Autosomal Monosomies Research Articles

Prognostic Value of Monosomal Karyotype in Patients with Primary Acute Myeloid Leukemia On Behalf of Spanish CETLAM Group.

Abstract 1003Poster Board I-25 Introduction:Recently, the cooperative group HOVON-SAKK has refined the prognostic impact of cytogenetic abnormalities in acute myeloid leukemia (AML) by introducing the concept of monosomal karyotype (MK). This consists of ≥ 2 autosomal monosomies or one autosomal monosomy in addition to a structural alteration. In their experience, MK would explain the poor prognosis of AML with a complex karyotype. Objective:To investigate the prognostic impact of MK in patients with primary (de novo) AML enrolled in the Spanish CETLAM group protocols (AML 94/99/03). Also, to determine whether considering MK added predictive value to the cytogenetic classification of the Medical Research Council (MRC). Methods:Retrospective analysis of data from 1149 AML patients. Chromosomal formula was centrally reviewed with karyotypes being classified by the presence of MK and allocated into the MRC risk categories. Complete remission (CR) rate, disease-free survival (DFS) and overall survival (OS) were calculated. Results:The karyotype was assessable in 904 (79%) of the 1149 cases. In 145 of the 904 cases (16%), abnormalities involving CBF gene were detected and in 437 (48%) the karyotype was normal (NK). In 253 (28%) additional patients the karyotype was not monosomal; of them, 61 (24%) belonged to the unfavorable MRC with 17 cases harboring a complex karyotype ≥ 5 abnormalities, 7 cases with rearrangements 3q, 13 cases with -7, 9 cases with 5q abnormalities and 16 cases with t(6;9)). The remaining 69 (7.7%) patients had a MK; of them, 59 (85.5%) were from the unfavorable MRC category and included 43 cases with complex karyotype ≥ 5 abnormalities, 6 cases with rearrangements 3q, 5 cases with -7, 5 cases with alterations of 5q). The following table summarizes the results in terms of CR rate, DFS and OS:CBFNKMK- (IP)*MK- (UP)**MK+***pCR92%82%76%68%52%< 0,001DFS (4 years) median (month)58%± 5114.845%± 322.638%± 516.320%± 812.315%± 74.73<0,001OS (8 years) median (month)62%± 4-35%± 321.234%± 416.016%± 612.83%± 36.6<0,001*MK- (IP): Non-monosomal karyotype in the intermediate prognosis MRC cytogenetic group.**MK -(UP): Non-monosomal karyotype in the unfavorable prognosis MRC cytogenetic group.***MK+ : Cases with MK Conclusions:The addition of MK to the MRC cytogenetic classification refines the prognostic prediction. In our series, the dismal outcome of patients with MK is confirmed; these patients had worse prognosis than those with adverse cytogenetics without MK. Alternative treatment strategies are mandatory for MK+ patients.Supported in part by grants: GR1-01075, ECO07/90065, PI080672 and RD06/0020/0101. Disclosures:No relevant conflicts of interest to declare.

Monosomy Karyotype in Acute Myeloid Leukemia Predicts Adverse Treatment Outcome with Low Complete Remission Rate and Worse Event-Free and Overall Survival, and Associates with High Functional Activity of Multidrug Resistance (MDR).

