Research Of Pedigree-independent Haplotype Construction For Preimplantation Genetic Testing In Southeast Asian Deletion α-thalassemia

Part I:Study on the reference range of capture depth ratio of different thalasemia genotypes in embryo samplesResearch purposeTo explore the reference range of capture depth ratio of different thalasemia genotypes in biopsy embryo samples and find reference embryos.Research methodsFrom August 2019 to November 2021,the families underwent PGT in the Reproductive Center of the Third Affiliated Hospital of Guangzhou Medical University（hereinafter referred to as the Third Hospital of Guangzhou Medical University）due to thalassemia in both spouses were included.According to the genotype of biopsy embryos,they were divided into three groups:(αα/--^SEA)group,no mutation group,(--^SEA/--^SEA)group.High-throughput sequencing panel data related to chromosome 16 was selected,and the average sequencing depth ratio between target region（pathogenic gene region）and non-target region was calculated as capture depth ratio.The relationship between the thalassemia genotype and the capture depth ratio of embryos was inferred from the haplotype analysis of the family samples by the conventional method,and the reference range of capture depth ratios for different thalassemia genotype in the embryo samples was established.The research data was analyzed using statistical software SPSS25.0,and Kolmogorov-Smirnov was used to test the normality of the data,which conformed to the normal distribution and could be measured by mean and standard deviation.If the normal distribution is not met,the median and quartiles could be used to measure or the data could be transformed into variables.If the transformed data conforms to the normal distribution,the mean and standard deviation can also be used to measure.Research resultsIn the statistical analysis of capture depth ratio of the(αα/--^SEA)group,the significance level Sig<0.05,the data does not conform to the normal distribution,the skewness value（1.414）was more than 3 times of the standard error（0.2452）,showing a moderate positive skewed distribution,the data is transformed by logarithm（ln）,and the significance level of the transformed data is Sig>0.05,which conforms to the normal distribution,and the mean（-0.9280）and standard deviation can be used（0.5379）gage.It is estimated that the 95%reference value range of the logarithmic transformation of capture depth ratio of the(αα/--^SEA)group is（-1.9823,0.1262）,and the estimated 95%reference value range of the original data is（0.1378,1.1345）.In the statistical analysis of capture depth ratio of the non-mutation group,the significance level Sig>0.05 conforms to the normal distribution and can be measured by the mean（0.7213）and standard deviation（0.2468）.The range of the 95%reference value of capture depth ratio in the estimated non-mutation group was（0.2376,1.2049）.Capture depth ratios of the(--^SEA/--^SEA)group were all less than 0.01.Conclusion1.(αα/--^SEA)group and non-mutation group capture depth ratio 95%reference range of capture depth ratio intersection area is large,no obvious boundaries.Embryos with genotype of(αα/--^SEA)and no mutation could not be reference embryos used to construct haplotype of husband and wife for the detection of embryo pathogenicity..2.Capture depth ratio can predict the genotype of(--^SEA/--^SEA)as a reference embryo,which is used to construct the SNP haplotype analysis to infer the thalassemia genotype of the remaining embryos.Part Ⅱ: Research on clinical factors affecting the acquisition of reference embryosResearch purpose To explore the relationship between the reference embryos and the number of embryos available for biopsy of the subjects,and the related factors affecting the number of oocytes obtained from the subjects.Rasearch methods From August 2019 to February 2023,the families who underwent PGT at the Reproductive Center of the Third Hospital of Guangzhou Medical University due to thalassemia in both husband and wife were included.Families were divided into 2 groups according to whether there were reference embryos in the embryos available for biopsy(--^SEA/--^SEA): the group with reference embryos and the group without reference embryos.The relationship between the number of embryos for biopsy.The statistical software SPSS25.0 was used for statistical analysis of the research data,and the simple linear regression model was used to analyze the correlation between the number of embryos available for biopsy and the family coefficient without reference embryos.P<0.05 was considered statistically significant.According to