Risks and Complications of Monozygotic Triplet Pregnancy Following Single Blastocyst Transfer: Insights from a Case Report and Future Directions.

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Divyesh Upadhyay, Rajia Al Halaby, Sudha Anandt, Firas Albuz, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5055637/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background This case report explores the rare occurrence of monozygotic triplet pregnancies resulting from a single blastocyst transfer in Assisted Reproductive Technology (ART). The novelty lies in documenting this unusual outcome, which provides valuable insights into the potential for zygotic splitting and its implications for reproductive practices. Case Presentation The patient, a woman with diminished ovarian reserve, underwent in vitro fertilization with intracytoplasmic sperm injection (IVF-ICSI) and Preimplantation Genetic Testing for Aneuploidy (PGT-A). The testing identified one euploid and one aneuploid embryo. With informed consent, a single euploid frozen-thawed embryo was transferred. The pregnancy resulted in the development of monozygotic triplets, as confirmed by the presence of three yolk sacs and fetal poles. Unfortunately, the pregnancy ended in a spontaneous miscarriage at the 10th week of gestation. Conclusion The development of monozygotic triplets from a single embryo transfer underscores the unpredictable nature of ART and the potential for rare outcomes such as zygotic splitting. This case highlights the need for heightened awareness, early detection, and proactive management of such pregnancies to address associated risks. Further research is needed to better understand the mechanisms behind zygotic splitting in ART procedures and to improve clinical outcomes. Monozygotic Triplet Pregnancies Single Blastocyst Transfer Assisted Reproductive Technology (ART) Embryo Splitting In Vitro Fertilization (IVF). Figures Figure 1 Introduction Monozygotic multiple pregnancies occur when a single embryo splits to form genetically identical embryos around the time of blastocyst formation ( 1 ). Monozygotic twin pregnancy occurs as a result of the fertilization of a single egg with a single sperm ( 2 , 3 ). The embryo is then split at a later stage (usually in the blastocyst stage). It is more frequent that the embryo is divided into two (monozygotic twinning); but in certain cases, the inner cell mass can divide into three or four leading to the development of multiple fetuses or higher-order monozygotic multiple pregnancies ( 2 , 4 – 6 ). The timing of monozygotic splitting of the embryo is probably not fixed and the mechanism varies from one set to another ( 7 ). A zygote may split anytime during the first 14 days after fertilization, resulting in the various forms of monozygotic twins ( 8 ). That is, the exact mechanisms have not yet been established in natural or assisted human reproduction cases ( 9 ). Many theories have been put forward as to how and when this occurs. The likelihood of occurrence is at any point in terms of the establishment of polarity and axes ( 7 ). Familial (i.e., genetic) and infertility treatment are considered potential factors leading to multiple pregnancies ( 10 ). In this case report, we conducted a thorough literature review on monozygotic triplet pregnancies. Our search spanned from February 1994 to October 2023, using databases like PubMed and Google Scholar. We used keywords such as 'monozygotic triplets,' 'Single Embryo Transfer,' 'Assisted Reproductive Technology,' and 'Embryo Splitting,' along with relevant MeSH terms. This approach ensured a comprehensive examination of studies and case reports on monozygotic triplet pregnancies resulting from single blastocyst transfers. Incidence of Monozygotic Twinning and Triplet Pregnancies In the evolving field of Assisted Reproductive Technology (ART), there's been a notable increase in monozygotic twinning and triplet pregnancies, especially following blastocyst transfer. Studies have consistently shown a rise in such cases ( 11 – 15 ). While the global frequency of monozygotic twins remains constant at about 4:1000 births, ART conceptions demonstrate a 2.25 times higher rate than natural conceptions, with monozygotic triplet pregnancies occurring 100 times more frequently in IVF than in natural pregnancies, albeit still rare at 0.048% ( 3 , 15 ). Regionally, Japan reports a low prevalence of 0.04% for triplet pregnancies post-single blastocyst transfer, with 122 cases observed from 2007 to 2014 ( 16 ). A comprehensive study by ( 17 ) indicated a 1.36% prevalence of zygotic splitting in multiple pregnancies post-SET, analyzing 937,848 SET cycles. This study, along with others ( 12 , 18 , 19 ), identified factors like frozen-warmed embryo transfer cycles, blastocyst culture, and assisted hatching as risks for zygotic splitting. The paper underlines the importance of understanding the mechanisms of embryo splitting in ART to improve clinical outcomes and insights into high-order pregnancies like monozygotic triplets, particularly in the context of single blastocyst transfer using IVF-frozen embryo transfer. Monozygotic Triplet Pregnancies and Assisted Reproductive Technology The practice of Assisted Reproductive Technology (ART) has been implicated in increasing the incidence of monozygotic multiples, such as twins and triplets. Several key aspects of ART, including embryo manipulation, embryo culture, assisted hatching, and blastocyst transfer, have been postulated as significant contributing factors ( 13 , 17 ). Monozygotic twinning is an important phenomenon because monozygotic pregnancies are linked to increased obstetrical risks. Monozygotic pregnancies carry a higher risk of both, fetal and maternal morbidities prematurity, intrauterine growth restriction, and prenatal death ( 20 ); Maternal complications include an increased risk of pregnancy-induced hypertension, gestational diabetes, peripartum hemorrhage, operative delivery, postpartum depression and anxiety and parenting stress ( 21 , 22 ). The most commonly reported clinical complication during monozygotic pregnancy is chronic feto-fetal transfusion syndrome (twin-to-twin transfusion syndrome and twin anemia polycythemia sequence) resulting from unbalanced blood flow from one twin to the other ( 23 ). In addition, Twin-to-Twin Transfusion Syndrome (TTTS) is highly associated with the fetal death of one fetus due to a sudden drop in pressure and/or heart rate at one fetal end ( 23 ). Reducing the incidence of high-order multiple pregnancies associated with ART is one of the major objectives of reproductive medicine ( 24 ). The main strategy to reduce high-order pregnancies linked to ART is to promote Single Embryo Transfer (SET) after In Vitro Fertilization (IVF). But even by transferring one embryo, still there is a risk of multiple pregnancies due to monozygotic twinning. This is the reason why it’s very important to try to find the reason(s) behind this phenomenon, to try to prevent it. Ultrasonography is of utmost importance in early detection of monozygotic triplet, and pregnancy monitoring. Prenatal ultrasound (US) is essential in the management of monozygotic multiple pregnancies to allow determination of zygosity, chorionicity, evaluation of fetal anatomy, and serial assessment of cervical length and fetal growth ( 3 ). In addition, ultrasound aids in the detection of clinical complications during pregnancy such as Twin-to-Twin Transfusion Syndrome (TTTS) and intrauterine growth restriction (IUGR) ( 20 ). Hence, ultrasound assessment is very useful in managing high-risk pregnancies associated with ART. Case Presentation A 36-year-old woman (Gravida 4, Para 4) presented at our center (Al Ain Fertility Center, U.A.E.), with secondary infertility due to low ovarian reserve. No relevant patient medical history or family history was reported. The duration of infertility was reported to be 3 years. Her AMH level was found to be < 0.05ng/ml (the normal range of AMH is 2.0 to 6.8ng/ml). She had a normal karyotype and the test for fragile-X syndrome was negative. Semen analysis was found to be normal for her husband. Other relevant clinical investigations for the couple showed normal results. The patient had an IVF- Intracytoplasmic Sperm Injection (ICSI) treatment under standard antagonist protocol with preimplantation genetic testing for aneuploidy (PGT-A). Ovarian stimulation was started on the second day of the cycle with an injection of 150microgram single dose long-acting recombinant FSH (Elonva; Merck), 300IU daily dose of urine-derived FSH, and LH (Menopur; Ferring B.V), 5mg daily dose of letrozole (Femara; Novartis). When the leading follicles reached 17mm, she received a double trigger with r-hCG (Ovitrelle; Merck) and 0.2mg GnRH agonist (Gonapeptyl, Ferring) 36 hours before egg collection. Transvaginal ultrasound-guided aspiration under general anesthesia was performed and 3 follicles were aspirated, yielded 3 oocytes (MII). Semen analysis was conducted on the husband's frozen ejaculate sample using Kruger's strict criteria ( 25 ), which showed a sperm count of 56.6x106 per ml, with sperm motility at 60%, normal forms at 4%, and a DNA fragmentation rate of 19.5% (Halosperm®, ( 26 – 28 )). In addition, sperm preparation, sorting, and selection were done using magnetically activated sperm enrichment (MASE) protocol for ICSI ( 29 , 30 ). ICSI was performed on all of them. All 3 oocytes were fertilized (2PN) and resulting embryos were cultured in sequential media by using cleavage medium and blastocyst medium (Quinn’s Advantage medium, Sage biopharma). There were 3 high-quality embryos (graded 1,2 and 3 as per the Gardner embryo blastocyst grading system, ( 31 , 32 ) on day 3. One blastocyst (embryo no. 3) was biopsied and frozen on day 5, another blastocyst (embryo no. 2) was biopsied and frozen on day 6, and one blastocyst (embryo no. 1) was arrested on day 6. PGT-A was performed (Next Generation Sequencing - Ion ReproSeq™ PGS) for comprehensive chromosome aneuploidy screening which diagnosed a normal-euploid (46,XX) embryo (embryo no. 3) and an abnormal (45XX,-13) embryo (embryo no.2). Endometrial preparation for Frozen Embryo Transfer (FET) was done under standard protocol. The patient started with a daily dosage of 6mg estradiol valerate (Progynova; Bayer) from the second day of the cycle. On day 10, a transvaginal ultrasound scan was performed and triple lining endometrium was visualized measuring 7.5mm. She had a blood test that showed serum concentration of estradiol and progesterone to be 374pg/ml and 0.39 ng/ml respectively. Then, she started progesterone priming (day P1) with progesterone gel (Crinone gel, Merck) and 10mg/daily dydrogesterone (Duphaston; Abbott) to support the luteal phase. On day P + 5, serum concentrations of estradiol and progesterone were found to be 151.2pg/ml and 46.6ng/ml respectively. On day P + 6, a single euploid frozen-thawed embryo (embryo no. 3) was transferred under transvaginal ultrasound guidance (performed under standard protocol). She continued with the same medication after the embryo transfer. A pregnancy test done after 12 days showed a positive result (serum beta-hCG was 134.1mIU/ml). An ultrasound scan performed 2 weeks later, (6 weeks