Abstract 3112Poster Board III-49 BackgroundIt has been revealed that monosomy karyotype (MK), defined as 1) two or more distinct autosomal chromosome monosomies or 2) one single autosomal monosomy in the presence of structural abnormalities, identifies the highly unfavorable cytogenetic risk group of patients with acute myeloid leukemia (Breems, J Clin Oncol 2008), but lacking validation data. The current study aimed to validate the unfavorable impact of MK not only on overall survival (OS) but also on complete remission (CR) rate and event free survival (EFS) in AML patients. MethodsA total of 370 consecutive AML (excluding APL) patients with available cytogenetic data who received treatment between 1995 and 2008 at the Samsung Medical Center, Seoul, Korea were included in this retrospective study, among whom 169 patients (45.7%) showed normal karyotype; 65 patients (17.6%), core binding factor (CBF) positive AML; and 136 patients (36.7%), non-CBF AML, respectively. Karyotypes were scored according to their structural abnormalities, monosomy, trisomy, deletion and marker chromosome. The CR rate, EFS and OS were compared according to the presence of each cytogenetic abnormality. In addition, multidrug resistance (MDR) functional assay (P-glycoprotein assay) was performed and MDR functional activity was calculated by verapamil-inhibited rhodamine-123 efflux activity minus uninhibited rhodamine-123 efflux activity, and positivity was determined as equal to or over 5% MDR activity. ResultsAmong treated non-CBF AML group with any kind of cytogenetic abnormalities (n=136), 95 patients (69.9%) had structural cytogenetic abnormalities, 29 pts (21.3%), autosomal monosomy, 18 pts (13.2%), sex chromosome abnormalities, 59 pts (43.4%), autosomal trisomy, 41 pts (30.1%), deletion of part of a chromosome, 18 pts (13.2%), addition and 18 pts (13.2%), marker chromosome(s). MK was noted in 23 patients (16.9%), and complex karyotype (≥3 abnormalities) were found in 40 pts (29.%), -5 in 5 pts (3.7%), -7 in 12 pts (8.8%), del(5q) in 4 pts (2.9%), del(7q) in 8 pts (5.9%), inv(3) or t(3;3) in 4 pts (2.9%), t(6;9) in 5 pts (3.7%), and t(9;22) in 2 pts (1.5%).In univariate analyses, MK+ group was revealed to be associated with shorter OS and (median 10 vs 31months, p=0.044) EFS duration (median 1.3 vs 10.1 months, p=0.002), and a lower CR rate (70.8% vs 34.8%, p=0.002). In a multivariate analysis, MK was associated with lower CR rate (HR of non-CR 0.33, 95% C.I. 0.12-0.93, p=0.036).MK has been defined as a single monosomy with structural abnormalities or multiple monosomies in a previous study. However, there were no significant difference in survival and CR rate between a single monosomy with (n=9) or without(n=6) structural abnormalities (OS, 23 vs 8 months; p =0.349; EFS, 1 vs 9months; p=0.078; CR rate 33% vs 56%; p=0.608). The group with single autosomal monosomy showed a trend of better survival (n=15, median OS 23 months, EFS 1month) than multiple autosomal monosomy group (n=14, OS 6months, EFS 1month), but it was not significantly different (p = 0.322, p=0.221).The functional MDR activity was measured in 40 patients, and positive MDR activity was found to be significantly associated with the presence of MK (87.5% vs 33.3%, p=.013). In addition, the functional MDR activity was significantly higher in MK+ group (n=8, 45.9±17.8%, mean±S.E.) than in MK- group (n=32, 4.3 ±2.7%, p=0.005 by Mann-Whitney U-test). ConclusionThe current study demonstrated that the AML patients harboring MK showed a poor outcome in terms of lower CR rate and worse EFS/OS in an independent cohort of Korean AML patients, and that MK karyotype was associated with high MDR functional activity of leukemic blasts. DisclosuresNo relevant conflicts of interest to declare.

AML Patients with Monosomal Karyotype Are Characterized by Absence of NPM1 and FLT3 Mutations and Worse Clinical Outcome.

