A Stepwise, Prevalence-Driven Framework to Resolve Prenatal Testing Discrepancies: Integrating Institutional Expertise and a β-Thalassemia Case Study

A Stepwise, Prevalence-Driven Framework to Resolve Prenatal Testing Discrepancies: Integrating Institutional Expertise and a β-Thalassemia Case Study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Case Report A Stepwise, Prevalence-Driven Framework to Resolve Prenatal Testing Discrepancies: Integrating Institutional Expertise and a β-Thalassemia Case Study Ahmad Anjomshoaa, Ahmad Enhesari, Shahriar Dabiri, Mahla Hoseinzadeh Mehrabi, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6400994/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Background: While direct testing in prenatal diagnosis provides superior specificity, incorporating indirect methods is also valuable. Concordant results between direct and indirect methods reinforce diagnostic accuracy, while discordant results must be carefully assessed, as they may indicate technical errors or reveal underlying biological mechanisms which would ultimately advance our understanding of inherited disorders. Despite their clinical and diagnostic significance, no standardized protocol currently exists for resolving such conflicts. Based on years of experience in prenatal testing, we have proposed a prevalence-driven, stepwise framework that accounts for both the frequency and significance of such discrepancies in prenatal diagnostics. We validated this protocol using a representative case of β-thalassemia, demonstrating its effectiveness in addressing complex diagnostic scenarios. Case Presentation: we used a systematic approach to identify a fetus genotype using a combination of direct and indirect molecular methods for β-thalassemia disease. The results of the direct molecular testing showed a heterozygous state while RFLP and haplotype analyses showed that the fetus is affected with β-thalassemia in homozygous state. After ruling out the possible causes including technical errors, maternal contamination, non-paternity, mosaicism in the placenta and chimerism, we concluded that the discrepancy was caused by a balanced recombination event within the β-globin gene cluster during parental meiosis. Direct testing results were ultimately deemed definitive due to their higher specificity and directness. Conclusions: This report underscores the necessity of addressing such inconsistencies systematically and proposes a systematic resolution framework to ensure diagnostic accuracy. By adopting the outlined approach, laboratories can confidently resolve such discrepancies and build confidence in the molecular testing process even in complex or rare cases. Prenatal Diagnosis Stepwise Protocol Discrepancy in Molecular Testing Figures Figure 1 Figure 2 Background Resolving the inconsistency between molecular testing results for prenatal diagnosis is critical for accurate genetic counseling and appropriate clinical management of the pregnancy. Molecular testing methods, including Amplification-Refractory Mutation System (ARMS-PCR) and Sanger sequencing as direct methods, and Restriction Fragment Length Polymorphism (RFLP) and short tandem repeats (STR) profiling as indirect methods, are commonly used in combination to confirm fetal genotype. However, inconsistencies between these methods can lead to diagnostic uncertainty [1, 2]. Such discrepancies are rarely reported but may arise due to the biological or technical reasons. These challenges necessitate a systematic approach to validate results, maintain laboratory accuracy, and ensure appropriate genetic counseling. Here, we present a rare case of β-thalassemia prenatal diagnosis complicated by conflicting results. While direct methods indicated a heterozygous carrier state, indirect methods suggested the fetus was affected with β-thalassemia major. Our multidisciplinary investigation revealed a rare recombination event within the β-globin gene cluster as the cause of the discrepancy. This report underscores the importance of recognizing such inconsistencies and outlines a practical resolution strategy. Case Presentation A couple, both heterozygous for the IVSI-5 (G>C) mutation in the β-globin gene, was referred to the prenatal diagnostic laboratory of Afzalipour Hospital, Kerman, Iran for prenatal diagnosis during their third pregnancy. Their first child was affected with β-thalassemia major, while the second was a carrier (Fig1A). Chorionic villus sampling (CVS) was performed at 12 weeks of gestation. The CVS sample was dissected under a stereo microscope, and DNA was extracted using salting-out method as previously described [3]. DNA was also extracted from the peripheral blood leukocytes of the parents, the affected child and the carrier sibling using the same method. ARMS-PCR identified the fetus as heterozygous for the mutation indicating carrier status. The homozygous state of the affected child and the carrier status of both parents and the sibling were simultaneously confirmed. The β -globin gene haplotypes were also determined by RFLP analysis using six RFLPs, including HindIII Gγ, HinfI β, HincII ψ β, RsaI β, AvaII β, and HindIII Aγ(Fig1B). The phase of the RFLPs was established using the homozygous offspring. AvaII β (within β globin gene) was uninformative, as all family members were +/+. Three RFLPs (RsaI β, HindIII Aγ and HinfI β) were 50% informative suggesting the fetus was either a carrier or affected. However, two RFLPs (HincII ψ β and HindIII Gγ) were 100% informative and suggested the fetus was homozygous for disease alleles (Fig1C). The RFLP pattern for the fetus mirrored that of the affected sibling, creating a discrepancy between the results of direct and indirect testing. Our proposed stepwise protocol was applied to investigate discordant results. 