Preimplantation genetic testing for aneuploidy: The management of mosaic embryos

Eun Jeong Yu; Min Jee Kim; Eun A Park; Inn Soo Kang

doi:10.5653/cerm.2022.05393

Clin Exp Reprod Med > Volume 49(3); 2022 > Article

Yu, Kim, Park, and Kang: Preimplantation genetic testing for aneuploidy: The management of mosaic embryos

Review Article

Clinical and Experimental Reproductive Medicine 2022;49(3):159-167.

Published online: August 31, 2022

DOI: https://doi.org/10.5653/cerm.2022.05393

Preimplantation genetic testing for aneuploidy: The management of mosaic embryos

Eun Jeong Yu¹

, Min Jee Kim^2,³

, Eun A Park^2,⁴

, Inn Soo Kang⁵

¹Department of Obstetrics and Gynecology, CHA Fertility Center Seoul Station, CHA University School of Medicine, Seoul, Republic of Korea

²Department of Biomedical Sciences, College of Life Sciences, CHA University, Seongnam, Republic of Korea

³Laboratory of Reproductive Genetics, CHA Biotech, Seongnam, Republic of Korea

⁴Fertility Research Lab, CHA Fertility Center Seoul Station, Seoul, Republic of Korea

⁵Department of Obstetrics and Gynecology, CHA Fertility Center Daegu, CHA University School of Medicine, Daegu, Republic of Korea

Corresponding author: Inn Soo Kang Department of Obstetrics and Gynecology, CHA Fertility Center Daegu, CHA University School of Medicine, 2095 Dalgubeol-daero, Jung-gu, Daegu 41936, Korea
Tel: +82-53-222-4200, Fax: +82-53-214-6611 E-mail: ikang67pgd@gmail.com

Received March 31, 2022 Revised June 15, 2022 Accepted June 24, 2022

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

As the resolution and accuracy of diagnostic techniques for preimplantation genetic testing for aneuploidy (PGT-A) are improving, more mosaic embryos are being identified. Several studies have provided evidence that mosaic embryos have reproductive potential for implantation and healthy live birth. Notably, mosaic embryos with less than 50% aneuploidy have yielded a live birth rate similar to euploid embryos. This concept has led to a major shift in current PGT-A practice, but further evidence and theoretically relevant data are required. Proper guidelines for selecting mosaic embryos suitable for transfer will reduce the number of discarded embryos and increase the chances of successful embryo transfer. We present an updated review of clinical outcomes and practice recommendations for the transfer of mosaic embryos using PGT-A.

Keywords: Embryo transfer; Mosaicism; Preimplantation genetic testing

Introduction

In the field of preimplantation genetic testing for aneuploidy (PGT-A), mosaicism was first identified 25 years ago in a validation study, where it was thought to be caused by an insufficient trophectoderm (TE) sample size [1]. Technological innovations, such as next-generation sequencing (NGS), have significantly improved the identification and quantification of mosaicism. Some authors recently proposed that an “intermediate copy number” of individual chromosomes is a more accurate term than mosaicism [2].

Mosaic embryos have the potential to implant and develop into genetically normal babies [3,4]. Greco et al. [5] first reported in 2015 that 18 women who had mosaic embryo transfers gave birth to six healthy euploid newborns. In a recent prospective study, the authors demonstrated that mosaic embryos had a similar implantation rate (55% vs. 55.8%, p=0.86) and live birth rate (43.4% vs. 42.9%, p=0.82), as well as equivalent developmental potential to that of euploid embryos [6]. In addition, multicenter studies found no significant differences between euploid and mosaic embryo transfers in terms of the preterm delivery rate, birth weight, or risk of congenital malformations [3,7,8].

However, arguments for and against transferring mosaic embryos still exist [9]. The International Do No Harm Group in in vitro fertilization (IVF) argued against the 2019 Preimplantation Genetic Diagnosis International Society (PGDIS) guideline for mosaic embryo transfer [10] on the basis that the interpretation of mosaicism in PGT-A was misleading [9,11]. On the contrary, a recently published prospective non-selection study reported that the risk of clinical error in the diagnosis of uniform aneuploidy by NGS-based PGT-A was exceedingly low (0%–2%), suggesting that PGT-A has high predictive power [12]. Given the variability in the management of mosaic embryos, it is important for clinicians to have informative genetic counseling resources available when informing their patients of PGT-A results and giving recommendations for mosaic embryo transfer. Therefore, we aimed to provide the latest clinical outcomes following mosaic embryo transfers in PGT-A cycles and a summary of updated practice recommendations.

Definition and types of mosaicism

Mosaicism is the presence of more than one genotypically distinct cell population within a single zygote [13]. Mosaic cellular populations are thought to arise from post-zygotic mitotic errors during post-zygotic cell division [13]. In PGT-A, mosaicism is defined as a mixture of 20% to 80% aneuploid and euploid DNA content; those with less than 20% aneuploid DNA are called euploid, and those with more than 80% aneuploid DNA are called aneuploid. [14]. The incidence of mosaic embryos has been reported to be 5%, but some have found rates of 20%–30% using PGT-A [15]. Mosaicism is more frequently found in cleavage-stage embryos (30%–70%) [16] compared with blastocyst-stage embryos (5%–15%) [17,18].

