Advertisement

An evidence-based scoring system for prioritizing mosaic aneuploid embryos following preimplantation genetic screening

Published:February 01, 2018DOI:https://doi.org/10.1016/j.rbmo.2018.01.005

      Abstract

      The aim of this study was to devise an evidence-based scoring system for prioritizing mosaic aneuploid embryos for transfer. A retrospective analysis was performed of all sequential cytogenetic and molecular results on chorionic villi samples (n = 72,472) and products of conception (n = 3806) analysed at a single centre. The likelihood that a mosaic aneuploidy detected in chorionic villi samples will involve the fetus, the incidence of clinically significant fetal uniparental disomy in the presence of a mosaic in chorionic villi and the chance of the mosaicism culminating in miscarriage were used to generate a scoring system for prioritizing mosaic aneuploid embryos detected by preimplantation genetic screening. A composite score was obtained for each individual mosaic aneuploidy after assignment of an individual risk score based on the incidence/likelihood of each adverse outcome. A final additional score was assigned to viable full or mosaic aneuploidies with a well-defined phenotype. The higher the composite score the lower the priority for embryo transfer. In conclusion, due to the paucity of prospective studies on the actual transfer of mosaic aneuploid embryos, we suggest using this evidence-based scoring system to provide a useful tool for clinicians, embryologists and patients.

      Keywords

      Introduction

      Aneuploidy is the most common type of chromosome abnormality and is the leading cause of implantation failure, miscarriage and congenital abnormalities in humans (
      • Hassold T.
      • Abruzzo M.
      • Adkins K.
      • Griffin D.
      • Merrill M.
      • Millie E.
      • Saker D.
      • Shen J.
      • Zaragoza M.
      Human aneuploidy: incidence, origin, and aetiology.
      ). This fact prompted the introduction of preimplantation genetic screening (PGS). The hypothesis was that if embryos obtained by IVF were screened for aneuploidy prior to transfer, implantation and pregnancy rates would improve and miscarriage rates decrease (
      • Munné S.
      • Lee A.
      • Rosenwaks Z.
      • Grifo J.
      • Cohen J.
      Diagnosis of major chromosome aneuploidies in human preimplantation embryos.
      ). This approach would be particularly useful in patients at an increased risk of having aneuploid embryos, such as patients of advanced age, those with recurrent implantation failure or cases with repeated miscarriage. Initially, PGS was performed by fluorescence in-situ hybridization (FISH) on fixed cells and day 3 biopsy. However, the effectiveness of this approach has been questioned by several randomized control trials (
      • Mastenbroek S.
      • Twisk M.
      • van Echten-Arends J.
      • Sikkema-Raddatz B.
      • Korevaar J.C.
      • Verhoeve H.R.
      • Vogel N.E.
      • Arts E.G.
      • de Vries J.W.
      • Bossuyt P.M.
      • Buys C.H.
      • Heineman M.J.
      • Repping S.
      • van der Veen F.
      In vitro fertilization with preimplantation genetic screening.
      ,
      • Mastenbroek S.
      • Twisk M.
      • van der Veen F.
      • Repping S.
      Preimplantation genetic screening: a systematic review and meta-analysis of RCTs.
      ,
      • Twisk M.
      • Mastenbroek S.
      • Bossuyt P.M.
      • Korevaar J.C.
      • Heineman M.J.
      • Repping S.
      • van der Veen F.
      The effectiveness of preimplantation genetic screening.
      ,
      • Twisk M.
      • Mastenbroek S.
      • van Wely M.
      • Heineman M.J.
      • Van der Veen F.
      • Repping S.
      Preimplantation genetic screening for abnormal number of chromosomes (aneuploidies) in in vitro fertilization or intracytoplasmic sperm injection.
      ). One of the reasons why PGS with FISH may not have been successful is that only a limited number of chromosomes were analysed. Other reasons may include technical proficiency with biopsy and fixation of cells for FISH analysis (
      • Cohen J.
      • Grifo J.
      Multicentre trial of preimplantation genetic screening reported in the New England Journal of Medicine: an in-depth look at the findings.
      ,
      • Munné S.
      • Cohen J.
      • Simpson J.L.
      In vitro fertilization with preimplantation genetic screening.
      ,
      • Munné S.
      • Gianaroli L.
      • Tur-Kaspa I.
      • Magli C.
      • Sandalinas M.
      • Grifo J.
      • Cram D.
      • Kahraman S.
      • Verlinsky Y.
      • Simpson J.L.
      Sub-standard application of PGS may interfere with its clinical success.
      ,
      • Simpson
      The Randomized Clinical Trial in assessing Preimplantation Genetic Screening (PGS): necessary but not sufficient.
      ). The development of novel molecular approaches has ushered in the concept of PGS 2.0. In this approach, comprehensive chromosomal screening (CCS) of all 24 chromosomes is performed by array comparative genomic hybridization (aCGH), real-time quantitative PCR (qPCR) or, more recently, next-generation sequencing (NGS) (
      • Forman E.J.
      • Hong K.H.
      • Franasiak J.M.
      • Scott Jr., R.T.
      Obstetrical and neonatal outcomes from the BEST Trial: single embryo transfer with aneuploidy screening improves outcomes after in vitro fertilization without compromising delivery rates.
      ,
      • Rubio C.
      • Bellver J.
      • Rodrigo L.
      • Castillón G.
      • Guillén A.
      • Vidal C.
      • Giles J.
      • Ferrando M.
      • Cabanillas S.
      • Remohí J.
      • Pellicer A.
      • Simón C.
      In vitro fertilization with preimplantation genetic diagnosis for aneuploidies in advanced maternal age: a randomized, controlled study.
      ,
      • Scott Jr., R.T.
      • Upham K.M.
      • Forman E.J.
      • Hong K.H.
      • Scott K.L.
      • Taylor D.
      • Tao X.
      • Treff N.R.
      Blastocyst biopsy with comprehensive chromosome screening and fresh embryo transfer significantly increases in vitro fertilization implantation and delivery rates: a randomized controlled trial.
      ,
      • Yang Z.
      • Liu J.
      • Collins G.S.
      • Salem S.A.
      • Liu X.
      • Lyle S.S.
      • Peck A.C.
      • Sills E.S.
      • Salem R.D.
      Selection of single blastocysts for fresh transfer via standard morphology assessment alone and with array CGH for good prognosis IVF patients: results from a randomized pilot study.
      ). The analysis is usually performed on several trophectoderm (TE) cells removed from a day 5–6 blastocyst. However, when such genome-wide approaches are employed, particularly when several cells are analysed, mosaic aneuploidy is occasionally detected, specifically in 4% of embryos by aCGH (
      • Greco E.
      • Minasi M.G.
      • Fiorentino F.
      Healthy babies after intrauterine transfer of mosaic aneuploid blastocysts.
      ) and 21% of embryos by NGS (
      • Munné S.
      • Wells D.
      Detection of mosaicism at blastocyst stage with the use of high-resolution next-generation sequencing.