Abstract 2638Poster Board II-614 Background:The importance of cytogenetics in prognosis of AML is now widely recognized and accepted in clinical practice. A recent study found that autosomal chromosomal monosomy predicted for an adverse outcome. The goal of this study is to characterize patients with monosomal karyotype by mutation status and clinical features. Methods:One-hundred forty consecutive AML patients diagnosed at Stanford University Hospital between 2005 and 2008 with adequate material for mutation analysis were studied. Cases were classified using the 2008 WHO criteria. Diagnostic cytogenetic findings were reviewed and patients were stratified into risk groups using Southwest Oncology Group criteria. An abnormality was considered clonal when at least two metaphases had the same aberration, except for clonal monosomy, which required at least three metaphases. The karyotype analysis was based on 20 or more metaphases. All samples were tested for NPM, FLT3 (ITD and D835) and CEBPA mutations. Clinical parameters including hemogram data at time of diagnosis were reviewed. Clinical follow-up including overall survival (OS), progression free survival (PFS) and complete remission (CR) rates were retrospectively determined. Kaplan-Meier methods and univariate Cox proportional hazards regression analysis were used to compare the clinical data. Results:The cases included 77 males and 63 females with a median age of 58 (range 17-83). Cytogenetic risk-group stratification resulted in 14 patients with favorable, 88 with intermediate and 28 with unfavorable risk status. Loss of one or more autosomal chromosomes was present in 18 /130 patients (13.8%) with available cytogenetic studies. A single autosomal monosomy was found in 5 patients while 13 patients had two or more autosomal monosomies. The most common chromosomes lost in these 18 patients included 7 (55% of 18 cases), 5 (50%), 17 (33%), 21 (22%), 20 (22%), 22 (17%) and 18 (11%). Using the 2008 WHO criteria, there were 66 AML with myelodysplasia-related changes (AML-MRC), 55 AML not otherwise specified (AML-NOS), 14 AML with either t(8;21), inv(16) or t(15;17) and 5 therapy related AMLs. Overall, 35 patients (25% of all patients) had a NPM1 mutation (19 of which were FLT3 mutated), 33 had FLT3-ITD mutation (24%), 11 had FLT3-D835 (8%) and 11 had a CEBPA mutation (8%) (4 of which were FLT3 mutated). Patients with monosomal karyotype were significantly older (83 vs. 59 years, p=0.0125) and presented with lower WBC (34 vs. 66 K/uL, p=0.0006), lower platelets (41 vs. 64 K/uL, p=0.0111), and lower blasts (38% vs. 65%, p=0.0030) as compared to the rest of AML patients. In addition, patients with monosomal karyotype were more frequently diagnosed with AML-MRC (16/18 vs. 48/107, p=0.0034) and exhibited a decreased frequency of NPM1 mutation (0/18 vs. 28/107, p=0.0138) and FLT3-ITD mutation (0/18 vs. 29/107, p=0.0117). Clinical outcome data showed that patients with monosomal karyotype had a significantly worse OS, PFS and CR compared to the rest of AML patients (OS p=0.001, PFS p=0.002 and CR p=0.0262). Dividing patients by number of monosomies showed that patients with 2 or more monosomies had a significantly worse OS (p=0.0001) and PFS (p=0.0045) than patients without any monosomies. However, no difference in OS or PFS was seen when comparing patients with 1 monosomy to those with 2 or more monosomies. Within the AML-MRC group, monosomal karyotype correlated with lower WBC (17 vs. 37 K/uL, p=0.0005), lower platelets (21 vs. 35 K/uL, p=0.0095), lower blasts (19% vs. 36%, p=0.0015) and shorter OS (p=0.0322) and PFS (p=0.0084). Conclusion:AML patients with monosomal karyotype exhibit a significantly worse OS, PFS and lower CR as compared to other AML patients. Most of patients fall within the newly defined AML-MRC group and are characterized by significant absence of NPM1 and FLT3-ITD mutations. Disclosures:No relevant conflicts of interest to declare.

Prognostic Value of the ‘Monosomal Karyotype' Entity in Elderly Patients with AML: a GOELAMS Study of 186 Patients with Unfavourable Cytogenetic Abnormalities.

