Application and value of multi-technique integration in the prenatal diagnosis of mosaicism derived of ICSI blastocyst

Application and value of multi-technique integration in the prenatal diagnosis of mosaicism derived of ICSI blastocyst | 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 Research Article Application and value of multi-technique integration in the prenatal diagnosis of mosaicism derived of ICSI blastocyst Yun Huang, Shuping Zhang, Peng Zou, Limin Huang, Xiaofeng Li, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8634923/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Background. To investigate the antenatal diagnosis of a pregnant fetus after intracytoplasmic sperm injection (ICSI) and blastocyst transplantation. By integrating multiple technologies, we clarified the specific characteristics of the 20p mosaic duplication, evaluated its pathogenicity and clinical significance, and provided evidence for prenatal genetic counseling. Methods. Amniocentesis was performed on a pregnant woman identified as high-risk by non-invasive prenatal testing (NIPT) suggesting a 15q26.3 deletion.Fetal amniotic fluid exfoliative cells were analyzed using an integrated approach combining G-banding karyotyping, chromosomal microarray analysis (CMA), and fluorescence in situ hybridization (FISH). Results. NIPT showed a 3.04 Mb deletion at 15q26.3.CMA showed a 20.51 Mb mosaic duplication in the 20p13p11.23 region (approximately 50% mosaicism),while G-banding karyotype analysis showed no abnormality in amniotic fluid cells.Mid-term FISH analysis of amniotic fluid cells in our hospital showed a der(15)t(15;20)(qter;p13) mosaicism, meaning that a portion of the 20p13 segment was translocated to the end of chromosome 15. FISH analysis from outer hospital indicated that 49% of cells carried a 20p13 duplication signal. Both parents had normal peripheral blood karyotypes. Conclusion. This case is a complex chromosomal rearrangement in the early cleavage stage of ICSI blastocyst transfer. The high proportion of 20p13p11.23 mosaic duplication has potential serious pathogenicity, and the risk of poor prognosis in continued pregnancy is high. Termination of pregnancy is a reasonable decision. This study highlights the core value of multi-technology integration in the prenatal diagnosis of complex chromosomal abnormalities after ICSI. At the same time, it is suggested that couples with abnormal sperm parameters such as male UU infection, abnormal sperm morphology, or a history of repeated fertilization/implantation failure should be alert to the risk of mosaic chromosomal abnormalities. Strengthening preimplantation genetic testing (PGT) protocols before assisted reproduction is recommended, providing a reference for optimizing prenatal diagnosis strategies in ICSI-assisted pregnancies. ICSI blastocyst Chromosome structural rearrangement Chromosome microarray analysis 20p mosaic duplication prenatal diagnosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction As the core technology of assisted reproduction, intracytoplasmic sperm injection (ICSI) provides the possibility of fertility for infertile families, but the process of in vitro operation and culture of embryos may increase the risk of chromosomal abnormalities, especially causing mosaic structural rearrangement, which poses challenges to prenatal diagnosis [ 1 ] . Prenatal diagnosis is an important gateway to prevent birth defects. Non-Invasive Prenatal Testing (NIPT) has been widely used in clinical practice as an effective method to screen fetal aneuploidy. However, its detection efficacy for micro-deletions/micro-duplications and complex structural rearrangements is limited, and its positive predictive value is low. Abnormal results need to be verified by interventional diagnosis [ 2 ] . In interventional prenatal diagnosis, G banding karyotype analysis has low resolution and is difficult to identify minor or low-proportion mosaic abnormalities; The detection range of Fluorescence in situ hybridization (FISH) is limited to preset targets; Chromosome microarray analysis (CMA) can detect genome-wide copy number variation (CNV) at the submicroscopic level, with a resolution of 10-100kb, and can also identify homozygous regions. It has become a first-line diagnostic technology for high-risk populations [ 3 ] . The short arm of human chromosome 20 (20p) carries several key genes, such as those involved in neural development and metabolic regulation, and its fragment abnormalities are closely related to a variety of clinical phenotypes [ 4 ] .From the perspective of chimerism ratio, according to the group standard of "Prenatal Diagnosis and Genetic Counseling of Chromosomal Mosaicism" [ 5 ] ,when the chimerism ratio exceeds 30%, the impact of abnormal cell lines on fetal phenotype is significantly increased, and the clinical prognosis Increased risk of poor. This paper reports a pregnant fetus with 15q26. 3 deletion after ICSI blastocyst transplantation. The complicated case of 20p13p11. 23 mosaic duplication was evaluated by NIPT, CMA, FISH and other integrated diagnosis techniques. It provides basis for clinical decision-making and genetic counseling, and also provides reference for optimizing prenatal diagnosis strategy after ICSI. Materials and methods 1.1 Research Subjects The couple failed their first in vitro fertilization (IVF) treatment in 2024 due to the man's history of Ureaplasma urealyticum (UU) infection, abnormal sperm morphology, and the woman's ovarian dysfunction. After comprehensive evaluation, considering that the man's sperm factor may affect fertilization and embryonic development, he chose to receive intracytoplasmic sperm injection (ICSI) technology for assisted pregnancy. One blastocyst was obtained in the second ICSI cycle and a successful pregnancy was achieved after transplantation. 1.2 Auxiliary inspection At the 13th week of pregnancy, the thickness of the nuchal transparent layer was 1.4 mm in the normal range, but the placenta previa was low; No abnormalities were found in the ultrasound during second trimester of pregnancy. Prenatal serological screening: There was no abnormality in the early Down syndrome screening at 13 +1 weeks of pregnancy, and no serological screening at the middle stage; At 13 weeks of gestation, non-invasive prenatal genetic testing (NIPT) was performed, and the screening suggested a high risk of 3.04 Mb deletion in the 15q26. 3 region, as shown in Fig. 1. In order to further confirm the diagnosis, amniocentesis was performed at 18 weeks of gestation, and 20mL amniotic fluid was collected for related tests. Fetal 3-D color Doppler ultrasound was performed at 21 weeks of gestation, and no serious structural deformities were found in the fetus. 1.3 Experimental Methods 1.3. 1 Specimen collection Before assisted pregnancy, the peripheral blood chromosome karyotype analysis was carried out for reproductive assisted pregnant couples. After full informed consent during pregnancy, 20mL amniotic fluid was extracted for chromosome karyotype analysis and 10mL for chromosome microarray analysis. As the results of chromosome microarray analysis showed that there were partial fragmental duplications in the short arm of chromosome 20, on the premise of full informed consent, 2mL peripheral blood of both pregnant women and couples was extracted for fetal chromosome microarray pedigree analysis. The study was approved by the Ethics Committee of Prenatal Diagnostic Techniques of our hospital. 1.3. 2 Cell culture and karyotype analysis After centrifugation, 1 ~ 2mL of amniotic fluid was inoculated into amniotic fluid cell culture medium. After standard methods of culture, fluid exchange, passage, harvesting, chromosome preparation and G-banding karyotype analysis, the number of more than 20 mitotic phases was counted in double-person and double-line, and at least 5 metaphase karyotype mitotic phases were analyzed. The karyotype description was carried out according to the "International Nomenclature System of Human Cytogenetics" (ISCN2020). The diagnostic criteria for detecting chimerism were carried out according to "Prenatal Genetic Diagnosis and Genetic Counseling for Chromosomal Chimerism". 1.3. 2 Chromosome microarray analysis (CMA) 10 mL amniotic fluid samples with qualified quality control were collected aseptically, and the amniotic fluid genomic DNA was extracted using QIAamp ® DNA Blood Mini Kit (QIAGEN, Germany). The genomic DNA was digested into short fragments by enzyme, then amplified by PCR and purified by magnetic bead method. The purified product was fragmented into 25-125bp fragments and labeled with biotin. After the product was mixed with the hybridization solution for denaturation, the chip hybridization, washing and staining were carried out, and the chip scan was used to detect DNA copy number variation (CNV) using the Affymetrix CytoScan 750K chip platform (Thermo Scientific, USA). 1.3. 3 Detection of chromosome fluorescence in situ hybridization (FISH) in amniotic fluid cells Metaphase cells were detected using 20q12 (red)/20p13 (green) specific probes and 15q24. 1 (red)/17q21 (green) specific site DNA probes (both from Wuhan Kanglu Co., Ltd.), and chromosomes 20 and 15 were analyzed. All operations were performed in accordance with the FISH procedure, including: dropping the cell suspension onto the glass slide, aging, adding probes, hybridization overnight, elution, staining, and microscopic examination. S500-24 in situ hybridization instrument (Thermo Company, USA was used for hybridization, and the multi-color fluorescence image processing and analysis were performed with a fluorescence microscope (model: BX51, from Olympus Company, Japan). 1.3. 4 Sperm FISH detection Under aseptic conditions, fresh semen samples of 1-2ml were taken, and 20q12 (red)/20p13 (green) specific site DNA probes (Wuhan Kanglu Co., Ltd.) were used to analyze chromosome 20. All operations were performed according to the FISH operation procedure, including: sperm liquefaction, hypotonic, prefixation, fixation,dropping the cell suspension onto the glass slide, aging, alkaline expansion, addition of probes, overnight hybridization, elution, staining, and microscopic examination. The remaining steps are the same as 1.3. 