      ). This usually implies that aneuploidy is present in only some of the cells whereas others are normal (euploid). Following PGS, preference is obviously given to euploid over mosaic embryos. In some cases, however, there are no euploid embryos, and only mosaic aneuploid embryos are available for transfer. The possibility that viable embryos may be discarded due to concerns over mosaicism represents one of the greatest challenges currently facing PGS, because there are several reports of healthy children being born following the transfer of such mosaic embryos (
      • Fragouli E.
      • Alfarawati S.
      • Spath K.
      • Babariya D.
      • Tarozzi N.
      • Borini A.
      • Wells D.
      Analysis of implantation and ongoing pregnancy rates following the transfer of mosaic diploid-aneuploid blastocysts.
      ,
      • Greco E.
      • Minasi M.G.
      • Fiorentino F.
      Healthy babies after intrauterine transfer of mosaic aneuploid blastocysts.
      ,
      • Munné S.
      • Wells D.
      Detection of mosaicism at blastocyst stage with the use of high-resolution next-generation sequencing.
      ). Nonetheless, the transfer of mosaic embryos is associated with significantly poorer outcomes than those of the control euploid embryos, having lower implantation and ongoing pregnancy rates and higher rates of miscarriage. It thus remains to be determined whether all mosaic embryos should be considered for transfer, and if so, what types of mosaic aneuploidy are more likely than others to be associated with adverse outcomes.
      While it is not yet common practice to transfer mosaic embryos, it has been suggested that this may be considered under some circumstances, as proposed by some authors (
      • Munné S.
      • Grifo J.
      • Wells D.
      Mosaicism: ‘survival of the fittest’ versus ‘no embryo left behind’.
      ). A recent Preimplantation Genetic Diagnosis International Society (PGDIS) Position Statement on chromosome mosaicism in PGS has suggested a guideline to prioritize mosaic embryos for transfer, based on the level of mosaicism and the specific chromosome involved (
      • PGDIS
      PGDIS position statement on chromosome mosaicism and preimplantation aneuploidy testing at the blastocyst stage.
      ). Likewise, following the 2016 CoGEN meeting in Barcelona, an updated position statement was issued (CoGEN Statement). Subsequently, it has also been established that there are, in fact, no differences in pregnancy outcomes between monosomic and trisomic mosaics (
      • Munné S.
      • Blazek J.
      • Large M.
      • Martinez-Ortiz P.A.
      • Nisson H.
      • Liu E.
      • Tarozzi N.
      • Borini A.
      • Becker A.
      • Zhang J.
      • Maxwell S.
      • Grifo J.
      • Babariya D.
      • Wells D.
      • Fragouli E.
      Detailed investigation into the cytogenetic constitution and pregnancy outcome of replacing mosaic blastocysts detected with the use of high-resolution next-generation sequencing.
      ). While these recommendations provide some framework for clinical decision making, there are scant prospective follow-up studies on the outcome of pregnancies achieved following transfer of mosaic aneuploid embryos. Until such data become available, it is possible to extrapolate from cytogenetic analyses of chorionic villus samples (CVS) performed for prenatal diagnosis.
      The gold standard for cytogenetic analysis of CVS is by investigating both the cytotrophoblast by direct preparation (DP) and the placental mesenchyme by long-term culture (LTC) (
      • Grati F.R.
      • Grimi B.
      • Frascoli G.
      • Di Meco A.M.
      • Liuti R.
      • Milani S.
      • Trotta A.
      • Dulcetti F.
      • Grosso E.
      • Miozzo M.
      • Maggi F.
      • Simoni G.
      Confirmation of mosaicism and uniparental disomy in amniocytes, after detection of mosaic chromosome abnormalities in chorionic villi.
      ,
      • Ledbetter D.H.
      • Zachary J.M.
      • Simpson J.L.
      • Golbus M.S.
      • Pergament E.
      • Jackson L.
      • Mahoney M.J.
      • Desnick R.J.
      • Schulman J.
      • Copeland K.L.
      • Verlinsky Y.
      • Yang-Feng T.
      • Schonberg S.A.
      • Babu A.
      • Tharapel A.
      • Dorfmann A.
      • Lubs H.A.
      • Rhoads G.G.
      • Fowler S.E.
      • De La Cruz F.
      Cytogenetic results from the U.S. Collaborative Study on CVS.
      ). Using this approach, placental mosaic aneuploidy can be detected in about 2% of cases (
      • Hsu L.Y.
      • Yu M.T.
      • Neu R.L.
      • Van Dyke D.L.
      • Benn P.A.
      • Bradshaw C.L.
      • Shaffer L.G.
      • Higgins R.R.
      • Khodr G.S.
      • Morton C.C.
      • Wang H.
      • Brothman A.R.
      • Chadwick D.
      • Disteche C.M.
      • Jenkins L.S.
      • Kalousek D.K.
      • Pantzar T.J.
      • Wyatt P.
      Rare trisomy mosaicism diagnosed in amniocytes, involving an autosome other than chromosomes 13, 18, 20, and 21: karyotype/phenotype correlations.
      ,
      • Malvestiti F.
      • Agrati C.
      • Grimi B.
      • Pompilii E.
      • Izzi C.
      • Martinoni L.
      • Gaetani E.
      • Liuti M.R.
      • Trotta A.
      • Maggi F.
      • Simoni G.
      • Grati F.R.
      Interpreting mosaicism in chorionic villi: results of a monocentric series of 1001 mosaics in chorionic villi with follow-up amniocentesis.
      ). When mosaicism is detected on CVS, it is necessary to follow up with confirmatory amniocentesis to assess whether the mosaic state involves the fetus itself or is only confined to the placenta. The likelihood of aneuploidy also being present in the fetus depends on: (i) the chromosome involved; (ii) the type of aneuploidy; (iii) the percentage of abnormal cells; and (iv) the tissue distribution (cytotrophoblast, mesenchyme, or both).
      Thus, both CVS and PGS attempt to predict the chromosomal status of the embryo by analysing the cells of the trophoblast. In fact, the TE cells removed for PGS at the blastocyst stage are the precursors of the placental cytotrophoblast. One may therefore view TE biopsy for PGS as a ‘very early’ direct preparation CVS.
      In order to devise an evidence-based scoring system for prioritizing mosaic aneuploid embryos for transfer, the likelihood that a mosaic aneuploidy detected in the trophoblast by CVS is also present in the fetus was analysed. The impact of mosaicism on the occurrence of uniparental disomy (UPD) was also reviewed. This is because a clinically significant UPD has been reported in 2.1% of fetuses with a normal karyotype on amniocentesis following the detection of a mosaic aneuploidy on CVS (
      • Malvestiti F.
      • Agrati C.
      • Grimi B.
      • Pompilii E.
      • Izzi C.
      • Martinoni L.
      • Gaetani E.
      • Liuti M.R.
      • Trotta A.
      • Maggi F.
      • Simoni G.
      • Grati F.R.
      Interpreting mosaicism in chorionic villi: results of a monocentric series of 1001 mosaics in chorionic villi with follow-up amniocentesis.
      ). To further assess the impact of mosaic aneuploidy on pregnancy outcome, its incidence in products of conception (POC) was also studied, as these would more likely be associated with non-viability. Finally, an additional risk score was assigned to those mosaic or full aneuploidies that can lead to viable affected births with a well-characterized phenotype.