Abstract 1577▪▪This icon denotes an abstract that is clinically relevant.Poster Board I-603 INTRODUCTIONThe prognosis of acute myeloid leukemia (AML) in patients over 60 years of age remains very poor, especially in those with unfavorable cytogenetic abnormalities. Breems et al. recently identified in AML patients under 60 years old, a monosomal karyotype (MK), defined by the presence of at least two autosomal monosomies or one monosomy plus one structural abnormality. MK provides a significantly better prognostic prediction than the conventionally defined complex karyotype (CK) in such young patients [J Clin Oncol 2008;26:4791-7]. METHODSIn order to investigate the prognostic relevance of MK in the elderly, a retrospective study of 186 previously untreated AML patients with classically defined unfavourable cytogenetic was performed. All were aged 60 years or more and were selected among patients enrolled in 3 prospective GOELAMS trials (SA4, SA2002 and R04) between 1996 and 2006. In the three consecutive trials, patients were treated with standard dose cytarabine + idarubicin, followed by monthly re-inductions and maintenance. Poor-risk cytogenetics' patients were well-balanced between the treatment's arms and no significant influence of the hypotheses tested in the trials could be demonstrated (beneficial effect of fludarabine, androgenotherapy or gemtuzumab-ozogamicin, respectively) [manuscripts submitted]. RESULTSThe median age of the patients was 68 years (range: 60-79 y) and the M/F ratio 95/91. Thirteen percent of the patients had secondary AML. Leukocytosis >30 G/L was noted in 15% of the cases. Poor cytogenetic features included monosomy 5 or del 5q in 89 patients (48%) and monosomy 7 or del 7q in 86 (46%). Only 9 patients (<5%) had 11q23 abnormalities, 15 (8%) 3q21q26 abnormalities and 3 t(6;9). CK (defined by three or more abnormalities) was found in 129 patients (69%) and 109 patients had abnormalities corresponding to MK (58%). Only 9 patients had MK without CK. The overall complete remission rate was 48%, induction failure 35% and induction death 17%. With a median follow-up of 43 months for survivors, overall survival (OS) was 13.7% at 2 years (95% confidence interval [CI], 12.4%-14.9%). Patients with MK had a significantly lower CR rate at 37% (41/109) vs 65% (49/75) for patients with poor-risk AML without MK (p=.0004) and a significantly increased rate of persistent leukemia at 40% (44/109) vs 20% (15/75) (p=.006). OS was significantly impaired in MK patients: 7% vs 22% at 2 years (p< .0001). In multivariate analysis using a Cox model, MK appeared as the major independent prognostic factor for CR achievement (OR 3.26, CI: 1.75-6.07, p=.0002) and OS (HR 1.84, CI: 1.34-2.52, p=.0001). Age (60-69, ≥70 years) and leukocytosis (<30, ≥30 G/L) did not stand as independent prognostic factors. By comparison with the standard cytogenetic classification, CK carries a worse prognostic prediction for OS than MK (HR 1.53, CI: 1.10-2.12, p=.01). In the subgroup of 129 CK patients, survival was also significantly impaired in MK+ patients: 8% OS at 2 years, by comparison with 25% in MK- patients, (p=.03). CONCLUSIONThese results, obtained within a cohort of elderly patients with cytogenetically unfavourable AML, show that MK, according to the criteria proposed by Breems et al., is also an independent factor of very poor prognosis in older age, further stratifying prognostic subgroups. DisclosuresNo relevant conflicts of interest to declare.

Impact of Cytogenetics and New Molecular Markers On the Achievement of Complete Remission in Patients with Primary Acute Myeloid Leukemia. On Behalf of CETLAM Cooperative Group. Spain.