1. Technical validation: A comprehensive review of RFLP and ARMS procedures, reagents, and equipments was performed. DNA was re-extracted from all family members and molecular testing was repeated. Methodological errors were ruled out. ARMS-PCR results were confirmed by Sanger sequencing. 2. Maternal Cell Contamination (MCC)/Sample misidentification/non-paternity STR amplification analysis (AmpFLSTR®Identifiler®PCR kit, Applied Biosystems, USA) was conducted using genomic DNA from the parents, the affected child and the fetus to rule out MCC, sample misidentification, and confirm the paternity. Fragment sizes were analyzed using GeneMapper software and an ABI 3130 Genetic Analyzer. STR amplification analysis confirmed the absence of MCC and the presence of pure fetal DNA. STR profiling also excluded sample misidentification, contamination, and confirmed paternity. 3. Amniocentesis testing: At 16 weeks of gestation, amniocentesis was performed to exclude Confined Placental Mosaicism (CPM) and provide another line of results’ validation. Amniotic fluid-derived DNA was tested and ARMS and RFLP testing results were concordant with those from CVS. 4. Haplotype Analysis: To validate the RFLP results and examine possible recombination events within the β globin gene cluster, haplotype analysis was conducted using the HBB SegCheck™ v1.2 kit (Kowsar Biotech, Iran) designed to help identification of β -thalassemia carriers and aneuploidy of chromosomes 13, 18 and 21, simultaneously. This kit employs 12 highly polymorphic STR markers, five of which map to the 11p15.4 region encompassing β-globin gene cluster. Two markers (D11SD3.3 and D11SD11.2) map downstream of the β-globin gene, while three markers (D11SU2.9, D11SU6.1 and D11SU11) target the upstream region (Fig1B). Haplotype analysis showed that the fetal haplotype matched that of the affected sibling, corroborating the RFLP findings of homozygosity (Fig1C). Finally, a combination of direct and indirect molecular testing suggested a recombination event during the meiosis in the father (Fig1D). Multiplex ligation-dependent probe amplification (MLPA) analysis using SALSA ® MLPA ® Probemix P102 HBB (MRC, Holland) identified no deletions or duplications in β-globin gene cluster indicating that the recombination event was balanced. Direct testing results were ultimately considered definitive. Discussion Resolving discrepancies between direct and indirect prenatal molecular testing is critical to ensuring diagnostic accuracy and guiding clinical decisions. Our proposed framework, derived from two decades of institutional expertise resolving many discordant cases, addresses this challenge through a prevalence-driven, stepwise protocol that prioritizes common technical and biological errors before investigating rare genetic mechanisms. This hierarchy mirrors diagnostic workflows in resource-constrained settings, where laboratories must balance accuracy with efficiency. In the presented case, the findings suggest that during meiosis in the father, a recombination event disrupted linkage between the mutation and the polymorphic markers and masked the fetal carrier status in RFLP analysis. Such events are well-documented in the literature for regions of high homology, such as the β-globin gene cluster, where sequence similarity facilitates mispairing and non-reciprocal exchange during homologous recombination [4,5,6]. This study emphasizes the role of recombination events in the evolution of the human β-like globin genes, highlighting how such events can lead to genetic variations within this gene cluster and how multiple haplotypes can arise from a single mutation through mechanisms like recombination or gene conversion [7,8,9]. As it is essential to carefully analyze the data to resolve the discrepancy, here we outline the potential reasons and propose a practical framework to resolve such discrepancies (Fig 2). Step 1: Technical errors in molecular testing Errors in the analysis of the samples may lead to conflicting results between direct and indirect methods. The laboratory should ensure high quality DNA extraction and amplification conditions and repeat the molecular testing to rule out technical errors. If concerns regarding sample quality persist, reassessment using newly collected samples with proper controls is recommended. Step 2: Maternal contamination/Failure to correctly identify patient or specimens/ Misattributed paternity and gamete donation Maternal DNA contamination, particularly in CVS, can lead to false heterozygous results in direct molecular methods if the fetus is homozygous for a mutation. It is essential to perform a maternal contamination assay such as STR profiling to determine if maternal DNA is present in the sample [10]. The laboratory should ensure rigorous separation of maternal decidua from fetal villi during sample preparation to minimize contamination. Inaccurate patient identification, collection of biological specimens from the wrong person, specimen labeling errors and inaccurate entry or transmission and exchange of the specimens are potential dangers during the assessment of a prenatal sample. Tracing sample processing workflows is critical to exclude errors. The proper use of genetic fingerprinting methods such as STR profiling may be necessary to ensure that inaccurate patient identification and specimens mislabeling or exchange not been happened during the process of molecular testing. Discrepancies in prenatal molecular testing may arise due to misattributed paternity or the use of donor gametes as the presumed genetic relationships do not align with the actual biological relationships. To address this, it is essential to confirm the genetic relationships among the fetus, mother, and presumed father. Employing genetic fingerprinting methods, such as STR profiling, can verify these relationships and ensure accurate interpretation of test results. Additionally, obtaining detailed reproductive history, including information on the use of donor sperm or eggs, is