Mosaicism between trophectoderm and inner cell mass Antonio Capalbo, Ph.D.a,b and Laura Rienzi, M.Sc.a,b a GENERA, Centre for Reproductive Medicine, Clinica Valle Giulia, Rome; and b GENETYX, Molecular Genetics Laboratory, Vicenza, Italy
Defining the actual incidence and prevalence of mosaicism in human blastocysts still remains a difficult task. The small amount of evidence generated by animal and human studies does not support the existence of mechanisms involved in developmental arrest, clonal depletion, or aneuploidy rescue for abnormal cells in euploid/aneuploid embryos during preimplantation development. However, studies in humans are mainly descriptive and lack functional evidence. Understanding the biological mechanisms that beset preimplantation differentiation holds the potential to reveal the role of aneuploidies and gene dosage imbalances in cell fate decision, providing important clues on the origin and evolution of embryonic mosaicism. The evidence on human blastocysts suggests that a mosaic euploid/aneuploid configuration is detected in around 5% of embryos. This figure supports the extremely low level of mosaicism reported in natural and IVF pregnancies. Similarly, the clinical management of patterns consistent with the presence of mosaicism in a trophectoderm biopsy during preimplantation genetic diagnosis cycles (PGD-A) is still a controversial issue. Despite the facts that some contemporary comprehensive chromosomal screening platforms can detect mosaic samples in cell mixture models with variable accuracy and many reproductive genetics laboratories are now routinely including embryonic mosaicism on their genetic reports, a diagnosis of certainty for mosaicism in PGD-A cycles is conceptually impracticable. Indeed, several technical and biological sources of errors clearly exist when trying to estimate mosaicism from a single trophectoderm biopsy in PGD-A cycles and must be understood to adequately guide patients during clinical care. (Fertil SterilÒ 2017;107:1098–106. Ó2017 by American Society for Reproductive Medicine.) Key Words: Chromosomal mosaicism, blastocyst, inner cell mass, trophectoderm, aneuploidies, preimplantation genetic screening Discuss: You can discuss this article with its authors and with other ASRM members at https://www.fertstertdialog.com/users/ 16110-fertility-and-sterility/posts/15693-23856
T
he unbalanced transmission of chromosomes in human gametes and early preimplantation embryos causes aneuploidy, which is a major cause of infertility, pregnancy loss, and intellectual disability in humans (1). In the preimplantation and prenatal setting, chromosome abnormalities span a wide range of genomic imbalances, from polyploidy, to whole chromosome and large structural aneuploidies, down to submicroscopic deletions and duplications. Whole chromosome aneuploidies, monosomies and trisomies for the entire chromosomes, are the far more prevalent abnormalities and have been extensively investigated due to their high incidence in human conceptions and their clear association with clinical phenotypes and infertility. Undoubtedly, they represent
the single most common form of aneuploidy and the primary cause for implantation failure in IVF cycles and miscarriages in human pregnancies. Another well-defined characteristic of human aneuploidies is their strict correlation with female age. As women age, oocytes become increasingly susceptible to chromosome segregation errors during the meiotic process. Extensive analysis of main autosomal trisomies in clinical pregnancies revealed that more than 90% had a meiotic origin. The majority are due to maternal errors, with >75% due to errors in meiosis I and 50%) have full developmental potential, while when the proportion of normal blastomeres was decreased to one third, there was a partial rescue of embryonic lethality. In humans, both functional and prospective nonselection studies aimed at modeling the clinical fate and the reproductive potential of embryos showing intermediate copy number profiles are lacking. Prospective cohort studies in PGD-A cycles are also of poor value to determine the predictive value of patterns consistent with mosaicism as these are subjected to a patient and embryo selection bias. In conclusion, despite the fact that some contemporary CCS platforms can detect mosaic samples in cell mixture models with variable accuracy (59–61), a diagnosis of certainty for mosaicism in PGD-A cycles is conceptually impracticable. Many reproductive genetics laboratories are now routinely including embryonic mosaicism on their diagnostic reports. However, for the arguments discussed above, both biological and technological limitations exist and must be understood in order to adequately counsel patients during clinical care. Furthermore, it should be stressed that the chromosomal variations observed in a TE biopsy will not unequivocally represent the mosaicism rate present within the whole embryo. The existence of a sample bias has to be acknowledged in patients consent forms as a limitation for mosaicism detection in PGD-A cycles. In the light of the arguments discussed above, mosaicism should be recognized as a biological limitation for PGD-A programs as it is for prenatal diagnosis. In addition, it appears reasonable to avoid reporting mosaicism in PGD-A cycles
1103
1104
until its impact on clinical treatment has been clarified. A more conservative approach for reporting this type of result may be therefore preferred until evidence showing correlation between intermediate copy number profiles and mosaicism in embryos is available (i.e., ‘‘analysis showing a pattern that is consistent with the potential presence of mosaicism’’). Similarly, if future nonselection studies and randomized controlled trials show a lower reproductive potential for blastocyst with intermediate copy number profiles, clinically validated criteria should be introduced into clinical practice. In this case, on top of their genetic diagnosis, additional viability scores could be assigned to embryos. Nonetheless, extensive genetic counseling detailing all the specific limitations should be given to patients.