3. 1.3. 5 Data analysis All detection data of chromosome microarrays were processed and analyzed using Chromosome AnalysisSuite (ChAS) V4.3 software, which is capable of detecting clinically relevant genes and copy number variants (CNVS) and homozygous regions (LOH) with genomic resolutions greater than 100 kb. The My POD Finder software (from the Swedish Clinical Genetics in Lund development software) was used for analysis of family genotypes and for evaluation of duplicate sources. Refer to the 2021 "Technical Standards for Interpretation and Reporting of Primary Copy Number Variations: Common Consensus Recommendations of the American College of Medical Genetics and Genomics (ACMG) and Clinical Genome Resources (ClinGen)" [6] And "Guidelines for the Application of Chromosome Microarray Analysis in Prenatal Diagnosis (2023)" [7] to evaluate copy number variants and homozygous regions. Results 2.1 Karyotype analysis of amniotic fluid cells The chromosomal karyotype of amniotic fluid cells in pregnant women was 46, XN by counting 20 mitotic phases and analyzing 5-10 karyotypes. In addition, the results of karyotype analysis of peripheral blood chromosomes of both spouses were normal (Fig. 2). Fig. 2 Karyotype map of G-banding chromosomes Note: A: Chromosome karyotype analysis of amniotic fluid cells in pregnant women; B: Female peripheral blood karyotype analysis; C: Male peripheral blood karyotype analysis 2.2 Results of chromosome microarray pedigree analysis The CMA detection results of fetal amniotic fluid cells showed arr20p13p11.23 (61,662_20,573,017) x2-3 mos, the size was about 20.51 Mb, and the chimerism ratio was about 50%. There was no obvious variation in the chromosome CMA detection results of the peripheral blood of the couple, as shown in Figure 3. Fig. 3 Genealogical results of chromosome microarray analysis Note: A: Chromosome microarray analysis of amniotic fluid cells in pregnant women; B: Chromosome microarray analysis of female peripheral blood; C: Male peripheral blood chromosome microarray analysis 2.3 Results of chromosome fluorescence in situ hybridization (FISH) in amniotic fluid cells In the metaphase phase of amniotic fluid cells, G-banding reanalysis of chromosome karyotype analysis showed that there might be occult translocation of 15q26. 3 in some karyotypes. Therefore, FISH detection of the metaphase division phase of amniotic fluid cells was carried out, and the 20p13 (green)/20q12 (red) probe was used to verify chromosome 20. The signal result was 3 green and 2 red (3G2R), suggesting that there was an increase in 20p13 copy number. Using the 17q21 (green)/15q24. 1 (red) probe for the same karyotype, the results verified the 20p13 translocation to 15qter (Fig. 4). In addition, FISH detection in the later stage of the hospital showed that the proportion of cells carrying 20p13 repeat signal was 49%. Fig. 4 Chromosome karyotype and fluorescence in situ hybridization (FISH) detection results of amniotic fluid cells Note: A: Chromosome karyotype analysis of amniotic fluid cell karyotype map of pregnant women; B: amniotic fluid cell chromosome metaphase FISH-20p13 (green)/20q12 (red) probe; C: Metaphase FISH-17q21 (green)/15q24. 1 (red) probe of amniotic fluid cell chromosome 2.4 MyPODFinder Results According to the chromosome microarray analysis results of the couple and the fetus, the genotyping information derived from the ChAS software for the duplicate 20p13p11.23 fragment of the fetus was imported into the MyPODFinder software for analysis, and the results showed that most of the duplicate fragments came from paternal origin (Fig. 5). Fig. 5 MyPODFinder pedigree analysis of fetal 20p13p11.23 fragment Note: Desired AB calls in child are selected for Allele Peak values satisfying x 0.2. If AP value x > 0.2, AB is ordered as a AAB call and ordered the parent that has a BB call is NOT the origin of the duplicated allele.If AP value x <-0.2, AB is ordered as a ABB call and ordered the parent that has a AA call is NOT the origin of the duplicated allele. 2.6 Sperm FISH test results According to the source of the duplicate fragment suggested by MyPODFinder, the male sperm results were tested by DNA probe interval FISH at the specific site of 20p13 (green)/20q12 (red), but there were no 2 green and 1 red signals in the results, all of which were signal patterns 1 green 1 red (1G1R) (Figure 6). Discussion In this case, we report the case of a pregnant woman conceived by the intracytoplasmic sperm injection technique whose fetus suggested a high risk of a 3.04 Mb deletion in the 15q26. 3 region in the NIPT. However, the deletion of 15q26. 3 was not clearly detected in further amniotic fluid cell chromosome microarray analysis and chromosome karyotype analysis. On the contrary, 20.51 Mb mosaic duplication was found in the 20p13p11. 23 segment of the fetus through CMA, and the mosaic ratio was about 50%. Notably, in the amniotic fluid cell FISH assay, we found that the 20p13 mosaic repeat fragment translocated to the 15qter (that is the 15q26. 3 region), which suggested that there may be structural abnormalities in the 15q26.3 region, although the CMA did not clearly detect deletions. These experimental results suggest that there are complex chromosomal structural rearrangements in this fetus, especially significant duplications in the short arm of chromosome 20, which may have a major impact on the fetal phenotype. According to group standards, when the chimerism ratio exceeds 30%, the risk of fetal phenotypic abnormalities and poor clinical prognosis is significantly increased [ 5 ] , and the short arm of chromosome 20 carries key genes related to neurodevelopment and metabolic regulation, and its fragment abnormalities are closely related to various clinical phenotypes [ 4 ] .Previous studies have shown that the short arm segment duplication of chromosome 20 is a rare chromosome abnormality, and its clinical phenotype is highly heterogeneous, which can be accompanied by many aspects of neurodevelopment, metabolism and skeletal system abnormalities, excessive developmental retardation, and facial deformities. Theoretically, the high proportion of mosaic duplication in the 20p13p11.23 segment of the fetus may also affect the phenotype. However, in this case, the three-dimensional color Doppler ultrasound examination of the fetus at the 21st week of pregnancy did not show serious structural abnormalities, which was inconsistent with the potential phenotypic risk suggested by genetic testing to form the "phenotype-genotype" at the current stage. It is speculated that it may be related to mosaic characteristics–The proportion of 50% abnormal cells in amniotic fluid cells may not be completely equal to the distribution proportion of abnormal cells in various tissues and organs of the fetus. Some organs may have normal karyotypes, or due to the early gestational age, 20p repeat-related phenotypes, such as developmental delay, etc. Functional abnormalities have not yet appeared, such needs to be combined with subsequent pregnancy monitoring and further follow-up observation of the fetus after birth. Although the diagnosis was clarified through multi-technology integration (CMA, FISH, karyotyping), this study has several limitations. Although CMA can detect non-equilibrium rearrangements, it cannot identify balanced translocations; FISH only targeted specific targets (e.g., 20p13, 15q24) and was not validated for deletion on 15q26. 3 due to the lack of probes. In addition, amniotic fluid cells are heterogeneous in morphology, in vitro, and in vivo properties, and are mainly derived from fetal tissues, such as skin, respiratory system, intestinal tract, urinary system, amniotic membrane, and connective tissue [ 9 ] , and their mosaic ratio may not Really reflect the distribution of abnormal cells in fetal core organs (such as brain, heart). In this case, the sample size of sperm FISH detection is limited, and whole genome sequencing is not used to further verify whether there is a low proportion of mosaic or structural variation in sperm. Failure to obtain fetal tissue for multi-organ mosaic ratio verification or autopsy due to pregnancy termination limits in-depth analysis of genotype-phenotype associations. In addition, the positive predictive value of NIPT for microdeletions/microduplicates was low, and the 15q26.3 deletion suggested in this case was finally judged as false positive, reflecting the detection limitations of current screening techniques for complex structural rearrangements. In order to identify the origin of the 20p13 repeat, we analyzed the origin of the repeat by MyPOD Finder software, and the results showed that most of the alleles of the repeat fragment originated from paternal lines. Based on this bioinformatic conjecture, we further performed FISH detection on male semen samples to verify whether the abnormalities were directly derived from paternal germ cells, but no 20p13 repeat signal was detected in sperm FISH results. This negative result contradicts MyPOD Finder's paternal conjecture, which may be due to the following reasons: First, there may be a very low proportion of 20p13 duplicate cells in paternal sperm (such as 10% lower than the lower limit of conventional detection by FISH technology), which is affected by random sampling error and was not captured. According to the 2019 statement of PGDIS (International Society for Preimplantation Diagnosis) [ 10 ] , chimerism diagnosis is usually limited by the proportion of abnormal cells of 20%-80%, and a low proportion of abnormal cells below this range may be missed due to technical limitations. Detection, and FISH detection results are easily disturbed by factors such as the number of biopsy cells, cell damage, etc. When the proportion of abnormal cells is extremely low, false negatives are prone to occur. Although higher resolution technologies such as NGS can improve the detection rate, sperm detection in this study This method may lead to missed diagnosis. Second, the 20p13 mosaic duplication is more likely to be a de novo mutation in the early cleavage stage after fertilization, rather than directly inherited from the paternal sperm. Studies