Mosaicism can be classified into four types based on cell lineage and the timing of mitotic errors in the blastocyst stage [19,20]. An embryo is defined as “total mosaic” when both the inner cell mass (ICM) and TE contain aneuploid and euploid cells. If the mosaic population is exclusively ICM, the embryo is defined as “ICM mosaic,” and if exclusively TE, the embryo is “TE mosaic.” Finally, if all cells in the ICM are aneuploid and all cells in the TE are euploid (or vice versa), the embryo is “ICM/TE mosaic”.

Factors contributing to the diagnosis of mosaicism

Mosaicism may not be associated with maternal age [21]. Some authors suggested a slight increase in mosaicism in younger patients compared to women over 37 years of age [22]. In cases of low-degree mosaicism and segmental aneuploidies, the incidence of mosaicism showed a negative correlation with maternal age [23,24]. Contrary to the effects of maternal age, ovarian response to stimulation was positively related to the occurrence of segmental aneuploidy. In one study, the oocyte vitrification and ovarian response showed no effect on the mosaicism rate [22].

A high proportion of mosaic embryos was found in couples with low sperm concentrations [25,26]. The prevalence of mosaic and chaotic aneuploidy in blastomeres ranges from 35% to 68% in oligozoospermic and azoospermic men [27,28]. There is a higher proportion of mosaic embryos in PGT-A cycles with male infertility compared to patients with normal sperm parameters. The highest mosaicism rates were related to the severity of male infertility [25,26].

Technical laboratory factors may affect the quality of a biopsy and thus may affect the occurrence of mosaicism within the TE. The differences in platform specificity and sensitivity, the protocols for DNA amplification, and the threshold settings established for interpretation can lead to differences in the proportion of mosaicism and the number of euploid embryos to transfer [29]. Other factors associated with the biopsy technique, including the conditions surrounding cell loading and the number of cells biopsied, can also affect the results [30]. The method of fertilization [31] and laboratory conditions, such as oxygen concentration, pH and osmolality in the embryo culture medium, and temperature are related to an increased rate of mosaicism [30].

Management: transfer of mosaic embryos

Multiple factors determine the fate and viability of mosaic embryos, such as the degree of mosaicism in the biopsied sample, the specific type and number of chromosomes involved, and the type of mosaicism.

1. Priority for mosaic embryo transfer

In 2016, the position statement of the PGDIS recommended priorities for mosaic embryo transfers based on the specific chromosome involved and the level of mosaicism [32]. In 2017, the World Congress on Controversies in Preconception, Preimplantation and Prenatal Genetic Diagnosis highlighted the need for PGT-A in IVF practice and updated the PGDIS position statement on recommendations for clinical practice [33]. In 2018, Grati et al. [34] published a study on the chorionic villi samples (CVS) and products of conception (POC) after natural pregnancy to provide a practice guideline whereby mosaic embryos could lead to healthy live births. In 2020, Munne et al. [35] suggested classifying mosaic embryos into high- (>50%) and low-level (<50%) groups, with preference for transferring single segmental mosaic embryos over other types of mosaicism. In 2021, Viotti et al. [4] formulated a ranking system using outcome data from one thousand mosaic embryo transfers for the prioritization of mosaic embryos in the clinical setting. They confirmed that combined mosaic embryos have significantly lower implantation and pregnancy rates than euploid embryos. They also found that the type and level of mosaicism had a significant impact on the embryo transfer outcomes. Their study helped to elucidate the problems presented by mosaic transfer and attempted to provide firm conclusions. Relevant medical society practice guidelines and recommendations, including the recent PGDIS 2021 guidelines [36], are summarized in Table 1.

Despite these diverse ranking approaches, attempts to provide clinical recommendations for patients may yet be in early stages. Uncertainty remains regarding related factors affecting the clinical outcome data of mosaic embryo transfer. Some studies have suggested differences in live-birth rates based on the type and level of mosaicism [38] or involvement of a full versus partial chromosome [39], while others have failed to find such significance using the same classification system [40].

2. The degree of mosaicism

Chromosomal mosaicism has been defined as low-level mosaicism if abnormal cells are in the 30%–50% range and high-level mosaicism if abnormal cells are in the 50%–70% range using the NGS validation algorithm [41]. Clinical outcome data related to high- versus low-level mosaicism still show conflicting results. Some studies found that low-level mosaicism was related to improvement in ongoing pregnancy rates [38], while others did not find statistically significant results [40,42,43]. Embryos with low-level mosaicism are more likely to develop into healthy babies than high-level mosaic embryos, whereas high-level mosaic embryos increase the risk of miscarriage [35,38,41,44]. A recent prospective study found that embryos with more than 50% mosaicism have a significantly lower implantation rate (24.4% vs. 54.6%; p<0.002), clinical pregnancy rate (15.2% vs. 46.4%; p<0.001), and live birth rate (15.2% vs. 46.6%; p<0.001) compared to euploid embryos in the NGS profile [38]. Capalbo et al. [6] showed that low- (20%–30%) or moderate-degree (30%–50%) mosaic embryo transfer yielded similar clinical and neonatal outcomes in a prospective double-blinded non-selection trial.

