Construction of a Quality Control System for PGT Based on SNP Genotyping Technology: A Novel Method for Embryo Traceability Verification in Assisted Reproductive Technology

Construction of a Quality Control System for PGT Based on SNP Genotyping Technology: A Novel Method for Embryo Traceability Verification in Assisted Reproductive Technology | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 23 March 2026 V1 Latest version Share on Construction of a Quality Control System for PGT Based on SNP Genotyping Technology: A Novel Method for Embryo Traceability Verification in Assisted Reproductive Technology Authors : Jiahui Ma , Kexin Shi , Hongqiang Xie , Ming Gao , Yuping Niu , Li-Juan Wang , Yifang Jia , … Show All … , Ping Sun , Yingxin Zhang , Yu Wang , Xuan Gao , Zi-Jiang Chen , Yang Zou , and Yuan Gao 0000-0002-7644-2994 [email protected] Show Fewer Authors Info & Affiliations https://doi.org/10.22541/au.177429475.58772285/v1 135 views 110 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Objective: To clarify the specific causes of inconsistencies between prenatal fetal genetic results and preimplantation genetic testing (PGT) outcomes via ECIT. Design: A retrospective clinical study focusing on embryo origin tracing and PGT result discrepancy analysis. Setting: Institute of the Women, Children, and Reproductive Health, Shandong University. Sample: 16 patients with successful pregnancies after PGT-selected embryo transfer (April 2021–June 2024); clinical samples with discordant fetal genetic and PGT results were analyzed. Methods: PGT results were first rechecked to confirm accurate embryo transfer. ECIT was then used for pregnancy embryo origin tracing and discrepancy clarification. Main Outcome Measures: Identification of PGT-prenatal diagnosis inconsistency causes (natural conception, embryonic mosaicism); confirmation of embryo origin in sequential transfers and optimal implantation window. Results: Among 15 discordant cases, 2 gestational embryos were not PGT-selected or cycle-matched, but genetically related to the mother, confirming natural conception. The remaining 13 cases involved consistent embryo origin between gestation and PGT transfer, verifying accurate targeted embryo transfer with discrepancies likely attributed to embryonic mosaicism. In one sequential PGT transfer case, miscarriage tissue was confirmed as the day-5 transferred embryo. Conclusions: This study introduces a novel pregnancy embryo tracing technology applicable to a variety of clinical scenarios. It can be used to analyze the causes of fetal abnormalities and pregnancy loss, define the optimal implantation window in sequential transfer, and further refine and optimize the clinical PGT quality control system. Funding: This study was supported by Key R&D Program of Shandong Province, China（2023ZLGX02）; Key R&D Program of Shandong Province, China （2023CXPT010）. Key words: Embryo Traceability | Kinship Identification | Window of Implantation | PGT Quality Title Page Construction of a Quality Control System for PGT Based on SNP Genotyping Technology: A Novel Method for Embryo Traceability Verification in Assisted Reproductive Technology Jiahui Ma 1,2 , Kexin Shi 1,2 , Hongqiang Xie 1,2 , Ming Gao 1,2 , Yuping Niu 1.2 , Lijuan Wang 1,2 , Yifang Jia 3,4 , Ping Sun 5 , Ying-Xin Zhang 3,4 , Yu Wang 5 , Xuan Gao 1,2 , Zi-Jiang Chen 1,2 , Yang Zou 1,2, *, Yuan Gao 1,2, * 1 State Key Laboratory of Reproductive Medicine and Offspring Health, Center for Reproductive Medicine, Institute of Women, Children and Reproductive Health, Shandong University, Jinan 250012, Shandong, China | 2 Shandong Technology Innovation Center for Reproductive Health, Jinan, Shandong, 250012, China | 3 Prenatal Diagnosis Center, Shandong Provincial Hospital Affiliated to Shandong First Medical University, China | 4 Department of Clinical Laboratory, Shandong Provincial Hospital Affiliated to Shandong First Medical University, China | 5 Prenatal Diagnostic Center of Obstetrics and Gynecology Department, Qilu Hospital of Shandong University, Jinan, China *Correspondence: Yuan Gao, State Key Laboratory of Reproductive Medicine and Offspring Health, Center for Reproductive Medicine, Institute of Women, Children and Reproductive Health, Shandong University, Jinan 250012, Shandong, China (Email: [email protected] ) | Yang Zou, State Key Laboratory of Reproductive Medicine and Offspring Health, Center for Reproductive Medicine, Institute of Women, Children and Reproductive Health, Shandong University, Jinan 