have confirmed [ 10 – 11 ] that although ICSI technology is an important means of assisting pregnancy for couples with abnormal sperm quality, the potential interference with zygotic spindle assembly during the operation may increase the risk of chromosome segregation errors-if the imbalanced distribution of chromosomal segments occurs in the first 2–3 mitoses of the embryo (such as delayed sister chromatid segregation in the 20p13 region), it can directly form mosaic abnormalities. Fragouli and other scholars mentioned [ 12 ] that the incidence of chimerism in the pre-implantation blastocyst stage reaches 5%-10%, and mitotic errors are the main mechanism for the formation of chimerism in human embryos, which is consistent with the result of this case where "the sperm was normal but the embryo showed chimeric duplication". Therefore, although bioinformatics suggests paternal sequence characteristics, the negative FISH results of sperm support that the abnormality is caused by newborn mitotic errors during embryo self-development. In this case, the couple finally chose ICSI technology to assist pregnancy because of the history of Ureaplasma urealyticum (UU) infection in the man, abnormal sperm morphology (malformation rate > 96%) and ovarian dysfunction in the woman (AMH < 1.2 ng/mL). Clinical data show [ 13 – 15 ] that although blastocyst transfer can improve the pregnancy rate by screening embryos with higher developmental potential, which is higher than that of cleavage embryo transfer, the rate of embryonic chromosomal abnormalities (including chimerism, fragment duplication/deletion) in the ICSI cycle is significantly higher than that in the natural fertilization cycle, which is closely related to the slight disturbance of the oocyte cytoplasmic microenvironment and the impact of ICSI operation on the stability of the spindle [ 11 ] . According to the research of Marin and other scholars [ 16 ] , the frequency of blastocyst chimerism varies significantly due to different detection methods and classification criteria: comprehensive chromosome screening high-throughput sequencing (NGS/CCS) studies based on a single sampling of trophoblast biopsy show that the chimerism ratio ranges from 4.8% to 44% (typical value is about 15%); While studies integrating data by multi-biopsy reanalysis (1271 blastocysts) showed [ 17 ] that the overall chimerism frequency reached 35.7%, of which 32.0% were classified as mosaic due to inconsistent results, and 3.7% were reciprocal translocation aneuploidy. In addition, the potential impact of UU infection in men needs to be focused on: Although this case has not directly confirmed the causal relationship between UU and 20p13p11. 23 duplication, studies have shown [ 18 – 19 ] that urogenital tract mycoplasma infection can indirectly increase the risk of embryonic chromosomal abnormalities through two ways: First, UU adsorption on the surface of sperm destroys the integrity of the cell membrane, leading to an increase in the fragmentation rate of sperm DNA, thereby increasing the probability of mitotic errors in embryos after fertilization; Second, UU infection may induce local inflammation of endometrium, reduce endometrial receptivity, and indirectly affect the stability of chromosomes during embryo implantation. Although this case successfully diagnosed fetal 20p13 mosaic duplication through the multi-technology integration strategy of "NIPT primary screening chromosome karyotype analysis/CMA confirmed FISH verification family traceability", its limitations still need to be analyzed objectively: First, in this case NIPT initially suggested an abnormal signal on chromosome 15, but subsequent verification only identified a duplication in the 20p13p11. 23 fragment, which was related to the resolution limitation of NIPT for fragmentation abnormalities. According to ACMG guidelines [ 20 ] , NIPT, as a screening method, has a false positive rate of about 0.02%-0.17%, mainly due to maternal chromosomal abnormalities, placental mosaicism (CPM) or detection technology noise. Second, although CMA and FISH have significantly improved the detection rate of abnormalities, neither of them can fully cover all types of chromosomal abnormalities: CMA is less sensitive to abnormalities without fragment increase or decrease such as balanced translocations, while FISH is less than 5%. There is still a risk of missed diagnosis for extremely low proportions of chimerism, which suggests that NGS technology (such as low-depth whole genome sequencing) needs to be combined in the future to achieve more accurate detection of low proportions of chimerism and small fragment abnormalities. Third, the association mechanism between male UU infection and embryonic chromosomal abnormalities is not yet clear. This case only recorded the history of UU infection, but did not detect the impact of infection on the expression of sperm spindle-related proteins, nor did it analyze the mutations of chromosome segregation-related genes in embryonic cleavage cells. The complete evidence chain of "pathway-chromosomal abnormality" needs to be further elucidated in subsequent cohort studies with larger samples. Taken together, this case highlights the risk of complex chromosomal rearrangements in embryos after ICSI and the need for multi-technique integration in prenatal diagnosis. Future research can combine single-cell sequencing, long-read sequencing and multi-tissue verification to more fully reveal the formation mechanism and distribution of chimerism. For couples assisted by ICSI, it is recommended to strengthen preimplantation genetic testing (PGT) and prenatal diagnosis strategies, especially for those with abnormal sperm parameters or repeated failure, the possibility of mosaic abnormalities should be vigilant. Declarations Funding Declaration The study presented in this manuscript was supported in part by several funding sources. Specifically, it received financial support from the Shenzhen Science and Technology Program under grants JCYJ20230807095202004, JCYJ20240813120016022, and JCYJ20250604183845061, awarded to X.F. Li.Additionally, the Applied Basic Research Foundation (2023A1515010618) provided funding to X.F. Li.The project also benefited from resources allocated by the Shenzhen High-level Hospital Construction Fund and the Guangdong Provincial Clinical Research Center for Laboratory Medicine (2023B110008). Ethics Declaration This study was conducted in accordance with ethical principles and was approved by the Ethics Committee of Prenatal Diagnostic Techniques at Peking University Shenzhen Hospital [Research] [2026] No. 102.All participants provided full informed consent before participating in the study, and all procedures adhered to the relevant guidelines and regulations. Author Contribution Yun Huang (Y.H.) served as the first author and played a pivotal role in the study by conducting experimental operations, performing data analysis, writing the manuscript, and conducting literature searches.My comprehensive involvement ensured the integrity and progression of the research from inception to completion.He Wang (H.W.) provided expert guidance on the professional aspects of the article, ensuring that the research content adhered to the highest standards of the field and offering invaluable insights throughout the manuscript preparation process.Xiaofeng Li (X.L.) contributed by offering support for manuscript submission and securing funding for the project, which was crucial for the successful execution and dissemination of the research findings.Fang Li (F.L.) and Peng Zou (P.Z.) participated actively in chromosome karyotype detection and report review, ensuring the accuracy and reliability of the genetic data presented in the study. Their meticulous work was essential for the interpretation of the genetic results.Shuping Zhang (S.Z.) provided technical support for the FISH (Fluorescence In Situ Hybridization) experiments, which were critical for validating specific genetic abnormalities. Her expertise contributed significantly to the experimental design and execution.Limin Huang (L.M.) offered technical support for NIPT (Non-Invasive Prenatal Testing) experiments, which played a key role in the prenatal diagnosis aspect of the study. Her contributions ensured the smooth running of these complex experiments.All authors reviewed and approved the final version of the manuscript, ensuring that it accurately reflects the collective contributions and findings of the research team. References Kovaleva NV, Cotter PD. Somatic/gonadal mosaicism for structural autosomal rearrangements: female predominance among carriers of gonadal mosaicism for unbalanced rearrangements [J]. Molecular Cytogenetics, 2016, 9 (1): 8. Wang Luming, Zhou Chiyan, Hu Yue, et al. Clinical application of non-invasive prenatal genetic detection in 12085 cases in the screening of fetal chromosomal abnormalities [J]. Chinese Journal of Medical Genetics, 2020, 37 (10): 1069–1073. Wang, L., Zhou, C., Hu, Y., Jin, Y., & Liu, X. (2020). Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics, 37 (10), 1069–1073. https://doi.org/10.3760/cma.j.cn511374-20190930-00503 Prenatal Screening and Diagnostics Group of Birth Defect Prevention and Control Professional Committee of Chinese Preventive Medicine Association, Prenatal Diagnostics Group of Medical Genetics Branch of Chinese Medical Association. Guidelines for the Application of Chromosome Microarray Analysis Technology in Prenatal Diagnosis (2023) [J]. Chinese Journal of Obstetrics and Gynecology, 2023, 58 (8): 565–575. Liu Mian, Zhang Rui, He Yi, et al. Research progress on aneuploidy and uniparental disomy of chromosome 20 [J]. Chinese Journal of Prenatal Diagnosis (Electronic Edition), 2021, 13 (3): 6–11. Guangdong Precision Medicine Application Society. Prenatal Genetic Diagnosis and Genetic Counseling for Chromosomal Mosaicism [S]. T/GDPMAA 0007-2021, 2021. Riggs, E. R., Andersen, E. F., Cherry, A. M., Kantarci, S., Kearney, H., Patel, A., Raca, G., Ritter, D. I., South, S. T., Thorland, E. C., Pineda-Alvarez, D., Aradhya, S., & Martin, C. L. (2021). Correction: Technical standards for the interpretation and reporting of constitutional copy-number variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics (ACMG) and the Prenatal Screening and Diagnostics Group of Birth Defect Prevention and Control Professional Committee of Chinese Preventive Medicine Association, Prenatal Diagnostics Group of Medical Genetics Branch of Chinese Medical Association, Liu Juntao, et al. Application of chromosome microarray analysis technology in prenatal diagnosis Guidelines (2023) [J]. Chinese Journal of Obstetrics and Gynecology, 2023, 58 (8): 565–575. Kennedy, B. J., Savage, S. K., & Kaler, S. G. (2024). Short report: Twins with 20p13 duplication. Case report and comprehensive literature review. Molecular genetics & genomic medicine, 12 (5), e2436. https://doi.org/10.1002/mgg3.2436 Amniotic Fluid Stem Cells and Their Secretomes as tools of generative medicine; Influence of Donor Characteristics on Standardization. J Stem Cells Regen Med.2024; 20 (1): 1–2. doi: 10.46582/jsrm.2001001 . PMID: 39044809; PMCID: PMC11262848. Cram, D. S., Leigh, D., Handyside, A., Rechitsky, L., Xu, K., Harton, G., Grifo, J., Rubio, C., Fragouli, E., Kahraman, S., Forman, E., Katz-Jaffe, M., Tempest, H., Thornhill, A., Strom, C., Escudero, T., Qiao, J., Munne, S., Simpson, J. L., & Kuliev, A. (2019). PGDIS Position Statement on the Transfer of Mosaic Embryos 2019. Reproductive West, J. D., & Everett, C. A. (2022). Preimplantation chromosomal mosaics, chimaeras and confined placental mosaicism. Reproduction & fertility, 3 (2), R66-R90. https://doi.org/10.1530/RAF-21-0095 Fragouli, E., Munne, S., & Wells, D. (2019). The cytogenetic constitution of human blastocysts: insights from comprehensive chromosome screening strategies. Human reproduction update, 25 (1), 15–33. https://doi.org/10.1093/humupd/dmy036 Thum, M. Y., Wells, V., & Abdalla, H. (2010). Patient selection criteria for blastocyst culture in IVF/ICSI treatment. Journal of assisted reproduction and genetics, 27 (9–10), 555–560. https://doi.org/10.1007/s10815-010-9457-9 Duan Li, Du Dan, Liu Haipeng, et al. Evaluation of the clinical application value of three embryo transfer strategies in IVF-ET [J]. Journal of Reproductive Medicine, 2020, 29: 1427–1432. Ma Jingwen, Wang Linlin, Zhang Yan, et al. Comparison of pregnancy outcomes between fresh-cycle single blastocyst transplantation and double blastocyst transplantation [J]. Journal of Reproductive Medicine, 2021, 30 (5): 594–599. Marin, D., Scott, R. T., Jr, & Treff, N. R. (2017). Preimplantation embryonic mosaicism: origin, sequences and the reliability of comprehensive chromosome screening. Current opinion in obstetrics & gynecology, 29 (3), 168–174. https://doi.org/10.1097/GCO.0000000000000358 Marin, D., Xu, J., & Treff, N. R. (2021). Preimplantation genetic testing for aneuploidy: A review of published blastocyst analysis agreement data. Prenatal diagnosis, 41 (5), 545–553. https://doi.org/10.1002/pd.5828 Xiang Li, Zhou Tiejun, Wang Guangxi, et al. Effect of Ureaplasma urealyticum on sperm morphology [J]. Journal of Luzhou Medical College, 2008, 31 (2): 148–150. Jiang Lan, Li Yidan, Xia Yan, et al. The impact of urogenital mycoplasma infection on different pregnancy outcomes [J]. Xinjiang Medicine, 2025, 55 (2): 153–157. Dungan, J. S., Klugman, S., Darilek, S., Malinowski, J., Akkari, Y. M. N., Monaghan, K. G., Erwin, A., Best, R. G., & ACMG Board of Directors.Electronic address: [email protected] (2023). Noninvasive prenatal screening (NIPS) for fetal chromosome abnormalities in a general-risk population: An epidemic-based clinical guideline of the American College of Medical Genetics and Genomics (ACMG). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 04 May, 2026 Reviews received at journal 27 Apr, 2026 Reviews received at journal 21 Apr, 2026 Reviewers agreed at journal 21 Apr, 2026 Reviewers agreed at journal 21 Apr, 2026 Reviewers invited by journal 21 Apr, 2026 Editor assigned by journal 21 Jan, 2026 Submission checks completed at journal 21 Jan, 2026 First submitted to journal 18 Jan, 2026 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-8634923","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":630162747,"identity":"a8b4b8ef-335c-415b-b02c-fa50b3f66953","order_by":0,"name":"Yun Huang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzElEQVRIiWNgGAWjYBAC+/kPGww+VPzn4WdvIFKLAUPygcIZZ5hlJHsOEK0lLeEzbxuzjcGNBCK1mDOcMdzAc4aNx+Dm4403GGpsoglqsWzsMTaQqODhkbydVmzBcCwtt4GgnsM8ZgYGZyR4+G7nmEkwNhwmQssxHvMfiW0GPAw3zxCpxeAMW4LBwbYEHoEbPERqkZzBfMCw4cwBHskeoF8SiPELP9Bk4z8VB+z52Q9vvPGhxoYIvyA7UiKBFOUQLaTqGAWjYBSMgpEBAEu9QVeucjfSAAAAAElFTkSuQmCC","orcid":"","institution":"Peking University Shenzhen Hospital","correspondingAuthor":true,"prefix":"","firstName":"Yun","middleName":"","lastName":"Huang","suffix":""},{"id":630162748,"identity":"088db321-e3f5-4f63-af3c-2a6858606d31","order_by":1,"name":"Shuping Zhang","email":"","orcid":"","institution":"Peking University Shenzhen Hospital","correspondingAuthor":false,"prefix":"","firstName":"Shuping","middleName":"","lastName":"Zhang","suffix":""},{"id":630162749,"identity":"522f2358-4076-4733-bbe8-754f8c5fb2fb","order_by":2,"name":"Peng Zou","email":"","orcid":"","institution":"Peking University Shenzhen Hospital","correspondingAuthor":false,"prefix":"","firstName":"Peng","middleName":"","lastName":"Zou","suffix":""},{"id":630162750,"identity":"167e2472-d1ed-42f1-8dec-378ea233d4c9","order_by":3,"name":"Limin Huang","email":"","orcid":"","institution":"Peking University Shenzhen Hospital","correspondingAuthor":false,"prefix":"","firstName":"Limin","middleName":"","lastName":"Huang","suffix":""},{"id":630162751,"identity":"e1d92d1a-5f30-44a8-bced-5c67ec423c11","order_by":4,"name":"Xiaofeng Li","email":"","orcid":"","institution":"Peking University Shenzhen Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiaofeng","middleName":"","lastName":"Li","suffix":""},{"id":630162752,"identity":"47f67c27-398d-42c0-9b93-bc1d4fbe1028","order_by":5,"name":"He Wang","email":"","orcid":"","institution":"Peking University Shenzhen Hospital","correspondingAuthor":false,"prefix":"","firstName":"He","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2026-01-19 04:23:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8634923/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8634923/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108493173,"identity":"1ede76c9-16f1-4e40-9238-6c985949a163","added_by":"auto","created_at":"2026-05-05 09:59:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":26559,"visible":true,"origin":"","legend":"\u003cp\u003eSequencing dot plot of NIPT chromosome 15 for non-invasive prenatal screening\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8634923/v1/6f1da5ade6c3c3ef99a60783.png"},{"id":108388612,"identity":"04298910-cef8-496f-8cda-c2e84bcc4d9b","added_by":"auto","created_at":"2026-05-04 06:43:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":662266,"visible":true,"origin":"","legend":"\u003cp\u003eKaryotype map of G-banding chromosomes\u003c/p\u003e\n\u003cp\u003eNote: A: Chromosome karyotype analysis of amniotic fluid cells in pregnant women; B: Female peripheral blood karyotype analysis; C: Male peripheral blood karyotype analysis\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8634923/v1/d9c8bff6c1f027f8e193e026.png"},{"id":108804078,"identity":"ebe84c5c-e582-4df6-837c-e6f72cbb7f7b","added_by":"auto","created_at":"2026-05-08 15:15:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1531254,"visible":true,"origin":"","legend":"\u003cp\u003eGenealogical results of chromosome microarray analysis\u003c/p\u003e\n\u003cp\u003eNote: A: Chromosome microarray analysis of amniotic fluid cells in pregnant women; B: Chromosome microarray analysis of female peripheral blood; C: Male peripheral blood chromosome microarray analysis\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8634923/v1/c6769be527a35037f8056e38.png"},{"id":108492500,"identity":"dfbb08d9-710a-4f9f-afdc-f56953f5e8b4","added_by":"auto","created_at":"2026-05-05 09:57:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":462965,"visible":true,"origin":"","legend":"\u003cp\u003eChromosome karyotype and fluorescence in situ hybridization (FISH) detection results of amniotic fluid cells\u003c/p\u003e\n\u003cp\u003eNote: A: Chromosome karyotype analysis of amniotic fluid cell karyotype map of pregnant women; B: amniotic fluid cell chromosome metaphase FISH-20p13 (green)/20q12 (red) probe; C: Metaphase FISH-17q21 (green)/15q24. 1 (red) probe of amniotic fluid cell chromosome\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8634923/v1/7d5b7e707601892a8761bd86.png"},{"id":108388615,"identity":"eec07059-b25b-4932-a6ce-753eca722763","added_by":"auto","created_at":"2026-05-04 06:43:06","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":21082,"visible":true,"origin":"","legend":"\u003cp\u003eMyPODFinder pedigree analysis of fetal 20p13p11.23 fragment\u003c/p\u003e\n\u003cp\u003eNote: Desired AB calls in child are selected for Allele Peak values satisfying x \u0026lt;-0.2 and x \u0026gt; 0.2.