      Materials and methods

      The study included cytogenetic samples analysed at a single centre (TOMA Advanced Biomedical Assays S.p.A., Busto Arsizio, Italy). The study received a notification of exempt determination from the TOMA Laboratory Institutional Review Board (approval #0000015) in December 2014. In order to evaluate the likelihood that a mosaic aneuploidy detected in the trophoblast is also present in the fetus we reviewed chorionic villus sampling performed between May 2000 and December 2016, including data previously published (
      • Grati F.R.
      Chromosomal mosaicism in human feto-placental development: implications for prenatal diagnosis.
      ,
      • Grati F.R.
      • Grimi B.
      • Frascoli G.
      • Di Meco A.M.
      • Liuti R.
      • Milani S.
      • Trotta A.
      • Dulcetti F.
      • Grosso E.
      • Miozzo M.
      • Maggi F.
      • Simoni G.
      Confirmation of mosaicism and uniparental disomy in amniocytes, after detection of mosaic chromosome abnormalities in chorionic villi.
      ,
      • Malvestiti F.
      • Agrati C.
      • Grimi B.
      • Pompilii E.
      • Izzi C.
      • Martinoni L.
      • Gaetani E.
      • Liuti M.R.
      • Trotta A.
      • Maggi F.
      • Simoni G.
      • Grati F.R.
      Interpreting mosaicism in chorionic villi: results of a monocentric series of 1001 mosaics in chorionic villi with follow-up amniocentesis.
      ). Cytogenetic analyses were performed in agreement with Italian and European guidelines (
      Linee Guida per la Diagnosi Citogenetica Consensus, 2007 and 2013.
      , www.sigu.net;
      Specific Constitutional Cytogenetic Guidelines ECA.
      , www.e-c-a.eu), which were progressively updated during the study period. Standard protocols were used to set up the cultures and chromosome preparations (
      • Babu A.
      • Verma R.S.
      Human Chromosomes Principles and Techniques.
      ) and a Q-banding technique (QFQ) was used for the entire series. Karyotype results were formulated according to the International System for Human Cytogenetic Nomenclature ((
      • Mitelman F.
      ISCN 1995.
      ,
      • Shaffer L.G.
      • Tommerup N.
      ISCN 2005.
      ,
      • Shaffer L.G.
      • Slovak M.L.
      • Campbell L.J.
      ISCN 2009.
      ,
      • Shaffer L.G.
      • McGowan-Jordan J.
      • Schmid M.
      ISCN 2013.
      ,
      • McGowan-Jordan J.
      • Simons A.
      • Schmid M.
      ISCN 2016.
      ). Methods used for karyotyping of chorionic villi (CV) and amniotic fluid (AF) and UPD investigation were described in detail by
      • Malvestiti F.
      • Agrati C.
      • Grimi B.
      • Pompilii E.
      • Izzi C.
      • Martinoni L.
      • Gaetani E.
      • Liuti M.R.
      • Trotta A.
      • Maggi F.
      • Simoni G.
      • Grati F.R.
      Interpreting mosaicism in chorionic villi: results of a monocentric series of 1001 mosaics in chorionic villi with follow-up amniocentesis.
      . Briefly, CV karyotyping was performed routinely by combining DP with LTC and at least 10 metaphases were scored and analysed for each method. Karyotyping on confirmatory AF was performed by scoring approximately 50 metaphases derived from 20–25 colonies from in-situ cultures. Mosaic trisomy in CV was defined as the presence of at least two cells showing the same abnormality and mosaic monosomy was defined in the presence of at least three cells with the same abnormality. The different types of mosaicism, depending on the tissues involved, are presented in Table 1.
      Table 1Different types of chromosomal mosaicism based on the tissue involvement.
      TypeNatureTrophoblast (direct preparation)Mesenchyme (long-term culture)Amniocytes
      ICPMAbnormalNormalNormal
      IICPMNormalAbnormalNormal
      IIICPMAbnormalAbnormalNormal
      IVTFMAbnormalNormalAbnormal
      VTFMNormalAbnormalAbnormal
      VITFMAbnormalAbnormalAbnormal
      CPM = confined placental mosaicism; TFM = true fetal mosaicism.
      True fetal mosaicism (TFM) was defined as the presence of at least two colonies from two AF cultures presenting the same abnormality previously observed in CV. To evaluate the risk of mosaicism resulting in clinically significant uniparental disomy (UPD), all UPD molecular analyses performed on CV samples in the same sample population (May 2000 to December 2016) were reviewed, some of which were previously reported (
      • Grati F.R.
      Chromosomal mosaicism in human feto-placental development: implications for prenatal diagnosis.
      ,
      • Grati F.R.
      • Grimi B.
      • Frascoli G.
      • Di Meco A.M.
      • Liuti R.
      • Milani S.
      • Trotta A.
      • Dulcetti F.
      • Grosso E.
      • Miozzo M.
      • Maggi F.
      • Simoni G.
      Confirmation of mosaicism and uniparental disomy in amniocytes, after detection of mosaic chromosome abnormalities in chorionic villi.
      ,
      • Malvestiti F.
      • Agrati C.
      • Grimi B.
      • Pompilii E.
      • Izzi C.
      • Martinoni L.
      • Gaetani E.
      • Liuti M.R.
      • Trotta A.
      • Maggi F.
      • Simoni G.
      • Grati F.R.
      Interpreting mosaicism in chorionic villi: results of a monocentric series of 1001 mosaics in chorionic villi with follow-up amniocentesis.
      ). Briefly, UPD testing was performed by segregation analysis of microsatellite markers located on the chromosome of interest using DNA from the fetus and the parents. UPD analysis was performed only in cases where the mosaic cytogenetic abnormality in CV involved a proposed or documented imprinted chromosome (6, 7, 11, 14, 15, 16), regardless of the number of abnormal metaphases (
      • Dawson A.J.
      • Chernos J.
      • McGowan-Jordan J.
      • Lavoie J.
      • Shetty S.
      • Steinraths M.
      • Wang J.C.
      • Xu J.
      CCMG guidelines: prenatal and postnatal diagnostic testing for uniparental disomy.
      ,
      • Kearney H.M.
      • Kearney J.B.
      • Conlin L.K.
      Diagnostic implications of excessive homozygosity detected by SNP-based microarrays: consanguinity, uniparental disomy, and recessive single-gene mutations.
      ). To further assess the impact of mosaicism on pregnancy outcome, we also reviewed cytogenetic abnormalities detected in first trimester miscarriages (POC) by karyotyping, collected from 1995 to 2015, including some cytogenetic data and methods previously published (
      • Grati F.R.
      • Gomes D.M.
      • Ganesamoorthy D.
      • Marcato L.
      • De Toffol S.
      • Blondeel E.
      • Malvestiti F.
      • Loeuillet L.
      • Ruggeri A.M.
      • Wainer R.
      • Maggi F.
      • Aboura A.
      • Dupont C.
      • Tabet A.C.
      • Guimiot F.
      • Slater H.R.
      • Simoni G.
      • Vialard F.
      Application of a new molecular technique for the genetic evaluation of products of conception.
      ).

      Results

      Likelihood that a mosaic aneuploidy detected in the trophoblast is also present in the fetus

      Of the 72,472 CVS samples analysed during the study period, chromosomal mosaicism was detected in 1524 cases (2.1%). Of these, 1166 cases were subsequently investigated by amniocentesis as well. In 1011 (86.7%) of the AF samples, there were no signs of aneuploidy. However, in 155 cases (13.3%), aneuploidy was demonstrated in the fetus as well (TFM).
      Mosaicism involving both placental layers (type III CVS mosaicism) has the highest likelihood of fetal involvement (37.7%) compared with mosaicism involving only one layer: 11.9% for mosaicism detected in the mesenchyme only (type II CVS mosaicism) and only 3.9% in mosaicism found in the cytotrophoblast only (type I CVS mosaicism).
      To more closely approximate PGS of trophectoderm (TE) cells, we focused specifically on mosaic aneuploidies presenting in the cytotrophoblast (CVS mosaicism types I and III). These included 280 cases of confined placental mosaicism (CPM) and 36 cases of TFM (Table 2). In these samples, no cases of trisomy for chromosomes 1, 6 and 17 were detected. This may be due to negative selection when present in the placenta and/or the fetus.
      Table 2The likelihood that a mosaicism (aneuploid/normal) detected in the cytotrophoblast is also found in the fetus.
      Abnormal cell line in mosaicismTotal (n = 316)CPM I and III (n = 280)TFM IV and VI (n = 36)Fetal involvement (%)
      Trisomy 1000
      Trisomy 2880
      Trisomy 326260
      Trisomy 4110
      Trisomy 5330
      Trisomy 6000
      Trisomy 747470
      Trisomy 816160
      Trisomy 9660
      Trisomy 10330
      Trisomy 11440
      Trisomy 12110
      Trisomy 13302913.3
      Trisomy 1487112.5
      Trisomy 1520200
      Trisomy 1643125.0
      Trisomy 17000
      Trisomy 181814422.2
      Trisomy 19110
      Trisomy 20181715.6
      Trisomy 211810844.4
      Trisomy 22110
      45,X66511522.7
      47,XXX74342.9
      47,XXY86225.0
      47,XYY220
      CPM = confined placental mosaicism; TFM = true fetal mosaicism.
      Mosaic aneuploidies show different likelihoods of fetal involvement and may therefore be assigned one of the following arbitrary risk scores:
      • 3. High risk (>15%): trisomy 16, 18, 21 and 45,X, 47,XXY, 47,XXX
      • 2. Intermediate risk (5–15%): trisomy 14 and 20
      • 1. Low risk (1–4%): trisomy 13
      • 0. No risk (<1%): trisomies 1–12, 15, 17, 19, 22 and 47,XYY