3. Specific chromosomes involved

The clinical outcomes of mosaicism can be highly dependent on the chromosomes involved. Autosomes were ranked in order of their risk of placental insufficiency, intrauterine growth restriction, and uniparental disomy (UPD). The mosaic trisomy 16 chromosome is commonly affected in preimplantation embryos and leads to a high risk of abnormal perinatal outcomes, such as intrauterine growth restriction, preterm birth, and hypertensive disorders [45]. Chromosomes X, 21, and 22 have been reported to be susceptible to whole chromosome errors [46-48]. Chromosomes 2, 6, 7, 11, 14, 15, 16, and 20 are known to be associated with UPD [49,50]. Recent findings showed that chromosome length had a positive correlation with the mitotic error of each chromosome, but a negative correlation with the meiotic error of the preimplantation embryo [47,51].

Grati et al. [34] devised a scoring system for prioritizing mosaic embryo transfers based on the mosaic patterns observed in prenatal samples and products of conception and on the involvement of specific chromosomes. Mosaic trisomies 1, 3, 10, 12, and 19 had top priority for embryo transfer because of their low risk of deleterious outcomes, whereas mosaic trisomies 13, 16, 18, 21, 45, and monosomy X had a high risk of nonviable births and should be avoided. However, this system was limited by the difficulty found in assessing the degree of mosaicism in preimplantation embryos based only on molecular and cytogenetic results.

4. Monosomies versus trisomies

Most monosomies arise from mitotic errors and most trisomies result from nondisjunction during maternal meiotic errors [17,52]. Since monosomic cells are less likely to be viable than trisomic cells [53], most monosomic cells are removed at the post-implantation phase [54,55]. Trisomic mosaicism can occur in live births with chromosomal aneuploidy and is associated with cognitive and physical impairments [56]. Although PGDIS recommended the transfer of embryos with mosaic monosomies over those with mosaic trisomies in 2016 [32], this statement was updated and removed in 2019 [10]. In addition, some authors did not find a significant difference in pregnancy rates between monosomic and trisomic mosaic embryos [40]. According to the PGDIS guidelines, mosaic trisomies 1, 3, 4, 5, 6, 8, 9, 10, 11, 12, 17, 19, 20, 22, X, and Y are preferred over mosaic trisomies 2, 7, 13, 14, 15, 16, 18, and 21 when mosaic trisomy is being considered for transfer [32]. Recent studies proposed that mosaic monosomies and mosaic trisomies have similar implantation rates (46% and 47.2%, respectively; p>0.05) and ongoing pregnancy rates (36% and 33%, respectively; p>0.05) [4,33].

5. Whole versus segmental aberrations

When duplication or deletion errors occur in a small portion of DNA during mitotic division, the embryo will have a mosaic of the segmental error, allowing some cells to have a normal copy number of chromosomes and others to have segmental deletion or duplication of the chromosomes [57]. In one study, segmental gain or loss was affected in 25% of mosaicism [58]. Some authors suggested that the incidence of segmental mosaicism may be overestimated due to biological and technical errors [59]. Clinical perspectives of embryo mosaicism, with respect to full versus partial aneuploidies, have been inconsistent. Some studies have reported a higher clinical pregnancy rate in partial aneuploid mosaicism [39,42,60], while others have not found a significant difference [40]. In the subgroup of segmental mosaic embryos, Viotti et al. [4] recently investigated clinical outcomes after the transfer of 1,000 mosaic embryos and reported similar implantation rates (51.6% vs. 57.2%; p=0.001) and ongoing pregnancy rates (43.1% vs. 52.3%; p=0.001) compared to euploid embryos. Other recent studies also revealed that segmental mosaic embryos had clinical outcomes comparable to euploid embryos [12,61].

Regarding chromosome type, large chromosomes such as chromosomes 1 to 9 are prone to breakage, resulting in segmental mosaicism [62,63], while a significantly lower percentage of copy number errors were observed in small chromosomes and acrocentric chromosomes (e.g., chromosomes 19, 21, 22, and Y) [64,65].

Segmental aneuploidies originate because of mitotic errors during preimplantation development [24]. This is related to blastocyst morphology, not to maternal age or clinical and embryological parameters [66]. A previous multicenter study of 822 mosaic embryo transfers demonstrated that the reproductive potential of mosaic embryos is affected by the number of euploid cells and the complexity in the TE biopsy sample [67]. The embryos with segmental aneuploidy had better clinical outcomes than mosaic embryos with one or two involved chromosomes (implantation rate: p<0.001, ongoing pregnancy rate/birth rate: p<0.001).

6. The number of chromosomes involved (single versus double versus complex aneuploidies)

Several studies found reduced pregnancy capacity in mosaic embryos that had three or more chromosomes involved [40] and in segmental mosaicism that had two or more chromosomes involved [42], whereas other studies did not report clinically significant differences between mosaic embryos involving one or two chromosomes [40,68]. Complex mosaic embryos had the lowest implantation rates among single aneuploid, double aneuploid, and segmental mosaic embryos [40].