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8634923/v1/ae73e323f2955c69415dad4a.png"},{"id":108493364,"identity":"b8a8d270-7df4-4a4a-96bb-28e97d944f0b","added_by":"auto","created_at":"2026-05-05 10:00:03","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":59733,"visible":true,"origin":"","legend":"\u003cp\u003eSperm interphase FISH-20p13 (green)/20q12 (red) specific probe signal results\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8634923/v1/89e82d48a73f93b578a4348e.png"},{"id":108809355,"identity":"ac31c621-81cd-4f73-a473-b29dad94ce1f","added_by":"auto","created_at":"2026-05-08 15:52:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3159832,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8634923/v1/e6bfcb6d-eaee-4175-b661-ad68f5ec94e5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Application and value of multi-technique integration in the prenatal diagnosis of mosaicism derived of ICSI blastocyst","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAs the core technology of assisted reproduction, intracytoplasmic sperm injection (ICSI) provides the possibility of fertility for infertile families, but the process of in vitro operation and culture of embryos may increase the risk of chromosomal abnormalities, especially causing mosaic structural rearrangement, which poses challenges to prenatal diagnosis \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Prenatal diagnosis is an important gateway to prevent birth defects. Non-Invasive Prenatal Testing (NIPT) has been widely used in clinical practice as an effective method to screen fetal aneuploidy. However, its detection efficacy for micro-deletions/micro-duplications and complex structural rearrangements is limited, and its positive predictive value is low. Abnormal results need to be verified by interventional diagnosis \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn interventional prenatal diagnosis, G banding karyotype analysis has low resolution and is difficult to identify minor or low-proportion mosaic abnormalities; The detection range of Fluorescence in situ hybridization (FISH) is limited to preset targets; Chromosome microarray analysis (CMA) can detect genome-wide copy number variation (CNV) at the submicroscopic level, with a resolution of 10-100kb, and can also identify homozygous regions. It has become a first-line diagnostic technology for high-risk populations \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe short arm of human chromosome 20 (20p) carries several key genes, such as those involved in neural development and metabolic regulation, and its fragment abnormalities are closely related to a variety of clinical phenotypes \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e.From the perspective of chimerism ratio, according to the group standard of \"Prenatal Diagnosis and Genetic Counseling of Chromosomal Mosaicism\"\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e,when the chimerism ratio exceeds 30%, the impact of abnormal cell lines on fetal phenotype is significantly increased, and the clinical prognosis Increased risk of poor.\u003c/p\u003e \u003cp\u003eThis paper reports a pregnant fetus with 15q26. 3 deletion after ICSI blastocyst transplantation. The complicated case of 20p13p11. 23 mosaic duplication was evaluated by NIPT, CMA, FISH and other integrated diagnosis techniques. It provides basis for clinical decision-making and genetic counseling, and also provides reference for optimizing prenatal diagnosis strategy after ICSI.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e1.1 Research Subjects\u003c/p\u003e\n\u003cp\u003eThe couple failed their first in vitro fertilization (IVF) treatment in 2024 due to the man\u0026apos;s history of Ureaplasma urealyticum (UU) infection, abnormal sperm morphology, and the woman\u0026apos;s ovarian dysfunction. After comprehensive evaluation, considering that the man\u0026apos;s sperm factor may affect fertilization and embryonic development, he chose to receive intracytoplasmic sperm injection (ICSI) technology for assisted pregnancy. One blastocyst was obtained in the second ICSI cycle and a successful pregnancy was achieved after transplantation.\u003c/p\u003e\n\u003cp\u003e1.2 Auxiliary inspection\u003c/p\u003e\n\u003cp\u003eAt the 13th week of pregnancy, the thickness of the nuchal transparent layer was 1.4 mm in the normal range, but the placenta previa was low; No abnormalities were found in the ultrasound during second trimester of pregnancy. Prenatal serological screening: There was no abnormality in the early Down syndrome screening at 13 +1 weeks of pregnancy, and no serological screening at the middle stage; At 13 weeks of gestation, non-invasive prenatal genetic testing (NIPT) was performed, and the screening suggested a high risk of 3.04 Mb deletion in the 15q26. 3 region, as shown in Fig. 1. In order to further confirm the diagnosis, amniocentesis was performed at 18 weeks of gestation, and 20mL amniotic fluid was collected for related tests. Fetal 3-D color Doppler ultrasound was performed at 21 weeks of gestation, and no serious structural deformities were found in the fetus.\u003c/p\u003e\n\u003cp\u003e1.3 Experimental Methods\u003c/p\u003e\n\u003cp\u003e1.3. 1 Specimen collection\u003c/p\u003e\n\u003cp\u003eBefore assisted pregnancy, the peripheral blood chromosome karyotype analysis was carried out for reproductive assisted pregnant couples. After full informed consent during pregnancy, 20mL amniotic fluid was extracted for chromosome karyotype analysis and 10mL for chromosome microarray analysis. As the results of chromosome microarray analysis showed that there were partial fragmental duplications in the short arm of chromosome 20, on the premise of full informed consent, 2mL peripheral blood of both pregnant women and couples was extracted for fetal chromosome microarray pedigree analysis. The study was approved by the Ethics Committee of Prenatal Diagnostic Techniques of our hospital.\u003c/p\u003e\n\u003cp\u003e1.3. 2 Cell culture and karyotype analysis\u003c/p\u003e\n\u003cp\u003eAfter centrifugation, 1 ~ 2mL of amniotic fluid was inoculated into amniotic fluid cell culture medium. After standard methods of culture, fluid exchange, passage, harvesting, chromosome preparation and G-banding karyotype analysis, the number of more than 20 mitotic phases was counted in double-person and double-line, and at least 5 metaphase karyotype mitotic phases were analyzed. The karyotype description was carried out according to the \u0026quot;International Nomenclature System of Human Cytogenetics\u0026quot; (ISCN2020). The diagnostic criteria for detecting chimerism were carried out according to \u0026quot;Prenatal Genetic Diagnosis and Genetic Counseling for Chromosomal Chimerism\u0026quot;.\u003c/p\u003e\n\u003cp\u003e1.3. 2 Chromosome microarray analysis (CMA)\u003c/p\u003e\n\u003cp\u003e10 mL amniotic fluid samples with qualified quality control were collected aseptically, and the amniotic fluid genomic DNA was extracted using QIAamp \u0026reg; DNA Blood Mini Kit (QIAGEN, Germany). The genomic DNA was digested into short fragments by enzyme, then amplified by PCR and purified by magnetic bead method. The purified product was fragmented into 25-125bp fragments and labeled with biotin. After the product was mixed with the hybridization solution for denaturation, the chip hybridization, washing and staining were carried out, and the chip scan was used to detect DNA copy number variation (CNV) using the Affymetrix CytoScan 750K chip platform (Thermo Scientific, USA).\u003c/p\u003e\n\u003cp\u003e1.3. 3 Detection of chromosome fluorescence in situ hybridization (FISH) in amniotic fluid cells\u003c/p\u003e\n\u003cp\u003eMetaphase cells were detected using 20q12 (red)/20p13 (green) specific probes and 15q24. 1 (red)/17q21 (green) specific site DNA probes (both from Wuhan Kanglu Co., Ltd.), and chromosomes 20 and 15 were analyzed. All operations were performed in accordance with the FISH procedure, including: dropping the cell suspension onto the glass slide, aging, adding probes, hybridization overnight, elution, staining, and microscopic examination. S500-24 in situ hybridization instrument (Thermo Company, USA was used for hybridization, and the multi-color fluorescence image processing and analysis were performed with a fluorescence microscope (model: BX51, from Olympus Company, Japan).\u003c/p\u003e\n\u003cp\u003e1.3. 4 Sperm FISH detection\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eUnder aseptic conditions, fresh semen samples of 1-2ml were taken, and 20q12 (red)/20p13 (green) specific site DNA probes (Wuhan Kanglu Co., Ltd.) were used to analyze chromosome 20. All operations were performed according to the FISH operation procedure, including: sperm liquefaction, hypotonic, prefixation, fixation,dropping the cell suspension onto the glass slide, aging, alkaline expansion, addition of probes, overnight hybridization, elution, staining, and microscopic examination. The remaining steps are the same as 1.3. 3.\u003c/p\u003e\n\u003cp\u003e1.3. 5 Data analysis\u003c/p\u003e\n\u003cp\u003eAll detection data of chromosome microarrays were processed and analyzed using Chromosome AnalysisSuite (ChAS) V4.3 software, which is capable of detecting clinically relevant genes and copy number variants (CNVS) and homozygous regions (LOH) with genomic resolutions greater than 100 kb. The My POD Finder software (from the Swedish Clinical Genetics in Lund development software) was used for analysis of family genotypes and for evaluation of duplicate sources. Refer to the 2021 \u0026quot;Technical Standards for Interpretation and Reporting of Primary Copy Number Variations: Common Consensus Recommendations of the American College of Medical Genetics and Genomics (ACMG) and Clinical Genome Resources (ClinGen)\u0026quot;\u003csup\u003e\u0026nbsp;[6]\u003c/sup\u003e And \u0026quot;Guidelines for the Application of Chromosome Microarray Analysis in Prenatal Diagnosis (2023)\u0026quot; \u003csup\u003e[7]\u003c/sup\u003e to evaluate copy number variants and homozygous regions.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e2.1 Karyotype analysis of amniotic fluid cells\u003c/p\u003e\n\u003cp\u003eThe chromosomal karyotype of amniotic fluid cells in pregnant women was 46, XN by counting 20 mitotic phases and analyzing 5-10 karyotypes. In addition, the results of karyotype analysis of peripheral blood chromosomes of both spouses were normal (Fig. 2).\u003c/p\u003e\n\u003cp\u003eFig. 2 Karyotype map of G-banding chromosomes\u003c/p\u003e\n\u003cp\u003eNote: A: Chromosome karyotype analysis of amniotic fluid cells in pregnant women; B: Female peripheral blood karyotype analysis; C: Male peripheral blood karyotype analysis\u003c/p\u003e\n\u003cp\u003e2.2 Results of chromosome microarray pedigree analysis\u003c/p\u003e\n\u003cp\u003eThe CMA detection results of fetal amniotic fluid cells showed arr20p13p11.23 (61,662_20,573,017) x2-3 mos, the size was about 20.51 Mb, and the chimerism ratio was about 50%. There was no obvious variation in the chromosome CMA detection results of the peripheral blood of the couple, as shown in Figure 3.