crucial for accurate prenatal diagnosis. Step 3: Mosaicism in the placenta Confined placental mosaicism (CPM), where the placenta contains genetically distinct cell populations, and vanishing twin syndrome where one embryo dies early, leaving residual fetal DNA in the placenta may lead to discrepancies between placental DNA and fetal genotypes [11]. Confirming the fetal genotype using amniocentesis-derived samples, which directly reflect fetal DNA, is necessary to resolve these cases. Rarely, chimerism may contribute to discordant results that can be evaluated using genetic markers across multiple loci. In addition, the possibility of low-level somatic mosaicism in the fetus cannot be entirely excluded. This could lead to a mixed population of cells with differing genetic profiles, causing discordance between testing methods. Step 4: Recombination events Recombination events between parental alleles may disrupt linkage-based analyses [12,13]. Employing alternative indirect methods, such as haplotyping using microsatellite markers, can validate findings and determine the extent of recombination.Evaluating the fetal haplotype in comparison to both parents and any affected or carrier siblings will identify potential recombination events. Step 5: SNPs changing restriction sites RFLP-based analyses rely on specific restriction enzyme sites, which may be altered by benign polymorphisms unrelated to the disease mutation. Such changes can result in misinterpretation. These may mimic a recombination-like pattern and disrupt the expected inheritance pattern. It may be necessary to review the sequence around the RFLP enzyme sites to check for polymorphisms in the specific alleles being analyzed. Step 6: Micro-rearrangements in the region of interest Subtle deletions, duplications, or insertions within the evaluated region that are too small to be detected by standard testing methods might interfere with linkage analysis. Such micro-rearrangements could theoretically disrupt the relationship between haplotypes and the mutation When discrepancies arise, it is logical to follow the principle that direct detection of the mutation carries higher diagnostic confidence, provided there is no contamination, technical errors, mosaicism, or other complicating factors. If uncertainty remains, sending the sample to a reference laboratory for independent validation may provide additional reassurance. The laboratory should consult with experts to interpret conflicting results comprehensively. It is recommended to maintain comprehensive records of the case to serve as a reference for future similar cases. Each prenatal laboratory should create guidelines that outline a stepwise approach to handle inconsistencies, emphasizing systematic investigations and advanced testing. Conclusion Inconsistent results between direct and indirect molecular testing methods in prenatal diagnosis can be attributed to the biological or technical reasons. The presented case shows a rare genetic mechanism leading to conflicting results between direct and indirect prenatal molecular testing and highlights the importance of a systematic resolution framework to ensure diagnostic accuracy. By prioritizing prevalent issues, our proposed framework ensures diagnostic accuracy and builds confidence in the molecular testing process, even in complex or rare cases. Abbreviations Chorionic Villus Sampling (CVS), Amplification Refractory Mutation System-Polymerase Chain Reaction (ARMS-PCR), Restriction Fragment Length Polymorphism (RFLP), Maternal Cell Contamination (MCC), Short Tandem Repeats (STR), Confined Placental Mosaicism (CPM), Multiplex ligation-dependent probe amplification (MLPA) Declarations Ethics approval and consent to participate: Written consent was obtained from the parents for inclusion in this report and is available upon request. Consent for publication: Consent for publication has been obtained from the parents and is available upon request. Availability of data and materials: Raw data for direct sequencing, STR amplification analysis, haplotype and MLPA analysis are available upon request. Competing interests: The authors declare no conflict of interest. Funding: Prenatal diagnostic costs were covered by Public Health Insurance Company. Authors' contributions: Conceptualization: A.A.; Data curation: A.A., M.H, J.S; Formal analysis: A.A.; Investigation: A.A., A.E., S.D; Methodology: A.A., A.E., S.D, M.H, J.S; Supervision: A.A.; Software: A.A.; Writing-original draft: A.A.; Writing-review and editing: A.A; Acknowledgments: We would like to thank Afzalipour Hospital for providing the facilities to conduct prenatal diagnosis and thankfully acknowledge the clinical and laboratory teams for their invaluable contributions to this case. References Camaschella C, Serra A, Saglio G, et al . Meiotic recombination in the beta globin gene cluster causing an error in prenatal diagnosis of beta thalassaemia. J Med Genet. 1988; 25: 307-10. DOI: 10.1136/jmg.25.5.307 Lin L., Zhang Y., Pan H., et al. Inconsistencies between prenatal diagnostic and genetic testing laboratories on variant validation of rare monogenic diseases. Prenat Diagn .2024; 44: 1053-61. DOI: 10.1002/pd.6628 Saleh-Gohari N., Saeidi K. & Ziaadini-Dashtkhaki S. Haplotype Analysis in Carriers of beta-Globin Gene Mutation Facilitates Genetic Counseling in beta-Thalassemia: A Cross-Sectional Study in Kerman Province, Iran. Iran J Public Health .2020; 49: 791-9. https://www.ncbi.nlm.nih.gov/pubmed/32548060 Papadakis M.N. & Patrinos G.P. Contribution of gene conversion in the evolution of the human beta-like globin gene family. Hum Genet .1999; 104: 117-25. DOI: 10.1007/s004390050923 Borg J, Georgitsi M, Aleporou-Marinou V, et al. Genetic recombination as a major cause of mutagenesis in the human globin gene clusters. Clin Biochem. 