Genetic Analysis Of Y Chromosomal AZFc Variants In Patients With Spermatogenic Failure And Assessment Of Their Clinical Outcomes After Assisted Reproductive Technology Treament

Infertile problems trouble about 10-15%of couples of child-bearing age throughout the world,and male factor contributed to 30%?50%among these cases.Primary spermatogenesis caused by genetic defects is the major reason of male infertility.Azoospermia factor(AZF)deletion of Y chromosome,generally known as the Y chromosome microdeletions,is the second most common genetic factor leading to male primary spermatogenesis disorders,preceded by Klinefelter's syndrome.At present,it is usually believed that AZF region can be divided into three sub-regions:AZFa,AZFb,and AZFc.And the frequency of AZFc deletion was highest among all Y chromosome microdeletions.Since high sequence similarities in the AZFc region promote frequent chromosome rearrangements via non-allelic homologous recombination,it is a challenge to accurately detect the genetic variations in this region.To date,the best undersood genetic structural variation in AZFc region was complete deletion type(i.e.b2/b4),and men carrying this mutation generally exhibit severe spermatogenesis failure.However,there are relatively few studies of partial deletion in AZFc region such as gr/gr,b2/b3,b1/b3 subtypes,and the medical consequences of these subtypes are controversial.In addition,the frequencies of other CNV(copy-number variants)and complex rearrangements in AZFc region and their contribution to spermatogenesis are unclear.To identify the common subtypes of partial AZFc deletion,two multiplex PCR systems of five sequence tagged sites(sY1189,sY1191,sY1192,sY1197,sY 1291)were established to detect the peripheral blood DNA from spermatogenic disorder group and the normal control group,respectively.Eighty-seven of 575(15.1%)patients with spermatogenic disorders were found to have AZFc partial deletion,among which gr/gr deletion accounted most(7.1%).Meanwhile,10 of 114(8.9%)normal control men were detected to have partial AZFc deletion,among which b2/b3 deletion accounted most(4.4%).In order to investigate the relationship between partial AZFc deletion and spermatogenesis failure,we compared the frequencies of deletions between the spermatogenesis disorder case group and the normal control group.We found the frequency of gr/gr deletion in the case group was significantly higher than that in the normal control group;furthermore,the frequency of gr/gr deletion either in the severe oligozoospermia subgroup or in the oligozoospermia subgroup was significantly higher than that in normal control group.However,thefrequency of b2/b3 deletion in the case group and that in the normal control.group were similar.These results suggest that gr/gr deletion might be an important risk factor for spermatogenesis failure.To determine the gene CNV in AZF region,we performed MLPA(Multiplex Ligation-dependent Probe Amplification)analysis on 55 patients with partial AZFc deletion.The copy numbers of critical genes such as DAZ,BPY2 and CDY in the AZFc region were obtained.A total of 13 different CNVs were identified,6 variants occurred in 26 cases with gr/gr deletion,3 variants in 23 cases with b2/b3 deletion,and the remaining 4 variants occurred in 6 cases with other rare subtypes.In addition,combining the gene copy-number variations with STS locus information,the genomic structural breakpoints of the 13 CNVs were located,and 8 complex rearrangements haboring both gene duplications and deletions which have not been reported previoulsy.The discovery of these CNVs provides experimental evidence for further study of genetic structure of the AZF region and its mechanism during spermatogenesis.To investigate the effect of partial AZFc deletion on assisted reproductive technology(ART)outcomes,we examined the embryonic and pregnant outcomes of couples with AZFc partial deletions after treatment in a case-control study.The fertilization rate and high-quality embryo rate in the case group of 25 couples treated with ICSI(In Vitro Fertilization/Intracytoplasmic Sperm Injection)were significantly lower than those in control group,while there was no significant difference in fertility rate,biochemical pregnancy rate and clinical pregnancy rate between the two groups;The fertility rate in gr/gr deletion subgroup was significantly lower than that in control group,but the cleavage rate was higher than control group.In addition,the cleavage rate in the gr/gr deletion subgroup was higher than that in the b2/b3 deletion subgroup.These results suggest that gr/gr deletion may affect the fertilization rate in ICSI treatment,whereas the b2/b3 deletion has a greater effect on the cleavage of embryos.