Abstract 4711 AimTo analyze the prognostic impact of cytogenetics and main molecular abnormalities on the achievement of complete remission (CR), in patients with primary (de novo) AML (M3 excluded). Patients and MethodsBetween december-2003 and july-2009, 605 patients up to 70 years-old were included in the CETLAM AML-03 protocol. Induction therapy consisted in one or two courses of idarubicin, intermediate dose ara-C and etoposide, in addition to G-CSF priming from day 0. Cytogenetics classification was as in the MRC studies: Favorable prognosis (FP), intermediate (IP) and adverse (AP). In the IP group, molecular analysis of NPM1 (NPM1+) mutations, CEBPA mutations and internal tandem duplication of FLT3 gene (ITD/FLT3) was performed. In the AP group, the absence (MK-) or presence of monosomal karyotype (MK+) was studied; MK+ was defined as 2 or more autosomal monosomies, or one monosomy and ≥1 structural alteration. ResultsMedian age of the series was 53 years (range, 16-73). In 538 (89%) patients cytogenetic data were available; of them, 64 (10,5%) had FP, 375 (61,8) IP (included 255 with normal cytogenetics) and 99 (16,3%) AP. In the FP group, 33 (5,4%) had the AML1/ETO fusion and 31 (5,1%) the CBF/MYH11. In the IP group, 121 (31%) of 279 patients studied were NPM1+, 100 (26,7%) of 346 were DIT/FLT3+ and 22 (6%) of 235 analyzed were CEBPA+. In patients with AP, 47 were MK- and 33 MK+. Overall, 447 (73,8%) of 588 patients evaluable at the moment of the analysis achieved a CR. CR rates according cytogenetics were: FP 92,2%, IP 76,5% and AP 69,4%, p=0.01. In AML, with AML1-ETO+ and CBF/MYH11+, CR rates were 91% and 93.5%, respectively; of note no chemorefractory cases were observed. In the IP group, CR rates according to mutational status were: NPM1+/FLT3- 91.2%, CEBPA+/FLT3- 94.4%, no mutations 79.1% and DIT/FLT3+ 69.7% (p=0.003). Again, no refractoriness was observed in patients in IP patients belonging to the two groups with favourable molecular profile (p=0.003). In patients with AP, CR rate was 76.1% if MK- and 65.6% if MK+ (p=0.46). The prognostic impact of the herein described cytogenetic and molecular findings was confirmed in multivariate analyses. CommentsGenetic characterization is nowadays mandatory in AML. CR rate is high and refractoriness exceptional in the presence of AML1-ETO or CBF/MYH11 rearrangements, or NPM1 or CEBPA mutations without DIT/FLT3. In contrast, CR probability is lower in AP group patients, mainly in those with MK+. These patients require new strategies within clinical trials.Supported in part by grants: GR1-01075, ECO07/90065, PI080672 and RD06/0020/0101. Disclosures:No relevant conflicts of interest to declare.

Monosomal Karyotype Is Predictive of Poor Response to Therapy and Worse Overall Survival in Secondary Acute Myeloid Leukemia (sAML); Analysis of a Multi-Center Phase II Study of Amonafide and Cytarabine Induction Therapy.

Abstract 2076Poster Board II-53 BackgroundRecently, further refinement of the “unfavorable” cytogenetic category in AML demonstrated that a “monosomal karyotype” (MK+) – defined as two or more distinct autosomal chromosome monosomies or one single autosomal monosomy in the presence of structural abnormalities – carries a markedly inferior prognosis to that of a “non-monosomal karyotype” (MK-) – defined as a group with various non-core binding factor (CBF) cytogenetic abnormalities but who are MK negative (J. Clin Onc 26:4791). This finding was based on data from patients predominantly with de novo AML. We analysed data from a phase II trial of patients with sAML to examine whether the monosomal karyotype is also predictive of poor outcome in sAML patients. MethodsThe karyotypes of pre-treatment blast cells from previously untreated sAML patients on a phase II study were reviewed to identify categories of core binding factor karyotype (CBF), normal karyotype (CN), MK+ and MK- (as defined above), and compared to clinical outcomes. Patients on the study all received induction therapy with amonafide 600 mg/m2/day IV days 1-5 and cytarabine 200 mg/m2/day IV days 1-7. Consolidation consisted of stem cell transplant (HSCT, n=10) or intermediate-dose (IDAC, n=13)/high-dose (HiDAC, n=7) cytarabine, depending on age and available HSCT donors. Results88 patients were treated. 2/88 (2%) were CBF, 29/88 (33%) were CN, 16/88 (18%) were MK+ and 31/88 (35%) were MK-. Patients with CBF and the 10 patients with unknown karyotype at baseline were excluded from the analysis. CR+CRi rate by karyotypic group was 19/29 (66%) for CN, 11/31 (35%) for MK-, 2/16 (13%) for MK+ (CN vs MK- p<0.05; CN vs MK+ p<0.05; MK- vs MK+ p=0.09). Median duration of remission was 282 days for CN, 304 days for MK-, and not reached for the 2 MK+ patients who achieved CR (p=NS). Median overall survival: 304 days CN, 145 days MK- and 144 days MK+ (Log rank test for survival: CN vs MK- p=0.07; CN vs MK+ p<0.05; MK- vs MK+ p=0.06). [Display omitted] ConclusionsIn this study of 88 patients with secondary AML, a monosomal karyotype was predictive of inferior response to therapy compared with both a normal karyotype and a non-monosomal “unfavorable” karyotype. Importantly, the 2-year overall survival in the MK+ group was significantly inferior to that in the CN group. Of note durable complete remissions were observed in responders regardless of karyotype. Though the number of patients in this study is relatively small, to our knowledge this is the only prospectively defined study of sAML patients demonstrating a significantly adverse impact of monosomal karyotype as compared with other “unfavorable” karyotypes. Further data to address the prognostic and predictive significance of specific cytogenetic abnormalities in sAML will be obtained from a currently enrolling 450-patient phase III randomized trial of amonafide + cytarabine vs daunorubicin + cytarabine in patients with sAML (ACCEDE). Disclosures:Downie:Antisoma: Research Funding. Erba:Lilly: Research Funding; Antisoma: Research Funding; Wyeth: Research Funding; Cephalon: Honoraria, Research Funding; MGI Pharma: Honoraria; Pharmion: Honoraria; Celgene: Honoraria; BMS: Honoraria; Novartis: Honoraria, Research Funding; Genzyme: Consultancy, Honoraria, Research Funding; Gemin-X: Research Funding; Kanisa: Research Funding. Stone:Celgene: Consultancy, Speakers Bureau; Merck: Consultancy; Genzyme: Consultancy; Eisai: Consultancy. Rizzieri:Antisoma: Research Funding. Foran:Antisoma: Research Funding.