Genetic counseling

A recent statement by the American Society for Reproductive Medicine highlighted the importance of patient education prior to PGT-A [37]. Before the transfer of mosaic embryos, counseling should include a discussion of the potential challenges in interpreting mosaic results, the potential risks of mosaic embryo transfers, and the limited neonatal outcome data available. In addition, counseling should provide information regarding the genetic advantages, risks, and limitations of a prenatal diagnosis. Thus far, most prenatal testing results after mosaic embryo transfers have shown normal healthy fetuses with no specific chromosomal abnormalities [3]. However, we found two reports of babies with abnormal karyotypes: a baby with 15q duplication syndrome after transfer of a 57% segmental mosaic embryo [69] and a healthy baby with 2% mosaic monosomy 2 after transfer of a 35% mosaic monosomy 2 embryo [70].

Patients should be informed about the risk of mosaicism in a biopsy specimen, the complexities of the various possible outcomes after transfer of a mosaic embryo, and the need for close prenatal monitoring, including amniocentesis. Until definitive data is available, patients should be advised to go through additional cycles if possible to obtain euploid embryos instead of transferring a mosaic embryo. A schematic prioritization of mosaic embryos according to clinical outcomes is shown in Table 2.

Prenatal diagnosis after transfer of mosaic embryos

If a pregnancy has been confirmed after mosaic embryo transfer, prenatal diagnosis is recommended to identify fetal chromosomes and other genetic conditions. Although evidence-based guidance for prenatal testing after mosaic embryo transfer is still lacking, most practice statements consistently recommend amniocentesis as the gold standard for prenatal diagnosis [32,33,37,71]. Karyotyping of the amniocytes obtained by amniocentesis is done to diagnose aneuploidy in the fetus [72]. CVS can be useful for patients seeking a diagnosis during the first trimester; however, CVS results represent placental cells derived from the TE. Thus, mosaic findings detected using CVS may indicate placental mosaicism, and follow-up amniocentesis is required to clarify the results. The major advantage of amniocentesis is the ability to analyze fetal cells directly, but it may miss low-level mosaicism. Therefore, amniocentesis best represents the chromosome complement within fetal tissues, but patients should know that some mosaicism may not be detectable. Depending on the PGT-A result, further analysis of prenatal samples should also be considered; chromosomal microarray can be performed if segmental aneuploidy or UPD is involved [37,73]. Cell-free DNA (cfDNA) testing, also known as noninvasive prenatal testing (NIPT), has not been validated to detect mosaicism because NIPT analyzes circulating cfDNA fragments in the maternal plasma derived from both the mother’s and apoptotic trophoblasts, but not from the fetus itself [74].

Conclusion

Although interest in mosaic embryo transfers is increasing, the debate over whether mosaic embryos can be transferred is ongoing. In practice, the identification of mosaic subgroups that are viable and worthy of transfer is very important, but it is also vital to inform patients that the data on postnatal and neonatal outcomes following mosaic embryo transfers are still limited and that clinical outcomes have been mixed. We emphasize the need for further research on the genetic and clinical outcomes of mosaic embryo transfers. Large-scale multicenter studies would be of particular value in collecting data for the risk evaluation of mosaic embryo transfers and could potentially reduce the disposal of viable embryos for implantation and live births.

Conflict of interest

No potential conflict of interest relevant to this article was reported.

Author contributions

Conceptualization: ISK. Writing–original draft: EJY. Writing–review & editing: all authors.

Table 1.

A list of professional medical society guidelines and recommendations regarding mosaic embryo transfer

Variable	PGDIS 2016 [32]	CoGEN 2017 [33]	Grati 2018 [34]	PGDIS 2019 [10]	ASRM 2020 [37]	PGDIS 2021 [36]
More favorable clinical outcomes in euploid than mosaic embryos	Yes	Yes	Yes	Yes	Yes	Yes
More favorable clinical outcomes in low than high levels of mosaicism	Not assessed	Yes (20%-40% vs. 40%–70%)	Not assessed	Yes (<40% vs. >40%)	Yes (but controversial)	Yes
Specific chromosome(s) involved	Lowest priority: chr 13, 18, 21	Lowest priority: chr 13, 18, 21, 22	Lowest priority: chr 13, 18, 21 and 45, X	Embryos mosaic for chromosomes that are associated with potential for uniparental disomy, severe intrauterine growth restriction, or liveborn syndromes may be given lower priority.	Some studies have found risky outcomes depending on the specific chromosome numbers involved; while others have reported that mosaic aneuploidies involving most chromosomes have pregnancies and live births with an abnormal phenotype.	No specific comments
Specific chromosome(s) involved	Lesser priority: potential for uniparental disomy (chr 14, 15), intrauterine growth restriction (chr 2, 7, 16)	Low priority: uniparental disomy (chr 14, 15), intrauterine growth restriction (chr 2, 7, 16)	Lesser priority: potential for uniparental disomy (chr 14, 15), intrauterine growth restriction (chr 2, 7, 16)			No specific comments
More favorable clinical outcomes in monosomies than trisomies	Yes	Yes	Yes	Yes	Yes	Yes
Different clinical outcomes between mosaic types (segmental vs. whole chromosome vs. complex	Not assessed	In the case of complex mosaicism, transfer is not recommended	Not assessed	Not assessed	Controversial	Yes
Recommendation of prenatal test method	Amniocentesis	Amniocentesis	Amniocentesis	Amniocentesis	Amniocentesis	Amniocentesis
Special considerations		If a decision is made to transfer a non-complex, low-level mosaic embryo, one can prioritize selection based on the specific chromosome involved.		If a decision is made to transfer embryos mosaic for a single chromosome, one can prioritize selection primarily based on the level of mosaicism and then the specific chromosome involved.	Before transfer of mosaic embryos, comprehensive genetic counseling should be provided.	The relative percentage of mosaicism seems to be a better predictor of outcome than the specific chromosomes involved.

