The Use Of Whole-genome Sequencing To Expand Diatgnosis Of Chromosomal Abnormalities For Couples With Recurrent Pregnancy Loss And Their Impacts On Meiotic Segregation Patterns

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Whole-genome sequencing expands diagnosis of chromosomal abnormalities for couples with recurrent pregnancy lossRecurrent pregnancy loss(PRL)is a common obstetric complication,which adversely affects about 1%of couples seeking pregnancy.The causes of RPL are complicated,among them genetic factors,antiphospholipid syndrome(APS)and congenital uterine malformations being directly related.Factors like infections,autoimmunity and hormonal or metabolic disorders(diabetes mellitus,thyroid dysfunction)have been associated with miscarriage,whereas their roles in RPL are unclear.About half of patients has no identifiably causative factor,which is classified as idiopathic or unexplained RPL(uRPL).To these uRPL patients,some prophylactic treatments are provided,but the effectiveness is unsatisfied.Currently,peripheral blood G-banded chromosome analysis is the standard assay to detect chromosomal rearrangements in couples with RPL despite its limited resolution(320-400 bands).The prevalence of chromosomal abnormalities in RPL couples is approximately 5%,among which autosomal reciprocal balanced translocations being the most commonly observed,followed by Robertsonian translocations and inversions.These patients could yield a livebirth outcome by using preimplantation genetic testing for structural rearrangement to select normal or balanced embryos.Recently,whole-genome sequencing(WGS)shows its capability in yielding additional etiologic diagnoses with improved resolution.Objective:We applied a well-established WGS approach in uRPL couples to expand etiology of chromosomal abnormality among uRPL couples and to determine the extent of additional chromosomal structural rearrangements that would provide valuable information for clinical management.Methods:We performed low coverage WGS retrospectively for 1024 uRPL couples,all of whom had routine G-banding chromosome analysis.Inclusion criteria included:(1)two or more first trimester clinical pregnancy losses;(2)normal karyotypes for both partners via G-banding chromosome analysis;(3)no anatomical malformations;(4)no APS or auto-antibodies positive;(5)no endocrine disorders or metabolic disorders.Chromosomal translocations and inversions were confirmed by polymerase chain reaction(PCR)and Sanger sequencing,with some cases being confirmed by resubmitted fresh blood using fluorescence in-situ hybridization(FISH)and high-resolution chromosome analysis.Copy number variants(CNVs)were validated using chromosomal microarray analysis(CMA)chips.Results:Overall,low-coverage WGS yielded results for 98.7%(1011/1024)of the uRPL couples as 13 samples were excluded because of poor DNA quality,resulting in a final cohort of 1011 couples.Among this cohort,49 chromosomal rearrangements were detected in 48(4.75%)couples including 22 balanced chromosomal translocations and 27 inversions.Among these 22 balanced translocations,14 cases were established in original or high-resolution chromosome analysis,leaving 8 cases only identified by WGS due to cryptic translocation or the similar size and banding pattern of the reciprocally exchanged segments.Among the 27 inversions,four cases were established by chromosome analysis while 23 cases were not due to cryptic segment(one case)or size of involved segments below the resolution limits of G-banding chromosome analysis.All chromosomal translocations and inversions were validated by PCR and Sanger sequencing,with FISH being performed in some fresh samples.Besides,one pathogenic and five likely pathogenic CNVs were reported by low-coverage WGS,all being validated using CMA chips.Conclusion:In the context of chromosome analysis for RPL couples,low-coverage WGS identified additional etiologic chromosomal abnormalities,including balanced translocations,inversions and CNVs,compared to standard karyotype results.It is highly recommended for patients with RPL to pursue WGS as this study demonstrates its ability to expand diagnosis of chromosomal abnormalities.Chapter ? The impacts of chromosomal abnormalities on meiotic segregation patterns Section ? Interaction of acrocentric ch romosome involved in translocation and sex of the carrier influences the proportion of alternate segregation in autosomal reciprocal translocationsBalanced reciprocal translocation is defined as the result of an exchange of segments between two non-homologous chromosomes.As one type of commonly structural chromosomal rearrangements in human beings,the prevalence of balanced reciprocal translocation is 0.74-1.52 per 1000 newborns and 2.4%in couples with recurrent pregnancy loss.During meiosis,the two translocated chromosomes and their two homologous normal chromosomes form a quadrivalent and subsequently segregate at anaphase I.There are five types of segregation patterns:alternate segregation,adjacent-1 segregation,adjacent-2 segregation,3:1 or 4:0 segregation.Theoretically,gametes with 36 different karyotypes are produced in carriers of a reciprocal translocation.Normal or balanced gametes are produced by an alternate mode of segregation.Gametes produced by the other segregation patterns have unbalanced karyotypes.Carriers of reciprocal translocations are at a significantly increased risk of fertility problems such as infertility,pregnancy loss and newborns with congenital anomalies,due to the generation of unbalanced gametes in meiotic segregation of a quadrivalent.Previous studies have reported that meiotic segregation patterns of a quadrivalent can be affected by factors such as a carrier's sex and age and the chromosome type.In these studies,the proportions