250012, Shandong, China (Email: [email protected] ). Running title: Embryo Consistency Identification Technology ABSTRACT Objective: To clarify the specific causes of inconsistencies between prenatal fetal genetic results and preimplantation genetic testing (PGT) outcomes via ECIT. Design: A retrospective clinical study focusing on embryo origin tracing and PGT result discrepancy analysis. Setting: Institute of the Women, Children, and Reproductive Health, Shandong University. Sample: 16 patients with successful pregnancies after PGT-selected embryo transfer (April 2021–June 2024); clinical samples with discordant fetal genetic and PGT results were analyzed. Methods: PGT results were first rechecked to confirm accurate embryo transfer. ECIT was then used for pregnancy embryo origin tracing and discrepancy clarification. Main Outcome Measures: Identification of PGT-prenatal diagnosis inconsistency causes (natural conception, embryonic mosaicism); confirmation of embryo origin in sequential transfers and optimal implantation window. Results: Among 15 discordant cases, 2 gestational embryos were not PGT-selected or cycle-matched, but genetically related to the mother, confirming natural conception. The remaining 13 cases involved consistent embryo origin between gestation and PGT transfer, verifying accurate targeted embryo transfer with discrepancies likely attributed to embryonic mosaicism. In one sequential PGT transfer case, miscarriage tissue was confirmed as the day-5 transferred embryo. Conclusions: This study introduces a novel pregnancy embryo tracing technology applicable to a variety of clinical scenarios. It can be used to analyze the causes of fetal abnormalities and pregnancy loss, define the optimal implantation window in sequential transfer, and further refine and optimize the clinical PGT quality control system. Funding: This study was supported by Key R&D Program of Shandong Province, China（2023ZLGX02）; Key R&D Program of Shandong Province, China （2023CXPT010）. Key words: Embryo Traceability | Kinship Identification | Window of Implantation | PGT Quality 1 | Introduction Birth defects, which are primarily attributed to genetic factors, are a leading cause of early abortion, stillbirth, perinatal death, infant death, and congenital disability 1 2 . Preimplantation Genetic Testing (PGT) is a technique that screens embryos for genetic variations or chromosomal abnormalities before implantation. It allows the selection of genetically normal embryos for transfer, thus reducing the risk of inherited genetic disorders, chromosomal anomalies and recurrent pregnancy loss 3 . PGT has been widely applied in the detection of monogenic disorders and the identification of carriers with chromosomal structural variations 4 . Its role in enhancing the success rate of in vitro fertilization (IVF) and preventing the transmission of genetic diseases is well recognized 5 . However，Several studies have reported inconsistent results in pregnancies following PGT application 6,7 . There is still a risk of miscarriage due to chromosomal abnormalities even after the transfer of euploid embryos 8 . Additionally, there have been reports of healthy infants being born from embryos identified as abnormal following PGT 9 . Multiple factors can contribute to these inconsistencies, such as natural conception 10 , embryonic mosaicism 11,12 , testing errors or transfer mistakes. The clinical quality control system for PGT is currently imperfect. This is mainly due to the lack of conclusive evidence and the inability to promptly identify transfer errors or contamination, which poses risks to medical safety of assisted reproduction. It may even lead to serious medical accidents and ethical issues. Therefore, determining whether the gestational embryo and the tested embryo are the same individual is crucial for analyzing the reasons behind these inconsistent results. To date, there have been no reported studies on consistency identification technologies. Given this, there has been a significant need for a simple and sensitive identification technology for embryo traceability within the quality control work-flow of assisted reproductive technology (ART). Traditional identification technologies based on short tandem repeat (STR) analysis are limited in their ability to address these issues due to restricted number of loci they can analyze. Single nucleotide polymorphism (SNP) analysis, an emerging technology, can overcome some of the limitations of STR technology to a certain extent 13,14 . However, until now, there have been no studies reporting on embryo traceability technology. In this study, we developed a novel technology for tracing gestational embryos, termed Embryo Consistency Identification Technology (ECIT). This technology can be used to establish a PGT quality control process and identify the gestational embryos in sequential transfer. 