      Incidence of clinically significant fetal UPD in cases with a mosaic aneuploidy in CV

      During the same study period, UPD investigations were performed on a subgroup of 169 cases due to increased risk of clinically significant UPD of chromosomes 6, 7, 11, 14, 15 and 16, according to the current guidelines (
      • Dawson A.J.
      • Chernos J.
      • McGowan-Jordan J.
      • Lavoie J.
      • Shetty S.
      • Steinraths M.
      • Wang J.C.
      • Xu J.
      CCMG guidelines: prenatal and postnatal diagnostic testing for uniparental disomy.
      ,
      • Kearney H.M.
      • Kearney J.B.
      • Conlin L.K.
      Diagnostic implications of excessive homozygosity detected by SNP-based microarrays: consanguinity, uniparental disomy, and recessive single-gene mutations.
      ). Results presented in Table 3 demonstrate that UPD was detected in nine cases (5.3%): three cases (21.4%) with trisomy 14 CPM (two cases of CPM type I and one case of CPM type III); two cases (6.5%) of trisomy 15 CPM (one CPM type II and one CPM type III), and in four cases (16%) of trisomy 16 CPM (three cases of CPM type III and one case of TFM type VI). The following risk scores for clinically significant UPD resulting from mosaic aneuploidy were assigned:
      • 3. High risk (>10%): trisomy 14 and 16
      • 2. Intermediate risk (1–10%): trisomy 15
      • 1. Low risk (<1%): trisomy 6, 7, 11
      • 0. No risk – all others
        Table 3The likelihood of clinically significant UPD as a result of mosaicism (aneuploid/normal) detected in the cytotrophoblast.
        Abnormal cell line in mosaicismCases investigatedCases with UPDType of mosaicismUPD incidence (%)
        Trisomy 630
        Trisomy 7900
        Trisomy 1160
        Trisomy 141432 (CPM I) + 1 (CPMII)21.4
        Trisomy 153121 (CPMII) + 1 (CPMIII)6.5
        Trisomy 162543 (CPM III) + 1 (TFM VI)16.0
        Total16998 CPM and 1 TFM5.3
        CPM = confined placental mosaicism; TFM = true fetal mosaicism; UPD = uniparental disomy.

      Incidence of mosaic aneuploidies in POC

      During the study period, 3806 samples from POC were cytogenetically analysed. Of these, 1242 (32.6%) did not provide a result because of culture failure, maternal cell contamination or poor chromosome morphology. Of the remaining 2564 reportable samples, an abnormal karyotype was noted in 1269 (49.5%) cases. Of these, 75 cases demonstrated mosaic chromosomal aberrations. In 11 cases, however, there was no normal cell line and in seven others, the abnormal cell line was complex, non-aneuploid (e.g. isochromosome, deletion, translocation, additional euchromatic material, inversion). The remaining 57 cases are presented in Table 4 and included 14 cases (25%) of mosaic tetraploidy, probably representing a culture artefact due to cell endo-reduplication, eight cases (14%) of mosaic 45,X, five cases (9%) of mosaic trisomy 16, and three cases (5%) each of mosaic trisomy 2, 8 and 20. Mosaic aneuploidies likely to be associated with miscarriage may be assigned one of four risk scores based on the incidence of each individual mosaic aneuploidy in POC:
      • 3. High risk (>10%): mosaic 45,X/46,XX
      • 2. Intermediate risk (4–10%): trisomies 2, 6, 8, 16, 17, 20, 21, 22
      • 1. Low risk (1–3%): trisomies 4, 5, 7, 9, 11, 13, 14, 15, 18, 47,XXY, and mosaic 45,X/46,XY
      • 0. Very low risk (<1%): trisomies 1, 3, 10, 12, 19
        Table 4The incidence of mosaicism (aneuploid/normal) detected in products of conception (POC).
        Abnormal cell line in mosaicismNumberIncidence (%)
        Trisomy 235
        Trisomy 412
        Trisomy 512
        Trisomy 624
        Trisomy 712
        Trisomy 835
        Trisomy 912
        Trisomy 1112
        Trisomy 1312
        Trisomy 1412
        Trisomy 1512
        Trisomy 1659
        Trisomy 1724
        Trisomy 1812
        Trisomy 2035
        Trisomy 2135
        Trisomy 2224
        45,X/46,XX814
        45,X/46,XY12
        47,XXY/46,XY12
        Monosomy 2112
        Tetraploidy1425
        Total57100

      Generating an evidence-based scoring system for prioritizing mosaic aneuploid embryos

      Table 5 summarizes the composite score for each type of mosaic aneuploidy based on (i) the likelihood that the mosaicism detected in the trophoblast is also present in the fetus; (ii) the incidence of clinically significant fetal UPD resulting from the mosaic aneuploidy; and (iii) the incidence of the mosaic aneuploidy in POC. Additional risk scores were assigned to viable aneuploidies: a score of 4 for trisomy 13, 18, 21 and 45,X; a score of 3 for the well-described viable mosaic trisomy 16; a score of 2 for viable mosaic aneuploidies of chromosomes 8 and 9; and a score of 1 for other sex chromosome aneuploidy (47,XXX, 47,XXY and 47,XYY).
      Table 5An evidence-based scoring system for prioritizing mosaic aneuploid/normal embryos for transfer following PGS.
      Abnormal cell line in mosaicismRisk of fetal involvementRisk of UPDRisk of miscarriageRisk of viable aneuploidyComposite score
      Trisomy 10
      Trisomy 222
      Trisomy 30
      Trisomy 411
      Trisomy 511
      Trisomy 6123
      Trisomy 7112
      Trisomy 8224
      Trisomy 9123
      Trisomy 100
      Trisomy 11112
      Trisomy 120
      Trisomy 131146
      Trisomy 142316
      Trisomy 15213
      Trisomy 16332311
      Trisomy 1722
      Trisomy 183148
      Trisomy 190
      Trisomy 20224
      Trisomy 213249
      Trisomy 2222
      45,X33410
      47,XXX314
      47,XXY3115
      47,XYY11