PGDIS, Preimplantation Genetic Diagnosis International Society; CoGEN, Congress on Controversies in Preconception, Preimplantation and Prenatal Genetic Diagnosis; ASRM, American Society for Reproductive Medicine; Chr, chromosome.

Table 2.

Schematic prioritization of mosaic embryo classified according to favorable clinical outcomes

Priority	Percentage of mosaicism	Monosomy vs. trisomy	Segmental vs. whole chromosome	Specific chromosomes involved	Number of Chr involved (single vs. double vs. complex)
Low clinical risk	Low (<50%)	Monosomy	Segmental	Chr 1, 3, 4, 5, 6, 10, 12, 17, 19, 20, 22, X, Y	Single
High clinical risk	High (>50%)	Trisomy	Whole	Chr 13, 18, 21: best-avoided	Complex
				Chr 6, 7, 11, 14, 15, 20: UPD risk
				Chr 2, 16: IUGR risk
				Chr 8, 9: aneuploidy viability

Chr, chromosome; UPD, uniparental disomy; IUGR, intrauterine growth restriction

References

1. Gutierrez-Mateo C, Colls P, Sanchez-Garcia J, Escudero T, Prates R, Ketterson K, et al. Validation of microarray comparative genomic hybridization for comprehensive chromosome analysis of embryos. Fertil Steril 2011;95:953-8.

2. Paulson RJ, Treff NR. Isn't it time to stop calling preimplantation embryos "mosaic"? F S Rep 2020;1:164-5.

3. Abhari S, Kawwass JF. Pregnancy and neonatal outcomes after transfer of mosaic embryos: a review. J Clin Med 2021;10:1369.

4. Viotti M, Victor AR, Barnes FL, Zouves CG, Besser AG, Grifo JA, et al. Using outcome data from one thousand mosaic embryo transfers to formulate an embryo ranking system for clinical use. Fertil Steril 2021;115:1212-24.

5. Greco E, Minasi MG, Fiorentino F. Healthy babies after intrauterine transfer of mosaic aneuploid blastocysts. N Engl J Med 2015;373:2089-90.

6. Capalbo A, Poli M, Rienzi L, Girardi L, Patassini C, Fabiani M, et al. Mosaic human preimplantation embryos and their developmental potential in a prospective, non-selection clinical trial. Am J Hum Genet 2021;108:2238-47.

7. Yakovlev P, Vyatkina S, Polyakov A, Pavlova M, Volkomorov V, Yakovlev M, et al. Neonatal and clinical outcomes after transfer of a mosaic embryo identified by preimplantation genetic testing for aneuploidies. Reprod Biomed Online 2022;45:88-100.

8. Zhang YX, Chen JJ, Nabu S, Yeung QS, Li Y, Tan JH, et al. The pregnancy outcome of mosaic embryo transfer: a prospective multicenter study and meta-analysis. Genes (Basel) 2020;11:973.

9. Gleicher N, Barad DH, Ben-Rafael Z, Glujovsky D, Mochizuki L, Modi D, et al. Commentary on two recently published formal guidelines on management of "mosaic" embryos after preimplantation genetic testing for aneuploidy (PGT-A). Reprod Biol Endocrinol 2021;19:23.

10. Cram DS, Leigh D, Handyside A, Rechitsky L, Xu K, Harton G, et al. PGDIS position statement on the transfer of mosaic embryos 2019. Reprod Biomed Online 2019;39 Suppl 1:e1-4.

11. Gleicher N, Albertini DF, Barad DH, Homer H, Modi D, Murtinger M, et al. The 2019 PGDIS position statement on transfer of mosaic embryos within a context of new information on PGT-A. Reprod Biol Endocrinol 2020;18:57.

12. Tiegs AW, Tao X, Zhan Y, Whitehead C, Kim J, Hanson B, et al. A multicenter, prospective, blinded, nonselection study evaluating the predictive value of an aneuploid diagnosis using a targeted next-generation sequencing-based preimplantation genetic testing for aneuploidy assay and impact of biopsy. Fertil Steril 2021;115:627-37.

13. Levy B, Hoffmann ER, McCoy RC, Grati FR. Chromosomal mosaicism: origins and clinical implications in preimplantation and prenatal diagnosis. Prenat Diagn 2021;41:631-41.