CONCLUDING REMARKS AND FUTURE PERSPECTIVES
Capalbo. Blastocyst mosaicism. Fertil Steril 2017.
92/181 (51.7) 77/181 (42.8) 41/51 (80) 9/51 (18) 4/10 (40) 6/10 (60) 29/50 (58) 13/50 (26)
Note: Abnormal cells in euploid-aneuploid mosaic embryos are similarly distributed between ICM and TE, indicating no sign of preferential allocation or confinement of chromosomally abnormal cells. The fact that results were almost identical for samples from the TE and ICM indicates that data obtained from a clinical TE sample can generally be considered diagnostic of the ICM chromosomal complement. CGH ¼ comparative genomic hybridization.
– – Not applicable
TE-confined mosaicism (%) ICM-confined mosaicism (%) Mosaic blastocysts (both ICM and TE) (%) Most frequent mosaic chromosomes Fully euploid blastocyst (%) Fully aneuploid blastocyst (%)
chr16; chr22
19/70 (70) 49/70 (27)
4/181 (2.2) 2/181 (1.1) 5/181 (2.8) 0/51 (0) 1/51 (2) 0/51 (0)
chr16; chr17; chr22; chr21
– 181 71 Range, 26–42 – SNP array with parental support 51 17 31 Donated blastocysts
FISH þ CGH reanalysis 10 4 33 Blastocysts diagnosed as aneuploid by FISH-based blastomere analysis 0/10 (0) 0/10 (0) 0/10 (0) Method No. of samples No. of patients Mean female age Origin of embryos
FISH þ SNP array reanalysis 50 24 35 Blastocysts diagnosed as aneuploid by FISH-based blastomere analysis 4/50 (8) 1/50 (2) 3/50 (6)
aCGH þ FISH reanalysis 70 26 36 Blastocysts diagnosed as aneuploid by aCGH-based TE analysis 0/70 (0) 0/70 (0) 2/70 (3)
Overall Johnson et al. (2010) Capalbo et al. (2013) Fragouli et al. (2008) Northrop et al. (2010)
Summary table of methodology and main findings from relevant studies on the cytogenetic constitution of ICM and TE samples from disaggregated human blastocysts.
TABLE 1
VIEWS AND REVIEWS
The small amount of evidence gathered from animal and human studies suggests the absence of mechanisms involved in embryo arrest, clonal depletion, or aneuploidy rescue for abnormal cells in euploid/aneuploid embryos during preimplantation development and blastocyst differentiation. Thus, it is likely that abnormal cells in mosaic diploid/aneuploid blastocysts are randomly allocated to the ICM and TE. However, our understanding of human preimplantation development is limited due to ethical and legal restrictions on embryo research and scarcity of materials. Studies in humans are mainly descriptive and lack functional evidence. Most of the information on embryo development is inferred from animal models and embryonic stem cell cultures, which should therefore be applied to the human model with caution. Understanding the biological mechanisms that beset preimplantation differentiation might reveal how aneuploidies and related gene dosage imbalances impact cell fate decisions and provide important clues on the biology of mosaicism in preimplantation embryos. The lack of correction for expected false positives and the use of nonstandardized criteria for classification of embryos as mosaic have likely resulted in an overestimation of mosaicism in IVF embryos. The little evidence available on humans suggests that mosaic euploid/aneuploid configuration is a relatively uncommon feature and should not impact the effectiveness of PGD-A programs appreciably. Further studies are needed to corroborate these findings and to better characterize at the single-cell level the incidence and prevalence of mosaicism at the blastocyst stage. Undoubtedly, novel CCS technologies able to distinguish between meiotic and mitotic errors in embryonic biopsies will deliver powerful research and clinical tools for the comprehensive analysis of aneuploidies in human blastocyst and consequent PGD-A protocols improvement. At the present, reports of mosaicism should be avoided in blastocyst PGD-A cycles due to the several technical and biological issues present when attempting this type of diagnosis and the lack of evidence from nonselection studies. The observation of intermediate chromosome copy number profiles would be better reported as a ‘‘pattern consistent with the presence of mosaicism,’’ and genetic counseling should be provided to guide patients' decisions. VOL. 107 NO. 5 / MAY 2017
Fertility and Sterility®
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VOL. 107 NO. 5 / MAY 2017
Defining the actual incidence and prevalence of mosaicism in human blastocysts still remains a difficult task. The small amount of evidence generated by animal and human studies does not support the existence of mechanisms involved in developmental arrest, clonal depletion, or aneuploidy rescue for abnormal cells in euploid/aneuploid embryos during preimplantation development. However, studies in humans are mainly descriptive and lack functional evidence. Understanding the biological mechanisms that beset preimplantation differentiation holds the potential to reveal the role of aneuploidies and gene dosage imbalances in cell fate decision, providing important clues on the origin and evolution of embryonic mosaicism. The evidence on human blastocysts suggests that a mosaic euploid/aneuploid configuration is detected