2009;42:1839-50. DOI: 10.1016/j.clinbiochem.2009.07.014 Gerhard D.S., Kidd K.K., Kidd J.R., et al. Identification of a recent recombination event within the human beta-globin gene cluster. Proc Natl Acad Sci U S A . 1984; 81: 7875-9. DOI: 10.1038/nrg2193 Smith R.A., Ho P.J., Clegg J.B., et al. Recombination breakpoints in the human beta-globin gene cluster. Blood .1998; 92: 4415-21. https://www.ncbi.nlm.nih.gov/pubmed/9834248 Chen J.M., Cooper D.N., Chuzhanova N, et al. Gene conversion: mechanisms, evolution and human disease. Nat Rev Genet. 2007;8: 762-75. DOI: 10.1038/nrg2193 Pirastu M., Galanello R., Doherty M.A., et al. The same beta-globin gene mutation is present on nine different beta-thalassemia chromosomes in a Sardinian population. Proc Natl Acad Sci U S A .1987;84: 2882-5. DOI: 10.1073/pnas.84.9.2882 Nagan N., Faulkner N.E., Curtis C., et al. Laboratory guidelines for detection, interpretation, and reporting of maternal cell contamination in prenatal analyses a report of the association for molecular pathology. J Mol Diagn. 2011; 13: 7-11. DOI: 10.1016/j.jmoldx.2010.11.013 Hayata K., Hiramatsu Y., Masuyama H., et al. Discrepancy between Non-invasive Prenatal Genetic Testing (NIPT) and Amniotic Chromosomal Test due to Placental Mosaicism: A Case Report and Literature Review. Acta Med Okayama. 2017; 71: 181-5. DOI: 10.18926/AMO/54988 Old J.M., Heath C., Fitches A., et al. Meiotic recombination between two polymorphic restriction sites within the beta globin gene cluster. J Med Genet .1986; 23: 14-8. DOI: 10.1136/jmg.23.1.14 Zago M.A., Silva W.A., Jr., Gualandro S., et al. Rearrangements of the beta-globin gene cluster in apparently typical betaS haplotypes. Haematologica .2001; 86: 142-5. https://www.ncbi.nlm.nih.gov/pubmed/11224482 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviewers invited by journal 06 May, 2025 Editor invited by journal 10 Apr, 2025 Editor assigned by journal 09 Apr, 2025 Submission checks completed at journal 09 Apr, 2025 First submitted to journal 08 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6400994","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Case Report","associatedPublications":[],"authors":[{"id":453705522,"identity":"37c31140-152c-4997-a166-74c6d795591b","order_by":0,"name":"Ahmad Anjomshoaa","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3UlEQVRIiWNgGAWjYHCCBCA+AMTMh6ECjA3EamFLJloLA1QLjzFxruKf3fDw0Y0/d+T42898NrrZxiDP38Dc9gGfFok7B5KNc9ueGUucyd2cnNvGYDjjAGPzDLzW3EhIk85tOJzYcCB382GgFsYNDIzNeHXIg7Tk/DmcOP/8m8cgLfYEtRiAtbAdTtxwI4cZ5LBEgloMbySA/HLY2PDGM2PjnHMSyTMOE9AidyMn8THQYXJy55MfS+eU2dj2t7c/xqsFGB0JyDwJYCogoIGBgf0AQSWjYBSMglEwwgEAi25OBQYMV5sAAAAASUVORK5CYII=","orcid":"","institution":"Kerman University of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Ahmad","middleName":"","lastName":"Anjomshoaa","suffix":""},{"id":453705523,"identity":"c571c1f6-ef7c-4ac3-9842-207f1b5e5585","order_by":1,"name":"Ahmad Enhesari","email":"","orcid":"","institution":"Kerman University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Ahmad","middleName":"","lastName":"Enhesari","suffix":""},{"id":453705524,"identity":"1524ac38-c00d-4de1-849f-4549957e4743","order_by":2,"name":"Shahriar Dabiri","email":"","orcid":"","institution":"Kerman University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Shahriar","middleName":"","lastName":"Dabiri","suffix":""},{"id":453705525,"identity":"6fbcc1a5-fe48-403e-b3ae-93234340dc62","order_by":3,"name":"Mahla Hoseinzadeh Mehrabi","email":"","orcid":"","institution":"Kerman University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Mahla","middleName":"Hoseinzadeh","lastName":"Mehrabi","suffix":""},{"id":453705526,"identity":"a59cd231-1824-4b1e-a20c-4d6010ef8fbe","order_by":4,"name":"Javad Shokrian","email":"","orcid":"","institution":"Kerman University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Javad","middleName":"","lastName":"Shokrian","suffix":""}],"badges":[],"createdAt":"2025-04-08 08:23:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6400994/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6400994/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82560927,"identity":"5b526534-0b1f-4a44-8b61-e09727cb757c","added_by":"auto","created_at":"2025-05-13 01:37:29","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":457721,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA beta-thalassemia PND case with discordant results evaluated according to our proposed framework. 1A) \u003c/strong\u003eFamily pedigree. \u003cstrong\u003e1B) \u003c/strong\u003eMap of beta-globin gene cluster, RFLP restriction sites and STRs used for haplotype analysis. \u003cstrong\u003e1C) \u003c/strong\u003eDetected haplotypes based on the RFLP and haplotype analysis. Inherited pattern of the fetus and the affected sibling is the same. \u003cstrong\u003e1D) \u003c/strong\u003eRecombination event in parental meiosis. Different haplotypes are shown with different colors with the approximate breaking point of the recombination event shown as a dotted line in the father.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6400994/v1/3cb34d747bf246d4e937f675.png"},{"id":82560931,"identity":"7d8eca01-b3fe-4242-9734-4917e29128b7","added_by":"auto","created_at":"2025-05-13 01:37:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":310814,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe proposed systematic framework to resolve prenatal testing discrepancies\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6400994/v1/b6f2697ffcf6772fc6f2f136.png"},{"id":82562494,"identity":"11563fc5-7527-4b8f-b7f4-646e834051c3","added_by":"auto","created_at":"2025-05-13 01:45:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1836642,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6400994/v1/e9537e8a-a316-4a8d-b972-f629cfa3b811.