of pregnancy) showed a single gestational sac with three yolk sacs and fetal poles. Crown–rump lengths were 0.37cm, 0.40cm, and 0.37cm with positive heartbeats (Fig. 1) . She continued the same medication and the ultrasound performed at 7 gestational weeks revealed a single intrauterine gestational sac and the presence of three yolk sacs. Crown–rump lengths were 0.80cm, 0.96cm, and 0.82 cm with positive heartbeats. The couple was counseled on the higher risk involved in monozygotic multiple pregnancies and the patient was further referred for pregnancy follow-up to an obstetric unit in a tertiary hospital. In the presented case, the patient adhered to a meticulously planned IVF-ICSI treatment protocol, including ovarian stimulation, embryo development, and endometrial preparation for frozen embryo transfer (FET). She tolerated the interventions well, with no adverse reactions reported to the medications or procedures. The regimen's effectiveness was initially confirmed by a positive pregnancy test and the development of monozygotic triplets, though the pregnancy, unfortunately, ended in a spontaneous miscarriage at the 10th week of pregnancy. Discussion This case report and literature review focus on monozygotic (MZ) triplet pregnancies, happening sometimes after Assisted Reproductive Technologies (ART) such as In Vitro Fertilization (IVF) and Intracytoplasmic Sperm Injection (ICSI). Conducted through systematic searches of the PubMed database, the review offers a comprehensive understanding of these pregnancies, particularly those arising from single blastocyst transfers. It includes a detailed case description of a specific MZ triplet pregnancy resulting from a single blastocyst transfer via IVF-Frozen Embryo Transfer (IVF-FET), encompassing the patient's history, treatment protocols, and outcomes, to provide a complete view of the unique aspects of the case. Furthermore, the report critically examines potential risk factors associated with monozygotic multiple pregnancies, such as blastocyst transfer and assisted hatching, emphasizing the need for further research into their role in zygotic splitting. Complementing this is a comparative analysis within the ART context, contrasting various cases to shed light on the potential mechanisms behind monozygotic twinning or tripling. A summarized literature search is presented in Table 1 . The patient in the present case had a low ovarian reserve, which is a common characteristic among several cases in the literature. For example, Gurunath et al. ( 33 ) described a 29-year-old woman undergoing IVF, and Schlueter et al. ( 34 ) reported on a 34-year-old woman undergoing ICSI, both with no relevant medical history, similar to our case. The treatment protocols varied, with some cases utilizing fresh embryo transfers and others frozen, as in the case of Ota et al. ( 35 ), where a frozen blastocyst transfer resulted in the live birth of triplets. In the realm of assisted reproductive technologies like IVF and ICSI, the trend towards single blastocyst transfer is notable, as it aims to balance the reduction of multiple pregnancy risks with favorable pregnancy rates. This approach is reflected in various studies: Saravelos et al. ( 36 ) reported monozygotic triplets from both single morula and blastocyst transfers, while Sutherland et al. ( 37 ) noted a miscarriage following a single blastocyst transfer. Our study, involving a 36-year-old undergoing IVF/ICSI with a single Day 5 blastocyst, also ended in miscarriage at 10 weeks, contrasting with Dessolle et al. ( 38 ) and Saravelos et al. ( 36 ), where single embryo transfers resulted in the birth of twins and triplets, respectively. Ota et al. ( 35 ) further documented a successful triplet birth from a single Day 5 blastocyst. These findings underscore the unpredictability and complexity inherent in monozygotic triplet pregnancies post-single embryo transfer, emphasizing the importance of careful monitoring and management in such scenarios. In the context of monozygotic triplet pregnancies following single blastocyst transfer in Assisted Reproductive Technology (ART), outcomes and complications exhibit considerable variability. The present case, characterized by the presence of three yolk sacs and fetal poles, unfortunately, culminated in a miscarriage at 10 weeks, underscoring the heightened risk of adverse outcomes. This contrasts with other reported cases, such as those by Dessolle et al. ( 38 ), where a successful birth of two healthy females was achieved after selective fetal reduction, and by Saravelos et al. ( 36 ), who documented both a live birth at 35 + 6 weeks following selective reduction and an ongoing twin pregnancy after similar intervention. Other reports, like those from Sutherland et al. ( 37 ), Schlueter et al. ( 34 ), and Ota et al. ( 35 ), also indicate a range of outcomes, including miscarriages and live births at various gestational ages, with complications such as Intrauterine Growth Restriction (IUGR), Twin-to-Twin Transfusion Syndrome (TTTS), early delivery, and maternal issues like hypertension and bleeding. These cases collectively highlight the complex and high-risk nature of monozygotic triplet pregnancies in ART. The mechanisms behind monozygotic multiples in Assisted Reproductive Technology (ART), particularly triplet pregnancies following Single Embryo Transfer (SET), remain elusive. Procedures such as Intracytoplasmic Sperm Injection (ICSI), blastocyst culture, and assisted hatching, which involve breaching the zona pellucida, are thought to increase the likelihood of these occurrences. This is evidenced by studies like those of Salat-Baroux et al. ( 39 ) and Belaisch-Allart et al. ( 40 ). Moreover, a literature search, as presented in Table 1 , shows case reports of monozygotic triplets following single embryo transfer. Various factors, including the type of ART procedure, the stage of embryo development, and embryo morphology, are implicated in these findings. The research by Ferri et al., ( 15 ) Ikemoto et al. ( 17 ) Faraj et al. ( 41 ), Risquez et al. ( 42 ), and Peramo et al.( 13 ) suggests a multifactorial process, influenced by a combination of treatment protocols and patient characteristics. Despite the increased incidence after ART, monozygotic triplets are still a rare phenomenon. Ongoing research is critical to understand these mechanisms better and to develop strategies that minimize their occurrence while optimizing patient outcomes. Mechanisms that may contribute to the occurrence of monozygotic triplet pregnancies: The inner cell mass (ICM) is a collection of cells within the blastocyst that eventually develops into the embryo. A monozygotic triplet pregnancy arises from two distinct ICM splitting events. The timing of these splits determines the configuration of the chorion and amnion compartments, which are the protective membranes encasing the embryo ( 12 , 33 , 36 , 43 ). Recognizing and assessing such ICM splitting occurrences is vital, as emphasized by ( 37 ), who suggests that these evaluations should be integrated as a standard procedure in clinical embryology. Factors potentially involved in embryo splitting are: 1. Blastocyst Culture and In-Vitro Culture Conditions: Advances in cell culture media have enabled the in-vitro development of embryos to the blastocyst stage. This transition from the cleavage stage to blastocyst stage embryo transfer may influence the likelihood of monozygotic splitting. The extended culture period and varying media components can impact the embryo's development and hatching behavior. Additionally, the in-vitro progression to the blastocyst stage allows for prolonged observation and potentially improved embryo selection. However, this longer culture period might heighten the risk of monozygotic splitting due to changes in the embryo's microenvironment and prolonged exposure to artificial culture conditions. These alterations in the in-vitro culture environment are significant in inducing zygotic splitting ( 15 , 17 ). The specific conditions under which embryos are cultured in vitro play a crucial role in their development. Factors like increased glucose levels in the culture medium can lead to zygotic splitting by affecting the embryos' metabolic rates and division patterns, thereby influencing the incidence of monozygotic splitting ( 1 ). 2. Impact of Zona Pellucida Alterations: Extended periods of in-vitro culture can lead to the hardening of the zona pellucida, causing blastocyst herniation, where the blastocyst protrudes through the zona pellucida. This may result in the splitting of the inner cell mass and lead to monozygotic multiples. Additionally, any manipulation of the zona pellucida, whether mechanical or chemical, such as procedures aimed at thinning the zona pellucida to aid hatching, can induce embryo splitting ( 17 ). Furthermore, the exposure of the zona pellucida to culture medium, rather than oviductal or uterine secretions in IVF cycles, might also contribute to this hardening ( 40 ). Embryo manipulation in IVF includes techniques like, assisted hatching, and frozen-warmed embryo transfer, all of which can influence the embryo's division rates. Assisted hatching, involving a small incision in the zona pellucida, might induce the embryo to split ( 13 , 17 ). These procedures can modify the physical and biochemical properties of the zona pellucida. These alterations may influence the way the embryo hatches and potentially increase the likelihood of monozygotic splitting ( 40 ). 3. Artificial Shrinkage in Conjunction with Vitrification and Warming Vitrification, a quick-freezing method for embryo preservation, and warming, the corresponding thawing process for these embryos, introduce various factors that can complicate the identification of a specific cause for monozygotic triplet pregnancies. The stress and alterations caused by these techniques might influence the development and division of the embryo. Research indicates that artificial shrinkage significantly enhances the implantation rate, clinical pregnancy rate, and live birth rate of warmed blastocysts. However, it also increases the likelihood of monozygotic twin pregnancies ( 44 ). 4. Influence of Gonadotropin Concentrations and Ovarian Stimulation: Elevated levels of gonadotropins, which are hormones used in ovarian stimulation, can lead to the hardening of the zona pellucida, the outer layer of the embryo crucial for implantation. This hardening can affect the embryo's ability to hatch and implant into the uterine wall, potentially leading to monozygotic multiples. Ovarian stimulation, used in IVF to induce the ovaries to produce multiple eggs, may also impact the zona pellucida and the embryo itself, increasing the likelihood of embryo splitting and monozygotic multiples ( 15 , 17 , 45 ). 5. Younger Maternal Age Younger maternal age is often associated with higher fertility and better embryo quality. However, it is also considered a potential risk factor for monozygotic splitting. The exact physiological reasons are not entirely understood but could involve factors like higher hormonal levels or more robust uterine conditions conducive to embryo splitting ( 12 , 17 , 46 ). 