2 | Materials and Methods 2.1 | Study Subjects This study included five cases of patients who achieved successful pregnancies following PGT at the Institute of Women, Children and Reproductive Health, Shandong University but exhibited discrepancies between prenatal diagnosis or miscarriage product results and PGT embryo testing results. Additionally, one case of a patient who underwent sequential transplantation after PGT was included (Table 1 and Supplemental Table 1). 2.2 | Study Design Following PGT-A/PGT-M/PGT-SR treatment, transferable embryos were selected for single blastocyst transfer. When discrepancies were identified between prenatal diagnosis, miscarriage products, or live-born fetal results and the implanted embryo testing results, the retained PGT samples were first re-analyzed to confirm the accuracy of the PGT testing results. Consistency identification between the gestational samples and the retained samples of the transferred embryos was performed using ECIT. If the results indicated that the gestational embryo and the transferred embryo were indeed the same individual, further investigation was conducted to determine the reasons for the discrepancies. If the results indicated that the gestational embryo and the transferred embryo were not the same individual, consistency identification was performed between the sample and the parents as well as other embryos from the same cycle to further clarify the cause of the abnormalities. If the result shows that the pregnant embryo is the same individual as the father/mother, maternal/paternal contamination may have occurred during PGT. Patients underwent sequential transplantation following PGT, where one blastocyst was transferred on day 5 and another on day 7 within the same transfer cycle. Consistency identification between the submitted samples and the retained samples of the two transferred embryos was performed using ECIT to confirm the origin of the successfully gestated embryo, thereby further clarifying the window of implantation. 2.3 | Research Methods Parental genomic DNA (gDNA) was extracted from peripheral blood samples using a Blood/Tissue genomic DNA extraction kit (QIAGEN-69504) according to the manufacturer’s instruction. Then, the gDNA was double-digested with two restriction enzymes and ligated to adaptors. The combination of the two endonucleases was used to cut the gDNA to generate 2000-5000 endonuclease sites per 1-Mb region on human genome. Target DNA fragments were obtained using a magnetic bead-based selection system, followed by PCR amplification and magnetic bead purification to yield the final library products. The constructed libraries were sequenced on the DA500 sequencing platform (Suzhou Beike Medical Equipment Co., Ltd.) to obtain the total data volume. The most suitable data volume was selected based on sequencing data quality, and SNP analysis was performed using bioinformatics tools. The data were aligned to the hg19 genome using the bwa algorithm in the Sentieon software, followed by base quality calibration and SNP detection. The SNPs were further calibrated using public databases. The SNP data were quality-controlled using vcftools software, and data format conversion was performed using plink2/plink software to generate GWAS data files. The KING software (http://people.virginia.edu/~wc9c/KING) was used to compare kinship between two samples using the KING-robust method, and the Kinship value was outputted. The kinship value was used to determine the relationship of the gestational embryo. A Kinship value > 0.354 indicates that the sample and the gestational embryo are from the same source 15 (Figure 1). 