\u003c/p\u003e\n\u003cp\u003eFig. 3 Genealogical results of chromosome microarray analysis\u003c/p\u003e\n\u003cp\u003eNote: A: Chromosome microarray analysis of amniotic fluid cells in pregnant women; B: Chromosome microarray analysis of female peripheral blood; C: Male peripheral blood chromosome microarray analysis\u003c/p\u003e\n\u003cp\u003e2.3 Results of chromosome fluorescence in situ hybridization (FISH) in amniotic fluid cells\u003c/p\u003e\n\u003cp\u003eIn the metaphase phase of amniotic fluid cells, G-banding reanalysis of chromosome karyotype analysis showed that there might be occult translocation of 15q26. 3 in some karyotypes. Therefore, FISH detection of the metaphase division phase of amniotic fluid cells was carried out, and the 20p13 (green)/20q12 (red) probe was used to verify chromosome 20. The signal result was 3 green and 2 red (3G2R), suggesting that there was an increase in 20p13 copy number. Using the 17q21 (green)/15q24. 1 (red) probe for the same karyotype, the results verified the 20p13 translocation to 15qter (Fig. 4). In addition, FISH detection in the later stage of the hospital showed that the proportion of cells carrying 20p13 repeat signal was 49%.\u003c/p\u003e\n\u003cp\u003eFig. 4 Chromosome karyotype and fluorescence in situ hybridization (FISH) detection results of amniotic fluid cells\u003c/p\u003e\n\u003cp\u003eNote: A: Chromosome karyotype analysis of amniotic fluid cell karyotype map of pregnant women; B: amniotic fluid cell chromosome metaphase FISH-20p13 (green)/20q12 (red) probe; C: Metaphase FISH-17q21 (green)/15q24. 1 (red) probe of amniotic fluid cell chromosome\u003c/p\u003e\n\u003cp\u003e2.4 MyPODFinder Results\u003c/p\u003e\n\u003cp\u003eAccording to the chromosome microarray analysis results of the couple and the fetus, the genotyping information derived from the ChAS software for the duplicate 20p13p11.23 fragment of the fetus was imported into the MyPODFinder software for analysis, and the results showed that most of the duplicate fragments came from paternal origin (Fig. 5).\u003c/p\u003e\n\u003cp\u003eFig. 5 MyPODFinder pedigree analysis of fetal 20p13p11.23 fragment\u003c/p\u003e\n\u003cp\u003eNote: Desired AB calls in child are selected for Allele Peak values satisfying x \u0026lt;-0.2 and x \u0026gt; 0.2.\u003c/p\u003e\n\u003cp\u003eIf AP value x \u0026gt; 0.2, AB is ordered as a AAB call and ordered the parent that has a BB call is NOT the origin of the duplicated allele.If AP value x \u0026lt;-0.2, AB is ordered as a ABB call and ordered the parent that has a AA call is NOT the origin of the duplicated allele.\u003c/p\u003e\n\u003cp\u003e2.6 Sperm FISH test results\u003c/p\u003e\n\u003cp\u003eAccording to the source of the duplicate fragment suggested by MyPODFinder, the male sperm results were tested by DNA probe interval FISH at the specific site of 20p13 (green)/20q12 (red), but there were no 2 green and 1 red signals in the results, all of which were signal patterns 1 green 1 red (1G1R) (Figure 6).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this case, we report the case of a pregnant woman conceived by the intracytoplasmic sperm injection technique whose fetus suggested a high risk of a 3.04 Mb deletion in the 15q26. 3 region in the NIPT. However, the deletion of 15q26. 3 was not clearly detected in further amniotic fluid cell chromosome microarray analysis and chromosome karyotype analysis. On the contrary, 20.51 Mb mosaic duplication was found in the 20p13p11. 23 segment of the fetus through CMA, and the mosaic ratio was about 50%. Notably, in the amniotic fluid cell FISH assay, we found that the 20p13 mosaic repeat fragment translocated to the 15qter (that is the 15q26. 3 region), which suggested that there may be structural abnormalities in the 15q26.3 region, although the CMA did not clearly detect deletions. These experimental results suggest that there are complex chromosomal structural rearrangements in this fetus, especially significant duplications in the short arm of chromosome 20, which may have a major impact on the fetal phenotype.\u003c/p\u003e \u003cp\u003eAccording to group standards, when the chimerism ratio exceeds 30%, the risk of fetal phenotypic abnormalities and poor clinical prognosis is significantly increased \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e, and the short arm of chromosome 20 carries key genes related to neurodevelopment and metabolic regulation, and its fragment abnormalities are closely related to various clinical phenotypes\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e.Previous studies have shown that the short arm segment duplication of chromosome 20 is a rare chromosome abnormality, and its clinical phenotype is highly heterogeneous, which can be accompanied by many aspects of neurodevelopment, metabolism and skeletal system abnormalities, excessive developmental retardation, and facial deformities. Theoretically, the high proportion of mosaic duplication in the 20p13p11.23 segment of the fetus may also affect the phenotype. However, in this case, the three-dimensional color Doppler ultrasound examination of the fetus at the 21st week of pregnancy did not show serious structural abnormalities, which was inconsistent with the potential phenotypic risk suggested by genetic testing to form the \"phenotype-genotype\" at the current stage. It is speculated that it may be related to mosaic characteristics\u0026ndash;The proportion of 50% abnormal cells in amniotic fluid cells may not be completely equal to the distribution proportion of abnormal cells in various tissues and organs of the fetus. Some organs may have normal karyotypes, or due to the early gestational age, 20p repeat-related phenotypes, such as developmental delay, etc. Functional abnormalities have not yet appeared, such needs to be combined with subsequent pregnancy monitoring and further follow-up observation of the fetus after birth.\u003c/p\u003e \u003cp\u003eAlthough the diagnosis was clarified through multi-technology integration (CMA, FISH, karyotyping), this study has several limitations. Although CMA can detect non-equilibrium rearrangements, it cannot identify balanced translocations; FISH only targeted specific targets (e.g., 20p13, 15q24) and was not validated for deletion on 15q26. 3 due to the lack of probes. In addition, amniotic fluid cells are heterogeneous in morphology, in vitro, and in vivo properties, and are mainly derived from fetal tissues, such as skin, respiratory system, intestinal tract, urinary system, amniotic membrane, and connective tissue \u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e, and their mosaic ratio may not Really reflect the distribution of abnormal cells in fetal core organs (such as brain, heart). In this case, the sample size of sperm FISH detection is limited, and whole genome sequencing is not used to further verify whether there is a low proportion of mosaic or structural variation in sperm. Failure to obtain fetal tissue for multi-organ mosaic ratio verification or autopsy due to pregnancy termination limits in-depth analysis of genotype-phenotype associations. In addition, the positive predictive value of NIPT for microdeletions/microduplicates was low, and the 15q26.3 deletion suggested in this case was finally judged as false positive, reflecting the detection limitations of current screening techniques for complex structural rearrangements.\u003c/p\u003e \u003cp\u003eIn order to identify the origin of the 20p13 repeat, we analyzed the origin of the repeat by MyPOD Finder software, and the results showed that most of the alleles of the repeat fragment originated from paternal lines. Based on this bioinformatic conjecture, we further performed FISH detection on male semen samples to verify whether the abnormalities were directly derived from paternal germ cells, but no 20p13 repeat signal was detected in sperm FISH results. This negative result contradicts MyPOD Finder's paternal conjecture, which may be due to the following reasons: First, there may be a very low proportion of 20p13 duplicate cells in paternal sperm (such as 10% lower than the lower limit of conventional detection by FISH technology), which is affected by random sampling error and was not captured. According to the 2019 statement of PGDIS (International Society for Preimplantation Diagnosis) \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e, chimerism diagnosis is usually limited by the proportion of abnormal cells of 20%-80%, and a low proportion of abnormal cells below this range may be missed due to technical limitations. Detection, and FISH detection results are easily disturbed by factors such as the number of biopsy cells, cell damage, etc. When the proportion of abnormal cells is extremely low, false negatives are prone to occur. Although higher resolution technologies such as NGS can improve the detection rate, sperm detection in this study This method may lead to missed diagnosis. Second, the 20p13 mosaic duplication is more likely to be a de novo mutation in the early cleavage stage after fertilization, rather than directly inherited from the paternal sperm. Studies have confirmed \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e that although ICSI technology is an important means of assisting pregnancy for couples with abnormal sperm quality, the potential interference with zygotic spindle assembly during the operation may increase the risk of chromosome segregation errors-if the imbalanced distribution of chromosomal segments occurs in the first 2\u0026ndash;3 mitoses of the embryo (such as delayed sister chromatid segregation in the 20p13 region), it can directly form mosaic abnormalities. Fragouli and other scholars mentioned \u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e that the incidence of chimerism in the pre-implantation blastocyst stage reaches 5%-10%, and mitotic errors are the main mechanism for the formation of chimerism in human embryos, which is consistent with the result of this case where \"the sperm was normal but the embryo showed chimeric duplication\". Therefore, although bioinformatics suggests paternal sequence characteristics, the negative FISH results of sperm support that the abnormality is caused by newborn mitotic errors during embryo self-development.