6. Genetic Factors While the exact genetic factors are not well understood, it is believed that certain genetic predispositions may make an embryo more likely to split into monozygotic multiples. These could include specific gene mutations or epigenetic factors that influence how the embryo divides ( 17 , 47 ). Despite all the previous factors discussed, the precise causative mechanisms remain largely unelucidated ( 1 , 35 – 38 , 41 , 42 , 48 ). Complications and Risks Associated with Monochorionic Multiple Pregnancies Monochorionic multiple pregnancies, particularly those resulting from monozygotic splitting, are associated with a range of complications and risks that can affect both the fetuses and the mother. These pregnancies are characterized by shared placental circulation, which can lead to a variety of complications. Medical Complications Twin-to-Twin Transfusion Syndrome (TTTS): This is a condition where there is an imbalance in the blood supply between the fetuses, leading to one twin receiving too much blood and the other too little ( 15 , 38 ). Reversed Arterial Perfusion (TRAP): In this scenario, one fetus receives blood supply from the other, which can lead to heart failure in the supplying twin ( 49 ). Structural Anomalies: There is a higher risk of structural anomalies occurring in one of the fetuses in monozygotic multiple pregnancies ( 38 ). Vanishing Twin Syndrome: This phenomenon is characterized by the disappearance of one fetus during pregnancy, which is associated with adverse outcomes for the surviving fetus, including higher risks of low birth weight, preterm birth, being small for gestational age, and birth defects ( 50 , 51 , 52 ). Economic and Societal Complications Increased Economic Burden: The societal and economic costs of monochorionic multiple pregnancies are significantly higher compared to singleton pregnancies, with the cost of a triplet pregnancy being approximately 11 times higher than a singleton pregnancy ( 53 ). Management, Preventive Strategies, and Future Directions for multiple pregnancies in ART treatment Understanding and Addressing Complications Monozygotic multiple pregnancies, which can occur naturally or through In Vitro Fertilization (IVF) treatments, come with a range of complications and risks. These pregnancies are associated with specific complications such as twin-to-twin transfusion syndrome, and reversed arterial perfusion, with the latter occurring after a single intrauterine fetal death ( 15 , 38 , 49 , 54 ). Moreover, they are at a higher risk of structural anomalies, which usually affect only one fetus ( 38 ). The societal costs of monozygotic triplet pregnancies (MZTP) are substantial, with the cost of a triplet pregnancy being approximately 11 times higher than a singleton pregnancy ( 53 ). The phenomenon of the ‘vanishing embryo syndrome’ is also prevalent, where the pregnancy begins with a higher number of embryos but ends with fewer live births, posing risks such as low birth weight and birth defects for the surviving fetus(es) ( 50 , 55 , 56 ). Given these complications, it is vital to understand the associated risks thoroughly. Early Diagnosis and Counseling Early ultrasound diagnosis is indispensable in the management of multiple pregnancies in Assisted Reproductive Technology (ART) treatment, aiding in accurate diagnosis, counseling, and referral for appropriate management and care options from the early days of pregnancy ( 1 , 36 ). This diagnosis helps in determining vital details such as gestational age, chorionicity, and amnionicity, which are crucial in managing a multiple pregnancy ( 57 ). Early evaluations include checking the intrauterine location of the embryonic sac, the number of embryos in each sac, and the vitality and normality of embryo structures (58,59). Given the increased risk of high-order pregnancies in ART, early and regular ultrasound scan assessments are essential. Strategies for Managing Multiple Pregnancies Managing multiple pregnancies effectively involves a range of strategies, including the careful selection of high-potential embryos and limiting the number transferred to reduce high-order multiple pregnancies post-IVF ( 38 ). Patients should be informed of the increased risk of monozygotic pregnancy following a single blastocyst transfer ( 38 ). In cases of early diagnosis, there are two primary management options: waiting until the eighth week of pregnancy to observe potential spontaneous vanishing of a developing embryo, or opting for embryo reduction at the same stage to reduce miscarriage risks and pregnancy complications ( 60 ). Reducing the number of transferred embryos has been suggested to decrease the incidence of multiple pregnancies in IVF, enhancing outcomes such as live birth rates while reducing risks associated with high-order multiple pregnancies ( 41 , 61 ). The strategy of elective single-embryo transfer, possibly combined with frozen-embryo transfer and milder ovulation induction, presents a viable approach to reducing multiple birth rates while maintaining acceptable live birth rates ( 62 , 63 ). However, it is crucial to conduct prospective trials to identify definitive predictive factors for monozygotic multiple pregnancies post-IVF and to foster discussions and debates on this topic ( 42 ). Future research should aim to elucidate the relationship between zygotic splitting and Assisted Reproductive Technology (ART) procedures further. Conclusion In this case report and literature review, we delve into the rare occurrence of monozygotic triplet pregnancies following a single blastocyst transfer in IVF-FET, shedding light on ART-specific risk factors such as blastocyst transfer, assisted hatching, and in vitro culture that potentially increase zygotic splitting compared to natural conception. Despite strategies like elective single embryo transfer (SET) with frozen-embryo transfer and milder ovulation induction aimed at reducing multiple birth rates, our case, unfortunately, developed a monozygotic triplet pregnancy which ended in miscarriage. This case, along with others reported by ( 34 – 38 ), demonstrates a spectrum of outcomes, from successful births to miscarriages, emphasizing the high-risk nature of such pregnancies. The study, while offering current insights, also acknowledges the limitations in generalizability due to its singular nature and underscores the need for broader research. Various factors, including clinical and IVF mechanisms like High Gonadotrophin Concentrations and Zona hardening, alongside infertility etiology and maybe genetic factors, play significant roles, yet the precise mechanisms driving embryo splitting remain elusive. This gap in understanding calls for ongoing research to enhance clinical outcomes in this complex area of reproductive healthcare. Abbreviations AAFC: Al Ain Fertility Center AMH: Anti-Müllerian Hormone ART: Assisted Reproductive Technology FET: Frozen Embryo Transfer FSH: Follicle-Stimulating Hormone GnRH: Gonadotropin-Releasing Hormone hCG: Human Chorionic Gonadotropin ICSI: Intracytoplasmic Sperm Injection IUGR: Intrauterine Growth Restriction IVF: In Vitro Fertilization IVF-FET: In Vitro Fertilization-Frozen Embryo Transfer MASE: Magnetically Activated Sperm Enrichment MZTP: Monozygotic Triplet Pregnancies PGT-A: Preimplantation Genetic Testing for Aneuploidy QC: Quality Control SET: Single Embryo Transfer TTTS: Twin-to-Twin Transfusion Syndrome Declarations Ethics Approval and Consent to Participate Ethics approval for the present study was granted by the Al Ain Fertility Center - Research Ethics Committee (AAFC-REC) with Project ID: AAFC/CREC/2023/003. The AAFC-REC confirmed that the research adhered to all ethical guidelines for protecting human participants. This includes compliance with the ethical standards of the 1964 Declaration of Helsinki and its later amendments, local UAE health authority regulations (Department of Health, Abu Dhabi), policies, and standards, as well as those of the institutional research committee. Additionally, the requirement for informed consent was specifically waived by the AAFC-REC for this retrospective study due to the minimal risk posed by the study, the absence of adverse effects on participants' rights and welfare, and the maintenance of anonymity in the presentation of all data. This research was conducted in strict accordance with these comprehensive guidelines and regulations, ensuring adherence to the highest ethical standards for conducting human research. Consent for publication We confirm that all listed authors meet the authorship criteria and have approved the manuscript as well as the author order. We have duly considered the intellectual property aspects related to this work, confirming that there are no impediments to publication as per our institutional regulations. We acknowledge that the Corresponding Author is the primary contact during the editorial process and is tasked with updating the co-authors on manuscript progress, revisions, and proof approvals. A current and accessible email address for the Corresponding Author has been provided. Availability of data and material: The data that support the findings of this study are available from the corresponding author upon reasonable request. Due to the sensitive nature of the clinical information and to ensure patient confidentiality, data will not be publicly available. Requests for access to specific datasets used in this study will be considered by the authors, provided that they conform to ethical standards and regulations concerning patient data. Competing interests Dr. Braulio Peramo, as Principal Investigator and Chair of the Research Ethical Board at Al Ain Fertility Center, acknowledges a potential conflict of interest due to his dual role. This role is fully disclosed to ensure research transparency and integrity. Funding Not applicable, this study is not funded. Authors' contributions B.P.: Conceptualization, Methodology, Supervision, Investigation, Writing - Reviewing and Editing. D.U.: Data Curation, Investigation, Writing - Original Draft, Project Administration. R.A.H.: Investigation, Validation. S.A.: Resources, Data Curation, Visualization. F.A.: Resources, Data Curation. R.A.: Resources, Data Curation. Acknowledgments We would like to extend our heartfelt appreciation to the Al Ain Fertility Center (AAFC) management for their unwavering support and for providing the funding that made this research possible. Additionally, AAFC granted access to the essential data, which was crucial for this study. Dr. Saeed Al Dayeh, a Board Member at AAFC, provided strategic direction and governance and validated the research's alignment with the center's objectives. Mr. Mohd Al Hayek, Chief Financial Officer at AAFC, ensured budgetary compliance, facilitated the allocation of funds, and managed the financial aspects related to the research. Authors' information (optional) Divyesh Upadhyay, Research Assistant, Clinical Department, AAFC, Abu Dhabi, U.A.E. Rajia Al Halaby, OB/GYN Fertility Specialist, Clinical Department, AAFC, Abu Dhabi, U.A.E. Sudha Anandt, Genetics Lab Manager, Genetics Department, AAFC, Abu Dhabi, U.A.E. Firas Albuz, IVF Lab Manager, IVF Department, Al Ain Fertility Center, Abu Dhabi, U.A.E. Rawan Almekosh,Embryologist, IVF Department, Al Ain Fertility Center, Abu Dhabi, U.A.E. Braulio Peramo,Medical Director, Clinical Department, Al Ain Fertility Center, Abu Dhabi, U.A.E. Weblink Halosperm® | Halotech DNA. (n.d.). Retrieved January 17, 2024, from https://www.halotechdna.com/productos/halosperm References Su-Faye, L. E. E., Chapman, M. & Bowyer, L. Monozygotic triplets after single blastocyst transfer: case report and literature review. Aust N Z J Obstet Gynaecol 48, 583–586 (2008). Derom, C., Derom, R., Vlietinck, R., Berghe, H. Vanden & Thiery, M. Increased monozygotic twinning rate after ovulation induction. Lancet 1, 1236–1238 (1987). Fuchs, K. M. & D’Alton, M. E. Monochorionic Diamniotic Twin Gestations. Obstetric Imaging: Fetal Diagnosis and Care, 2nd Edition 645–648.e1 (2018) doi: 10.1016/B978-0-323-44548-1.00160-1 . Benirschke, K. & Kim, C. K. Multiple Pregnancy. 