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"A Stepwise, Prevalence-Driven Framework to Resolve Prenatal Testing Discrepancies: Integrating Institutional Expertise and a β-Thalassemia Case Study","fulltext":[{"header":"Background","content":"\u003cp\u003eResolving the inconsistency between molecular testing results for prenatal diagnosis is critical for accurate genetic counseling and appropriate clinical management of the pregnancy. Molecular testing methods, including Amplification-Refractory Mutation System (ARMS-PCR) and Sanger sequencing as direct methods, and Restriction Fragment Length Polymorphism (RFLP) and short tandem repeats (STR) profiling as indirect methods, are commonly used in combination to confirm fetal genotype. However, inconsistencies between these methods can lead to diagnostic uncertainty [1, 2].\u003c/p\u003e\n\u003cp\u003eSuch discrepancies are rarely reported but may arise due to the biological or technical reasons. These challenges necessitate a systematic approach to validate results, maintain laboratory accuracy, and ensure appropriate genetic counseling. Here, we present a rare case of \u0026beta;-thalassemia prenatal diagnosis complicated by conflicting results. \u0026nbsp;While direct methods indicated a heterozygous carrier state, indirect methods suggested the fetus was affected with \u0026beta;-thalassemia major. \u0026nbsp;Our multidisciplinary investigation revealed a rare recombination event within the \u0026beta;-globin gene cluster as the cause of the discrepancy. This report underscores the importance of recognizing such inconsistencies and outlines a practical resolution strategy.\u003c/p\u003e"},{"header":"Case Presentation","content":"\u003cp\u003eA couple, both heterozygous for the IVSI-5 (G\u0026gt;C) mutation in the β-globin gene, was referred to the prenatal diagnostic laboratory of Afzalipour Hospital, Kerman, Iran for prenatal diagnosis during their third pregnancy. Their first child was affected with β-thalassemia major, while the second was a carrier (Fig1A). Chorionic villus sampling (CVS) was performed at 12 weeks of gestation.\u0026nbsp;The CVS sample was dissected under a stereo microscope, and DNA was extracted using salting-out method as previously described [3]. DNA was also extracted from the peripheral blood leukocytes of the parents, the affected child and the carrier sibling using the same method. ARMS-PCR identified the fetus as heterozygous for the mutation indicating carrier status. \u0026nbsp;The homozygous state of the affected child and the carrier status of both parents and the sibling were simultaneously confirmed.\u003c/p\u003e\n\u003cp\u003eThe β -globin gene haplotypes were also determined by RFLP analysis using six RFLPs, including HindIII Gγ, HinfI β, HincII ψ β, RsaI β, AvaII β, and HindIII Aγ(Fig1B). The phase of the RFLPs was established using the homozygous offspring. AvaII β (within β globin gene) was uninformative, as all family members were +/+. Three RFLPs (RsaI β, HindIII Aγ and HinfI β) were 50% informative suggesting the fetus was either a carrier or affected. \u0026nbsp;However, two RFLPs (HincII ψ β and HindIII Gγ) were 100% informative and suggested the fetus was homozygous for disease alleles (Fig1C). The RFLP pattern for the fetus mirrored that of the affected sibling, creating a discrepancy between the results of direct and indirect testing.\u0026nbsp;Our proposed stepwise protocol was applied to investigate discordant results. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1. \u0026nbsp; Technical validation:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA comprehensive review of RFLP and ARMS procedures, reagents, and equipments was performed. DNA was re-extracted from all family members and molecular testing was repeated. Methodological errors were ruled out. ARMS-PCR results were confirmed by Sanger sequencing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. Maternal Cell Contamination (MCC)/Sample misidentification/non-paternity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSTR amplification analysis (AmpFLSTR®Identifiler®PCR kit, Applied Biosystems, USA) was conducted using genomic DNA from the parents, the affected child and the fetus to rule out MCC, sample misidentification, and confirm the paternity. Fragment sizes were analyzed using GeneMapper software and an ABI 3130 Genetic Analyzer. STR amplification analysis confirmed the absence of MCC and the presence of pure fetal DNA. STR profiling also excluded sample misidentification, contamination, and confirmed paternity. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. Amniocentesis testing:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt 16 weeks of gestation, amniocentesis was performed to exclude Confined Placental Mosaicism (CPM) and provide another line of results’ validation. Amniotic fluid-derived DNA was tested and ARMS and RFLP testing results were concordant with those from CVS.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4. Haplotype Analysis:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo validate the RFLP results and examine possible recombination events within the β globin gene cluster, haplotype analysis was conducted using the HBB SegCheck™ v1.2 kit (Kowsar Biotech, Iran) designed to help identification of β -thalassemia carriers and aneuploidy of chromosomes 13, 18 and 21, simultaneously. This kit employs 12 highly polymorphic STR markers, five of which map to the 11p15.4 region encompassing β-globin gene cluster. Two markers (D11SD3.3 and D11SD11.2) map downstream of the β-globin gene, while three markers (D11SU2.9, D11SU6.1 and D11SU11) target the upstream region (Fig1B). Haplotype analysis showed that the fetal haplotype matched that of the affected sibling, corroborating the RFLP findings of homozygosity (Fig1C). Finally, a combination of direct and indirect molecular testing suggested a recombination event during the meiosis in the father (Fig1D). Multiplex ligation-dependent probe amplification (MLPA) analysis using SALSA\u003csup\u003e®\u003c/sup\u003e MLPA\u003csup\u003e®\u003c/sup\u003e Probemix P102 HBB (MRC, Holland) identified no deletions or duplications in β-globin gene cluster indicating that the recombination event was balanced. Direct testing results were ultimately considered definitive.