in around 5% of embryos. This figure supports the extremely low level of mosaicism reported in natural and IVF pregnancies. Similarly, the clinical management of patterns consistent with the presence of mosaicism in a trophectoderm biopsy during preimplantation genetic diagnosis cycles (PGD-A) is still a controversial issue. Despite the facts that some contemporary comprehensive chromosomal screening platforms can detect mosaic samples in cell mixture models with variable accuracy and many reproductive genetics laboratories are now routinely including embryonic mosaicism on their genetic reports, a diagnosis of certainty for mosaicism in PGD-A cycles is conceptually impracticable. Indeed, several technical and biological sources of errors clearly exist when trying to estimate mosaicism from a single trophectoderm biopsy in PGD-A cycles and must be understood to adequately guide patients during clinical care. (Fertil SterilÒ 2017;107:1098–106. Ó2017 by American Society for Reproductive Medicine.) Key Words: Chromosomal mosaicism, blastocyst, inner cell mass, trophectoderm, aneuploidies, preimplantation genetic screening Discuss: You can discuss this article with its authors and with other ASRM members at https://www.fertstertdialog.com/users/ 16110-fertility-and-sterility/posts/15693-23856
T
he unbalanced transmission of chromosomes in human gametes and early preimplantation embryos causes aneuploidy, which is a major cause of infertility, pregnancy loss, and intellectual disability in humans (1). In the preimplantation and prenatal setting, chromosome abnormalities span a wide range of genomic imbalances, from polyploidy, to whole chromosome and large structural aneuploidies, down to submicroscopic deletions and duplications. Whole chromosome aneuploidies, monosomies and trisomies for the entire chromosomes, are the far more prevalent abnormalities and have been extensively investigated due to their high incidence in human conceptions and their clear association with clinical phenotypes and infertility. Undoubtedly, they represent
the single most common form of aneuploidy and the primary cause for implantation failure in IVF cycles and miscarriages in human pregnancies. Another well-defined characteristic of human aneuploidies is their strict correlation with female age. As women age, oocytes become increasingly susceptible to chromosome segregation errors during the meiotic process. Extensive analysis of main autosomal trisomies in clinical pregnancies revealed that more than 90% had a meiotic origin. The majority are due to maternal errors, with >75% due to errors in meiosis I and 50%) have full developmental potential, while when the proportion of normal blastomeres was decreased to one third, there was a partial rescue of embryonic lethality. In humans, both functional and prospective nonselection studies aimed at modeling the clinical fate and the reproductive potential of embryos showing intermediate copy number profiles are lacking. Prospective cohort studies in PGD-A cycles are also of poor value to determine the predictive value of patterns consistent with mosaicism as these are subjected to a patient and embryo selection bias. In conclusion, despite the fact that some contemporary CCS platforms can detect mosaic samples in cell mixture models with variable accuracy (59–61), a diagnosis of certainty for mosaicism in PGD-A cycles is conceptually impracticable. Many reproductive genetics laboratories are now routinely including embryonic mosaicism on their diagnostic reports. However, for the arguments discussed above, both biological and technological limitations exist and must be understood in order to adequately counsel patients during clinical care. Furthermore, it should be stressed that the chromosomal variations observed in a TE biopsy will not unequivocally represent the mosaicism rate present within the whole embryo. The existence of a sample bias has to be acknowledged in patients consent forms as a limitation for mosaicism detection in PGD-A cycles. In the light of the arguments discussed above, mosaicism should be recognized as a biological limitation for PGD-A programs as it is for prenatal diagnosis. In addition, it appears reasonable to avoid reporting mosaicism in PGD-A cycles
1103
1104
until its impact on clinical treatment has been clarified. A more conservative approach for reporting this type of result may be therefore preferred until evidence showing correlation between intermediate copy number profiles and mosaicism in embryos is available (i.e., ‘‘analysis showing a pattern that is consistent with the potential presence of mosaicism’’). Similarly, if future nonselection studies and randomized controlled trials show a lower reproductive potential for blastocyst with intermediate copy number profiles, clinically validated criteria should be introduced into clinical practice. In this case, on top of their genetic diagnosis, additional viability scores could be assigned to embryos. Nonetheless, extensive genetic counseling detailing all the specific limitations should be given to patients.