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H., Hammitt, D. G. & Van Voorhis, B. J. Increased risk of monochorionic twinning associated with assisted reproduction. Fertil Steril 60, 510–514 (1993). Knopman, J. M. et al. What makes them split? Identifying risk factors that lead to monozygotic twins after in vitro fertilization. Fertil Steril 102, 82–89 (2014). Peramo, B., Ricciarelli, E., Cuadros-Fernández, J. M., Huguet, E. & Hernández, E. R. Blastocyst transfer and monozygotic twinning. Fertil Steril 72, 1116–1117 (1999). Vitthala, S., Gelbaya, T. A., Brison, D. R., Fitzgerald, C. T. & Nardo, L. G. The risk of monozygotic twins after assisted reproductive technology: a systematic review and meta-analysis. Hum Reprod Update 15, 45–55 (2009). Ferri, G., Musto, M., Colombo, G. & Savasi, V. M. Monochorionic Triplet Gestation after Single Blastocyst Transfer Using Donor Oocytes: Case Report and Review. Case Rep Obstet Gynecol 2020, (2020). Yamashita, S. et al. Analysis of 122 triplet and one quadruplet pregnancies after single embryo transfer in Japan. Reprod Biomed Online 40, 374–380 (2020). Ikemoto, Y. et al. Prevalence and risk factors of zygotic splitting after 937 848 single embryo transfer cycles. Hum Reprod 33, 1984–1991 (2018). Mackenna, A., Schwarze, J. E., Crosby, J. & Zegers-Hochschild, F. Factors associated with embryo splitting and clinical outcome of monozygotic twins in pregnancies after IVF and ICSI. Hum Reprod Open 2020, (2020). Busnelli, A. et al. Risk factors for monozygotic twinning after in vitro fertilization: a systematic review and meta-analysis. Fertil Steril 111, 302–317 (2019). Dudenhausen, J. W. & Maier, R. F. Perinatal Problems in Multiple Births. Dtsch Arztebl Int 107, 663 (2010). Tal, R., Fridman, D. & Grazi, R. V. Monozygotic Triplets and Dizygotic Twins following Transfer of Three Poor-Quality Cleavage Stage Embryos. Case Rep Obstet Gynecol 2012, 1–4 (2012). El-Toukhy T, Bhattacharya S & Akande VA. Multiple Pregnancies Following Assisted Conception. BJOG (2018) doi: 10.1111/1471-0528.14974 . Gratacós, E., Ortiz, J. U. & Martinez, J. M. A systematic approach to the differential diagnosis and management of the complications of monochorionic twin pregnancies. Fetal Diagn Ther 32, 145–155 (2012). Crosignani, P. G. et al. Multiple gestation pregnancy. Human Reproduction 15, 1856–1864 (2000). Kruger, T. F. et al. Predictive value of abnormal sperm morphology in in vitro fertilization. Fertil Steril 49, 112–117 (1988). Fernández, J. L. et al. The Sperm Chromatin Dispersion Test: A Simple Method for the Determination of Sperm DNA Fragmentation. J Androl 24, 59–66 (2003). Fernández, J. L. et al. Simple determination of human sperm DNA fragmentation with an improved sperm chromatin dispersion test. Fertil Steril 84, 833–842 (2005). Sachdeva, Kabir et al. Semen Quality is Associated with Sperm Aneuploidy and DNA Fragmentation in the United Arab Emirates Population. Genetic testing and molecular biomarkers 24, 195–203 (2020). Palermo, G., Joris, H., Devroey, P. & Van Steirteghem, A. C. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 340, 17–18 (1992). WHO laboratory manual for the examination and processing of human semen. 5 Th Edition, (2010) https://apo.who.int/publications/i/item/9789241547789 Gardner, D. K., Lane, M. & Schoolcraft, W. B. Physiology and culture of the human blastocyst. J Reprod Immunol 55, 85–100 (2002). Gardner, D. K. & Balaban, B. Assessment of human embryo development using morphological criteria in an era of time-lapse, algorithms and ‘OMICS’: is looking good still important? Mol Hum Reprod 22, 704–718 (2016). Gurunath, S., Makam, A., Vinekar, S. & Biliangady, R. Monochorionic triamniotic triplets following conventional in vitro fertilization and blastocyst transfer. J Hum Reprod Sci 8, 54–57 (2015). Schlueter, R., Arnett, C., Huang, C. & Burlingame, J. Successful quintuplet pregnancy of monochorionic male quadruplets and single female after double embryo transfer: case report and review of the literature. Fertil Steril 109, 284–288 (2018). Ota, K. et al. Successful monozygotic triplet pregnancy after a single blastocyst transfer following in vitro maturation of oocytes from a woman with polycystic ovary syndrome: A case report. BMC Pregnancy Childbirth 20, 1–5 (2020). Saravelos, S. H. et al. Monochorionic quadramniotic and triamniotic pregnancies following single embryo transfers: two case reports and a review of the literature. J Assist Reprod Genet 33, 27–32 (2016). Sutherland, K., Leitch, J., Lyall, H. & Woodward, B. J. Time-lapse imaging of inner cell mass splitting with monochorionic triamniotic triplets after elective single embryo transfer: a case report. Reprod Biomed Online 38, 491–496 (2019). Dessolle, L. et al. Monozygotic triplet pregnancies after single blastocyst transfer: Two cases and literature review. Reprod Biomed Online 21, 283–289 (2010). Salat-baroux, J., Alvarez, S. & Antoine, J. M. A case of triple monoamniotic pregnancy combined with a bioamniotic twinning after in-vitro fertilization. Hum Reprod 9, 374–375 (1994). Belaisch-allart, J. et al. Monozygotic triplet pregnancy following transfer of frozen-thawed embryos. Hum Reprod 10, 3064–3066 (1995). Faraj, R., Evbuomwan, I., Sturgiss, S. & Aird, I. Monozygotic triplet pregnancy following egg donation and transfer of single frozen-thawed embryo. Fertil Steril 89, 1260.e9-1260.e12 (2008). Rísquez, F. et al. Monochorionic triplets after single embryo transfer. Reprod Biomed Online 9, 370–371 (2004). Ghulmiyyah, L. M. et al. Monochorionic-triamniotic triplet pregnancy after intracytoplasmic sperm injection, assisted hatching, and two-embryo transfer: first reported case following IVF. BMC Pregnancy Childbirth 3, (2003). Wang, C. et al. Effect of different artificial shrinkage methods, when applied before blastocyst vitrification, on perinatal outcomes. Reprod Biol Endocrinol 15, (2017). Cohen, J., Alikani, M., Trowbridge, J. & Rosenwaks, Z. Implantation enhancement by selective assisted hatching using zona drilling of human embryos with poor prognosis. Hum Reprod 7, 685–691 (1992). Franasiak, J., Dondik, Y., Molinaro, T., sterility, K. H.-F. and & 2015, undefined. Blastocyst transfer is not associated with increased rates of monozygotic twins when controlling for embryo cohort quality. Elsevier . Machin, G. Non-identical monozygotic twins, intermediate twin types, zygosity testing, and the non-random nature of monozygotic twinning: A review. Am J Med Genet C Semin Med Genet 151, 110–127 (2009). Yanaihara, A., Yorimitsu, T., Motoyama, H., Watanabe, H. & Kawamura, T. Monozygotic multiple gestation following in vitro fertilization: analysis of seven cases from Japan. J Exp Clin Assist Reprod 4, 4 (2007). Unger, S., Hoopmann, M., Bald, R., Foth, D. & Nawroth, F. Monozygotic triplets and monozygotic twins after ICSI and transfer of two blastocysts: case report. Human Reproduction 19, 110–113 (2004). Pinborg, A., Lidegaard, Ø. & Andersen, A. N. The vanishing twin: a major determinant of infant outcome in IVF singleton births. Br J Hosp Med (Lond) 67, 417–420 (2006). Dickey, R. P. et al. Spontaneous reduction of multiple pregnancy: Incidence and effect on outcome. Am J Obstet Gynecol 186, 77–83 (2002). Evron, E., Sheiner, E., Friger, M., Sergienko, R. & Harlev, A. Vanishing twin syndrome: is it associated with adverse perinatal outcome? Fertil Steril 103, 1209–1214 (2015). Crosignani, P. G. et al. Multiple gestation pregnancy. Human Reproduction 15, 1856–1864 (2000). Yakín, K., Kahraman, S. & Cömert, S. Three blastocyst stage embryo transfer resulting in a quintuplet pregnancy. Hum Reprod 16, 782–784 (2001). Barton, S. E., Missmer, S. A. & Hornstein, M. D. Twin pregnancies with a ‘vanished’ embryo: a higher risk multiple gestation group? Human Reproduction 26, 2750–2753 (2011). Magnus, M. C. et al. Vanishing twin syndrome among ART singletons and pregnancy outcomes. Hum Reprod 32, 2298–2304 (2017). Antsaklis, P. et al. Early Pregnancy Ultrasound Assessment of Multiple Pregnancy. Multiple Pregnancy - New Challenges (2018) doi: 10.5772/INTECHOPEN.81498 . Hernandez-Andrade, E., Patwardhan, M., Cruz-Lemini, M. & Luewan, S. Early Evaluation of the Fetal Heart. Fetal Diagn Ther 42, 161–173 (2017). Detti, L. et al. Early pregnancy ultrasound measurements and prediction of first trimester pregnancy loss: A logistic model. Sci Rep 10, (2020). Cai, P., Ouyang, Y., Gong, F. & Li, X. Pregnancy outcomes of dichorionic triamniotic triplet pregnancies after in vitro fertilization-embryo transfer: Multifoetal pregnancy reduction versus expectant management. BMC Pregnancy Childbirth 20, 1–9 (2020). Newman, R. B. & Ellings, J. M. Antepartum management of the multiple gestation: the case for specialized care. Semin Perinatol 19, 387–403 (1995). Tobias, T., Sharara, F. I., Franasiak, J. M., Heiser, P. W. & Pinckney-Clark, E. Promoting the use of elective single embryo transfer in clinical practice. Fertil Res Pract 2, (2016). Bergh, C., Kamath, M. S., Wang, R. & Lensen, S. Strategies to reduce multiple pregnancies during medically assisted reproduction. Fertil Steril 114, 673–679 (2020). Tables Table 1 is available in the Supplementary Files section. Additional Declarations Competing interest reported. Disclosure Statement: Dr. Braulio Peramo, as Principal Investigator and Chair of the Research Ethical Board at Al Ain Fertility Center, acknowledges a potential conflict of interest due to his dual role. This role is fully disclosed to ensure research transparency and integrity. Furthermore, all authors confirm no financial conflicts of interest could influence this study. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5055637","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Case Report","associatedPublications":[],"authors":[{"id":376483632,"identity":"894d87e9-6592-47ca-8993-654529fdc407","order_by":0,"name":"Divyesh Upadhyay","email":"","orcid":"","institution":"Al Ain Fertility Center","correspondingAuthor":false,"prefix":"","firstName":"Divyesh","middleName":"","lastName":"Upadhyay","suffix":""},{"id":376483633,"identity":"d1a8d048-079f-482f-aec3-03576e0bdcf8","order_by":1,"name":"Rajia Al Halaby","email":"","orcid":"","institution":"Al Ain Fertility Center","correspondingAuthor":false,"prefix":"","firstName":"Rajia","middleName":"Al","lastName":"Halaby","suffix":""},{"id":376483634,"identity":"cda48682-76f0-473e-acf6-98f90fea7e8b","order_by":2,"name":"Sudha Anandt","email":"","orcid":"","institution":"Al Ain Fertility Center","correspondingAuthor":false,"prefix":"","firstName":"Sudha","middleName":"","lastName":"Anandt","suffix":""},{"id":376483635,"identity":"bda121eb-52d5-48d4-8c9c-332765206631","order_by":3,"name":"Firas Albuz","email":"","orcid":"","institution":"Al Ain Fertility Center","correspondingAuthor":false,"prefix":"","firstName":"Firas","middleName":"","lastName":"Albuz","suffix":""},{"id":376483636,"identity":"7bc34e64-ea11-400e-ad87-d5fb0fcb5676","order_by":4,"name":"Rawan Almekosh","email":"","orcid":"","institution":"Al Ain Fertility Center","correspondingAuthor":false,"prefix":"","firstName":"Rawan","middleName":"","lastName":"Almekosh","suffix":""},{"id":376483637,"identity":"d9349135-a58e-4846-8f73-7eef8a0ee9b1","order_by":5,"name":"Braulio Peramo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAo0lEQVRIiWNgGAWjYBACAygtR7oWY9K1JDYQrcVcuvnYh585Nulr2xsYP3zMIUKL5ZxjyTN7t6XlbjtzgFly5jZiHHYjx5iBd9vh3G03EtiYeYnTkv+Z8e+2w+lm9x8QrSWHGajycILZDQYitQD9Yswsuy3NcNuZxGbi/AIMsceMb7fZyJsdP3zww0ditDBIwFmMDcSoR9EyCkbBKBgFowAHAAA5FTbSg9jEIwAAAABJRU5ErkJggg==","orcid":"","institution":"Al Ain Fertility Center","correspondingAuthor":true,"prefix":"","firstName":"Braulio","middleName":"","lastName":"Peramo","suffix":""}],"badges":[],"createdAt":"2024-09-09 06:22:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5055637/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5055637/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":69837227,"identity":"1a14997f-6c6c-400d-b26d-9f1d2525f252","added_by":"auto","created_at":"2024-11-25 16:34:43","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":274253,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5055637/v1/97b587f7dcadaddb408907f4.jpg"},{"id":82261889,"identity":"d0429d1a-b281-47cb-84f2-d658eaa08629","added_by":"auto","created_at":"2025-05-08 12:17:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1132361,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5055637/v1/e2a94c61-55e4-46d2-afd1-4bdb332753fb.pdf"},{"id":69837229,"identity":"7f486338-d3e8-4e0b-bcff-0bbdd8b9ebdb","added_by":"auto","created_at":"2024-11-25 16:34:43","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":38271,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-5055637/v1/4f4cede73e3bda4806241a3e.docx"}],"financialInterests":"Competing interest reported. Disclosure Statement:\nDr. Braulio Peramo, as Principal Investigator and Chair of the Research Ethical Board at Al Ain Fertility Center, acknowledges a potential conflict of interest due to his dual role. This role is fully disclosed to ensure research transparency and integrity. Furthermore, all authors confirm no financial conflicts of interest could influence this study.","formattedTitle":"Risks and Complications of Monozygotic Triplet Pregnancy Following Single Blastocyst Transfer: Insights from a Case Report and Future Directions.","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMonozygotic multiple pregnancies occur when a single embryo splits to form genetically identical embryos around the time of blastocyst formation (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Monozygotic twin pregnancy occurs as a result of the fertilization of a single egg with a single sperm (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). The embryo is then split at a later stage (usually in the blastocyst stage). It is more frequent that the embryo is divided into two (monozygotic twinning); but in certain cases, the inner cell mass can divide into three or four leading to the development of multiple fetuses or higher-order monozygotic multiple pregnancies (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe timing of monozygotic splitting of the embryo is probably not fixed and the mechanism varies from one set to another (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). A zygote may split anytime during the first 14 days after fertilization, resulting in the various forms of monozygotic twins (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). That is, the exact mechanisms have not yet been established in natural or assisted human reproduction cases (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Many theories have been put forward as to how and when this occurs. The likelihood of occurrence is at any point in terms of the establishment of polarity and axes (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Familial (i.e., genetic) and infertility treatment are considered potential factors leading to multiple pregnancies (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). In this case report, we conducted a thorough literature review on monozygotic triplet pregnancies. Our search spanned from February 1994 to October 2023, using databases like PubMed and Google Scholar. We used keywords such as 'monozygotic triplets,' 'Single Embryo Transfer,' 'Assisted Reproductive Technology,' and 'Embryo Splitting,' along with relevant MeSH terms. This approach ensured a comprehensive examination of studies and case reports on monozygotic triplet pregnancies resulting from single blastocyst transfers.\u003c/p\u003e\n\u003ch3\u003eIncidence of Monozygotic Twinning and Triplet Pregnancies\u003c/h3\u003e\n\u003cp\u003eIn the evolving field of Assisted Reproductive Technology (ART), there's been a notable increase in monozygotic twinning and triplet pregnancies, especially following blastocyst transfer. Studies have consistently shown a rise in such cases (\u003cspan additionalcitationids=\"CR12 CR13 CR14\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). While the global frequency of monozygotic twins remains constant at about 4:1000 births, ART conceptions demonstrate a 2.25 times higher rate than natural conceptions, with monozygotic triplet pregnancies occurring 100 times more frequently in IVF than in natural pregnancies, albeit still rare at 0.048% (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Regionally, Japan reports a low prevalence of 0.04% for triplet pregnancies post-single blastocyst transfer, with 122 cases observed from 2007 to 2014 (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). A comprehensive study by (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e) indicated a 1.36% prevalence of zygotic splitting in multiple pregnancies post-SET, analyzing 937,848 SET cycles. This study, along with others (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e), identified factors like frozen-warmed embryo transfer cycles, blastocyst culture, and assisted hatching as risks for zygotic splitting. The paper underlines the importance of understanding the mechanisms of embryo splitting in ART to improve clinical outcomes and insights into high-order pregnancies like monozygotic triplets, particularly in the context of single blastocyst transfer using IVF-frozen embryo transfer.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMonozygotic Triplet Pregnancies and Assisted Reproductive Technology\u003c/h2\u003e \u003cp\u003eThe practice of Assisted Reproductive Technology (ART) has been implicated in increasing the incidence of monozygotic multiples, such as twins and triplets. Several key aspects of ART, including embryo manipulation, embryo culture, assisted hatching, and blastocyst transfer, have been postulated as significant contributing factors (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMonozygotic twinning is an important phenomenon because monozygotic pregnancies are linked to increased obstetrical risks. Monozygotic pregnancies carry a higher risk of both, fetal and maternal morbidities prematurity, intrauterine growth restriction, and prenatal death (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e); Maternal complications include an increased risk of pregnancy-induced hypertension, gestational diabetes, peripartum hemorrhage, operative delivery, postpartum depression and anxiety and parenting stress (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). The most commonly reported clinical complication during monozygotic pregnancy is chronic feto-fetal transfusion syndrome (twin-to-twin transfusion syndrome and twin anemia polycythemia sequence) resulting from unbalanced blood flow from one twin to the other (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). In addition, Twin-to-Twin Transfusion Syndrome (TTTS) is highly associated with the fetal death of one fetus due to a sudden drop in pressure and/or heart rate at one fetal end (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eReducing the incidence of high-order multiple pregnancies associated with ART is one of the major objectives of reproductive medicine (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). The main strategy to reduce high-order pregnancies linked to ART is to promote Single Embryo Transfer (SET) after In Vitro Fertilization (IVF). But even by transferring one embryo, still there is a risk of multiple pregnancies due to monozygotic twinning. This is the reason why it\u0026rsquo;s very important to try to find the reason(s) behind this phenomenon, to try to prevent it. Ultrasonography is of utmost importance in early detection of monozygotic triplet, and pregnancy monitoring. Prenatal ultrasound (US) is essential in the management of monozygotic multiple pregnancies to allow determination of zygosity, chorionicity, evaluation of fetal anatomy, and serial assessment of cervical length and fetal growth (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). In addition, ultrasound aids in the detection of clinical complications during pregnancy such as Twin-to-Twin Transfusion Syndrome (TTTS) and intrauterine growth restriction (IUGR) (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Hence, ultrasound assessment is very useful in managing high-risk pregnancies associated with ART.\u003c/p\u003e \u003c/div\u003e"},{"header":"Case Presentation","content":"\u003cp\u003eA 36-year-old woman (Gravida 4, Para 4) presented at our center (Al Ain Fertility Center, U.A.E.), with secondary infertility due to low ovarian reserve. No relevant patient medical history or family history was reported. The duration of infertility was reported to be 3 years. Her AMH level was found to be \u0026lt;\u0026thinsp;0.05ng/ml (the normal range of AMH is 2.0 to 6.8ng/ml). She had a normal karyotype and the test for fragile-X syndrome was negative. Semen analysis was found to be normal for her husband. Other relevant clinical investigations for the couple showed normal results.