      Discussion

      Mosaic aneuploidy has no doubt always existed in preimplantation embryos. In fact, it has been suggested that most human preimplantation embryos are mosaics of euploid and aneuploid cells (
      • van Echten-Arends J.
      • Mastenbroek S.
      • Sikkema-Raddatz B.
      • Korevaar J.C.
      • Heineman M.J.
      • van der Veen F.
      • Repping S.
      Chromosomal mosaicism in human preimplantation embryos: a systematic review.
      ). The survival of blastocyst mosaics to term depends, among other factors, on the abnormal cell load. Using a mouse model,
      • Bolton H.
      • Graham S.J.
      • Van der Aa N.
      • Kumar P.
      • Theunis K.
      • Fernandez Gallardo E.
      • Voet T.
      • Zernicka-Goetz M.
      Mouse model of chromosome mosaicism reveals lineage-specific depletion of aneuploid cells and normal developmental potential.
      demonstrated that in chimeric embryos, containing aneuploid and euploid cells, the aneuploid cells in the fetal lineage are eliminated by apoptosis, whereas the aneuploid cells in the placental lineage show severe proliferative defects. They have shown that the aneuploid cells are progressively depleted from the blastocyst stage onwards. They concluded that mosaic embryos may have full developmental potential if they contain a sufficient proportion of euploid cells.
      However, the new high-resolution techniques used for CCS, particularly NGS, can now detect its presence. It is obvious that some mosaic aneuploid embryos, if transferred, may result in the birth of healthy children (
      • Fragouli E.
      • Alfarawati S.
      • Spath K.
      • Babariya D.
      • Tarozzi N.
      • Borini A.
      • Wells D.
      Analysis of implantation and ongoing pregnancy rates following the transfer of mosaic diploid-aneuploid blastocysts.
      ,
      • Greco E.
      • Minasi M.G.
      • Fiorentino F.
      Healthy babies after intrauterine transfer of mosaic aneuploid blastocysts.
      ,
      • Munné S.
      • Wells D.
      Detection of mosaicism at blastocyst stage with the use of high-resolution next-generation sequencing.
      ). In a prospective study,
      • Greco E.
      • Minasi M.G.
      • Fiorentino F.
      Healthy babies after intrauterine transfer of mosaic aneuploid blastocysts.
      described the transfer of mosaic embryos in 18 consecutive women who had no euploid embryos. These resulted in eight pregnancies, six of which resulted in a euploid live birth as confirmed by CVS, and two were biochemical pregnancies. Using our scoring system, cases resulting in a euploid live birth had a lower mean composite score compared with those that had not (2.5 versus 5.7), suggesting that they would have had higher priority.
      • Fragouli E.
      • Alfarawati S.
      • Spath K.
      • Babariya D.
      • Tarozzi N.
      • Borini A.
      • Wells D.
      Analysis of implantation and ongoing pregnancy rates following the transfer of mosaic diploid-aneuploid blastocysts.
      used NGS to retrospectively analyse 44 archived trophectoderm biopsies from 39 patients. Compared with the control group, mosaic embryos were associated with significantly lower implantation (30.1% versus 55.8%) and ongoing pregnancy rates (15.4% versus 46.2%) and higher miscarriage rates (55.6% versus 17.2%). Of these, 12 were mosaics for one or more segmental aneuploidies, with a pregnancy rate of 66%; 20 had mosaic aneuploidy of more than one chromosome resulting in one ongoing pregnancy (5%) and two miscarriages (10%). Of the 12 embryos with a single whole-chromosome aneuploidy, three embryos with mosaic aneuploidy of chromosomes 14, 20 and 7 resulted in ongoing pregnancies (25%) and three embryos with mosaicism for chromosomes 11, 17 and 19 resulted in miscarriage (25%). Finally, in a retrospective study,
      • Munné S.
      • Blazek J.
      • Large M.
      • Martinez-Ortiz P.A.
      • Nisson H.
      • Liu E.
      • Tarozzi N.
      • Borini A.
      • Becker A.
      • Zhang J.
      • Maxwell S.
      • Grifo J.
      • Babariya D.
      • Wells D.
      • Fragouli E.
      Detailed investigation into the cytogenetic constitution and pregnancy outcome of replacing mosaic blastocysts detected with the use of high-resolution next-generation sequencing.
      described the transfer of 143 mosaic embryos as determined by NGS in four different centres (44 of which were previously reported by
      • Fragouli E.
      • Alfarawati S.
      • Spath K.
      • Babariya D.
      • Tarozzi N.
      • Borini A.
      • Wells D.
      Analysis of implantation and ongoing pregnancy rates following the transfer of mosaic diploid-aneuploid blastocysts.
      ). Compared with matched controls, the implantation rate (IR) was lower (53% versus 71%), the fetal loss rate was significantly higher (24% versus 10%; P = 0.002) and the ongoing IR significantly lower (41% versus 63%; P < 0.006). Complex abnormal mosaics, involving three or more chromosomes, had a significantly reduced ongoing IR (10%) compared with mosaic single aneuploid (50%), double mosaic (45%) and mosaic segmental (41%). There was no difference in ongoing IR between mosaic monosomy and mosaic trisomy. Likewise, no significant differences were noted in ongoing IR between single-chromosome mosaic embryos and double-chromosome mosaic embryos (46% versus 45%). Embryos with 40–80% abnormal cells had an ongoing pregnancy rate of 22% compared with 56% in those with <40% abnormal cells. Nonetheless, despite these initial studies it is currently unclear what type of mosaic aneuploidy is tolerable and what degree of mosaicism is acceptable.
      A recent PGDIS Position Statement on chromosome mosaicism in PGS has suggested a guideline to prioritize mosaic embryos for transfer, based on the level of mosaicism and the specific chromosome involved (
      • PGDIS
      PGDIS position statement on chromosome mosaicism and preimplantation aneuploidy testing at the blastocyst stage.
      ). Accordingly, euploid/monosomic mosaic embryos are preferred over euploid/trisomic ones because the former (with the exception of 45,X) are not viable. Types of mosaic aneuploidy to be avoided include: (i) mosaic aneuploidies that may lead to a viable affected birth (chromosomes 13, 18, 21); (ii) those implicated in intrauterine growth restriction (chromosomes 2, 7, 16); or (iii) those that may be associated with uniparental disomy (UPD) (chromosomes 14, 15). Mosaic aneuploid embryos to be considered for transfer include those trisomic for chromosomes 1, 3, 4, 5, 6, 8, 9, 10, 11, 12, 17, 19, 20, 22, X, Y.
      The subsequent CoGEN Position Statement on Chromosomal Mosaicism Detected in Preimplantation Blastocyst Biopsies (
      • CoGEN Statement
      COGEN position statement on chromosomal mosaicism detected in preimplantation blastocyst biopsies.
      ) includes the following recommendations for prioritizing mosaic embryos for transfer: (i) embryos mosaic for trisomies capable of live born viability (chromosomes 13, 18, 21, 22) are of lowest priority; (ii) embryos mosaic for trisomies associated with uniparental disomy (chromosomes 14, 15) are low priority; (iii) embryos mosaic for trisomies associated with intrauterine growth retardation (chromosomes 2, 7, 16) are low priority; (iv) mosaicism involving chromosomes 1, 3, 4, 5, 6, 8, 9, 10, 11, 12, 17, 19, 20, have not been associated with the aforementioned adverse outcomes; and (v) mosaic monosomies should be considered to have similar risk as their counterpart trisomies.
      Such recommendations provide some guidance for clinical decision making until sufficient prospective data accumulate. However, in the presence of several mosaic aneuploid embryos, it may be difficult to assign the correct priority, as no clear scoring system is provided.
      In this study we sought to develop a scoring system for prioritizing preimplantation mosaic aneuploid embryos, based on clinical evidence derived from actual cytogenetic analyses of first trimester CVS and from POC. This approach was chosen because the trophectoderm (TE) cells used for PGS correspond embryologically to the cytotrophoblast that is analysed in a CVS direct preparation (
      • Bianchi D.W.
      • Wilkins-Haug L.E.
      • Enders A.C.
      • Hay E.D.
      Origin of extraembrionic mesoderm in experimental animals: relevance to chorionic mosaicism in humans.
      ). The likelihood that a mosaic aneuploidy detected in CVS will involve the fetus, the risk for clinically significant UPD and the chance of the mosaicism culminating in miscarriage was used to generate a scoring system for prioritizing mosaic aneuploid embryos detected by PGS. Table 2, Table 3, Table 4 describe the incidence of each of these adverse outcomes, based on which an arbitrary score was assigned for each chromosome. A higher score implies a higher likelihood of an adverse outcome. Additional scores were assigned to viable full or mosaic aneuploidies. Table 5 summarizes the composite scores for all these adverse outcomes. The lower the composite score the higher the priority for embryo transfer.