Foreword: Coping with sex chromosome imbalance

This special issue is devoted to the topic of dosage compensation. The dramatic phenotypes that are associated with most autosomal monosomies or trisomies illustrate the importance of maintaining appropriate gene dosage in diploid organisms. Buffering and compensating for such genetic imbalance clearly represents a challenge for complex eukaryotes. In sexually dimorphic species with chromosome-based mechanisms of sex determination (XX/XY and ZW/ZZ), the contrasting doses of sex chromosomes in males and females would be expected to introduce a large-scale imbalance in levels of gene expression. This imbalance would be at two levels. First, the dosage differences between the sexes (XX versus XY or ZW versus ZZ); second, the dosage difference between sex chromosomes and autosomes in the heterogametic sex (XY males, or ZW females) where only a single copy of the X or Z chromosome is present, in contrast to two copies of all autosomes. However, sex chromosome imbalances are usually well tolerated—how can this be?

Monosomal Karyotype in Acute Myeloid Leukemia: A Better Indicator of Poor Prognosis Than a Complex Karyotype

To investigate the prognostic value of various cytogenetic components of a complex karyotype in acute myeloid leukemia (AML). Cytogenetics and overall survival (OS) were analyzed in 1,975 AML patients age 15 to 60 years. Besides AML with normal cytogenetics (CN) and core binding factor (CBF) abnormalities, we distinguished 733 patients with cytogenetic abnormalities. Among the latter subgroup, loss of a single chromosome (n = 109) conferred negative prognostic impact (4-year OS, 12%; poor outcome). Loss of chromosome 7 was most common, but outcome of AML patients with single monosomy -7 (n = 63; 4-year OS, 13%) and other single autosomal monosomies (n = 46; 4-year OS, 12%) did not differ. Structural chromosomal abnormalities influenced prognosis only in association with a single autosomal monosomy (4-year OS, 4% for very poor v 24% for poor). We derived a monosomal karyotype (MK) as a predictor for very poor prognosis of AML that refers to two or more distinct autosomal chromosome monosomies (n = 116; 4-year OS, 3%) or one single autosomal monosomy in the presence of structural abnormalities (n = 68; 4-year OS, 4%). In direct comparisons, MK provides significantly better prognostic prediction than the traditionally defined complex karyotype, which considers any three or more or five or more clonal cytogenetic abnormalities, and also than various individual specific cytogenetic abnormalities (eg, del[5q], inv[3]/t[3;3]) associated with very poor outcome. MK enables (in addition to CN and CBF) the prognostic classification of two new aggregates of cytogenetically abnormal AML, the unfavorable risk MK-negative category (4-year OS, 26% +/- 2%) and the highly unfavorable risk MK-positive category (4-year OS, 4% +/- 1%).