\u003c/p\u003e \u003cp\u003eThe patient had an IVF- Intracytoplasmic Sperm Injection (ICSI) treatment under standard antagonist protocol with preimplantation genetic testing for aneuploidy (PGT-A). Ovarian stimulation was started on the second day of the cycle with an injection of 150microgram single dose long-acting recombinant FSH (Elonva; Merck), 300IU daily dose of urine-derived FSH, and LH (Menopur; Ferring B.V), 5mg daily dose of letrozole (Femara; Novartis). When the leading follicles reached 17mm, she received a double trigger with r-hCG (Ovitrelle; Merck) and 0.2mg GnRH agonist (Gonapeptyl, Ferring) 36 hours before egg collection. Transvaginal ultrasound-guided aspiration under general anesthesia was performed and 3 follicles were aspirated, yielded 3 oocytes (MII). Semen analysis was conducted on the husband's frozen ejaculate sample using Kruger's strict criteria (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e), which showed a sperm count of 56.6x106 per ml, with sperm motility at 60%, normal forms at 4%, and a DNA fragmentation rate of 19.5% (Halosperm\u0026reg;, (\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e)). In addition, sperm preparation, sorting, and selection were done using magnetically activated sperm enrichment (MASE) protocol for ICSI (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). ICSI was performed on all of them. All 3 oocytes were fertilized (2PN) and resulting embryos were cultured in sequential media by using cleavage medium and blastocyst medium (Quinn\u0026rsquo;s Advantage medium, Sage biopharma). There were 3 high-quality embryos (graded 1,2 and 3 as per the Gardner embryo blastocyst grading system, (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e) on day 3. One blastocyst (embryo no. 3) was biopsied and frozen on day 5, another blastocyst (embryo no. 2) was biopsied and frozen on day 6, and one blastocyst (embryo no. 1) was arrested on day 6. PGT-A was performed (Next Generation Sequencing - Ion ReproSeq\u0026trade; PGS) for comprehensive chromosome aneuploidy screening which diagnosed a normal-euploid (46,XX) embryo (embryo no. 3) and an abnormal (45XX,-13) embryo (embryo no.2).\u003c/p\u003e \u003cp\u003eEndometrial preparation for Frozen Embryo Transfer (FET) was done under standard protocol. The patient started with a daily dosage of 6mg estradiol valerate (Progynova; Bayer) from the second day of the cycle. On day 10, a transvaginal ultrasound scan was performed and triple lining endometrium was visualized measuring 7.5mm. She had a blood test that showed serum concentration of estradiol and progesterone to be 374pg/ml and 0.39 ng/ml respectively. Then, she started progesterone priming (day P1) with progesterone gel (Crinone gel, Merck) and 10mg/daily dydrogesterone (Duphaston; Abbott) to support the luteal phase. On day P\u0026thinsp;+\u0026thinsp;5, serum concentrations of estradiol and progesterone were found to be 151.2pg/ml and 46.6ng/ml respectively. On day P\u0026thinsp;+\u0026thinsp;6, a single euploid frozen-thawed embryo (embryo no. 3) was transferred under transvaginal ultrasound guidance (performed under standard protocol). She continued with the same medication after the embryo transfer. A pregnancy test done after 12 days showed a positive result (serum beta-hCG was 134.1mIU/ml). An ultrasound scan performed 2 weeks later, (6 weeks of pregnancy) showed a single gestational sac with three yolk sacs and fetal poles. Crown\u0026ndash;rump lengths were 0.37cm, 0.40cm, and 0.37cm with positive heartbeats \u003cb\u003e(Fig.\u0026nbsp;1)\u003c/b\u003e. She continued the same medication and the ultrasound performed at 7 gestational weeks revealed a single intrauterine gestational sac and the presence of three yolk sacs. Crown\u0026ndash;rump lengths were 0.80cm, 0.96cm, and 0.82 cm with positive heartbeats. The couple was counseled on the higher risk involved in monozygotic multiple pregnancies and the patient was further referred for pregnancy follow-up to an obstetric unit in a tertiary hospital.\u003c/p\u003e \u003cp\u003eIn the presented case, the patient adhered to a meticulously planned IVF-ICSI treatment protocol, including ovarian stimulation, embryo development, and endometrial preparation for frozen embryo transfer (FET). She tolerated the interventions well, with no adverse reactions reported to the medications or procedures. The regimen's effectiveness was initially confirmed by a positive pregnancy test and the development of monozygotic triplets, though the pregnancy, unfortunately, ended in a spontaneous miscarriage at the 10th week of pregnancy.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis case report and literature review focus on monozygotic (MZ) triplet pregnancies, happening sometimes after Assisted Reproductive Technologies (ART) such as In Vitro Fertilization (IVF) and Intracytoplasmic Sperm Injection (ICSI). Conducted through systematic searches of the PubMed database, the review offers a comprehensive understanding of these pregnancies, particularly those arising from single blastocyst transfers. It includes a detailed case description of a specific MZ triplet pregnancy resulting from a single blastocyst transfer via IVF-Frozen Embryo Transfer (IVF-FET), encompassing the patient's history, treatment protocols, and outcomes, to provide a complete view of the unique aspects of the case. Furthermore, the report critically examines potential risk factors associated with monozygotic multiple pregnancies, such as blastocyst transfer and assisted hatching, emphasizing the need for further research into their role in zygotic splitting. Complementing this is a comparative analysis within the ART context, contrasting various cases to shed light on the potential mechanisms behind monozygotic twinning or tripling. A summarized literature search is presented in \u003cb\u003eTable\u0026nbsp;1\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eThe patient in the present case had a low ovarian reserve, which is a common characteristic among several cases in the literature. For example, Gurunath et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e) described a 29-year-old woman undergoing IVF, and Schlueter et al. (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e) reported on a 34-year-old woman undergoing ICSI, both with no relevant medical history, similar to our case. The treatment protocols varied, with some cases utilizing fresh embryo transfers and others frozen, as in the case of Ota et al. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e), where a frozen blastocyst transfer resulted in the live birth of triplets.\u003c/p\u003e \u003cp\u003eIn the realm of assisted reproductive technologies like IVF and ICSI, the trend towards single blastocyst transfer is notable, as it aims to balance the reduction of multiple pregnancy risks with favorable pregnancy rates. This approach is reflected in various studies: Saravelos et al. (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e) reported monozygotic triplets from both single morula and blastocyst transfers, while Sutherland et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e) noted a miscarriage following a single blastocyst transfer. Our study, involving a 36-year-old undergoing IVF/ICSI with a single Day 5 blastocyst, also ended in miscarriage at 10 weeks, contrasting with Dessolle et al. (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e) and Saravelos et al. (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e), where single embryo transfers resulted in the birth of twins and triplets, respectively. Ota et al. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e) further documented a successful triplet birth from a single Day 5 blastocyst. These findings underscore the unpredictability and complexity inherent in monozygotic triplet pregnancies post-single embryo transfer, emphasizing the importance of careful monitoring and management in such scenarios.\u003c/p\u003e \u003cp\u003eIn the context of monozygotic triplet pregnancies following single blastocyst transfer in Assisted Reproductive Technology (ART), outcomes and complications exhibit considerable variability. The present case, characterized by the presence of three yolk sacs and fetal poles, unfortunately, culminated in a miscarriage at 10 weeks, underscoring the heightened risk of adverse outcomes. This contrasts with other reported cases, such as those by Dessolle et al. (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e), where a successful birth of two healthy females was achieved after selective fetal reduction, and by Saravelos et al. (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e), who documented both a live birth at 35\u0026thinsp;+\u0026thinsp;6 weeks following selective reduction and an ongoing twin pregnancy after similar intervention. Other reports, like those from Sutherland et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e), Schlueter et al. (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e), and Ota et al. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e), also indicate a range of outcomes, including miscarriages and live births at various gestational ages, with complications such as Intrauterine Growth Restriction (IUGR), Twin-to-Twin Transfusion Syndrome (TTTS), early delivery, and maternal issues like hypertension and bleeding. These cases collectively highlight the complex and high-risk nature of monozygotic triplet pregnancies in ART.\u003c/p\u003e \u003cp\u003eThe mechanisms behind monozygotic multiples in Assisted Reproductive Technology (ART), particularly triplet pregnancies following Single Embryo Transfer (SET), remain elusive. Procedures such as Intracytoplasmic Sperm Injection (ICSI), blastocyst culture, and assisted hatching, which involve breaching the zona pellucida, are thought to increase the likelihood of these occurrences. This is evidenced by studies like those of Salat-Baroux et al. (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e) and Belaisch-Allart et al. (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Moreover, a literature search, as presented in \u003cb\u003eTable\u0026nbsp;1\u003c/b\u003e, shows case reports of monozygotic triplets following single embryo transfer. Various factors, including the type of ART procedure, the stage of embryo development, and embryo morphology, are implicated in these findings. The research by Ferri et al., (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e) Ikemoto et al. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e) Faraj et al. (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e), Risquez et al. (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e), and Peramo et al.(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e) suggests a multifactorial process, influenced by a combination of treatment protocols and patient characteristics. Despite the increased incidence after ART, monozygotic triplets are still a rare phenomenon. Ongoing research is critical to understand these mechanisms better and to develop strategies that minimize their occurrence while optimizing patient outcomes.\u003c/p\u003e\n\u003ch3\u003eMechanisms that may contribute to the occurrence of monozygotic triplet pregnancies:\u003c/h3\u003e\n\u003cp\u003eThe inner cell mass (ICM) is a collection of cells within the blastocyst that eventually develops into the embryo. A monozygotic triplet pregnancy arises from two distinct ICM splitting events. The timing of these splits determines the configuration of the chorion and amnion compartments, which are the protective membranes encasing the embryo (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Recognizing and assessing such ICM splitting occurrences is vital, as emphasized by (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e), who suggests that these evaluations should be integrated as a standard procedure in clinical embryology. Factors potentially involved in embryo splitting are:\u003c/p\u003e \u003cp\u003e1. Blastocyst Culture and In-Vitro Culture Conditions:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eAdvances in cell culture media have enabled the in-vitro development of embryos to the blastocyst stage. This transition from the cleavage stage to blastocyst stage embryo transfer may influence the likelihood of monozygotic splitting. The extended culture period and varying media components can impact the embryo's development and hatching behavior. Additionally, the in-vitro progression to the blastocyst stage allows for prolonged observation and potentially improved embryo selection. However, this longer culture period might heighten the risk of monozygotic splitting due to changes in the embryo's microenvironment and prolonged exposure to artificial culture conditions. These alterations in the in-vitro culture environment are significant in inducing zygotic splitting (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e).