      Based on this scoring system, we suggest that mosaic aneuploid embryos should have the following priority for transfer:
      • Mosaic trisomies 1, 3, 10, 12 and 19 have a composite score of 0 and have the highest priority for transfer because of a very low risk of any of these adverse outcomes.
      • Mosaic trisomies 4 and 5 and 47,XYY have a composite score of 1 and are the second group to be considered for transfer, albeit with a disclaimer of a slightly increased likelihood of miscarriage (Table 4, Table 5) or a viable aneuploidy (47,XYY).
      • Mosaic trisomies 2, 7, 11, 17 and 22 have a composite score of 2 and are the third group to be considered for transfer, having a slightly higher risk of miscarriage or a relatively low risk for UPD (trisomies 7 and 11).
      • Mosaic trisomies 6, 9 and 15 have a composite score of 3 due to increased risk of miscarriage, UPD or viable aneuploidy. The possibility for transfer should be considered with caution and only after detailed discussion with the prospective parents.
      • Mosaic trisomies 8, 20, 47,XXX and 47,XXY have a composite score of 4–5, due to high risk of fetal involvement and a slightly increased risk for miscarriage and viable aneuploidy. The possibility for transfer could be considered after extensive discussion with prospective parents regarding the possible clinical manifestations thereof.
      The remaining mosaic aneuploidies are best avoided: trisomies 13, 14, 16, 18, 21 and 45,X.
      The aforesaid priority scores regarding autosomal trisomies should also apply to the respective mosaic autosomal monosomies. This is because these are most likely the result of post-zygotic non-disjunction which would give rise to two daughter cells: one trisomic and one monosomic, in addition to the euploid cell line already present in the conceptus. In such circumstances, the possibility of a complementary trisomic cell line in the embryo remains a possibility (
      • Turchetti D.
      • Pompilii E.
      • Magrini E.
      • Bonasoni M.P.
      • Pittalis M.C.
      • Segata M.
      • Pession A.
      • Santini D.
      • Pilu G.
      • Seri M.
      Persistence of a monosomic cell line in a fetus with mosaic trisomy 8.
      ).
      Retrospective studies on mosaic blastocysts and prenatal diagnosis data do not always match. As suggested by
      • Munné S.
      • Blazek J.
      • Large M.
      • Martinez-Ortiz P.A.
      • Nisson H.
      • Liu E.
      • Tarozzi N.
      • Borini A.
      • Becker A.
      • Zhang J.
      • Maxwell S.
      • Grifo J.
      • Babariya D.
      • Wells D.
      • Fragouli E.
      Detailed investigation into the cytogenetic constitution and pregnancy outcome of replacing mosaic blastocysts detected with the use of high-resolution next-generation sequencing.
      , this could be due to different mechanisms, such as cell load of abnormal cells determining the chance of producing a viable pregnancy (
      • Bolton H.
      • Graham S.J.
      • Van der Aa N.
      • Kumar P.
      • Theunis K.
      • Fernandez Gallardo E.
      • Voet T.
      • Zernicka-Goetz M.
      Mouse model of chromosome mosaicism reveals lineage-specific depletion of aneuploid cells and normal developmental potential.
      ,
      • Munné S.
      • Blazek J.
      • Large M.
      • Martinez-Ortiz P.A.
      • Nisson H.
      • Liu E.
      • Tarozzi N.
      • Borini A.
      • Becker A.
      • Zhang J.
      • Maxwell S.
      • Grifo J.
      • Babariya D.
      • Wells D.
      • Fragouli E.
      Detailed investigation into the cytogenetic constitution and pregnancy outcome of replacing mosaic blastocysts detected with the use of high-resolution next-generation sequencing.
      ) and the observation that later on the trophoblast acquires aneuploid cells that become invasive and implant (
      • Weier J.F.
      • Weier H.U.G.
      • Jung C.J.
      • Gormley M.
      • Zhou Y.
      • Chu L.W.
      • Genbacev O.
      • Wright A.A.
      • Fisher S.J.
      Human cytotrophoblasts acquire aneuploidies as they differentiate to an invasive phenotype.
      ).
      Additional consideration should also be given to the detected degree of mosaicism. In a recent survey held by CoGEN on the IVF-Worldwide Website, the views and practices regarding mosaicism in PGS were assessed among 102 IVF centres from 32 countries, representing a total of 108,900 annual IVF cycles (
      • Weissman A.
      • Shoham G.
      • Shoham Z.
      • Fishel S.
      • Leong M.
      • Yaron Y.
      Chromosomal mosaicism detected during preimplantation genetic screening (PGS): results of a worldwide web-based survey.
      ). Of all respondents, 31% consider embryonic mosaic aneuploidy when detected in >20% of the cells, fewer consider mosaicism when detected in >30% (12.6–14.6%) and only a minority consider mosaicism when the percentage of aneuploid cells is >50%. The results of this survey represent the relevant opinions of PGS practitioners and specialists. However, the clinical utility of the detected degree of mosaicism is generally unknown. In fact, it might only reflect a technical bias due to the random sampling of a restricted trophoblastic area rather than the real degree of mosaicism in all of the embryos.
      • Munné S.
      • Blazek J.
      • Large M.
      • Martinez-Ortiz P.A.
      • Nisson H.
      • Liu E.
      • Tarozzi N.
      • Borini A.
      • Becker A.
      • Zhang J.
      • Maxwell S.
      • Grifo J.
      • Babariya D.
      • Wells D.
      • Fragouli E.
      Detailed investigation into the cytogenetic constitution and pregnancy outcome of replacing mosaic blastocysts detected with the use of high-resolution next-generation sequencing.
      reported that embryos with 40–80% abnormal cells had an ongoing pregnancy rate of 22% compared with 56% in those with <40% abnormal cells. However, the correlation between the chromosome-specific degree of mosaicism and the implantation outcome is limited and prospective studies are needed to explore these aspects. Therefore, until sufficient follow-up data are available, the degree of mosaicism should be used with caution as a criterion for prioritizing embryos. If such studies demonstrate a predictive role for this criterion, the suggested scheme could then assign an additional score for the degree of mosaicism to be included in the embryo-specific composite score. Future studies will probably discover further biological predictive criteria whose introduction will be beneficial for the refining of the composite score for the prioritization of mosaic embryos, with a progressively more complex algorithm.
      In case of an ongoing pregnancy after the transfer of mosaic aneuploid embryos, prenatal diagnosis on amniocytes should be highly recommended due to the a priori increased risk of fetal aneuploidy. CVS is not recommended due to the higher likelihood of encountering the same mosaic aneuploidy detected at blastocyst stage by PGS. In case of a normal karyotype after a standard analysis, an extended analysis and/or cell count on amniocytes should be performed to exclude the presence of low-level mosaicism in the fetus. This is the same laboratory procedure that is recommended for the analysis of amniocytes after the detection of a mosaic condition in the CV (
      • Grati F.R.
      • Malvestiti F.
      • Grimi B.
      • Liuti R.
      • Agrati C.
      • Gaetani E.
      • Milani S.
      • Martinoni L.
      • Zanatta V.
      • Gallazzi G.
      • Maggi F.
      • Simoni G.
      Increased risk after noninvasive prenatal screening on cell-free DNA circulating in maternal blood: does a new indication for invasive prenatal diagnosis require new criteria for confirmatory cytogenetic analysis?.
      ,
      • Munné S.
      • Wells D.
      Detection of mosaicism at blastocyst stage with the use of high-resolution next-generation sequencing.
      ). Likewise, non-invasive prenatal testing by cell-free DNA is not recommended as the cell-free DNA fragments are not derived from the fetus itself but from the apoptosis of the cytotrophoblastic cells.
      In conclusion, although the transfer of mosaic embryos may be associated with poorer outcome, there is also a compelling concern that viable embryos may be unjustifiably discarded due to concerns over mosaicism. Thus, until further prospective follow-up studies on the actual transfer of mosaic aneuploid embryos are available, we suggest using this scoring system as a tool for clinicians and embryologists, both for prioritizing embryos for transfer and as a counselling aid for discussing possible outcomes following transfer of mosaic aneuploid embryos detected by PGS.

