{"paper_id":"8ee85cd9-2d84-49fe-af40-5e6225009e31","body_text":"Recurrent pregnancy loss (RPL) is an important and\ncommon phenomenon in the reproductive system, which\naffects 2-5% of couples ( 1 ). According to the American\nSociety for Reproductive Medicine (ASRM), RPL is\ndefined as two or more consecutive pregnancy losses\nbefore 20 weeks while, minimum of three failed pregnancy\n(<20 weeks gestation) is determined by European Society of Human Reproduction and Embryology\n(ESHRE) and the Royal College of Obstetricians and\nGynecologists (RCOG) ( 2 ). RPL occurrence , a highly\nheterogeneous condition , was attributed to several causes\nincluding endocrine dysfunction, auto immune disorders, thrombophilia,\ngenetic abnormalities, infectious\ndiseases, uterine anomalies, sperm DNA fragmentation\nand epigenetics ( 3 ,  4 ). However, the reason for half of\nRPL cases is still unclear ( 1 ).\nGenetic factors such as chromosomal rearrangements\nand gene mutations are responsible for 2-5% of the defined causes of RPL ( 4 ). Chromosomal balanced structural rearrangements, mainly reciprocal and Robertsonian\ntranslocations , were identified more common in couples\nwith recurrent spontaneous abortions ( 5 ,  6 ). Also embryo chromosomal abnormalities such as, aneuploidy and\npolyploidy, were observed in 50-80% of aborted tissues,\nwhich are the most important reason for first-trimester\nspontaneous pregnancy loss ( 7 ). Nowadays, pre-implantation genetic screening (PGS) is performed to improve\nthe in vitro fertilization (IVF) success rate ( 8 ) by embryo\nchromosomal abnormalities detection.\nBefore advent of array comparative genomic hybridization (array-CGH), fluorescence in situ hybridization\n(FISH) technique was performed as screening approach\nfor over two decades. Recently, due to FISH limitations,\nit has been recommended that this technique should be\nreplaced by developed screening methods such as next\ngeneration sequencing (NGS) and array-CGH ( 9 ). Unlike FISH, array-CGH could analyze all 24 chromosomes\nand shows high accuracy for aneuploidy detection ( 10 ).\nRecent advances in NGS technology, enable to use this\ntechnique for chromosomal screening in preimplantation\nembryos ( 11 ). Moreover, using this technique is challenging due to detecting large insertions and deletions (indels)\n(>1 kb) and complex structural variations ( 12 ).\nThe present study is to find out the relation between\nchromosomal abnormality and RPL among patients referred to the Royan Reproductive Clinic (Tehran-Iran)\nfrom 2006 to 2018.\n\nThis retrospective descriptive study includes a total of\n1204 individuals (602 couples) with RPL history (more\nthan two consecutive pregnancy losses before 20 weeks\nof gestation) referring to the Royan Reproductive Clinic,\nTehran, Iran, during the period of 2006 to 2018. Informed\nconsent was obtained from all patients according to the\nRoyan Institute Ethical Committee Guidelines. The study\nwas performed in accordance with the Declaration of\nHelsinki and was approved by Institutional Review Board\nand Ethics Committees (Royan Institute: IR.ACECR.\nROYAN.REC.1397.213, Zanjan University of Medical\nSciences: ZUMS.REC.1396.182). Also, the most common RPL causes,\nsuch as hysterosalpingography, immunological tests, semen analysis, clotting assay, and blood\ntests for diabetes mellitus, hypothyroidism and infectious\ncause history were investigated for each couple.\nIn this study, severe intrauterine adhesions and Mullerian tract anomalies described as anatomical abnormalities\nin female reproduction system. Also, we categorized diabetes type II, polycystic ovarian syndrome, hypothyroidism, endometriosis and hyperprolactinemia as endocrine\nproblems Thrombophilic genetic factors such as homozygous mutations in each\nof the  MTHFR  (C677T), Factor\nV Leiden (1691G > A),  PAI-1  (4G/4G) and prothrombin\n(G20210A) genes were reported. According to ESHRE\nguideline ( 3 ), thrombophilia-related mutations were evaluated for the patients who had additional risk factors for\nthrombophilia or had a family history. Also, sperm DNA\nfragmentation index (DFI) and high DNA stainability\n(HDS) were assessed by the sperm chromatin structure\nassay (SCSA). For the determination of sperm DNA damage, we considered DFI >25% or HDS >15%.\nKaryotyping from peripheral blood lymphocytes\nwas performed for both male and female partners,\naccording to standard cytogenetic techniques ( 12 ).\nBriefly, at least 25 metaphase cells were analyzed\nfor each patient while every suspected mosaic cases\nreceived extensive work-up, additional cells were\nexamined to exclude 10% mosaicism at a 95% confidence level.\nPolymorphic rearrangements including heterochromatin variants were considered normal\nkaryotypes. Karyotypes were described according to\nthe International System for Human Cytogenetic Nomenclature criteria (ISCN) ( 13 ).\nUsing array-CGH, PGS was performed to identify embryos chromosomal\naneuploidy during assisted reproductive technology (ART) treatment.\nFollowing the long protocol ovarian stimulation ( 14 ), the mature oocytes were\nfertilized by intracytoplasmic sperm injection (ICSI) and\ncycles testing of blastomeres was performed in 3-day embryos.\nUsing array-CGH, single-cell numerical chromosomal\nabnormalities were verified that those cells removed on\nday 3 to 5 in early embryo stages. In this aim, the 24 sure\n® Microarray Pack version 3.0 (Illumina®; cat. #: PR-10-\n408702-PK, USA) was applied.