\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eThe specific conditions under which embryos are cultured in vitro play a crucial role in their development. Factors like increased glucose levels in the culture medium can lead to zygotic splitting by affecting the embryos' metabolic rates and division patterns, thereby influencing the incidence of monozygotic splitting (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003e2. Impact of Zona Pellucida Alterations:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eExtended periods of in-vitro culture can lead to the hardening of the zona pellucida, causing blastocyst herniation, where the blastocyst protrudes through the zona pellucida. This may result in the splitting of the inner cell mass and lead to monozygotic multiples. Additionally, any manipulation of the zona pellucida, whether mechanical or chemical, such as procedures aimed at thinning the zona pellucida to aid hatching, can induce embryo splitting (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Furthermore, the exposure of the zona pellucida to culture medium, rather than oviductal or uterine secretions in IVF cycles, might also contribute to this hardening (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e).\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eEmbryo manipulation in IVF includes techniques like, assisted hatching, and frozen-warmed embryo transfer, all of which can influence the embryo's division rates. Assisted hatching, involving a small incision in the zona pellucida, might induce the embryo to split (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). These procedures can modify the physical and biochemical properties of the zona pellucida. These alterations may influence the way the embryo hatches and potentially increase the likelihood of monozygotic splitting (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e).\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003e3. Artificial Shrinkage in Conjunction with Vitrification and Warming\u003c/p\u003e \u003cp\u003eVitrification, a quick-freezing method for embryo preservation, and warming, the corresponding thawing process for these embryos, introduce various factors that can complicate the identification of a specific cause for monozygotic triplet pregnancies. The stress and alterations caused by these techniques might influence the development and division of the embryo. Research indicates that artificial shrinkage significantly enhances the implantation rate, clinical pregnancy rate, and live birth rate of warmed blastocysts. However, it also increases the likelihood of monozygotic twin pregnancies (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e4. Influence of Gonadotropin Concentrations and Ovarian Stimulation:\u003c/p\u003e \u003cp\u003eElevated levels of gonadotropins, which are hormones used in ovarian stimulation, can lead to the hardening of the zona pellucida, the outer layer of the embryo crucial for implantation. This hardening can affect the embryo's ability to hatch and implant into the uterine wall, potentially leading to monozygotic multiples. Ovarian stimulation, used in IVF to induce the ovaries to produce multiple eggs, may also impact the zona pellucida and the embryo itself, increasing the likelihood of embryo splitting and monozygotic multiples (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e5. Younger Maternal Age\u003c/p\u003e \u003cp\u003eYounger maternal age is often associated with higher fertility and better embryo quality. However, it is also considered a potential risk factor for monozygotic splitting. The exact physiological reasons are not entirely understood but could involve factors like higher hormonal levels or more robust uterine conditions conducive to embryo splitting (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e6. Genetic Factors\u003c/p\u003e \u003cp\u003eWhile the exact genetic factors are not well understood, it is believed that certain genetic predispositions may make an embryo more likely to split into monozygotic multiples. These could include specific gene mutations or epigenetic factors that influence how the embryo divides (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). Despite all the previous factors discussed, the precise causative mechanisms remain largely unelucidated (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan additionalcitationids=\"CR36 CR37\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eComplications and Risks Associated with Monochorionic Multiple Pregnancies\u003c/h3\u003e\n\u003cp\u003eMonochorionic multiple pregnancies, particularly those resulting from monozygotic splitting, are associated with a range of complications and risks that can affect both the fetuses and the mother. These pregnancies are characterized by shared placental circulation, which can lead to a variety of complications.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eMedical Complications\u003c/span\u003e \u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eTwin-to-Twin Transfusion Syndrome (TTTS): This is a condition where there is an imbalance in the blood supply between the fetuses, leading to one twin receiving too much blood and the other too little (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e).\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eReversed Arterial Perfusion (TRAP): In this scenario, one fetus receives blood supply from the other, which can lead to heart failure in the supplying twin (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e).\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eStructural Anomalies: There is a higher risk of structural anomalies occurring in one of the fetuses in monozygotic multiple pregnancies (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e).\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eVanishing Twin Syndrome: This phenomenon is characterized by the disappearance of one fetus during pregnancy, which is associated with adverse outcomes for the surviving fetus, including higher risks of low birth weight, preterm birth, being small for gestational age, and birth defects (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e).\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEconomic and Societal Complications\u003c/h2\u003e \u003cp\u003eIncreased Economic Burden: The societal and economic costs of monochorionic multiple pregnancies are significantly higher compared to singleton pregnancies, with the cost of a triplet pregnancy being approximately 11 times higher than a singleton pregnancy (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eManagement, Preventive Strategies, and Future Directions for multiple pregnancies in ART treatment\u003c/h3\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eUnderstanding and Addressing Complications\u003c/h2\u003e \u003cp\u003eMonozygotic multiple pregnancies, which can occur naturally or through In Vitro Fertilization (IVF) treatments, come with a range of complications and risks. These pregnancies are associated with specific complications such as twin-to-twin transfusion syndrome, and reversed arterial perfusion, with the latter occurring after a single intrauterine fetal death (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). Moreover, they are at a higher risk of structural anomalies, which usually affect only one fetus (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe societal costs of monozygotic triplet pregnancies (MZTP) are substantial, with the cost of a triplet pregnancy being approximately 11 times higher than a singleton pregnancy (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). The phenomenon of the \u0026lsquo;vanishing embryo syndrome\u0026rsquo; is also prevalent, where the pregnancy begins with a higher number of embryos but ends with fewer live births, posing risks such as low birth weight and birth defects for the surviving fetus(es) (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e). Given these complications, it is vital to understand the associated risks thoroughly.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eEarly Diagnosis and Counseling\u003c/h2\u003e \u003cp\u003eEarly ultrasound diagnosis is indispensable in the management of multiple pregnancies in Assisted Reproductive Technology (ART) treatment, aiding in accurate diagnosis, counseling, and referral for appropriate management and care options from the early days of pregnancy (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). This diagnosis helps in determining vital details such as gestational age, chorionicity, and amnionicity, which are crucial in managing a multiple pregnancy (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e). Early evaluations include checking the intrauterine location of the embryonic sac, the number of embryos in each sac, and the vitality and normality of embryo structures (58,59). Given the increased risk of high-order pregnancies in ART, early and regular ultrasound scan assessments are essential.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStrategies for Managing Multiple Pregnancies\u003c/h2\u003e \u003cp\u003eManaging multiple pregnancies effectively involves a range of strategies, including the careful selection of high-potential embryos and limiting the number transferred to reduce high-order multiple pregnancies post-IVF (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Patients should be informed of the increased risk of monozygotic pregnancy following a single blastocyst transfer (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). In cases of early diagnosis, there are two primary management options: waiting until the eighth week of pregnancy to observe potential spontaneous vanishing of a developing embryo, or opting for embryo reduction at the same stage to reduce miscarriage risks and pregnancy complications (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eReducing the number of transferred embryos has been suggested to decrease the incidence of multiple pregnancies in IVF, enhancing outcomes such as live birth rates while reducing risks associated with high-order multiple pregnancies (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e). The strategy of elective single-embryo transfer, possibly combined with frozen-embryo transfer and milder ovulation induction, presents a viable approach to reducing multiple birth rates while maintaining acceptable live birth rates (\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e). However, it is crucial to conduct prospective trials to identify definitive predictive factors for monozygotic multiple pregnancies post-IVF and to foster discussions and debates on this topic (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Future research should aim to elucidate the relationship between zygotic splitting and Assisted Reproductive Technology (ART) procedures further.