14. Gleicher N, Patrizio P, Brivanlou A. Preimplantation genetic testing for aneuploidy: a castle built on sand. Trends Mol Med 2021;27:731-42.

15. Capalbo A, Rienzi L. Mosaicism between trophectoderm and inner cell mass. Fertil Steril 2017;107:1098-106.

16. Mertzanidou A, Wilton L, Cheng J, Spits C, Vanneste E, Moreau Y, et al. Microarray analysis reveals abnormal chromosomal complements in over 70% of 14 normally developing human embryos. Hum Reprod 2013;28:256-64.

17. Johnson DS, Cinnioglu C, Ross R, Filby A, Gemelos G, Hill M, et al. Comprehensive analysis of karyotypic mosaicism between trophectoderm and inner cell mass. Mol Hum Reprod 2010;16:944-9.

18. Capalbo A, Wright G, Elliott T, Ubaldi FM, Rienzi L, Nagy ZP. FISH reanalysis of inner cell mass and trophectoderm samples of previously array-CGH screened blastocysts shows high accuracy of diagnosis and no major diagnostic impact of mosaicism at the blastocyst stage. Hum Reprod 2013;28:2298-307.

19. Liu J, Wang W, Sun X, Liu L, Jin H, Li M, et al. DNA microarray reveals that high proportions of human blastocysts from women of advanced maternal age are aneuploid and mosaic. Biol Reprod 2012;87:148.

20. Vera-Rodriguez M, Rubio C. Assessing the true incidence of mosaicism in preimplantation embryos. Fertil Steril 2017;107:1107-12.

21. Daphnis DD, Delhanty JD, Jerkovic S, Geyer J, Craft I, Harper JC. Detailed FISH analysis of day 5 human embryos reveals the mechanisms leading to mosaic aneuploidy. Hum Reprod 2005;20:129-37.

22. Rubio C, Rodrigo L, Garcia-Pascual C, Peinado V, Campos-Galindo I, Garcia-Herrero S, et al. Clinical application of embryo aneuploidy testing by next-generation sequencing. Biol Reprod 2019;101:1083-90.

23. Girardi L, Serdarogullari M, Patassini C, Poli M, Fabiani M, Caroselli S, et al. Incidence, origin, and predictive model for the detection and clinical management of segmental aneuploidies in human embryos. Am J Hum Genet 2020;106:525-34.

24. Babariya D, Fragouli E, Alfarawati S, Spath K, Wells D. The incidence and origin of segmental aneuploidy in human oocytes and preimplantation embryos. Hum Reprod 2017;32:2549-60.

25. Tarozzi N, Nadalini M, Lagalla C, Coticchio G, Zaca C, Borini A. Male factor infertility impacts the rate of mosaic blastocysts in cycles of preimplantation genetic testing for aneuploidy. J Assist Reprod Genet 2019;36:2047-55.

26. Kahraman S, Sahin Y, Yelke H, Kumtepe Y, Tufekci MA, Yapan CC, et al. High rates of aneuploidy, mosaicism and abnormal morphokinetic development in cases with low sperm concentration. J Assist Reprod Genet 2020;37:629-40.

27. Silber S, Escudero T, Lenahan K, Abdelhadi I, Kilani Z, Munne S. Chromosomal abnormalities in embryos derived from testicular sperm extraction. Fertil Steril 2003;79:30-8.

28. Rodrigo L, Peinado V, Mateu E, Remohi J, Pellicer A, Simon C, et al. Impact of different patterns of sperm chromosomal abnormalities on the chromosomal constitution of preimplantation embryos. Fertil Steril 2010;94:1380-6.

29. Garcia-Pascual CM, Navarro-Sanchez L, Navarro R, Martinez L, Jimenez J, Rodrigo L, et al. Optimized NGS approach for detection of aneuploidies and mosaicism in PGT-A and imbalances in PGT-SR. Genes (Basel) 2020;11:724.

30. Swain JE. Controversies in ART: can the IVF laboratory influence preimplantation embryo aneuploidy? Reprod Biomed Online 2019;39:599-607.

31. Palmerola KL, Vitez SF, Amrane S, Fischer CP, Forman EJ. Minimizing mosaicism: assessing the impact of fertilization method on rate of mosaicism after next-generation sequencing (NGS) preimplantation genetic testing for aneuploidy (PGT-A). J Assist Reprod Genet 2019;36:153-7.

32. PGDIS. PGDIS position statement on chromosome mosaicism and preimplantation aneuploidy testing at the blastocyst stage [Internet]. Northbrook: PGDIS; 2016 [cited 2022 Aug 10]. Available from: http://www.pgdis.org/docs/newsletter_071816.html

33. IVF-Worldwide.com. COGEN position statement on chromosomal mosaicism detected in preimplantation blastocyst biopsies [Internet]. IVF-Worldwide.com; 2017 [cited 2022 Aug 10]. Available from: https://ivf-worldwide.com/cogen/oep/publications/cogen-position-statement-on-chromosomal-mosaicism-detected-in-preimplantation-blastocyst-biopsies.html

34. Grati FR, Gallazzi G, Branca L, Maggi F, Simoni G, Yaron Y. An evidence-based scoring system for prioritizing mosaic aneuploid embryos following preimplantation genetic screening. Reprod Biomed Online 2018;36:442-9.