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eResolving discrepancies between direct and indirect prenatal molecular testing is critical to ensuring diagnostic accuracy and guiding clinical decisions. Our proposed framework, derived from two decades of institutional expertise resolving many discordant cases, addresses this challenge through a prevalence-driven, stepwise protocol that prioritizes common technical and biological errors before investigating rare genetic mechanisms. This hierarchy mirrors diagnostic workflows in resource-constrained settings, where laboratories must balance accuracy with efficiency. In the presented case, the findings suggest that during meiosis in the father, a recombination event disrupted linkage between the mutation and the polymorphic markers and masked the fetal carrier status in RFLP analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSuch events are well-documented in the literature for regions of high homology, such as the \u0026beta;-globin gene cluster, where sequence similarity facilitates mispairing and non-reciprocal exchange during homologous recombination [4,5,6]. This study emphasizes the role of recombination events in the evolution of the human \u0026beta;-like globin genes, highlighting how such events can lead to genetic variations within this gene cluster and how multiple haplotypes can arise from a single mutation through mechanisms like recombination or gene conversion [7,8,9].\u003c/p\u003e\n\u003cp\u003eAs it is essential to carefully analyze the data to resolve the discrepancy, here we outline the\u0026nbsp;\u003c/p\u003e\n\u003cp\u003epotential reasons and propose a practical framework to resolve such discrepancies (Fig 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStep 1: Technical errors in molecular testing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eErrors in the analysis of the samples may lead to conflicting results between direct and indirect methods. The laboratory should ensure high quality DNA extraction and amplification conditions and repeat the molecular testing to rule out technical errors. \u0026nbsp;If concerns regarding sample quality persist, reassessment using newly collected samples with proper controls is recommended.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStep 2: Maternal contamination/Failure to correctly identify patient or specimens/ Misattributed paternity and gamete donation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMaternal DNA contamination, particularly in CVS, can lead to false heterozygous results in direct molecular methods if the fetus is homozygous for a mutation. It is essential to perform a maternal contamination assay such as STR profiling to determine if maternal DNA is present in the sample [10]. The laboratory should ensure rigorous separation of maternal decidua from fetal villi during sample preparation to minimize contamination.\u003c/p\u003e\n\u003cp\u003eInaccurate patient identification, collection of biological specimens from the wrong person, specimen labeling errors and inaccurate entry or transmission and exchange of the specimens are potential dangers during the assessment of a prenatal sample. Tracing sample processing workflows is critical to exclude errors. The proper use of genetic fingerprinting methods such as STR profiling may be necessary to ensure that inaccurate patient identification and specimens mislabeling or exchange not been happened during the process of molecular testing.\u003c/p\u003e\n\u003cp\u003eDiscrepancies in prenatal molecular testing may arise due to misattributed paternity or the use of donor gametes as the presumed genetic relationships do not align with the actual biological relationships. To address this, it is essential to confirm the genetic relationships among the fetus, mother, and presumed father. Employing genetic fingerprinting methods, such as STR profiling, can verify these relationships and ensure accurate interpretation of test results. Additionally, obtaining detailed reproductive history, including information on the use of donor sperm or eggs, is crucial for accurate prenatal diagnosis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStep 3: Mosaicism in the placenta\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConfined placental mosaicism (CPM), where the placenta contains genetically distinct cell populations, and vanishing twin syndrome where one embryo dies early, leaving residual fetal DNA in the placenta may lead to discrepancies between placental DNA and fetal genotypes [11]. Confirming the fetal genotype using amniocentesis-derived samples, which directly reflect fetal DNA, is necessary to resolve these cases. Rarely, chimerism may contribute to discordant results that can be evaluated using genetic markers across multiple loci. \u0026nbsp;In addition, the possibility of low-level somatic mosaicism in the fetus cannot be entirely excluded. This could lead to a mixed population of cells with differing genetic profiles, causing discordance between testing methods.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStep 4: Recombination events\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRecombination events between parental alleles may disrupt linkage-based analyses [12,13]. Employing alternative indirect methods, such as haplotyping using microsatellite markers, can validate findings and determine the extent of recombination.Evaluating the fetal haplotype in comparison to both parents and any affected or carrier siblings will identify potential recombination events.