      References

        • Babu A.
        • Verma R.S.
        Human Chromosomes Principles and Techniques.
        2nd ed. McGraw Hill, Austin, TX1995
        • Bianchi D.W.
        • Wilkins-Haug L.E.
        • Enders A.C.
        • Hay E.D.
        Origin of extraembrionic mesoderm in experimental animals: relevance to chorionic mosaicism in humans.
        Am. J. Med. Genet. 1993; 46: 542-550
        • Bolton H.
        • Graham S.J.
        • Van der Aa N.
        • Kumar P.
        • Theunis K.
        • Fernandez Gallardo E.
        • Voet T.
        • Zernicka-Goetz M.
        Mouse model of chromosome mosaicism reveals lineage-specific depletion of aneuploid cells and normal developmental potential.
        Nat. Commun. 2016; 7: 11165
        • CoGEN Statement
        COGEN position statement on chromosomal mosaicism detected in preimplantation blastocyst biopsies.
        • Cohen J.
        • Grifo J.
        Multicentre trial of preimplantation genetic screening reported in the New England Journal of Medicine: an in-depth look at the findings.
        Reprod. Biomed Online. 2007; 15: 365-366
        • Dawson A.J.
        • Chernos J.
        • McGowan-Jordan J.
        • Lavoie J.
        • Shetty S.
        • Steinraths M.
        • Wang J.C.
        • Xu J.
        CCMG guidelines: prenatal and postnatal diagnostic testing for uniparental disomy.
        Clin. Genet. 2011; 79: 118-124
        • Forman E.J.
        • Hong K.H.
        • Franasiak J.M.
        • Scott Jr., R.T.
        Obstetrical and neonatal outcomes from the BEST Trial: single embryo transfer with aneuploidy screening improves outcomes after in vitro fertilization without compromising delivery rates.
        Am. J. Obstet. Gynecol. 2014; 210 (e1–e6; Epub 2013 Oct 18): 157https://doi.org/10.1016/j.ajog.2013.10.016
        • Fragouli E.
        • Alfarawati S.
        • Spath K.
        • Babariya D.
        • Tarozzi N.
        • Borini A.
        • Wells D.
        Analysis of implantation and ongoing pregnancy rates following the transfer of mosaic diploid-aneuploid blastocysts.
        Hum. Genet. 2017; 136: 805-819
        • Grati F.R.
        Chromosomal mosaicism in human feto-placental development: implications for prenatal diagnosis.
        J. Clin. Med. 2014; 3: 809-837
        • Grati F.R.
        • Grimi B.
        • Frascoli G.
        • Di Meco A.M.
        • Liuti R.
        • Milani S.
        • Trotta A.
        • Dulcetti F.
        • Grosso E.
        • Miozzo M.
        • Maggi F.
        • Simoni G.
        Confirmation of mosaicism and uniparental disomy in amniocytes, after detection of mosaic chromosome abnormalities in chorionic villi.
        Eur. J. Hum. Genet. 2006; 14: 282-288
        • Grati F.R.
        • Gomes D.M.
        • Ganesamoorthy D.
        • Marcato L.
        • De Toffol S.
        • Blondeel E.
        • Malvestiti F.
        • Loeuillet L.
        • Ruggeri A.M.
        • Wainer R.
        • Maggi F.
        • Aboura A.
        • Dupont C.
        • Tabet A.C.
        • Guimiot F.
        • Slater H.R.
        • Simoni G.
        • Vialard F.
        Application of a new molecular technique for the genetic evaluation of products of conception.
        Prenat. Diagn. 2013; 33: 32-41
        • Grati F.R.
        • Malvestiti F.
        • Grimi B.
        • Liuti R.
        • Agrati C.
        • Gaetani E.
        • Milani S.
        • Martinoni L.
        • Zanatta V.
        • Gallazzi G.
        • Maggi F.
        • Simoni G.
        Increased risk after noninvasive prenatal screening on cell-free DNA circulating in maternal blood: does a new indication for invasive prenatal diagnosis require new criteria for confirmatory cytogenetic analysis?.
        Prenat. Diagn. 2015; 35 (No abstract available): 308-309https://doi.org/10.1002/pd.4483
        • Greco E.
        • Minasi M.G.
        • Fiorentino F.
        Healthy babies after intrauterine transfer of mosaic aneuploid blastocysts.
        N. Engl. J. Med. 2015; 373: 2089-2090
        • Hassold T.
        • Abruzzo M.
        • Adkins K.
        • Griffin D.
        • Merrill M.
        • Millie E.
        • Saker D.
        • Shen J.
        • Zaragoza M.
        Human aneuploidy: incidence, origin, and aetiology.
        Environ. Mol. Mutagen. 1996; 28: 167-175
        • Hsu L.Y.
        • Yu M.T.
        • Neu R.L.
        • Van Dyke D.L.
        • Benn P.A.
        • Bradshaw C.L.
        • Shaffer L.G.
        • Higgins R.R.
        • Khodr G.S.
        • Morton C.C.
        • Wang H.
        • Brothman A.R.
        • Chadwick D.
        • Disteche C.M.
        • Jenkins L.S.
        • Kalousek D.K.
        • Pantzar T.J.
        • Wyatt P.
        Rare trisomy mosaicism diagnosed in amniocytes, involving an autosome other than chromosomes 13, 18, 20, and 21: karyotype/phenotype correlations.
        Prenat. Diagn. 1997; 17: 201-242
        • Kearney H.M.
        • Kearney J.B.
        • Conlin L.K.
        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
        • Ledbetter D.H.
        • Zachary J.M.
        • Simpson J.L.
        • Golbus M.S.
        • Pergament E.
        • Jackson L.
        • Mahoney M.J.
        • Desnick R.J.
        • Schulman J.
        • Copeland K.L.
        • Verlinsky Y.
        • Yang-Feng T.
        • Schonberg S.A.
        • Babu A.
        • Tharapel A.
        • Dorfmann A.
        • Lubs H.A.
        • Rhoads G.G.
        • Fowler S.E.
        • De La Cruz F.
        Cytogenetic results from the U.S. Collaborative Study on CVS.
        Prenat. Diagn. 1992; 12: 317-345
      1. Linee Guida per la Diagnosi Citogenetica Consensus, 2007 and 2013.
        • Malvestiti F.
        • Agrati C.
        • Grimi B.
        • Pompilii E.
        • Izzi C.
        • Martinoni L.
        • Gaetani E.
        • Liuti M.R.
        • Trotta A.
        • Maggi F.
        • Simoni G.
        • Grati F.R.
        Interpreting mosaicism in chorionic villi: results of a monocentric series of 1001 mosaics in chorionic villi with follow-up amniocentesis.
        Prenat. Diagn. 2015; 35: 1117-1127
        • Mastenbroek S.
        • Twisk M.
        • van Echten-Arends J.
        • Sikkema-Raddatz B.
        • Korevaar J.C.
        • Verhoeve H.R.
        • Vogel N.E.
        • Arts E.G.
        • de Vries J.W.
        • Bossuyt P.M.
        • Buys C.H.
        • Heineman M.J.
        • Repping S.
        • van der Veen F.
        In vitro fertilization with preimplantation genetic screening.
        N. Engl. J. Med. 2007; 357: 9-17
        • Mastenbroek S.
        • Twisk M.
        • van der Veen F.
        • Repping S.
        Preimplantation genetic screening: a systematic review and meta-analysis of RCTs.
        Hum. Reprod. Update. 2011; 17: 454-466
        • McGowan-Jordan J.
        • Simons A.
        • Schmid M.
        ISCN 2016.
        (An International System for Human Cytogenomic Nomenclature (2016); ISBN: 978-3-318-05857-4)2016
        • Mitelman F.
        ISCN 1995.
        (An International System for Human Cytogenetic Nomenclature (1995) Recommendations of the International Standing Committee on Human Cytogenetic Nomenclature, Memphis, Tenn., October 1994. Published in collaboration with ‘Cytogenetics and Cell Genetics‘; Plus fold-out: ‘The Normal Human Karyotype G- and R-bands‘; ISBN: 978-3-8055-6226-3)1995