35. Munne S, Spinella F, Grifo J, Zhang J, Beltran MP, Fragouli E, et al. Clinical outcomes after the transfer of blastocysts characterized as mosaic by high resolution next generation sequencing-further insights. Eur J Med Genet 2020;63:103741.

36. Leigh D, Cram DS, Rechitsky S, Handyside A, Wells D, Munne S, et al. PGDIS position statement on the transfer of mosaic embryos 2021. Reprod Biomed Online 2022;45:19-25.

37. Practice Committee and Genetic Counseling Professional Group (GCPG) of the American Society for Reproductive Medicine. Clinical management of mosaic results from preimplantation genetic testing for aneuploidy (PGT-A) of blastocysts: a committee opinion. Fertil Steril 2020;114:246-54.

38. Spinella F, Fiorentino F, Biricik A, Bono S, Ruberti A, Cotroneo E, et al. Extent of chromosomal mosaicism influences the clinical outcome of in vitro fertilization treatments. Fertil Steril 2018;109:77-83.

39. Fragouli E, Alfarawati S, Spath K, Babariya D, Tarozzi N, Borini A, et al. Analysis of implantation and ongoing pregnancy rates following the transfer of mosaic diploid-aneuploid blastocysts. Hum Genet 2017;136:805-19.

40. Munne S, Blazek J, Large M, Martinez-Ortiz PA, Nisson H, Liu E, et al. Detailed investigation into the cytogenetic constitution and pregnancy outcome of replacing mosaic blastocysts detected with the use of high-resolution next-generation sequencing. Fertil Steril 2017;108:62-71.e8.

41. Lin PY, Lee CI, Cheng EH, Huang CC, Lee TH, Shih HH, et al. Clinical outcomes of single mosaic embryo transfer: high-level or low-level mosaic embryo, does it matter? J Clin Med 2020;9:1695.

42. Victor AR, Tyndall JC, Brake AJ, Lepkowsky LT, Murphy AE, Griffin DK, et al. One hundred mosaic embryos transferred prospectively in a single clinic: exploring when and why they result in healthy pregnancies. Fertil Steril 2019;111:280-93.

43. Kushnir VA, Darmon SK, Barad DH, Gleicher N. Degree of mosaicism in trophectoderm does not predict pregnancy potential: a corrected analysis of pregnancy outcomes following transfer of mosaic embryos. Reprod Biol Endocrinol 2018;16:6.

44. Lee CI, Cheng EH, Lee MS, Lin PY, Chen YC, Chen CH, et al. Healthy live births from transfer of low-mosaicism embryos after preimplantation genetic testing for aneuploidy. J Assist Reprod Genet 2020;37:2305-13.

45. Grau Madsen S, Uldbjerg N, Sunde L, Becher N, Danish Fetal Medicine Study Group; Danish Clinical Genetics Study Group. Prognosis for pregnancies with trisomy 16 confined to the placenta: a Danish cohort study. Prenat Diagn 2018;38:1103-10.

46. Rius M, Daina G, Obradors A, Ramos L, Velilla E, Fernandez S, et al. Comprehensive embryo analysis of advanced maternal age-related aneuploidies and mosaicism by short comparative genomic hybridization. Fertil Steril 2011;95:413-6.

47. McCoy RC, Demko ZP, Ryan A, Banjevic M, Hill M, Sigurjonsson S, et al. Evidence of selection against complex mitotic-origin aneuploidy during preimplantation development. PLoS Genet 2015;11:e1005601.

48. Harton GL, Cinnioglu C, Fiorentino F. Current experience concerning mosaic embryos diagnosed during preimplantation genetic screening. Fertil Steril 2017;107:1113-9.

49. Dawson AJ, McGowan-Jordan J, Chernos J, Xu J, Lavoie J, Wang JC, et al. Canadian college of medical geneticists guidelines for the indications, analysis, and reporting of cancer specimens. Curr Oncol 2011;18:e250-5.

50. Kearney HM, Kearney JB, Conlin LK. Diagnostic implications of excessive homozygosity detected by SNP-based microarrays: consanguinity, uniparental disomy, and recessive single-gene mutations. Clin Lab Med 2011;31:595-613.

51. Chuang TH, Chang YP, Lee MJ, Wang HL, Lai HH, Chen SU. The incidence of mosaicism for individual chromosome in human blastocysts is correlated with chromosome length. Front Genet 2021;11:565348.

52. Johnson DS, Gemelos G, Baner J, Ryan A, Cinnioglu C, Banjevic M, et al. Preclinical validation of a microarray method for full molecular karyotyping of blastomeres in a 24-h protocol. Hum Reprod 2010;25:1066-75.

53. Bunnell ME, Wilkins-Haug L, Reiss R. Should embryos with autosomal monosomy by preimplantation genetic testing for aneuploidy be transferred? Implications for embryo selection from a systematic literature review of autosomal monosomy survivors. Prenat Diagn 2017;37:1273-80.