3 | Results 3.1 | Traceability of Gestational Embryos in Cases of Discrepancy Between Prenatal Diagnosis and PGT In Case-1, a 39-year-old patient presented to our center for consultation and treatment due to indications including secondary infertility and chromosomal balanced translocation. The female patient has a medical history of one healthy female infant delivered via cesarean section, one induced abortion, and two spontaneous abortions. Cytogenetic analysis revealed a karyotype of 46,XX with a balanced translocation between chromosomes 8 and 11, specifically t(8;11)(q24.1;q13). The patient has requested PGT to assist in achieving a successful pregnancy. Ultimately, the couple opted to undergo both PGT-A and PGT-SR. During the embryo transfer cycle, a total of six embryos were obtained without euploid. Following clinical genetic counseling, a D5 blastocyst graded as 4BB, with a result indicating ”+ (mosaic)(4)(46%), balanced translocation carrier”, was selected for transfer. The embryo transfer resulted in a successful pregnancy (Figure S1A-B). At 20 weeks and 4 days of gestation, the patient underwent prenatal diagnosis at Shandong Provincial Hospital. The diagnostic results indicated that the fetal karyotype was normal and karyotyping showed no abnormalities (Figure S1C). We collected the patient’s amniotic fluid sample and performed concordance analysis with the embryo selected by PGT to verify consistency. Initially, we conducted a re-evaluation of the embryo selected by PGT, and the results remained consistent with the previous findings, indicating ”+(mosaic)(4)(46%), balanced translocation carrier”(Figure S1D). Subsequently, consistency analysis was performed using ECIT. The results demonstrated that a total of 202,085 valid SNP loci were identified in both the amniotic fluid sample and the embryo selected by PGT. The kinship was 0.2258, indicating that the embryo resulting in the successful pregnancy was not of the same origin as the embryo selected by PGT. Subsequently, we performed concordance analysis between the collected amniotic fluid sample and the remaining 5 blastocysts obtained in the same cycle. The results revealed that none of the other embryos shared the same origin as the embryo responsible for the successful pregnancy (Figure S1E) (Table 2). In Case-2, a 32-year-old patient had a history of missed abortion managed with medical termination (Table 2). Cytogenetic analysis revealed a karyotype of 46,XX with a balanced translocation of t(2;7)(q13;q32). Following genetic counseling, the patient underwent PGT-A and PGT-SR for assisted reproduction. During the embryo transfer cycle, 3 embryos were obtained. A D6 euploid blastocyst graded as 4BB was selected for transfer, which subsequently led to a successful pregnancy. At 22 weeks and 2 days of gestation, prenatal diagnosis performed at Qilu Hospital confirmed Trisomy 21 in the fetus, resulting in the termination of the pregnancy (Figure S2A-B). We collected samples of fetal tissue, placenta, and amniotic fluid from the aborted fetus and performed consistency analysis with the embryo selected by PGT for genetic concordance verification. Initially, we conducted a re-evaluation of the embryo selected by PGT, and the results remained a euploid blastocyst (Figure S2D). Subsequently, consistency analysis was performed using ECIT. The results demonstrated that a total of 560,572 effective SNP loci were identified between the fetal tissue sample and the embryo selected by PGT, with a kinship value of 0.4739. Similarly, 552,023 effective SNP loci were detected between the placental sample and the embryo selected by PGT, with a kinship value of 0.4740, while 536,995 effective SNP loci were identified between the amniotic fluid sample and the embryo selected by PGT, with a kinship value of 0.4725. These findings indicate that the fetus, placenta, and amniotic fluid share the same origin as the embryo selected by PGT (Figure S2C). Subsequently, we performed copy number variation (CNV) analysis on each sample. The fetal sample exhibited trisomy 21, while the placental sample showed mosaic trisomy 21 with a mosaicism ratio of 33% (Figure S2D) (Table 2). 3.2 | Traceability of Gestational Embryos in Cases of Discrepancy Between Miscarriage Sample and PGT In Case-3, a 28-year-old patient underwent dilation and curettage due to a missed abortion, with cytogenetic analysis revealing a karyotype of 45,XX,der(13;21)(q10;q10) (Table 2). Following genetic counseling, PGT-SR was recommended. During the embryo transfer cycle, a total of 13 embryos were obtained, and a D5 euploid blastocyst graded as 4AA was selected for transfer, resulting in a successful pregnancy (Figure S3A-B). At 7 weeks and 6 days of gestation, the patient experienced an early miscarriage. These findings were inconsistent with the results obtained from PGT. We collected chorionic villus tissue samples from the patient and performed genetic concordance analysis to compare them with the embryo selected by PGT. Initially, we conducted a