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStep 5: SNPs changing restriction sites\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRFLP-based analyses rely on specific restriction enzyme sites, which may be altered by benign polymorphisms unrelated to the disease mutation. Such changes can result in misinterpretation. These may mimic a recombination-like pattern and disrupt the expected inheritance pattern. It may be necessary to review the sequence around the RFLP enzyme sites to check for polymorphisms in the specific alleles being analyzed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStep 6: Micro-rearrangements in the region of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSubtle deletions, duplications, or insertions within the evaluated region that are too small to be detected by standard testing methods might interfere with linkage analysis. Such micro-rearrangements could theoretically disrupt the relationship between haplotypes and the mutation\u003c/p\u003e\n\u003cp\u003eWhen discrepancies arise, it is logical to follow the principle that direct detection of the mutation carries higher diagnostic confidence, provided there is no contamination, technical errors, mosaicism, or other complicating factors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIf uncertainty remains, sending the sample to a reference laboratory for independent validation may provide additional reassurance. The laboratory should consult with experts to interpret conflicting results comprehensively. \u0026nbsp;It is recommended to maintain comprehensive records of the case to serve as a reference for future similar cases. Each prenatal laboratory should create guidelines that outline a stepwise approach to handle inconsistencies, emphasizing systematic investigations and advanced testing.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eInconsistent results between direct and indirect molecular testing methods in prenatal diagnosis can be attributed to the biological or technical reasons. The presented case shows a rare genetic mechanism leading to conflicting results between direct and indirect prenatal molecular testing and highlights the importance of a systematic resolution framework to ensure diagnostic accuracy. By prioritizing prevalent issues, our proposed framework ensures diagnostic accuracy and builds confidence in the molecular testing process, even in complex or rare cases.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eChorionic Villus Sampling (CVS), Amplification Refractory Mutation System-Polymerase Chain Reaction (ARMS-PCR), Restriction Fragment Length Polymorphism (RFLP), Maternal Cell Contamination (MCC), Short Tandem Repeats (STR), Confined Placental Mosaicism (CPM), Multiplex ligation-dependent probe amplification (MLPA)\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u0026nbsp;\u003c/strong\u003eWritten consent was obtained from the parents for\u0026nbsp;\u003c/p\u003e\n\u003cp\u003einclusion in this report and is available upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e Consent for publication has been obtained from the parents and is available upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u0026nbsp;\u003c/strong\u003eRaw data for direct sequencing, STR amplification analysis, haplotype and MLPA analysis are available upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e The authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e Prenatal diagnostic costs were covered by Public Health Insurance Company.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions:\u0026nbsp;\u003c/strong\u003eConceptualization: A.A.;\u0026nbsp;Data curation: A.A., M.H, J.S;\u0026nbsp;Formal analysis: A.A.; Investigation: A.A., A.E., S.D; Methodology: A.A., A.E., S.D, M.H, J.S; Supervision: A.A.; Software: A.A.; Writing-original draft: A.A.; Writing-review and editing: A.A;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u0026nbsp;\u003c/strong\u003eWe would like to thank Afzalipour Hospital for providing the facilities to conduct prenatal diagnosis and thankfully acknowledge the clinical and laboratory teams for their invaluable contributions to this case.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCamaschella C, Serra A, Saglio G, et al . Meiotic recombination in the beta globin gene cluster causing an error in prenatal diagnosis of beta thalassaemia.\u003cem\u003e J Med Genet.\u003c/em\u003e 1988; 25: 307-10. DOI: 10.1136/jmg.25.5.307\u003c/li\u003e\n\u003cli\u003eLin L., Zhang Y., Pan H., et al. Inconsistencies between prenatal diagnostic and genetic testing laboratories on variant validation of rare monogenic diseases. \u003cem\u003ePrenat Diagn\u003c/em\u003e.2024; 44: 1053-61. DOI: 10.1002/pd.6628 \u003c/li\u003e\n\u003cli\u003eSaleh-Gohari N., Saeidi K. \u0026amp; Ziaadini-Dashtkhaki S. Haplotype Analysis in Carriers of beta-Globin Gene Mutation Facilitates Genetic Counseling in beta-Thalassemia: A Cross-Sectional Study in Kerman Province, Iran. \u003cem\u003eIran J Public Health\u003c/em\u003e.2020; 49: 791-9. https://www.ncbi.nlm.nih.gov/pubmed/32548060\u003c/li\u003e\n\u003cli\u003ePapadakis M.N. \u0026amp; Patrinos G.P. Contribution of gene conversion in the evolution of the human beta-like globin gene family. \u003cem\u003eHum Genet\u003c/em\u003e.1999; 104: 117-25. DOI: 10.1007/s004390050923\u003c/li\u003e\n\u003cli\u003eBorg J, Georgitsi M, Aleporou-Marinou V, et al. Genetic recombination as a major cause of mutagenesis in the human globin gene clusters. \u003cem\u003eClin Biochem.\u003c/em\u003e 2009;42:1839-50. DOI: 10.1016/j.clinbiochem.2009.07.014\u003c/li\u003e\n\u003cli\u003eGerhard D.S., Kidd K.K., Kidd J.R., et al. Identification of a recent recombination event within the human beta-globin gene cluster. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e. 1984; 81: 7875-9. DOI: 10.1038/nrg2193\u003c/li\u003e\n\u003cli\u003eSmith R.A., Ho P.J., Clegg J.B., et al. Recombination breakpoints in the human beta-globin gene cluster. \u003cem\u003eBlood\u003c/em\u003e.1998; 92: 4415-21. https://www.ncbi.nlm.nih.gov/pubmed/9834248\u003c/li\u003e\n\u003cli\u003eChen J.M., Cooper D.N., Chuzhanova N, et al. Gene conversion: mechanisms, evolution and human disease.