Blastocyst formation rates in chromosomally normal versus abnormal embryos as analyzed by 24 chromosome microarray-based anueploidy screening (mPGD)

OBJECTIVE: Blastocyst culture and transfer is utilized as a tool to select embryos with higher implantation rates. The addition of mPGD may enhance embryo selection and improve patient outcomes. mPGD was utilized to evaluate the relationship between anueploidy and blastulation rates of embryos in extended culture.DESIGN: Retrospective study of IVF patients undergoing extended culture with embryo biopsy and mPGD.MATERIALS AND METHODS: Oocyte retrieval and ICSI were performed according to standard protocols. Embryos were biopsied on day 3 or 5 of development and ploidy status was determined by 24 chromosome mPGD as previously described. Embryos were classified by mPGD as having a normal or abnormal complement of chromosomes. Abnormal embryos were further classified according to their errors: sex chromosomal, autosomal trisomy, autosomal monosomy or multiple errors. Blastocyst formation rates were compared between groups and statistical analysis was performed using Chi-square tests.Table 1Aneuploidy StatusBlastocyst FormationNormal60% (21/35)Abnormal Sex70.6% (12/17)Trisomy50% (9/18)Monosomy47% (9/19)Multiple21% (6/29) Open table in a new tab CONCLUSIONS: This study evaluating the full karyotype of developing human embryos demonstrates that ploidy status is associated with blastulation. While aneuploid embryos are significantly less likely to blastulate, it is still quite common. This suggests that blastulation rates alone may not be sufficient to fully prognosticate the reproductive potential of an individual embryo. Improvements in embryo selection using blastocyst transfer in tandem with mPGD may increase the possibility of selecting embryos with higher implantation rates. A randomized clinical trial is currently underway. OBJECTIVE: Blastocyst culture and transfer is utilized as a tool to select embryos with higher implantation rates. The addition of mPGD may enhance embryo selection and improve patient outcomes. mPGD was utilized to evaluate the relationship between anueploidy and blastulation rates of embryos in extended culture. DESIGN: Retrospective study of IVF patients undergoing extended culture with embryo biopsy and mPGD. MATERIALS AND METHODS: Oocyte retrieval and ICSI were performed according to standard protocols. Embryos were biopsied on day 3 or 5 of development and ploidy status was determined by 24 chromosome mPGD as previously described. Embryos were classified by mPGD as having a normal or abnormal complement of chromosomes. Abnormal embryos were further classified according to their errors: sex chromosomal, autosomal trisomy, autosomal monosomy or multiple errors. Blastocyst formation rates were compared between groups and statistical analysis was performed using Chi-square tests. CONCLUSIONS: This study evaluating the full karyotype of developing human embryos demonstrates that ploidy status is associated with blastulation. While aneuploid embryos are significantly less likely to blastulate, it is still quite common. This suggests that blastulation rates alone may not be sufficient to fully prognosticate the reproductive potential of an individual embryo. Improvements in embryo selection using blastocyst transfer in tandem with mPGD may increase the possibility of selecting embryos with higher implantation rates. A randomized clinical trial is currently underway.

Array comparative genomic hybridization profiling of first‐trimester spontaneous abortions that fail to grow in vitro

Cytogenetic analysis of spontaneous abortion samples can be limited by culture failure. Failure to grow in vitro has traditionally been suspected to be due to in vivo death of tissue associated with spontaneous abortion (SAB) or simply technical factors of growth in culture. We used array comparative genomic hybridization (array CGH) to investigate chromosomal imbalances in products of conception that failed to grow in vitro. Our data on 26 cases of SABs that failed to grow in culture are compared and contrasted with published data on cytogenetic findings following in vitro culture. The results revealed abnormalities uncommonly seen by classic cytogenetic methods. These abnormalities include high rates of double aneuploidy and autosomal monosomy. The data taken together suggest that classic cytogenetics of spontaneous abortion may yield normal karyotypes or selected abnormal karyotypes that permit cell proliferation in vitro while Array CGH detects other abnormalities. Array CGH is becoming an important clinical assay for unbalanced chromosome abnormalities whether cells grow in culture or not and in cases of analysis on one or few cells.

Features of chromosomal abnormalities in spontaneous abortion cell culture failures detected by interphase FISH analysis.