re-evaluation of the embryo selected by PGT, and the results remained a euploid blastocyst (Figure S3D). Subsequently, consistency analysis was performed using ECIT. The results demonstrated that a total of 209,150 effective SNP loci were identified between the chorionic villus tissue sample and the embryo selected by PGT, with a kinship value of 0.4449. These findings indicate that the gestational embryo originated from the embryo selected by PGT (Figure S3E) (Table 2). In Case-4, a 42-year-old patient experienced a total of 10 pregnancies (1 following ovulation induction and 9 spontaneous pregnancies) during 2006 to February 2017 (Table 2). During the embryo transfer cycle, a total of 3 embryos were obtained, and a D5 euploid blastocyst graded as 4BB was selected for transfer, resulting in a successful pregnancy (Figure S4A). At 9 weeks and 4 days of gestation, the patient experienced an early miscarriage. Genetic analysis of the products of conception revealed a duplication of the chromosomal region seq[GRCh37] Xp22.33-Xq28×1 (Figure S4B). These findings were inconsistent with the results obtained from PGT. We collected chorionic villus tissue samples from the patient and performed genetic concordance analysis to compare them with the embryo selected by PGT. Initially, we conducted a re-evaluation of the embryo selected by PGT, and the results remained a euploid blastocyst (Figure S4C). Subsequently, consistency analysis was performed using ECIT. The results demonstrated that a total of 211,640 valid SNP loci were identified in both the amniotic fluid sample and the transferred embryo. The kinship was 0.2014, indicating that the embryo resulting in the successful pregnancy was not of the same origin as the embryo selected by PGT. Subsequently, we performed genetic concordance analysis between the collected chorionic villus sample and the other 2 blastocysts obtained in the same cycle. The results demonstrated that neither of the remaining embryos shared the same origin as the embryo selected by PGT (Figure S4D) (Table 2). Following communication with the patient, we learned that the patient had engaged in intercourse during the embryo implantation window. This indicates that the gestational embryo resulted from natural conception rather than the embryo transferred following PGT. In addition, we applied ECIT to another 10 cases, as detailed in Supplemental Table 2. The Kinship-level of these cases were the duplicate between aborted tissue and PGT sample. 3.3 | Traceability of Gestational Embryos in Cases of Discrepancy Between Fetus Sample and PGT In Case-5, a 33-year-old patient had previously undergone induced labor due to a prenatal diagnosis of ”anencephaly” and subsequently achieved a successful pregnancy through in IVF at our center (Table 2). At 10 weeks of gestation, prenatal diagnosis indicated ”absence of fetal heartbeat,” followed by medical termination of pregnancy. CNVs analysis of the aborted tissue revealed trisomy 16. After genetic counseling, PGT-A was recommended. During the embryo transfer cycle, a total of 7 embryos were obtained, and a euploid blastocyst with a grade of 4BB was selected for transfer (Figure 6, A). The patient delivered a full-term male infant; however, prenatal diagnostic testing via amniocentesis revealed a 10.29 Mb mosaic duplication in the q22.3q23.2 region of chromosome 5. Subsequent reanalysis at our center confirmed the accuracy of this finding (Figure S5, B). We collected tissue samples from the infant and performed consistency analysis to compare them with the embryo selected by PGT. Initially, we conducted a re-evaluation of the transferred embryo, and the results remained a euploid blastocyst (Figure S5C). Subsequently, consistency analysis was performed using ECIT. The results demonstrated that 253,899 effective SNP loci were identified in both the fetal sample and the transferred embryo, with a kinship value of 0.4564 (Figure S5D), indicating that the infant and the transferred embryo originated from the same source (Table 2). 