CONCLUDING REMARKS AND FUTURE PERSPECTIVES
Capalbo. Blastocyst mosaicism. Fertil Steril 2017.
92/181 (51.7) 77/181 (42.8) 41/51 (80) 9/51 (18) 4/10 (40) 6/10 (60) 29/50 (58) 13/50 (26)
Note: Abnormal cells in euploid-aneuploid mosaic embryos are similarly distributed between ICM and TE, indicating no sign of preferential allocation or confinement of chromosomally abnormal cells. The fact that results were almost identical for samples from the TE and ICM indicates that data obtained from a clinical TE sample can generally be considered diagnostic of the ICM chromosomal complement. CGH ¼ comparative genomic hybridization.
– – Not applicable
TE-confined mosaicism (%) ICM-confined mosaicism (%) Mosaic blastocysts (both ICM and TE) (%) Most frequent mosaic chromosomes Fully euploid blastocyst (%) Fully aneuploid blastocyst (%)
chr16; chr22
19/70 (70) 49/70 (27)
4/181 (2.2) 2/181 (1.1) 5/181 (2.8) 0/51 (0) 1/51 (2) 0/51 (0)
chr16; chr17; chr22; chr21
– 181 71 Range, 26–42 – SNP array with parental support 51 17 31 Donated blastocysts
FISH þ CGH reanalysis 10 4 33 Blastocysts diagnosed as aneuploid by FISH-based blastomere analysis 0/10 (0) 0/10 (0) 0/10 (0) Method No. of samples No. of patients Mean female age Origin of embryos
FISH þ SNP array reanalysis 50 24 35 Blastocysts diagnosed as aneuploid by FISH-based blastomere analysis 4/50 (8) 1/50 (2) 3/50 (6)
aCGH þ FISH reanalysis 70 26 36 Blastocysts diagnosed as aneuploid by aCGH-based TE analysis 0/70 (0) 0/70 (0) 2/70 (3)
Overall Johnson et al. (2010) Capalbo et al. (2013) Fragouli et al. (2008) Northrop et al. (2010)
Summary table of methodology and main findings from relevant studies on the cytogenetic constitution of ICM and TE samples from disaggregated human blastocysts.
TABLE 1
VIEWS AND REVIEWS
The small amount of evidence gathered from animal and human studies suggests the absence of mechanisms involved in embryo arrest, clonal depletion, or aneuploidy rescue for abnormal cells in euploid/aneuploid embryos during preimplantation development and blastocyst differentiation. Thus, it is likely that abnormal cells in mosaic diploid/aneuploid blastocysts are randomly allocated to the ICM and TE. However, our understanding of human preimplantation development is limited due to ethical and legal restrictions on embryo research and scarcity of materials. Studies in humans are mainly descriptive and lack functional evidence. Most of the information on embryo development is inferred from animal models and embryonic stem cell cultures, which should therefore be applied to the human model with caution. Understanding the biological mechanisms that beset preimplantation differentiation might reveal how aneuploidies and related gene dosage imbalances impact cell fate decisions and provide important clues on the biology of mosaicism in preimplantation embryos. The lack of correction for expected false positives and the use of nonstandardized criteria for classification of embryos as mosaic have likely resulted in an overestimation of mosaicism in IVF embryos. The little evidence available on humans suggests that mosaic euploid/aneuploid configuration is a relatively uncommon feature and should not impact the effectiveness of PGD-A programs appreciably. Further studies are needed to corroborate these findings and to better characterize at the single-cell level the incidence and prevalence of mosaicism at the blastocyst stage. Undoubtedly, novel CCS technologies able to distinguish between meiotic and mitotic errors in embryonic biopsies will deliver powerful research and clinical tools for the comprehensive analysis of aneuploidies in human blastocyst and consequent PGD-A protocols improvement. At the present, reports of mosaicism should be avoided in blastocyst PGD-A cycles due to the several technical and biological issues present when attempting this type of diagnosis and the lack of evidence from nonselection studies. The observation of intermediate chromosome copy number profiles would be better reported as a ‘‘pattern consistent with the presence of mosaicism,’’ and genetic counseling should be provided to guide patients' decisions. VOL. 107 NO. 5 / MAY 2017
Fertility and Sterility®
REFERENCES 1. 2. 3.
4.
5. 6.
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