\nThe array slids was scanned in InnoScan 900 microarray scanner (INNOPSYS Inc., Carbonne, France) and,\nData were analyzed using the BlueFuse Multi v3.1 software program (Illumina). Depending on the platform\nused, BlueFuse Multi software (BlueGnome Ltd, now Illumina) calculates median log 2 ratio for all the chromosomes, as the index of aneuploidy.\n\nFive hundred forty eight couples out of 602, (91.02%)\nhad a first trimester abortion experience (<13 weeks).\nAlso, the percentage of couples with ≥3 abortion was\n78.24% (the average abortion was 3.5 ± 1.6). Karyotype\nanalysis showed 8.13% (98/1204, 73 females and 25\nmales) chromosomal abnormalities in RPL patients. The\nreciprocal translocations were more frequent structural\nabnormality (2.74%) in 602 studied couples. The\nfrequency and types of chromosomal abnormalities are\nshown in Tables  1  and  2 .\nThe frequency and types of chromosomal abnormality in 602\ncouples (1204 cases)\nGynecologic structural abnormalities were identified\nin 16.77% (101/602) of the patients. Endocrine\ndisorder and thrombotic complications were observed\nin 26.07% (157/602) and 4.15% (25/602) of the\nfemales, respectively, while, sperm DNA damage\nwere detected in 14.95% of couples subjected\nto RPL ( Table 3 ).\nIn this study, only 83 couples (83/602) were undertaken\nPGS with array-CGH platform. Only the last cycle of\nPGS was considered for each couple.\nStructural chromosome abnormalities of the carrier couples with recurrent pregnancy loss (RPL)\nFrequency of factors associated with recurrent pregnancy loss (RPL) in 602 couples\nBased on the PGS-array-CGH results, of 13 abnormal karyotype couples, 20.68% (12/58) of\nanalyzed embryo were normal and all of them were transferred in 9 cycles. Finally, 33.33%\n(3/9) led to pregnancy and ended to live births. In the 70 normal karyotype couples, 70\ncycles PGS-array-CGH were performed, and 29.92% (85/284) of embryos were normal. In 72.85%\n(51/70) of cycles, embryo transfers (ETs) were carried out and 52.94% (27/51) of ETs lead\nto successful pregnancy. Noticeably, 70.37% of pregnancies was led to live births ( Fig .1 ,   Table\nS1B , See Supplementary Online Information in  www.ijfs. ir ). The\nfrequency of chromosomal abnormalities in PGSarray-CGH embryos is shown in Figure 2.\nTotally, 46 abnormal embryos were developed from\nabnormal karyotype couples; which among these, 16\nembryos (16/46-34.78%) showed a chaotic chromosomal\ncomplement. Abnormality in chromosomes 17 and 11\nwas not observed in the embryos. Also, 199 abnormal\nembryos were obtained from normal-karyotype couples;\nthe high rate of chaotic embryos is significant (45/199-\n22.61%). Also, the lowest frequencies were related to\nabnormality in chromosomes 17 (4/199-2.01%) and 11\n(6/199-3.01%).\nFlowchart of eligible subjects and their outcomes.\nPGS-array-CGH and embryos chromosomal abnormalities frequency. PGS; Pre-implantation genetic screening and Array-CGH; Array\ncomparative genomic hybridization.\n\nRPL is a multifactorial problem. Several studies were\nconducted to examine the prevalence of RPL risk factors\n(115-17). In this study, we evaluated five RPL associated\nfactors, including chromosomal abnormality, anatomical\ncharacter, endocrine, thrombotic defects and sperm DNA\ndamages. Here, we observed high frequency of Endocrine\ndisorder. Also, hypothyroidism was identified as the most\ncommon endocrine disorder, consistent with some previous\nreports ( 17 ,  18 ). The incidence of chromosomal abnormalities\nwas 15.61%, which is inconsistent with previous studies.\nThis different frequency was observed probably because of\nthe variety in sample size and nationality ( 19 ,  20 ). Here, we\nobserved translocation as a most common abnormality that\nis consistent with other investigations ( 21 ,  22 ). Noteworthy,\nthe chromosome 9 inversion was the most frequent structural\nchromosomal abnormality in the present study of, it is\nassociated with reproductive complications as described\npreviously ( 23 ,  24 ). Although, Merrion and Maisenbacher\n( 25 ) denied this association.\nPGS technology has improved the IVF success\nrate by improving embryo selection for transfer and\nsubsequently, reducing pregnancy loss. Recent molecular\ncytogenetics development, such as FISH and array-CGH,\nhave provided a rapid embryonic chromosomes screening\ntool at the preimplantation stage ( 26 ). Because of some\nlimitations, only small numbers of our participants could\nbenefit of PGS service.\nChromosome 16 disruption was observed more than\nother chromosomal abnormality in embryos of the normal\nkaryotype parents. It is consistent with previous studies\n( 21 ,  27 ).\n\nClinical examination of a large proportion of Iranian\ncouples with RPL history, indicated that hypothyroidism,\nanatomic factors and chromosomal anomalies are the major\nrisk factors for RPL phenotype. Therefore, assessment of\nthe mentioned factors would be useful for early diagnosis\nof RPL patients. Furthermore, identification of genetic\ncauses of RPL could be considered to predict the risk\nof next pregnancy loss and would assist physicians for\nprecise patient management in the clinic. Based on this\nretrospective study, it seems PGS platforms might provide\na better chance for RPL couples.","source_license":"CC-BY-4.0","license_restricted":false}