3.4 | Traceability of Gestational Embryos in Sequential Transplantation In Case-6, a-35-year-old patient had a history of missed abortion treated with dilation and curettage. In March 2020, she underwent IVF at our center. A total of 5 blastocysts were transferred sequentially, none of which resulted in a successful pregnancy. Following genetic counseling, a sequential embryo transfer strategy was adopted. A euploid blastocyst graded 4AB on day 5 and a euploid blastocyst graded 4BB on day 7 were selected for transfer (Figure S6A). At 8 weeks and 2 days of gestation, the patient experienced an early miscarriage. Genetic analysis of the miscarriage product confirmed that the embryo remained euploid (Figure S6B). We collected chorionic tissue samples from the patient and performed concordance analysis with the two transferred embryos to determine the origin of the final pregnancy. The results demonstrated that the chorionic tissue sample and the transferred day 5 euploid blastocyst shared 220,282 valid SNP sites, with a kinship value of 0.4492. In contrast, the chorionic tissue sample and the transferred day 7 euploid blastocyst shared 226,286 valid SNP sites, with a kinship value of 0.2321 (Figure S6D) (Table 2). The results confirmed that the miscarried chorionic tissue originated from the day 5 transferred blastocyst, while the day 7 transferred embryo did not result in a successful pregnancy. 4 | Discussion 4.1 | Main Findings ECIT efficiently and accurately identifies the genetic relationship between the pregnancy embryo and the embryo selected for transfer via PGT. Its utility spans various clinical scenarios, including prenatal diagnosis, analysis of miscarriage tissues, neonatal testing, and sequential embryo transfer. ECIT overcomes the limitations of existing kinship analysis methods and significantly enhances the quality control process of PGT. 4.2 | Strengths and Limitations ECIT demonstrats high-precision detection capability (≥200,000 SNP loci at ≥10X sequencing depth) and broad clinical utility, including applications in prenatal diagnosis, miscarriage tissue analysis, and sequential embryo transfer. Although, ECIT has the potential applications in PGT quality control for scenarios such as embryo contamination and testing/transfer mistakes, no such cases were identified in this study. 4.3 | Interpretation Existing chromosomal abnormality detection methods are limited by low sequencing depth 16 , only capable of identifying chromosomal segment copy number variations (CNVs) without precise single SNP base resolution 17 . Monogenic disease screening assays typically target specific genomic regions rather than full-genome coverage 18 . Traditional STR technology analyzes 20+ autosomal STR loci, but IVF embryos from the same parents share highly similar genetic backgrounds, and whole-genome amplification (WGA) product STR testing suffers from high allele dropout and assay failure rates. 19–21 . This study develops the first SNP-based pregnancy embryo tracing technology—ECIT. Based on reduced-representation genome sequencing, ECIT delivers an average sequencing depth ≥10X and identifies ≥200,000 effective SNP loci, drastically boosting detection depth and accuracy; it enables precise embryonic consistency verification even for genetically similar samples, and requires only 3–5 trace cells, improving result reliability for low-quantity samples. The impact of sexual intercourse during IVF transfer cycles remains controversial, yet associated risks cannot be ignored 22 . Despite physician recommendations to avoid intercourse during the PGT transfer window, pre- and post-transfer natural conception remains possible 23 . Historically, it was difficult to distinguish whether chromosomally abnormal embryos originated from PGT transfer or natural conception, fueling patient doubts about testing and transfer quality control. This study confirms that cycle-stage natural conception is a key cause of PGT transfer outcome inconsistencies. ECIT verified that the successfully implanted embryos in Case 1 and Case 4 were not the PGT-transferred embryos nor other cycle embryos, eliminating testing/transfer errors and confirming natural conception; patient interviews confirmed non-compliance with intercourse restrictions during the implantation window, aligning with these findings. In PGT, 3–5 trophectoderm (TE) cells are biopsied for genetic testing, preserving the inner cell mass (ICM) 24 .The ICM develops into the fetus, while the TE forms part of the placenta 25,26 . Preimplantation mosaic embryos are common (5%–15% of blastocysts) 27–32 , and this study identifies mosaicism as a major driver of discrepancies between PGT results and post-transfer pregnancy outcomes 33–35 . In Case 2, the fetus and placenta derived from the PGT-selected euploid embryo, but amniotic fluid showed trisomy 21 and placental tissue exhibited 33% mosaicism, suggesting PGT false negativity due to confined placental mosaicism. Similarly, ECIT confirmed Case 3 and Case 5 