of adjacent-1,adjacent-2 or 3:1 segregation are significantly different;whereas the reported proportion of alternate segregation does not differ significantly,except in one study,and whether combined effects between these factors exist is unclear.Objective:The aim of this study is to investigate whether meiotic segregation patterns of reciprocal translocations are affected by the combined effect of chromosome type and a carrier's sex.Methods:A retrospective study of array comparative genomic hybridization outcome data from patients with autosomal reciprocal translocations was conducted to analyze meiotic segregation patterns and blastocyst euploidy rates.We enrolled 473 couples whose embryos were tested between January 2013 and September 2016.Meiotic segregation patterns of 2101 blastocysts from 243 female carriers,including 76 cases with translocations involving an acrocentric chromosome(Acr-ch),and 230 male carriers,including 88 cases with translocations involving Acr-ch,were analyzed according to chromosome type,carrier's sex and age.Results:In cases with translocations involving the Acr-ch subgroup,the proportion of alternate segregation(53.9 vs 33.4%,P<0.0001)was significantly higher in male carriers than in female carriers,with the proportion of 3:1 segregation(6.8 vs 16.3%,P<0.0001)being significantly lower.The proportions of alternate segregation were similar between sexes in cases with translocations not involving the Acr-ch subgroup.Meanwhile,in the female carrier subgroup,the proportion of alternate segregation(33.4 vs 45.2%,P<0.001)was significantly lower and the proportion of 3:1 segregation(16.3 vs 8.2%,P<0.001)was significantly higher in cases with translocations involving Acr-ch than in those not.In the male carrier subgroup,the proportion of alternate segregation(53.9 vs 46.9%,P = 0.031)was higher and the proportion of adjacent-1 segregation(27.1 vs 37.3%,P<0.001)was significantly lower in cases with translocations involving Acr-ch than in those not.Carrier's age did not affect the meiotic segregation patterns.However the euploidy rates were significantly lower in couples with advanced compared to young maternal age respectively.Conclusion:Interaction of an acrocentric chromosome involved in the translocation and sex of the carrier influences the proportion of alternate segregation for normal or balanced chromosome contents during meiotic segregation in autosomal reciprocal translocations.The findings of this study provide detailed information for genetic counselling of couples with autosomal reciprocal translocations on their chances of producing euploid gametes.Section ? Effects of a carrier's sex and age on segregation patterns of the trivalent of Robertsonian translocationsRobertsonian translocation is defined as the result of centric fusion of two acrocentric chromosomes.The incidence of Robertsonian translocations is 0.74-1.23 per 1000 newborns and 3.1%in couples with recurrent pregnancy loss.During the pachytene stage of prophase I in gametogenesis of Robertsonian translocations,the derivative chromosome and its two normal homologs form a trivalent structure.At anaphase I,the trivalent segregates in one of the three patterns:alternate segregation,adjacent segregation and 3:0 segregation.Theoretically,there are eight different types of gametes produced with respect to chromosomal constitution,among them two types generated from alternate segregation being normal or balanced.In section one,we have found that a significant difference of meiotic segregation patterns is existed between the carrier's sex in autosomal reciprocal translocations with an acrocentric chromosome involvement and this difference does not exist in translocations not involving an acrocentric chromosome.Objective:In this study,we further analyze the meiotic segregation patterns of the trivalent of Robertsonian translocations according to a carrier's sex and age and find some general characteristics shared by these two types of rearrangements.Methods:We designed a retrospective study to analyze the segregation patterns of the trivalent and euploidy rates of blastocyst.Data of 154 couples with Robertsonian translocation were collected.Embryos were detected using array comparative genomic hybridization between January 2013 and July 2017.Segregation patterns of the trivalent of 604 blastocysts from 77 female carriers and 77 male carriers were analyzed according to the carrier's sex and age.Results:The proportion of alternate segregation was significantly higher(82.9 vs 55.2%,P<0.001)in the male carriers than in the female carriers of Robertsonian translocation,with the proportion of adjacent segregation being significantly lower(16.8 vs 42.6%,P<0.001)and no difference in 3:0 segregation.The segregation patterns were similar in the same sex of carriers when analyzed according to type of the translocations.Carrier's age had no influence on the segregation patterns of the trivalent.This effect of maternal age on genomic abnormalities was related to abnormality of chromosomes unrelated to the translocations,especially whole chromosomal aneuploidy.Conclusion:The proportion of alternate segregation for normal or balanced chromosome contents is significantly higher in the male carriers than in the female carriers of Robertsonian translocation and the meiotic segregation patterns are independent of a carrier's age,as observed in autosomal reciprocal translocations with an acrocentric chromosome involvement.The findings from a joint analysis of Robertsonian translocations and autosomal reciprocal ones may provide some clues to understand the underlying mechanisms of meiotic segregation patterns of structural rearrangements in the female and male carriers in order to estimate their reproductive risks and counsel appropriately.