The Hereditary Traits Of Y Chromosome AZF Microdeletions And The Clinical Value Of Applying PGD

Objectives: The first part is to explore the clinical significance of AZF microdeletions, through analysising the incidence rate of north China male infertility patients with AZF microdeletions and the effect from different type of deletion on phenotype. In the second part we research the influence of AZF microdeletions on the reproductive hormone and the development of testicles. The third part we retrospective analysis the embryo fertilization rate, cleavage rate, good-embryo rate, implantation rate, pregnancy rate and early abortion rate and other indicators of AZF microdeletions the patients line ICSI-ET treatment. To discuss whether the AZF microdeletions would affect the outcome of ICSI treatment or not. In the forth part, for the male infertility patients getting ICSI we establish the method detecting the male embryos on cleavage stage through multiple displacement amplification(MDA) combined with polymerase chain reaction(PCR) method.Provide a new method to detect preimplantation embryos, provide the basis for a reasonable choice in patients with transplanted embryonic and more precise genetic counseling. The fifth part is to study the different pregnancy outcome comparing the patients accepting preimplantation genetic diagnosis(PGD) with those refusing it to screen the AZF microdeletions on embryos so that we can provide the clinical basis for the patients to get the PGD.Methods:In the first part, we collected 73 patients with non-obstructive azoospermia in Tianjin Central Hospital of Obstetrics and Gynecology, Center for Reproductive Medicine from Januage 2012 to August 2013 who undergoing reproductive treatment for the study excluding of vas deferens obstruction and other factors,90 patients with severe oligoasthenozoospermia and 63 patients with mild-to-moderate oligospermatism.( 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,sperm concentration 5~20 × 106/ml is mild-to-moderate oligospermatism). Thirty patients has normal fertility healthy male as control. Specimens from the patients with peripheral blood 2 m L ethylenediamine tetraacetate(EDTA) anticoagulated specimens for the detection of lead at-20 ℃. DNA was extracted using salting-out method, Select the 15 sites for detection of AZF microdeletions,included the AZFa region s Y82、s Y84、s Y86,the AZFb region s Y124、s Y127、s Y128、s Y133、s Y134、s Y143,AZFc area s Y239、s Y242、s Y254、s Y255,AZFd area s Y145、s Y152. These 15 bits composed of four sets of multiplex PCR system, respectively for the system I: of SRY, s Y254, s Y 143, s Y 242, s Y 255;System II: SRY, s Y 84 s Y 239, s Y 152;system III: SRY,s Y 86, s Y 127, s Y 145, s Y 124;system IV: SRY, SY 134, SY 82, SY 128, SY 133. Each system in the SRY gene as an internal control, both blank and normal fertility in male outside control.The second part,we stored 226 infertility males serum to assess the Follicle-stimulating hormone(FSH),Luteinizing hormone(LH),Testosterone(T) and studied the statistically significant difference.At the same time we also measured and compared the size of testicles.The third part, we collected 10 cases of AZF microdeletions in patients 13 ICSI cycles and the same period of treatment no AZF microdeletions in the serious oligoasthenozoospermia or azoospermia, a total of 82 cases, 98 ICSI cycles for study. Observation and analysis between the two groups of patients with fertilization rate, cleavage rate, good quality embryo, embryo implantation rate, clinical pregnancy rate and early abortion rate. The forth part,selecting the remaining 2PN/1PN cleavage stage embryos which is no freezing value,a total of 62 for study from 10 cases of AZF microdeletions in patients 13 ICSI cycles and the same situation with no AZF microdeletions we use micromanipulation technique to obtain single blastomere and MDA approach to amplification the whole genome from single blastomere. Then we use PCR to identify the sex.At last to detect 61 male embryos AZF microdeletions. The fifth part is to compare the pregnancy outcome after transplantation between embryos AZF microdeletions in 4 patients who accepted the PGD screening and 6 ICSI patients who refused the PGD screening.Result:Part one:This study were collected 226 infertility patients,and 23 cases with microdeletions of AZF gene,total absence rate was 10.18%(23/226). Azoospermia group, less serious weak azoospermia group and the oligozoospermia group of patients the missing rate was 12.33%(9/73),13.33%(12/90),3.17%(2/63).The control group, 30 healthy men were not found AZF gene microdeletion. Azoospermia group and the less serious asthenospermia group patients with Y chromosome AZF microdeletion was significantly higher than the mild-to-moderate oligospermatism group(P <0.05). 23 patients with AZF microdeletions, nine patients with AZFc deletions, eight cases AZFc+d missing,four cases of AZFa, one case of AZFb+c and one case of AZFa+b+c. Part two:Among the azoospermia and less serious weak azoospermia patients the level of FSH and LH in azoospermia group patients with Y chromosome AZF microdeletion was significantly higher than the control group(P <0.05);T level and testicular volume was significantly lower than the control group(P <0.05);For the mild-to-moderate oligospermatism patients,comparing AZF microdeletions group and control group there is no statistical difference of each index.(P>0.05). Part three: AZF microdeletions in patients fertilization rate was significantly lower than the no AZF microdeletions group(control group)(P <0.05);In AZF microdeletions patients the embryo cleavage rate, good quality embryo, the embryo implantation rate, clinical pregnancy rate are also lower than control group but there was not significantly different(P> 0.05);though early abortion rate in control group was a bit lower there was not significantly different(P> 0.05)Y chromosome microdeletion may have some impact on outcome of ICSI but we needs to be greater sample size of the study confirmed. Part four:(1) MDA amplification success rate is 93.5%(116/124).After Sex identification there are 61 male embryos and 55 female embryos.(2) AZF test results:(including 28 male embryos from AZF microdeletions patients and 33 male embryos from control patients.) All of 28 male embryos from the AZF microdeletions patients has different extent deletion:23 embryos has the same type of deletion with their parents and the missing area in 5 embryos expand. 9 male embryos from 6 patient among 33 male embryos in18 patients with no AZF deletions have de novo deletion,the deletion rate is 27.3%(9/33). Part five: We compared the pregnancy outcome after transplantation of embryos AZF microdeletions between 4 patients(5 ICSI cycles) who accepted the PGD screening and 6 patients(6 ICSI cycles) who refused the PGD screening. Between the two groups of patients with fertilization rate,embryo cleavage rate, good quality embryo was not significantly different(P> 0.05).In PGD group we total obtain 21 female embryos the embryo implantation rate is 27.3%, clinical pregnancy rate is 40% and no early abortion;in the other group the embryo implantation rate is 21.4%, clinical pregnancy rate is 42.9%,one case of early abortion.Conclusions: 1.In Tianjin AZF microdeletion rate in less serious weak azoospermia patients is highest and azoospermia patients is a little lower.AZFc deletion is the mainly loss area between them. Less serious weak azoospermia and mild-to-moderate oligospermatism are also AZFc deletion.AZFa deletion only exists in the azoospermia patients.2. This research confirmed AZF microdeletion has effect on the development of gonad lead to reduce testical volume, rise the FSH/LH level and obviously decrease the T level. 3. AZF 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 AZF microdeletions on the ICSI treatment needs to be greater sample size of the study confirmed. 4. AZF microdeletion can pass to the next generation through ICSI technology.In this process de novo microdeletion may happen and the area of lossing expand. 5. Accepting the PGD screening the female embryos won’t decrease clinical pregnancy rate, also reduce early abortion and avoid propagating to male offsprings.