pregnancies originated from PGT-selected embryos, with miscarriage tissue showing 73% and 74% mosaicism, respectively. A properly timed endometrial implantation window is critical for IVF success 36,37 . transfer timing is typically guided by ultrasound-measured endometrial thickness and receptivity, but personalized window assessment is needed for individual patients 38–40 . Sequential embryo transfer may extend the implantation window and improve pregnancy rates, but identifying the successfully implanted embryo has been challenging 41 . This study pioneeringly employs ECIT to pinpoint the implantation window for gestational embryos in patients with sequential embryo transfer. In our study’s Case 6, ECIT confirmed that the successful pregnancy resulted from a euploid blastocyst implanted on Day 5, not Day 7. Though this study has only one such case, future large-scale clinical research could further clarify the implantation window, holding great clinical value for optimizing assisted reproductive transfer strategies. 4.4 | Clinical Implications ECIT refines and optimizes the clinical quality control work-flow in PGT cycles (Figure 8). When discrepancies arise between prenatal diagnosis, miscarriage tissues, or live-birth infant results and the PGT results of the transferred embryo, the retained PGT samples must first be reanalyzed to confirm the initial PGT report’s accuracy. Then, ECIT should be performed between the gestational embryo and the PGT-selected embryo. The possible reasons are as follows, if ECIT confirms that the gestational embryo matches the PGT-selected embryo, the discrepancy may stem from embryonic mosaicism or limitations in biopsy and testing techniques. Conversely, if they don’t match, kinship analysis should be conducted on the couple’s samples and other embryos from the same cycle. Genetic unrelatedness of the gestational embryo to the couple implies potential transfer of a wrong embryo from another couple. If it is genetically related to the couple and matches another of their embryos, a testing or transfer error is likely. Genetic relatedness to the couple but no match with any other embryos may indicate a natural conception. If the gestational embryo matches the father or mother, maternal or paternal contamination during PGT may have occurred. 4.5 | Research Implications This study demonstrates the multifaceted research value of ECIT in reproductive medicine. The SNP data (≥200,000 loci, ≥10X depth) of ECIT overcomes the limitations of conventional STR technology (20 loci) and targeted sequencing. From a clinical translation perspective, the standardized quality control work-flow in PGT cycles (Figure 8) proposed in this study enables comprehensive analysis of potential causes when discrepancies occur between gestational embryo and the PGT-selected embryo. Future research could apply ECIT to more sequential embryo transfer cases to explore the optimal implantation window. Additionally, further investigations should explore potential applications of ECIT in broader clinical scenarios . 5 | Conclusion This study introduces a novel pregnancy embryo tracing technology applicable in various clinical scenarios. It can identify the origin of gestational embryos when discrepancies occur between prenatal diagnosis, miscarriage products, or live-birth fetus and PGT results. This aids in analyzing the causes of fetal abnormalities and pregnancy loss. Additionally, it can determine the origin of embryos responsible for a successful pregnancy in sequential embryo transfers and clarify the optimal implantation window offering valuable guidance for subsequent patient treatment. This technology refines and optimizes the clinical PGT quality control system. In summary, ECIT fills the current gap in embryo traceability technology and holds significant clinical value. Its potential applications also extend to forensic analysis of trace biological samples and archaeological studies involving ancient DNA identification, among other fields. We believe ECIT will find broader and more extensive applications in the future. Acknowledgments The authors are indebted to all staff in the reproductive medicine center for providing help. We thank all patients involved in this study. Conflicts of Interest All authors disclose no conflicts of interest related to this work. Author Contributions Y.G.: Conceptualization, Methodology, Funding acquisition, Supervision, Writing - review and editing. Y.Z.: Conceptualization, Methodology, Data curation, Formal analysis, Writing - original draft, Writing - review and editing. X.G.: Conceptualization, Methodology, Writing - review and editing. C.Z.J: Conceptualization, Methodology, Writing - review and editing. J.M.: Methodology, Data curation, Formal analysis, Writing - original draft, Writing - review and editing. 