\u003cem\u003e Nat Rev Genet.\u003c/em\u003e 2007;8: 762-75. DOI: 10.1038/nrg2193\u003c/li\u003e\n\u003cli\u003ePirastu M., Galanello R., Doherty M.A., et al. The same beta-globin gene mutation is present on nine different beta-thalassemia chromosomes in a Sardinian population. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e .1987;84: 2882-5. DOI: 10.1073/pnas.84.9.2882\u003c/li\u003e\n\u003cli\u003eNagan N., Faulkner N.E., Curtis C., et al. Laboratory guidelines for detection, interpretation, and reporting of maternal cell contamination in prenatal analyses a report of the association for molecular pathology. \u003cem\u003eJ Mol Diagn.\u003c/em\u003e2011; 13: 7-11. DOI: 10.1016/j.jmoldx.2010.11.013\u003c/li\u003e\n\u003cli\u003eHayata K., Hiramatsu Y., Masuyama H., et al. Discrepancy between Non-invasive Prenatal Genetic Testing (NIPT) and Amniotic Chromosomal Test due to Placental Mosaicism: A Case Report and Literature Review. \u003cem\u003eActa Med Okayama.\u003c/em\u003e2017; 71: 181-5. DOI: 10.18926/AMO/54988\u003c/li\u003e\n\u003cli\u003eOld J.M., Heath C., Fitches A., et al. Meiotic recombination between two polymorphic restriction sites within the beta globin gene cluster.\u003cem\u003e J Med Genet\u003c/em\u003e.1986; 23: 14-8. DOI: 10.1136/jmg.23.1.14\u003c/li\u003e\n\u003cli\u003eZago M.A., Silva W.A., Jr., Gualandro S., et al. Rearrangements of the beta-globin gene cluster in apparently typical betaS haplotypes. \u003cem\u003eHaematologica\u003c/em\u003e.2001; 86: 142-5. https://www.ncbi.nlm.nih.gov/pubmed/11224482\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-pregnancy-and-childbirth","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"prch","sideBox":"Learn more about [BMC Pregnancy and Childbirth](http://bmcpregnancychildbirth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/prch/default.aspx","title":"BMC Pregnancy and Childbirth","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Prenatal Diagnosis, Stepwise Protocol, Discrepancy in Molecular Testing ","lastPublishedDoi":"10.21203/rs.3.rs-6400994/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6400994/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e While direct testing in prenatal diagnosis provides superior specificity, incorporating indirect methods is also valuable. Concordant results between direct and indirect methods reinforce diagnostic accuracy, while discordant results must be carefully assessed, as they may indicate technical errors or reveal underlying biological mechanisms which would ultimately advance our understanding of inherited disorders. Despite their clinical and diagnostic significance, no standardized protocol currently exists for resolving such conflicts. Based on years of experience in prenatal testing, we have proposed a prevalence-driven, stepwise framework that accounts for both the frequency and significance of such discrepancies in prenatal diagnostics. We validated this protocol using a representative case of β-thalassemia, demonstrating its effectiveness in addressing complex diagnostic scenarios.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCase Presentation:\u003c/strong\u003e we used a systematic approach to identify a fetus genotype using a combination of direct and indirect molecular methods for β-thalassemia disease. The results of the direct molecular testing showed a heterozygous state while RFLP and haplotype analyses showed that the fetus is affected with β-thalassemia in homozygous state. After ruling out the possible causes including technical errors, maternal contamination, non-paternity, mosaicism in the placenta and chimerism, we concluded that the discrepancy was caused by a balanced recombination event within the β-globin gene cluster during parental meiosis. Direct testing results were ultimately deemed definitive due to their higher specificity and directness.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e This report underscores the necessity of addressing such inconsistencies systematically and proposes a systematic resolution framework to ensure diagnostic accuracy. By adopting the outlined approach, laboratories can confidently resolve such discrepancies and build confidence in the molecular testing process even in complex or rare cases.\u003c/p\u003e","manuscriptTitle":"A Stepwise, Prevalence-Driven Framework to Resolve Prenatal Testing Discrepancies: Integrating Institutional Expertise and a β-Thalassemia Case Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-13 01:37:25","doi":"10.21203/rs.3.rs-6400994/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2025-05-06T05:56:52+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-04-10T08:17:23+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-10T00:47:37+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-10T00:46:57+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Pregnancy and Childbirth","date":"2025-04-08T08:19:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-pregnancy-and-childbirth","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"prch","sideBox":"Learn more about [BMC Pregnancy and Childbirth](http://bmcpregnancychildbirth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/prch/default.aspx","title":"BMC Pregnancy and Childbirth","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4d134587-9f12-4fda-8353-a32439d2c055","owner":[],"postedDate":"May 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-05-13T01:37:25+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-13 01:37:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6400994","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6400994","identity":"rs-6400994","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}