The Research On Y Chromosome Microdeletion And Cytogenetic Analysis In Male Infertility Patients

Objective To find out the genetic reasons of male infertility,including abnormal karyotype and gene microdeletion of Y chromosome.At the same time, Multiplexed PCR was used to search for mutation hot spots and mutation frequency of AZF gene of Chinese infertile men.To provide theoretical basis and technical support for diagnosing and ftreating male infertility,and Assisted Reproductive Technologies.Materials and methods83samples are collected in the Central Hospital of Obsterics and Gynecology of Tianjin from2008to2011. The principle of choosing the patients were identical with WHO asking for, and we tried our best to exclude the factors of endocrine dyscrasia and obstruction of seminal passage and so on. karyotype analysis was performed83individuals with azoospermia or oligozoospermia by generalmicroscopes. We counted20karyotypes and analyze5karyotypes.Then we divided the83patients into two groups including normal karyotype group and abnormal karyotype group.Extracting DNA from2ml of EDTA-anticoagulant eripheral blood with phenol-chloroform extraction method and stored at-20℃.6-12sites were choosed to detected in most studies about AZF gene.In our study,we choosed15sites to detected,including sY82.sY84.sY86of AZFa region, sY124、sY127、sY128、sY133、 sY134、sY143of AZFb region, sY239、sY242、sY254、sY255of AZFc region and Y145、sY152of AZFd region.And15STSs consisted of four multiplexed PCR systems. SYR serving as inner control in every system. They were respectively system Ⅰ:SRY、sY254、sY143、sY242、sY255、system Ⅱ:SRY、sY84、sY239、 sY152; systemⅢ:SRY、s Y86、s Y127、sY145、sY124; system IV:SRY、sY134.sY82、sY128.sY133. We choose SRY as inner control to exclude false positive results.and choose blank and normal men as external controls.Results (1)Quality of extracted DNA:extracting DNA of peripheral blood with phenol-chloroform extraction method, DNA concentration ranges from44.61ul/to 89.68ul(96.2±8.6ng/ul),A260/A280ranges from1.65to1.85.(2)The analysis of13patients with abnormal karyotype:the karyotype of7azoospermia patients respectively were:3patients with47, XXY;1patient with45, XY,rob (13;21)(p10;q10);1patient with45,XY, rob (14;15) del (Y)(pter�'q11.2);1patient with48, XXYY;1patients with46,XY,inv (9),21pss,15p+. the karyotype of6oligozoospermia patients respectively were:2patients with47,XXY;1patients with46,XY,del(Y)(q11.23);1patient with,XY,t (6,22)(q15;p11);1patient with46,XY,t (15;22)(q11.2;q11.2).Although1patient had abnormal karyotype-46,XY,t (4;18)(q35;q11.2),his wife could be pregnant.The first child was induction of labor because of congenital heart disease.(3)Microdeletion regions:There were8AZF microdeletion in70patients with azoospermia and oligozoospermia, which accounted for11.4%.There were4AZF microdeletion in32azoospermic men,and the deletion rate was12.5%;there were4AZF microdeletion in38oligozoospermic men, and deletion rate was10.5%. As a whole,the deletion of AZF a region accounted for4.3%inclusive of AZF a region alone and AZFa+AZFb+AZFc regions;the deletion of AZFb region accounted for5.7%inclusive of AZFb alone、AZFb+AZFc regions AZFa+AZFb+AZFc regions and AZFb+AZFc+AZFd regions;the deletion of AZFc accounted for7.1%inclusive of AZFc region alone、AZFb+AZFc regions、 AZFa+AZFb+AZFc regions and AZFb+AZFc+AZFd regions;the deletion of AZFd accounted for1.4%inclusive of AZFb+AZFc+AZFd regions.(4)Microdeleltions of sites:There were14sites happening deletion in8patients.Deletion of AZFb+AZFc+AZFd was detected in1individual (including sY124、sY127、sY128、 sY133、sY134、sY143、sY145、sY152、sY239、sY242、sY254、sY255),which accounted for12.5%;deletion of AZFb+AZFc was detected in lindividual(including sY254、sY134),which accounted for12.5%; deletion of AZFa+AZFb+AZFcwas detected in1individual (incuding sY254、sY134、sY82),which accounted for12.5%; deletion of AZF a was detected in2individuals(sY84),which accounted for25.0%; deletion of AZFb was detected in1individual(sY127),which accounted for12.5%; deletion of AZFc was detected in2individuals(sY254),which accounted for25.0%.No one was detected the deletion of sY86.(5) The detection of1patient with46, XY, Yqh-:the karyotype of this patient was46.XY.del(Y)(q11.23),and clinical feature was oligozoospermia.The electrophoretogram show this patient exiting microdeletion of sY254(AZFc) and sY134(AZFb)Conclusion (1) In our study,the microdeletion rate of AZF gene was11.4%.This could show the detection if AZF gene microdeletion is necessary for the patients needing Assisted Reproductive Technologies,especially for the patients with azoospermia and oligozoospermia.At the same time.it explaned that15STSs we choose is right for Chinese people.(2)Abnormal chromosomes were also an important factor to cause male infertility.The infertilital men routinely performed karyotype analysis not only could help the patients find genetic factor about infertility,but also reduce probability of abnormal chromosome passing to their later generations.(3)To the people having hereditary disease,they should conduct prenatal consultation.They should conduct chromosome and gene diagnosis of amniotic or fine hair during the pregnancy.lt can decrease the birth probability of baby having hereditary disease.