\u003c/p\u003e \u003cp\u003eIn this case, the couple finally chose ICSI technology to assist pregnancy because of the history of Ureaplasma urealyticum (UU) infection in the man, abnormal sperm morphology (malformation rate\u0026thinsp;\u0026gt;\u0026thinsp;96%) and ovarian dysfunction in the woman (AMH\u0026thinsp;\u0026lt;\u0026thinsp;1.2 ng/mL). Clinical data show \u003csup\u003e[\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e that although blastocyst transfer can improve the pregnancy rate by screening embryos with higher developmental potential, which is higher than that of cleavage embryo transfer, the rate of embryonic chromosomal abnormalities (including chimerism, fragment duplication/deletion) in the ICSI cycle is significantly higher than that in the natural fertilization cycle, which is closely related to the slight disturbance of the oocyte cytoplasmic microenvironment and the impact of ICSI operation on the stability of the spindle \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. According to the research of Marin and other scholars \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e, the frequency of blastocyst chimerism varies significantly due to different detection methods and classification criteria: comprehensive chromosome screening high-throughput sequencing (NGS/CCS) studies based on a single sampling of trophoblast biopsy show that the chimerism ratio ranges from 4.8% to 44% (typical value is about 15%); While studies integrating data by multi-biopsy reanalysis (1271 blastocysts) showed\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e that the overall chimerism frequency reached 35.7%, of which 32.0% were classified as mosaic due to inconsistent results, and 3.7% were reciprocal translocation aneuploidy.\u003c/p\u003e \u003cp\u003eIn addition, the potential impact of UU infection in men needs to be focused on: Although this case has not directly confirmed the causal relationship between UU and 20p13p11. 23 duplication, studies have shown\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e that urogenital tract mycoplasma infection can indirectly increase the risk of embryonic chromosomal abnormalities through two ways: First, UU adsorption on the surface of sperm destroys the integrity of the cell membrane, leading to an increase in the fragmentation rate of sperm DNA, thereby increasing the probability of mitotic errors in embryos after fertilization; Second, UU infection may induce local inflammation of endometrium, reduce endometrial receptivity, and indirectly affect the stability of chromosomes during embryo implantation.\u003c/p\u003e \u003cp\u003eAlthough this case successfully diagnosed fetal 20p13 mosaic duplication through the multi-technology integration strategy of \"NIPT primary screening chromosome karyotype analysis/CMA confirmed FISH verification family traceability\", its limitations still need to be analyzed objectively: First, in this case NIPT initially suggested an abnormal signal on chromosome 15, but subsequent verification only identified a duplication in the 20p13p11. 23 fragment, which was related to the resolution limitation of NIPT for fragmentation abnormalities. According to ACMG guidelines \u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e, NIPT, as a screening method, has a false positive rate of about 0.02%-0.17%, mainly due to maternal chromosomal abnormalities, placental mosaicism (CPM) or detection technology noise. Second, although CMA and FISH have significantly improved the detection rate of abnormalities, neither of them can fully cover all types of chromosomal abnormalities: CMA is less sensitive to abnormalities without fragment increase or decrease such as balanced translocations, while FISH is less than 5%. There is still a risk of missed diagnosis for extremely low proportions of chimerism, which suggests that NGS technology (such as low-depth whole genome sequencing) needs to be combined in the future to achieve more accurate detection of low proportions of chimerism and small fragment abnormalities. Third, the association mechanism between male UU infection and embryonic chromosomal abnormalities is not yet clear. This case only recorded the history of UU infection, but did not detect the impact of infection on the expression of sperm spindle-related proteins, nor did it analyze the mutations of chromosome segregation-related genes in embryonic cleavage cells. The complete evidence chain of \"pathway-chromosomal abnormality\" needs to be further elucidated in subsequent cohort studies with larger samples.\u003c/p\u003e \u003cp\u003eTaken together, this case highlights the risk of complex chromosomal rearrangements in embryos after ICSI and the need for multi-technique integration in prenatal diagnosis. Future research can combine single-cell sequencing, long-read sequencing and multi-tissue verification to more fully reveal the formation mechanism and distribution of chimerism. For couples assisted by ICSI, it is recommended to strengthen preimplantation genetic testing (PGT) and prenatal diagnosis strategies, especially for those with abnormal sperm parameters or repeated failure, the possibility of mosaic abnormalities should be vigilant.\u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cb\u003eFunding Declaration\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe study presented in this manuscript was supported in part by several funding sources. Specifically, it received financial support from the Shenzhen Science and Technology Program under grants JCYJ20230807095202004, JCYJ20240813120016022, and JCYJ20250604183845061, awarded to X.F. Li.Additionally, the Applied Basic Research Foundation (2023A1515010618) provided funding to X.F. Li.The project also benefited from resources allocated by the Shenzhen High-level Hospital Construction Fund and the Guangdong Provincial Clinical Research Center for Laboratory Medicine (2023B110008).\u003c/p\u003e \u003cp\u003e \u003cb\u003eEthics Declaration\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThis study was conducted in accordance with ethical principles and was approved by the Ethics Committee of Prenatal Diagnostic Techniques at Peking University Shenzhen Hospital [Research] [2026] No. 102.All participants provided full informed consent before participating in the study, and all procedures adhered to the relevant guidelines and regulations.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eYun Huang (Y.H.) served as the first author and played a pivotal role in the study by conducting experimental operations, performing data analysis, writing the manuscript, and conducting literature searches.My comprehensive involvement ensured the integrity and progression of the research from inception to completion.He Wang (H.W.) provided expert guidance on the professional aspects of the article, ensuring that the research content adhered to the highest standards of the field and offering invaluable insights throughout the manuscript preparation process.Xiaofeng Li (X.L.) contributed by offering support for manuscript submission and securing funding for the project, which was crucial for the successful execution and dissemination of the research findings.Fang Li (F.L.) and Peng Zou (P.Z.) participated actively in chromosome karyotype detection and report review, ensuring the accuracy and reliability of the genetic data presented in the study. Their meticulous work was essential for the interpretation of the genetic results.Shuping Zhang (S.Z.) provided technical support for the FISH (Fluorescence In Situ Hybridization) experiments, which were critical for validating specific genetic abnormalities. Her expertise contributed significantly to the experimental design and execution.Limin Huang (L.M.) offered technical support for NIPT (Non-Invasive Prenatal Testing) experiments, which played a key role in the prenatal diagnosis aspect of the study. Her contributions ensured the smooth running of these complex experiments.All authors reviewed and approved the final version of the manuscript, ensuring that it accurately reflects the collective contributions and findings of the research team.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKovaleva NV, Cotter PD. Somatic/gonadal mosaicism for structural autosomal rearrangements: female predominance among carriers of gonadal mosaicism for unbalanced rearrangements [J]. Molecular Cytogenetics, 2016, 9 (1): 8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Luming, Zhou Chiyan, Hu Yue, et al. Clinical application of non-invasive prenatal genetic detection in 12085 cases in the screening of fetal chromosomal abnormalities [J]. Chinese Journal of Medical Genetics, 2020, 37 (10): 1069\u0026ndash;1073. Wang, L., Zhou, C., Hu, Y., Jin, Y., \u0026amp; Liu, X. (2020). Zhonghua yi xue yi chuan xue za zhi\u0026thinsp;=\u0026thinsp;Zhonghua yixue yichuanxue zazhi\u0026thinsp;=\u0026thinsp;Chinese journal of medical genetics, 37 (10), 1069\u0026ndash;1073. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3760/cma.j.cn511374-20190930-00503\u003c/span\u003e\u003cspan address=\"10.3760/cma.j.cn511374-20190930-00503\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePrenatal Screening and Diagnostics Group of Birth Defect Prevention and Control Professional Committee of Chinese Preventive Medicine Association, Prenatal Diagnostics Group of Medical Genetics Branch of Chinese Medical Association. Guidelines for the Application of Chromosome Microarray Analysis Technology in Prenatal Diagnosis (2023) [J]. Chinese Journal of Obstetrics and Gynecology, 2023, 58 (8): 565\u0026ndash;575.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu Mian, Zhang Rui, He Yi, et al. Research progress on aneuploidy and uniparental disomy of chromosome 20 [J]. Chinese Journal of Prenatal Diagnosis (Electronic Edition), 2021, 13 (3): 6\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuangdong Precision Medicine Application Society. Prenatal Genetic Diagnosis and Genetic Counseling for Chromosomal Mosaicism [S]. T/GDPMAA 0007-2021, 2021.