        • Munné S.
        • Blazek J.
        • Large M.
        • Martinez-Ortiz P.A.
        • Nisson H.
        • Liu E.
        • Tarozzi N.
        • Borini A.
        • Becker A.
        • Zhang J.
        • Maxwell S.
        • Grifo J.
        • Babariya D.
        • Wells D.
        • Fragouli E.
        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
        • Munné S.
        • Wells D.
        Detection of mosaicism at blastocyst stage with the use of high-resolution next-generation sequencing.
        Fertil. Steril. 2017; 107: 1085-1109
        • Munné S.
        • Lee A.
        • Rosenwaks Z.
        • Grifo J.
        • Cohen J.
        Diagnosis of major chromosome aneuploidies in human preimplantation embryos.
        Hum. Reprod. 1993; 8: 2185-2191
        • Munné S.
        • Cohen J.
        • Simpson J.L.
        In vitro fertilization with preimplantation genetic screening.
        N. Engl. J. Med. 2007; 357: 1769-1770
        • Munné S.
        • Gianaroli L.
        • Tur-Kaspa I.
        • Magli C.
        • Sandalinas M.
        • Grifo J.
        • Cram D.
        • Kahraman S.
        • Verlinsky Y.
        • Simpson J.L.
        Sub-standard application of PGS may interfere with its clinical success.
        Fertil. Steril. 2007; 88: 781-784
        • Munné S.
        • Grifo J.
        • Wells D.
        Mosaicism: ‘survival of the fittest’ versus ‘no embryo left behind’.
        Fertil. Steril. 2016; 105: 1146-1149
        • PGDIS
        PGDIS position statement on chromosome mosaicism and preimplantation aneuploidy testing at the blastocyst stage.
        (15th International Conference on Preimplantation Genetic Diagnosis, Bologna, Italy, Reprod Biomed Online)
        http://www.pgdis.org/docs/newsletter_071816.html
        Date: 2016
        Date accessed: February 1, 2018
        • Rubio C.
        • Bellver J.
        • Rodrigo L.
        • Castillón G.
        • Guillén A.
        • Vidal C.
        • Giles J.
        • Ferrando M.
        • Cabanillas S.
        • Remohí J.
        • Pellicer A.
        • Simón C.
        In vitro fertilization with preimplantation genetic diagnosis for aneuploidies in advanced maternal age: a randomized, controlled study.
        Fertil. Steril. 2017; 107: 1122-1129
        • Scott Jr., R.T.
        • Upham K.M.
        • Forman E.J.
        • Hong K.H.
        • Scott K.L.
        • Taylor D.
        • Tao X.
        • Treff N.R.
        Blastocyst biopsy with comprehensive chromosome screening and fresh embryo transfer significantly increases in vitro fertilization implantation and delivery rates: a randomized controlled trial.
        Fertil. Steril. 2013; 100: 697-703
        • Shaffer L.G.
        • Tommerup N.
        ISCN 2005.
        (An International System for Human Cytogenetic Nomenclature (2005) Recommendations of the International Standing Committee on Human Cytogenetic Nomenclature. Published in collaboration with ‘Cytogenetic and Genome Research‘ Plus fold-out: ‘The Normal Human Karyotype G- and R-bands‘; ISBN: 978-3-8055-8019-9)2005
        • Shaffer L.G.
        • Slovak M.L.
        • Campbell L.J.
        ISCN 2009.
        (An International System for Human Cytogenetic Nomenclature (2009) Recommendations of the International Standing Committee on Human Cytogenetic Nomenclature. Published in collaboration with ‘Cytogenetic and Genome Research ‘Plus fold-out: ‘The Normal Human Karyotype G- and R-bands‘; ISBN: 978-3-8055-8985-7)2009
        • Shaffer L.G.
        • McGowan-Jordan J.
        • Schmid M.
        ISCN 2013.
        (An International System for Human Cytogenetic Nomenclature (2013) Recommendations of the International Standing Committee on Human Cytogenetic Nomenclature. Published in collaboration with ‘Cytogenetic and Genome Research ‘Plus fold-out: ‘The Normal Human Karyotype G- and R-bands‘; ISBN: 978-3-318-02253-7)2013
        • Simpson
        The Randomized Clinical Trial in assessing Preimplantation Genetic Screening (PGS): necessary but not sufficient.
        Hum. Reprod. 2008; 23: 2179-2181
      2. Specific Constitutional Cytogenetic Guidelines ECA.
        (July)
        www.e-c-a.eu
        Date: 2012
        • Turchetti D.
        • Pompilii E.
        • Magrini E.
        • Bonasoni M.P.
        • Pittalis M.C.
        • Segata M.
        • Pession A.
        • Santini D.
        • Pilu G.
        • Seri M.
        Persistence of a monosomic cell line in a fetus with mosaic trisomy 8.
        Am. J. Med. Genet. A. 2011; 155A: 2791-2794
        • Twisk M.
        • Mastenbroek S.
        • Bossuyt P.M.
        • Korevaar J.C.
        • Heineman M.J.
        • Repping S.
        • van der Veen F.
        The effectiveness of preimplantation genetic screening.
        Reprod. Biomed. Online. 2005; 11: 519-520
        • Twisk M.
        • Mastenbroek S.
        • van Wely M.
        • Heineman M.J.
        • Van der Veen F.
        • Repping S.
        Preimplantation genetic screening for abnormal number of chromosomes (aneuploidies) in in vitro fertilization or intracytoplasmic sperm injection.
        Cochrane Database Syst. Rev. 2006; (CD005291)
        • van Echten-Arends J.
        • Mastenbroek S.
        • Sikkema-Raddatz B.
        • Korevaar J.C.
        • Heineman M.J.
        • van der Veen F.
        • Repping S.
        Chromosomal mosaicism in human preimplantation embryos: a systematic review.
        Hum. Reprod. Update. 2011; 17: 620-627
        • Weier J.F.
        • Weier H.U.G.
        • Jung C.J.
        • Gormley M.
        • Zhou Y.
        • Chu L.W.
        • Genbacev O.
        • Wright A.A.
        • Fisher S.J.
        Human cytotrophoblasts acquire aneuploidies as they differentiate to an invasive phenotype.
        Dev. Biol. 2005; 279: 420-432
        • Weissman A.
        • Shoham G.
        • Shoham Z.
        • Fishel S.
        • Leong M.
        • Yaron Y.
        Chromosomal mosaicism detected during preimplantation genetic screening (PGS): results of a worldwide web-based survey.
        Fertil. Steril. 2017; 107: 1092-1097
        • Yang Z.
        • Liu J.
        • Collins G.S.
        • Salem S.A.
        • Liu X.
        • Lyle S.S.
        • Peck A.C.
        • Sills E.S.
        • Salem R.D.
        Selection of single blastocysts for fresh transfer via standard morphology assessment alone and with array CGH for good prognosis IVF patients: results from a randomized pilot study.
        Mol. Cytogenet. 2012; 5: 24

      Biography

      Francesca Romana Grati graduated is R&D Director of Istituto TOMA Advanced Biomedical Assays and contract professor at the School of Medical Genetics in Milan. Her research interests are the epidemiology of fetal chromosome abnormalities, genetics and epigenetics of placenta and feto-placental mosaicisms.
      Key message
      Mosaic aneuploid embryos are occasionally encountered during PGS, and often these are the only embryos available for transfer. It is currently unclear whether mosaic embryos should be considered for transfer. The aim of this study was to devise an evidence-based scoring system for prioritizing mosaic aneuploid embryos for transfer.