54. Rubio C, Rodrigo L, Mercader A, Mateu E, Buendia P, Pehlivan T, et al. Impact of chromosomal abnormalities on preimplantation embryo development. Prenat Diagn 2007;27:748-56.

55. Barbash-Hazan S, Frumkin T, Malcov M, Yaron Y, Cohen T, Azem F, et al. Preimplantation aneuploid embryos undergo self-correction in correlation with their developmental potential. Fertil Steril 2009;92:890-6.

56. Sachdev NM, Maxwell SM, Besser AG, Grifo JA. Diagnosis and clinical management of embryonic mosaicism. Fertil Steril 2017;107:6-11.

57. Treff NR, Franasiak JM. Detection of segmental aneuploidy and mosaicism in the human preimplantation embryo: technical considerations and limitations. Fertil Steril 2017;107:27-31.

58. Coll L, Parriego M, Mateo S, Garcia-Monclus S, Rodriguez I, Boada M, et al. Prevalence, types and possible factors influencing mosaicism in IVF blastocysts: results from a single setting. Reprod Biomed Online 2021;42:55-65.

59. Lai HH, Chuang TH, Wong LK, Lee MJ, Hsieh CL, Wang HL, et al. Identification of mosaic and segmental aneuploidies by next-generation sequencing in preimplantation genetic screening can improve clinical outcomes compared to array-comparative genomic hybridization. Mol Cytogenet 2017;10:14.

60. Zhang L, Wei D, Zhu Y, Gao Y, Yan J, Chen ZJ. Rates of live birth after mosaic embryo transfer compared with euploid embryo transfer. J Assist Reprod Genet 2019;36:165-72.

61. Wang L, Wang X, Liu Y, Ou X, Li M, Chen L, et al. IVF embryo choices and pregnancy outcomes. Prenat Diagn 2021;41:1709-17.

62. Nakhuda G, Jing C, Butler R, Guimond C, Hitkari J, Taylor E, et al. Frequencies of chromosome-specific mosaicisms in trophoectoderm biopsies detected by next-generation sequencing. Fertil Steril 2018;109:857-65.

63. Capalbo A, Rienzi L, Cimadomo D, Maggiulli R, Elliott T, Wright G, et al. Correlation between standard blastocyst morphology, euploidy and implantation: an observational study in two centers involving 956 screened blastocysts. Hum Reprod 2014;29:1173-81.

64. Franasiak JM, Forman EJ, Hong KH, Werner MD, Upham KM, Treff NR, et al. Aneuploidy across individual chromosomes at the embryonic level in trophectoderm biopsies: changes with patient age and chromosome structure. J Assist Reprod Genet 2014;31:1501-9.

65. Iwarsson E, Malmgren H, Inzunza J, Ahrlund-Richter L, Sjoblom P, Rosenlund B, et al. Highly abnormal cleavage divisions in preimplantation embryos from translocation carriers. Prenat Diagn 2000;20:1038-47.

66. Fragouli E, Alfarawati S, Spath K, Jaroudi S, Sarasa J, Enciso M, et al. The origin and impact of embryonic aneuploidy. Hum Genet 2013;132:1001-13.

67. Escriba MJ, Vendrell X, Peinado V. Segmental aneuploidy in human blastocysts: a qualitative and quantitative overview. Reprod Biol Endocrinol 2019;17:76.

68. Lledo B, Morales R, Ortiz JA, Blanca H, Ten J, Llacer J, et al. Implantation potential of mosaic embryos. Syst Biol Reprod Med 2017;63:206-8.

69. Mounts EL, Zhu SO, Sanderson RK, Coates A, Hesla JS. Mosaic embryo diagnosis correlated with abnormal 15q duplication syndrome in offspring. Fertil Steril 2019;112:e241-2.

70. Kahraman S, Cetinkaya M, Yuksel B, Yesil M, Pirkevi Cetinkaya C. The birth of a baby with mosaicism resulting from a known mosaic embryo transfer: a case report. Hum Reprod 2020;35:727-33.

71. Besser AG, Mounts EL. Counselling considerations for chromosomal mosaicism detected by preimplantation genetic screening. Reprod Biomed Online 2017;34:369-74.

72. Anchan RM, Quaas P, Gerami-Naini B, Bartake H, Griffin A, Zhou Y, et al. Amniocytes can serve a dual function as a source of iPS cells and feeder layers. Hum Mol Genet 2011;20:962-74.

73. Del Gaudio D, Shinawi M, Astbury C, Tayeh MK, Deak KL, Raca G, ACMG Laboratory Quality Assurance Committee. Diagnostic testing for uniparental disomy: a points to consider statement from the American College of Medical Genetics and Genomics (ACMG). Genet Med 2020;22:1133-41.

74. Choi H, Lau TK, Jiang FM, Chan MK, Zhang HY, Lo PS, et al. Fetal aneuploidy screening by maternal plasma DNA sequencing: 'false positive' due to confined placental mosaicism. Prenat Diagn 2013;33:198-200.