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRiggs, E. R., Andersen, E. F., Cherry, A. M., Kantarci, S., Kearney, H., Patel, A., Raca, G., Ritter, D. I., South, S. T., Thorland, E. C., Pineda-Alvarez, D., Aradhya, S., \u0026amp; Martin, C. L. (2021). Correction: Technical standards for the interpretation and reporting of constitutional copy-number variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics (ACMG) and the\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePrenatal Screening and Diagnostics Group of Birth Defect Prevention and Control Professional Committee of Chinese Preventive Medicine Association, Prenatal Diagnostics Group of Medical Genetics Branch of Chinese Medical Association, Liu Juntao, et al. Application of chromosome microarray analysis technology in prenatal diagnosis Guidelines (2023) [J]. Chinese Journal of Obstetrics and Gynecology, 2023, 58 (8): 565\u0026ndash;575.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKennedy, B. J., Savage, S. K., \u0026amp; Kaler, S. G. (2024). Short report: Twins with 20p13 duplication. Case report and comprehensive literature review. Molecular genetics \u0026amp; genomic medicine, 12 (5), e2436. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/mgg3.2436\u003c/span\u003e\u003cspan address=\"10.1002/mgg3.2436\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmniotic Fluid Stem Cells and Their Secretomes as tools of generative medicine; Influence of Donor Characteristics on Standardization. J Stem Cells Regen Med.2024; 20 (1): 1\u0026ndash;2. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.46582/jsrm.2001001\u003c/span\u003e\u003cspan address=\"10.46582/jsrm.2001001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. PMID: 39044809; PMCID: PMC11262848.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCram, D. S., Leigh, D., Handyside, A., Rechitsky, L., Xu, K., Harton, G., Grifo, J., Rubio, C., Fragouli, E., Kahraman, S., Forman, E., Katz-Jaffe, M., Tempest, H., Thornhill, A., Strom, C., Escudero, T., Qiao, J., Munne, S., Simpson, J. L., \u0026amp; Kuliev, A. (2019). PGDIS Position Statement on the Transfer of Mosaic Embryos 2019. Reproductive\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWest, J. D., \u0026amp; Everett, C. A. (2022). Preimplantation chromosomal mosaics, chimaeras and confined placental mosaicism. Reproduction \u0026amp; fertility, 3 (2), R66-R90. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1530/RAF-21-0095\u003c/span\u003e\u003cspan address=\"10.1530/RAF-21-0095\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFragouli, E., Munne, S., \u0026amp; Wells, D. (2019). The cytogenetic constitution of human blastocysts: insights from comprehensive chromosome screening strategies. Human reproduction update, 25 (1), 15\u0026ndash;33. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/humupd/dmy036\u003c/span\u003e\u003cspan address=\"10.1093/humupd/dmy036\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThum, M. Y., Wells, V., \u0026amp; Abdalla, H. (2010). Patient selection criteria for blastocyst culture in IVF/ICSI treatment. Journal of assisted reproduction and genetics, 27 (9\u0026ndash;10), 555\u0026ndash;560. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10815-010-9457-9\u003c/span\u003e\u003cspan address=\"10.1007/s10815-010-9457-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuan Li, Du Dan, Liu Haipeng, et al. Evaluation of the clinical application value of three embryo transfer strategies in IVF-ET [J]. Journal of Reproductive Medicine, 2020, 29: 1427\u0026ndash;1432.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa Jingwen, Wang Linlin, Zhang Yan, et al. Comparison of pregnancy outcomes between fresh-cycle single blastocyst transplantation and double blastocyst transplantation [J]. Journal of Reproductive Medicine, 2021, 30 (5): 594\u0026ndash;599.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarin, D., Scott, R. T., Jr, \u0026amp; Treff, N. R. (2017). Preimplantation embryonic mosaicism: origin, sequences and the reliability of comprehensive chromosome screening. Current opinion in obstetrics \u0026amp; gynecology, 29 (3), 168\u0026ndash;174. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1097/GCO.0000000000000358\u003c/span\u003e\u003cspan address=\"10.1097/GCO.0000000000000358\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarin, D., Xu, J., \u0026amp; Treff, N. R. (2021). Preimplantation genetic testing for aneuploidy: A review of published blastocyst analysis agreement data. Prenatal diagnosis, 41 (5), 545\u0026ndash;553. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/pd.5828\u003c/span\u003e\u003cspan address=\"10.1002/pd.5828\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXiang Li, Zhou Tiejun, Wang Guangxi, et al. Effect of Ureaplasma urealyticum on sperm morphology [J]. Journal of Luzhou Medical College, 2008, 31 (2): 148\u0026ndash;150.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang Lan, Li Yidan, Xia Yan, et al. The impact of urogenital mycoplasma infection on different pregnancy outcomes [J]. Xinjiang Medicine, 2025, 55 (2): 153\u0026ndash;157.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDungan, J. S., Klugman, S., Darilek, S., Malinowski, J., Akkari, Y. M. N., Monaghan, K. G., Erwin, A., Best, R. G., \u0026amp; ACMG Board of Directors.Electronic address: [email protected] (2023). Noninvasive prenatal screening (NIPS) for fetal chromosome abnormalities in a general-risk population: An epidemic-based clinical guideline of the American College of Medical Genetics and Genomics (ACMG).\u003c/span\u003e\u003c/li\u003e\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":"archives-of-gynecology-and-obstetrics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arch","sideBox":"Learn more about [Archives of Gynecology and Obstetrics](https://www.springer.com/journal/404)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/arch/default.aspx","title":"Archives of Gynecology and Obstetrics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"ICSI blastocyst, Chromosome structural rearrangement, Chromosome microarray analysis, 20p mosaic duplication, prenatal diagnosis","lastPublishedDoi":"10.21203/rs.3.rs-8634923/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8634923/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground.\u003c/strong\u003e To investigate the antenatal diagnosis of a pregnant fetus after intracytoplasmic sperm injection (ICSI) and blastocyst transplantation. By integrating multiple technologies, we clarified the specific characteristics of the 20p mosaic duplication, evaluated its pathogenicity and clinical significance, and provided evidence for prenatal genetic counseling.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods. \u003c/strong\u003eAmniocentesis was performed on a pregnant woman identified as high-risk by non-invasive prenatal testing (NIPT) suggesting a 15q26.3 deletion.Fetal amniotic fluid exfoliative cells were analyzed using an integrated approach combining G-banding karyotyping, chromosomal microarray analysis (CMA), and fluorescence in situ hybridization (FISH).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults.\u003c/strong\u003e NIPT showed a 3.04 Mb deletion at 15q26.3.CMA showed a 20.51 Mb mosaic duplication in the 20p13p11.23 region (approximately 50% mosaicism),while G-banding karyotype analysis showed no abnormality in amniotic fluid cells.Mid-term FISH analysis of amniotic fluid cells in our hospital showed a der(15)t(15;20)(qter;p13) mosaicism, meaning that a portion of the 20p13 segment was translocated to the end of chromosome 15. FISH analysis from outer hospital indicated that 49% of cells carried a 20p13 duplication signal. Both parents had normal peripheral blood karyotypes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion.\u003c/strong\u003e This case is a complex chromosomal rearrangement in the early cleavage stage of ICSI blastocyst transfer. The high proportion of 20p13p11.23 mosaic duplication has potential serious pathogenicity, and the risk of poor prognosis in continued pregnancy is high. Termination of pregnancy is a reasonable decision. This study highlights the core value of multi-technology integration in the prenatal diagnosis of complex chromosomal abnormalities after ICSI. At the same time, it is suggested that couples with abnormal sperm parameters such as male UU infection, abnormal sperm morphology, or a history of repeated fertilization/implantation failure should be alert to the risk of mosaic chromosomal abnormalities. Strengthening preimplantation genetic testing (PGT) protocols before assisted reproduction is recommended, providing a reference for optimizing prenatal diagnosis strategies in ICSI-assisted pregnancies.\u003c/p\u003e","manuscriptTitle":"Application and value of multi-technique integration in the prenatal diagnosis of mosaicism derived of ICSI blastocyst","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-04 06:43:01","doi":"10.21203/rs.3.rs-8634923/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-04T08:10:47+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-27T08:40:14+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-21T17:00:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"225814767509322296532923730302662348529","date":"2026-04-21T11:20:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"259792148429534818827378752596045770623","date":"2026-04-21T10:17:39+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-21T10:00:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-21T16:31:36+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-21T16:21:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Gynecology and Obstetrics","date":"2026-01-19T04:08:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"archives-of-gynecology-and-obstetrics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arch","sideBox":"Learn more about [Archives of Gynecology and Obstetrics](https://www.springer.com/journal/404)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/arch/default.aspx","title":"Archives of Gynecology and Obstetrics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0b240be2-1247-45c8-84f8-37d3468d9c27","owner":[],"postedDate":"May 4th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-04T08:10:47+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-18T06:53:50+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-04 06:43:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8634923","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8634923","identity":"rs-8634923","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}