Recurrent Spontaneous Abortions: A Cytogenetic Approach to study Genomic Imbalance due to Chromosomal Abnormalities in Products of Conception

Abstract

Background: Recurrent spontaneous abortion is defined as the occurrence of two or more consecutive abortions in a clinically recognized pregnancy. An abnormal embryonic chromosome complement is one of the major cause for recurrent abortions. Most commonly found chromosomal abnormalities in products of conception are aneuploidies followed by structural rearrangements. These abnormalities can be de-novo or could be inherited from either of the parents, as 4-5% of couples with two or more miscarriages are balanced translocation carriers. This thesis is about the conventional and molecular cytogenetic combined approach in studying the genomic imbalance caused due to chromosomal abnormalities in products of conception from recurrent spontaneous abortions. Due to limitations of conventional cytogenetics studies molecular approach gives an insight of cryptic translocations and sub microscopic deletions and duplications. This era of high throughput advanced cytogenomic techniques also require next generation of clinical information, for better interpretation of results and establishing a correlation between genotype and phenotype (clinical indications) observed. This study also focuses on studying the pathogenicity of the variations observed in the products of conception with history of clinically significant ultrasound findings. Aim: This study was done to identify chromosomal abnormality in products of conception. The results from different copy number variations were studied for their pathogenicity and its relation with clinical phenotypic information. The chromosomal abnormality in products of conception is de novo or inherited was assessed by studying parental karyotype in some cases. Observations: Out of 300 products of conception samples about 113 (40.2%) samples were cultured successfully and their karyotype was analysed using CytoVision software and ISCN 2016. Interphase FISH was performed on 187 (66.5%) cases, which failed to grow in culture. Chromosomal microarray was performed on 20 (7.1%) cases, which resulted normal by FISH and karyotyping and had a significant clinical indication. Out of 281 cases, 55 abnormal (19.4%) and 226 normal (80.6%) were observed. Different chromosomal

anomalies were identified which consisted of 13 cases of monosomy X (23.6%), 9 with trisomy 16 (16.3%), 8 with trisomy 21 (14.5%), 3 with trisomy 13 (5.4%), 2 cases each with trisomy 15, trisomy 18, trisomy 20 and trisomy 22 (3.6%) followed by 1 case each of trisomy X and monosomy 21 (1.8%). Four cases showed mosaicism for different chromosomes, one case each showing mosaic XYY/XY[68/100] (1.8%), XXY/XY[70/100] (1.8%), XXY,+18/XY[60/100] (1.8%) XX,+21/XX[65/100] (1.8%). Two case were observed with presence of triploidy 69,XXY and 69,XXX (1.8%) each. Most frequently associated abnormality with recurrent spontaneous abortion is autosomal trisomy. In our present study, autosomal trisomy accounts for 50% of the total abnormal cases, which includes trisomy of chromosome 13, 15, 16, 18, 20, 21 and 22. This study shows eight cases of trisomy 21 followed by another often-found autosomal trisomy 16 (09 cases). Trisomy 16 is the most commonly reported aneuploidy after monosomy X, also known as the miscarriage chromosome. Trisomy 22 is reported with other uncommon autosomal trisomies. Mosaic case where 65% of the cells showed trisomy 21 and the rest had normal female chromosome complement, it could be a result of placental mosaicism or maternal cell contamination. Out of 55 abnormal findings, six cases showed structural abnormality, one by karyotyping 46,XX,der(13;14)(q10;q10),+14 (1.8%) and the remaining by chromosomal microarray which showed gain and losses on different chromosomes (9%). We observed four pathogenic variants on chromosome 16p31.3p13.2, 17q25.3, 11p15.5p15.1, and 9q22.32q31.3 and one variant of unknown significance on chromosome 15q26.3. Two of the unbalanced translocations in the fetus were the results from a balanced chromosomal translocation in the parents. These imbalances could not be detected on the karyotype due the size of rearrangements being 3.7 Mb and 529Kb. Phenotypically significant genes listed in OMIM database which are pathological and are associated with mental retardation, encephalopathy with brain atrophy and spasticity, specific facial anomalies, congenital cardiac and vascular defects, urinary malformations and hypoplastic distal phalanges of hands and multiple congenital anomalies. The genes which were not curated on different databases were then checked on ClinGen dosage sensitivity map, to understand whether the gene is pathogenic, likely pathogenic, benign or variant of unknown significance. The increased resolution of microarray technology over conventional cytogenetic analysis allows for identification of chromosomal imbalances with greater precision, accuracy, and technical sensitivity.

Age group 31-35 years showed maximum number of abnormal cases that is 28 (50%), followed by 26-30 years with 16 (29%) abnormal cases. Out of 20 couples with history of recurrent spontaneous abortions where the POC showed abnormal findings, four couples showed presence of balanced translocation of different chromosomes. Two cases were carrier for Robertsonian translocation 45,XX,der(13;14)(q10;q10) and 45,XY,der(14;21)(q10;q10), one case with balanced translocation of chromosome 2 and 16, 46,XX,t(2;16)(p25.3;p13.3) and one case with balanced translocation of chromosome 16 and 17, 46,XY,t(16:17)(p13.3;q25). One case showed paracentric inversion of chromosome 9 in one of the parent, 46,XY,inv9(q31q34.1). We observed the sex ratio as 1.3:1 between female and male fetus studied for chromosomal abnormality. The success rate of POC culture was 40.2%. Conclusion: Conventional cytogenetic provides a robust platform to study early pregnancy loss, whereas molecular cytogenetics methods have overcome the problems of tissue culture failure and provide more specific diagnostic yields. A combined approach of microarray and conventional cytogenetic methods to study POC can optimise findings of recurrent pregnancy loss. ClinGen Sequence Variant Interpretation (SVI) and the ACMG/ Association for Molecular Pathology (AMP) allowed to assess pathogenicity score of many genes not listed as phenotypically significant but when present in combination with a known dosage sensitive gene it may become pathogenic. Genetic counselling for such patients is must to understand the recurrence risk of chromosomal abnormaity in planning further pregnancies.