilization rate was significantly lower than the no Y chromosome microdeletions group (P<0.05), Between the two groups of patients with embryo cleavage rate, good quality embryo, the embryo implantation rate, biochemical pregnancy, clinical pregnancy rate and early abortion rate was not significantly different (P>0.05).Y chromosome microdeletion may have some impact on outcome of ICSI.Part three:(1) MDA amplification results:The single lymphocytes MDA amplification from86male patients are success,the rate is98.8%(85/86),30female patients with lymphocyte MDA amplification success, the rate is100%.(2) Sex identification results:lymphocytes MDA amplified sex identification with their blood source match, control and negative control showed no positive results.(3) AZF test results:9cases of Y chromosome microdeletions in86cases of male infertility patients, the deletion rate is10.5%(9/86). Single lymphocyte of AZF detection amplification were success,the rate is97.6%(83/85), Non-missing group of76male patients single lymphocytes the AZF detect SRY amplification success rate was98.7%(75/76), a case of missing AZFb (SY134), AZF detected results in accordance with the rate of98.7%(74/75);9cases deficits SRY amplification success rate was88.9%(8/9), AZF deletion type peripheral blood exactly the same, was100%.Nine cases of missing patients in three cases of AZFa (SY84) missing,four cases of AZFc+d deletion (sY254, SY255, SY242, SY239, SY125),one was AZFb deletion (SY134) and one was AZFc deletion (sY254SY255).Conclusions:1. Further define the Y chromosome microdeletions is an important reason to cause male infertility.This patients should be routinely performed detection and genetic counseling, and provide clinical basis for preimplantation genetic diagnosis. 2. Y chromosome microdeletions may have a certain impact on the outcome of ICSI-ET treatment.It can cause the reduction in fertilization rate, but cleavage rate, high-quality embryo rate, clinical pregnancy rate, early abortion rate was not significantly different,The effects of Y chromosome microdeletions on the ICSI treatment needs to be greater sample size of the study confirmed.3. The study was successed in the method of MDA-PCR-AZF detection of Y chromosome microdeletions with the proimplantation embtyo. Further improve the single blastomeres multiple displacement amplification methods and improved the success rate of amplification a single blastomere. Provide a new method for the clinical ICSI treatment of Y chromosome microdeletions.The study confirmed the vertical inheritance and even expand the area of genetic in the discarded embryos with Y chromosome microdeletions.The successful establishment of the method has important clinical value.