the 25 th percentile and 75 th percentile of the number of oocytes retrieved during the operation,the number of oocytes retrieved in the cycle was divided into three groups: ≤8 group,9-20 group,and ≥21 group.The measurement data conforming to the normal distribution were expressed as mean ± standard deviation,and the comparison between groups was performed by analysis of variance,while those not conforming to the normal distribution were expressed as the median（25th percentile,75 th percentile）,between groups Comparisons were made using the nonparametric Kruskal-Wallis H test.MμLtivariate logistic regression was used to analyze the factors affecting the number of oocytes retrieved during operation,including age,AMH,AFC,BMI,and FSH,and evaluate the odds ratio（OR）and 95% confidence interval of the influencing factors.ROC curve was used for data management and analysis referring to the clinical influencing factors of embryos.At the same time,the analysis of variance was performed for the oocyte retrieval under different ovulation induction schemes,and the measured data were expressed as mean ± standard deviation.SPSS25.0 software was used for statistical processing,and P<0.05 was considered statistically significant.Research results 1.The number of embryos available for biopsy had a statistically significant effect on the family coefficient without reference embryos（r=-0.810,P<0.001）.When the number of embryos available for biopsy ≥ 4,there is a 83.1% chance of finding a reference embryo,and as the number of embryos available for biopsy increases,the probability of containing a reference embryo in the family also increases.2.There was a significant positive correlation between the number of oocytes retrieved and the number of embryos available for biopsy（r=0.233,P<0.001）.3.There were no significant differences among the three groups in terms of age,BMI and FSH（all P>0.05）,but there were statistically significant differences between AMH and AFC（all P<0.05）.4.AMH and AFC were significant influencing factors in the comparison between the number of retrieved oocytes ≤8 and ≥21 groups（all P<0.05）;in the comparison between the number of retrieved oocytes between 9-20 groups and ≥21 groups,AMH was a significant factor Influencing factors（P<0.05）;AFC was a significant influencing factor（P<0.05）in the comparison between the 9-20 oocyte retrieval group and the ≤8 group.5.AMH is an independent factor affecting the number of retrieved oocytes（P≤8/≥21=0.001,P9-20/≥21=0.02）.AFC was also an independent factor affecting the number of oocytes retrieved（P≤8/≥21=0.001,P≤8/9-20=0.029）.6.The optimal critical values of the effects of AMH and AFC on reference embryos containing reference embryos were as follows: AMH≥2.955 ng/ml and AFC≥17.Conclusion 1.AMH and AFC can be used as indicators to predict the number of eggs or embryos biopsied.When AMH≥2.955 ng/ml and AFC≥17,the probability of reference embryo in biopsy was significantly increased.2.Increasing the number of embryos available for biopsy can increase the detection rate of reference embryos in biopsy embryos.Part Ⅲ:Validation of PGT method independent of pedigree Southeast Asian thalassemiaResearch purpose Capture depth ratio was used to predict the reference embryo.Exploring the accuracy of reference embryos in the genotype of inferred biopsied embryos in PGT-independent pedigree Southeast Asian thalassemia.Research methods From December 2021 to February 2023,the families were collected from families who underwent PGT in the Reproductive Center of the Third Hospital of Guangzhou Medical University due to thalassemia in both husband and wife.When the genotype of the embryo is unknown,capture depth ratio of each embryo in the family is calcμLated and compared with capture depth ratio reference value range in the first part.If capture depth ratio is <0.01,it can be used as a reference embryo to construct the SNP haplotype.Research results 1.Two of the 20 families do not contain reference embryos,and there is a 90% probability that reference embryos can be found in biopsy embryos,and the correct rate of genotype prediction of reference embryos by capture depth ratio is 100%.2.Among the 18 families,the accuracy rate of genotype prediction of the remaining embryos using reference embryos can reach 100%.Conclusion The reference embryo can be accurately found in the biopsy embryo through capture depth ratio,and the haplotype of the husband and wife can be constructed through the linkage analysis of the reference embryo and the samples of the husband and wife,and then the pathogenicity of other embryos can be successfμLly inferred.