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Efficacy of the endometrial receptivity array for repeated implantation failure in Japan: A retrospective, two‐centers study. Reprod Medicine & Biology 2017;16(3):290–6. 40. Bassil R, Casper R, Samara N, Hsieh T-B, Barzilay E, Orvieto R, et al. Does the endometrial receptivity array really provide personalized embryo transfer? J Assist Reprod Genet 2018;35(7):1301–5. 41. Zhou W, Cheng Z, Wang C, Feng Y. Clinical outcome analysis of sequential transplantation of frozen-thawed embryo transfer cycle: a retrospective study. Medicine 2023;102(8):e33042. Tables/Figures Caption List Table 1 Basic Information of the Study Subjects. Table 2 Summary of Research Findings. Table S1. Basic Information of the Supplemental Study Subjects. Table S2.Summary of the Supplemental Research Findings. Figure 1. ECIT Roadmap. Figure 2. Clinical Quality Control System for PGT. Figure S1. Case-1 Analysis of clinical sample testing results and kinship relationships. Figure S2. Case-2 Analysis of clinical sample testing results and kinship relationships. Figure S3. Case-3 Analysis of clinical sample testing results and kinship relationships. Figure S4. Case-4 Analysis of clinical sample testing results and kinship relationships. Figure S5. Case-5 Analysis of clinical sample testing results and kinship relationships. Figure S6. Case-6 Analysis of clinical sample testing results and kinship relationships. Supplementary Material File (figure 1. ecit roadmap.docx) Download 107.00 KB File (figure 2. clinical quality control system for pgt.docx) Download 464.55 KB File (table 1. basic information of the study subjects.docx) Download 18.33 KB File (table 2. summary of research findings.docx) Download 19.76 KB Information & Authors Information Version history V1 Version 1 23 March 2026 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords clinical guidelines early pregnancy loss: medical fertility control genetics Authors Affiliations Jiahui Ma Shandong University State Key Laboratory of Reproductive Medicine and Offspring Health View all articles by this author Kexin Shi Shandong University State Key Laboratory of Reproductive Medicine and Offspring Health View all articles by this author Hongqiang Xie Shandong University State Key Laboratory of Reproductive Medicine and Offspring Health View all articles by this author Ming Gao Shandong University State Key Laboratory of Reproductive Medicine and Offspring Health View all articles by this author Yuping Niu Shandong University State Key Laboratory of Reproductive Medicine and Offspring Health View all articles by this author Li-Juan Wang Shandong University State Key Laboratory of Reproductive Medicine and Offspring Health View all articles by this author Yifang Jia Shandong Provincial Hospital Affiliated to Shandong First Medical University View all articles by this author Ping Sun Qilu Hospital of Shandong University View all articles by this author Yingxin Zhang Shandong Provincial Hospital Affiliated to Shandong First Medical University View all articles by this author Yu Wang Qilu Hospital of Shandong University View all articles by this author Xuan Gao Shandong University State Key Laboratory of Reproductive Medicine and Offspring Health View all articles by this author Zi-Jiang Chen Shandong University State Key Laboratory of Reproductive Medicine and Offspring Health View all articles by this author Yang Zou Shandong University State Key Laboratory of Reproductive Medicine and Offspring Health View all articles by this author Yuan Gao 0000-0002-7644-2994 [email protected] Shandong University State Key Laboratory of Reproductive Medicine and Offspring Health View all articles by this author Metrics & Citations Metrics Article Usage 135 views 110 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Jiahui Ma, Kexin Shi, Hongqiang Xie, et al. Construction of a Quality Control System for PGT Based on SNP Genotyping Technology: A Novel Method for Embryo Traceability Verification in Assisted Reproductive Technology. Authorea . 23 March 2026. DOI: https://doi.org/10.22541/au.177429475.58772285/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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