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this case report and literature review, we delve into the rare occurrence of monozygotic triplet pregnancies following a single blastocyst transfer in IVF-FET, shedding light on ART-specific risk factors such as blastocyst transfer, assisted hatching, and in vitro culture that potentially increase zygotic splitting compared to natural conception. Despite strategies like elective single embryo transfer (SET) with frozen-embryo transfer and milder ovulation induction aimed at reducing multiple birth rates, our case, unfortunately, developed a monozygotic triplet pregnancy which ended in miscarriage. This case, along with others reported by (\u003cspan additionalcitationids=\"CR35 CR36 CR37\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e), demonstrates a spectrum of outcomes, from successful births to miscarriages, emphasizing the high-risk nature of such pregnancies. The study, while offering current insights, also acknowledges the limitations in generalizability due to its singular nature and underscores the need for broader research. Various factors, including clinical and IVF mechanisms like High Gonadotrophin Concentrations and Zona hardening, alongside infertility etiology and maybe genetic factors, play significant roles, yet the precise mechanisms driving embryo splitting remain elusive. This gap in understanding calls for ongoing research to enhance clinical outcomes in this complex area of reproductive healthcare.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAAFC: Al Ain Fertility Center\u003c/p\u003e\n\u003cp\u003eAMH: Anti-M\u0026uuml;llerian Hormone\u003c/p\u003e\n\u003cp\u003eART: Assisted Reproductive Technology\u003c/p\u003e\n\u003cp\u003eFET: Frozen Embryo Transfer\u003c/p\u003e\n\u003cp\u003eFSH: Follicle-Stimulating Hormone\u003c/p\u003e\n\u003cp\u003eGnRH: Gonadotropin-Releasing Hormone\u003c/p\u003e\n\u003cp\u003ehCG: Human Chorionic Gonadotropin\u003c/p\u003e\n\u003cp\u003eICSI: Intracytoplasmic Sperm Injection\u003c/p\u003e\n\u003cp\u003eIUGR: Intrauterine Growth Restriction\u003c/p\u003e\n\u003cp\u003eIVF: In Vitro Fertilization\u003c/p\u003e\n\u003cp\u003eIVF-FET: In Vitro Fertilization-Frozen Embryo Transfer\u003c/p\u003e\n\u003cp\u003eMASE: Magnetically Activated Sperm Enrichment\u003c/p\u003e\n\u003cp\u003eMZTP: Monozygotic Triplet Pregnancies\u003c/p\u003e\n\u003cp\u003ePGT-A: Preimplantation Genetic Testing for Aneuploidy\u003c/p\u003e\n\u003cp\u003eQC: Quality Control\u003c/p\u003e\n\u003cp\u003eSET: Single Embryo Transfer\u003c/p\u003e\n\u003cp\u003eTTTS: Twin-to-Twin Transfusion Syndrome\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics Approval and Consent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthics approval for the present study was granted by the Al Ain Fertility Center - Research Ethics Committee (AAFC-REC)\u0026nbsp;with Project ID: AAFC/CREC/2023/003. The AAFC-REC confirmed that the research adhered to all ethical guidelines for protecting human participants. This includes compliance with the ethical standards of the 1964 Declaration of Helsinki and its later amendments, local UAE health authority regulations (Department of Health, Abu Dhabi), policies, and standards, as well as those of the institutional research committee. Additionally, the requirement for informed consent was specifically waived by the AAFC-REC for this retrospective study due to the minimal risk posed by the study, the absence of adverse effects on participants\u0026apos; rights and welfare, and the maintenance of anonymity in the presentation of all data. This research was conducted in strict accordance with these comprehensive guidelines and regulations, ensuring adherence to the highest ethical standards for conducting human research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe confirm that all listed authors meet the authorship criteria and have approved the manuscript as well as the author order.\u0026nbsp;We have duly considered the intellectual property aspects related to this work, confirming that there are no impediments to publication as per our institutional regulations. We acknowledge that the Corresponding Author is the primary contact during the editorial process and is tasked with updating the co-authors on manuscript progress, revisions, and proof approvals. A current and accessible email address for the Corresponding Author has been provided.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request. Due to the sensitive nature of the clinical information and to ensure patient confidentiality, data will not be publicly available. Requests for access to specific datasets used in this study will be considered by the authors, provided that they conform to ethical standards and regulations concerning patient data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDr. Braulio Peramo, as Principal Investigator and Chair of the Research Ethical Board at Al Ain Fertility Center, acknowledges a potential conflict of interest due to his dual role. This role is fully disclosed to ensure research transparency and integrity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable, this study is not funded.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eB.P.: Conceptualization, Methodology, Supervision, Investigation, Writing - Reviewing and Editing. D.U.: Data Curation, Investigation, Writing - Original Draft, Project Administration. R.A.H.: Investigation, Validation. S.A.: Resources, Data Curation, Visualization. F.A.: Resources, Data Curation. R.A.: Resources, Data Curation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to extend our heartfelt appreciation to the Al Ain Fertility Center (AAFC) management for their unwavering support and for providing the funding that made this research possible. Additionally, AAFC granted access to the essential data, which was crucial for this study. Dr. Saeed Al Dayeh, a Board Member at AAFC, provided strategic direction and governance and validated the research\u0026apos;s alignment with the center\u0026apos;s objectives. Mr. Mohd Al Hayek, Chief Financial Officer at AAFC, ensured budgetary compliance, facilitated the allocation of funds, and managed the financial aspects related to the research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; information (optional)\u003c/strong\u003e\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eDivyesh Upadhyay, Research Assistant, Clinical Department, AAFC, Abu Dhabi, U.A.E.\u003c/li\u003e\n \u003cli\u003eRajia Al Halaby, OB/GYN Fertility Specialist, Clinical Department, AAFC, Abu Dhabi, U.A.E.\u003c/li\u003e\n \u003cli\u003eSudha Anandt, Genetics Lab Manager, Genetics Department, AAFC, Abu Dhabi, U.A.E.\u003c/li\u003e\n \u003cli\u003eFiras Albuz, IVF Lab Manager, IVF Department, Al Ain Fertility Center, Abu Dhabi, U.A.E.\u003c/li\u003e\n \u003cli\u003eRawan Almekosh,Embryologist, IVF Department, Al Ain Fertility Center, Abu Dhabi, U.A.E.\u003c/li\u003e\n \u003cli\u003eBraulio Peramo,Medical Director, Clinical Department, Al Ain Fertility Center, Abu Dhabi, U.A.E.\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u003cstrong\u003eWeblink\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHalosperm\u0026reg; | Halotech DNA. (n.d.). Retrieved January 17, 2024, from \u0026nbsp;\u003c/p\u003e\n\u003cp\u003ehttps://www.halotechdna.com/productos/halosperm\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eSu-Faye, L. E. E., Chapman, M. \u0026amp; Bowyer, L. Monozygotic triplets after single blastocyst transfer: case report and literature review. \u003cem\u003eAust N Z J Obstet Gynaecol\u003c/em\u003e 48, 583\u0026ndash;586 (2008).\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eDerom, C., Derom, R., Vlietinck, R., Berghe, H. Vanden \u0026amp; Thiery, M. Increased monozygotic twinning rate after ovulation induction. \u003cem\u003eLancet\u003c/em\u003e 1, 1236\u0026ndash;1238 (1987).\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eFuchs, K. M. \u0026amp; D\u0026rsquo;Alton, M. E. 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Strategies to reduce multiple pregnancies during medically assisted reproduction. \u003cem\u003eFertil Steril\u003c/em\u003e 114, 673\u0026ndash;679 (2020).\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Monozygotic Triplet Pregnancies, Single Blastocyst Transfer, Assisted Reproductive Technology (ART), Embryo Splitting, In Vitro Fertilization (IVF).","lastPublishedDoi":"10.21203/rs.3.rs-5055637/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5055637/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis case report explores the rare occurrence of monozygotic triplet pregnancies resulting from a single blastocyst transfer in Assisted Reproductive Technology (ART). The novelty lies in documenting this unusual outcome, which provides valuable insights into the potential for zygotic splitting and its implications for reproductive practices.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCase Presentation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe patient, a woman with diminished ovarian reserve, underwent in vitro fertilization with intracytoplasmic sperm injection (IVF-ICSI) and Preimplantation Genetic Testing for Aneuploidy (PGT-A). The testing identified one euploid and one aneuploid embryo. With informed consent, a single euploid frozen-thawed embryo was transferred. The pregnancy resulted in the development of monozygotic triplets, as confirmed by the presence of three yolk sacs and fetal poles. Unfortunately, the pregnancy ended in a spontaneous miscarriage at the 10th week of gestation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe development of monozygotic triplets from a single embryo transfer underscores the unpredictable nature of ART and the potential for rare outcomes such as zygotic splitting. This case highlights the need for heightened awareness, early detection, and proactive management of such pregnancies to address associated risks. Further research is needed to better understand the mechanisms behind zygotic splitting in ART procedures and to improve clinical outcomes.\u003c/p\u003e","manuscriptTitle":"Risks and Complications of Monozygotic Triplet Pregnancy Following Single Blastocyst Transfer: Insights from a Case Report and Future Directions.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-25 16:34:38","doi":"10.21203/rs.3.rs-5055637/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1e4154dc-b3f2-4277-8101-9954df0d84ba","owner":[],"postedDate":"November 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-05-08T12:09:08+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-25 16:34:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5055637","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5055637","identity":"rs-5055637","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}