Cytogenetic analysis of reproductive wastage is an important stage in understanding the genetic background of early embryogenesis. The results of conventional cytogenetic studies of spontaneous abortions depend on tissue culturing and are associated with a significant cell culture failure rate. We performed interphase dual-colour FISH analysis to detect chromosomal abnormalities in noncultured cells from two different tissues-cytotrophoblast and extraembryonic mesoderm-of 60 first-trimester spontaneous abortions from which cells had failed to grow in culture. An original algorithm was proposed to optimize the interphase karyotype screening with a panel of centromere-specific DNA probes for all human chromosomes. The overall rate of numerical chromosomal abnormalities in these cells was 53%. Both typical and rare forms of karyotype imbalance were found. The observation of six cases (19%) of monosomy 7, 15, 21 and 22 in mosaic form, with a predominant normal cell line, was the most unexpected finding. Cell lines with monosomies 21 and 22 were found both in cytotrophoblast and mesoderm, while cells with monosomy 7 and 15 were confined to the cytotrophoblast. The tissue-specific compartmentalization of cell lines with autosomal monosomies provides evidence that the aneuploidy of different human chromosomes may arise during different stages of intrauterine development. The effect of aneuploidy on selection may differ, however, depending on the specific chromosome. The abortions also revealed a high frequency of intratissue chromosomal mosaicism (94%), in comparison with that detected by conventional cytogenetic analysis (29%; P<0.001). Confined placental mosaicism was found in 25% of the embryos. The results of molecular cytogenetic analysis of these cell culture failures illustrate that the diversity and phenotypic effects of chromosomal abnormalities during the early stages of human development are underestimated.

An Evaluation of the Frequency of Spontaneous Aneuploidy in Human Somatic Cells Using the Technology of Interphase Cytogenetics

The frequency of spontaneous aneuploidy of the four autosomes and both sex chromosomes in the interphase nuclei of cultured and noncultured lymphocytes from clinically healthy men was examined by use of two-color fluorescent in situ hybridization (FISH). It was shown that in noncultured cells from the individuals examined autosomal nullisomies were practically not detected (the frequency 0 to 0.01%). At the same time, the frequency of such cells with either Y, or X nullisomy was at least an order of magnitude higher (about 0.15%). This frequency was comparable with the level of Y- or X-disomic cells, and also with autosomal monosomies, precluding from consideration of X-nullisomic cells as hybridization artifact. During lymphocyte cultivation, a statistically significant increase in the total frequency of Y- or X-nullisomic cells was observed already after the first cell division cycle. Thus, interphase FISH analysis is a sufficiently sensitive method enabling detection of higher, compared to the autosomes, loss of sex chromosomes in the process of cell division, a phenomenon observed during replicative cell aging, as well as during natural aging of the organism. Male cells with the de novo lost single X chromosome, probably, switch to apoptosis and do not survive during further life of a cell population. The frequency of total aneuploidy in human somatic cells with the correction for the resolution capacity of the interphase FISH analysis was estimated to be 5.62 and 6.90% for noncultured and cultured cells, respectively. This aneuploidy level is close to that in spermatozoa. The data obtained can serve as the basis for the examination of the aneugenic (aneuploidy-inducing) genotoxic effects and for the analysis of interindividual genetic instability.

Specific Features of Phenotypic Expression of Autosomal Monosomies during Pathological Postimplantation Development of Man

Autosomal monosomies represent a severe form of genomic disbalance which determines elimination of human embryos already at the preimplantation stages. As a rule, they occur very rarely in the materials of spontaneously aborted embryos and fetuses. Molecular-cytogenetic studies were carried out on the karyotype of cells of 60 spontaneous abortuses of I trimester of pregnancy with cell degeneration or absence of cell proliferation in the cultures, as a result of which the cells could not be studied using the standard metaphase analysis. The embryos were characterized by an unexpectedly high frequency of mosaic variants of monosomies for chromosomes 7, 15, 21, and 22, which amounted to 19% of all chromosome aberrations. Lethal forms of monosomies for human chromosomes 7 and 15 were described for the first time, since they are not found in spontaneous abortuses by standard cytogenetic methods. A hypothesis was proposed which accounts for the possibility of early postimplantation lethality of the embryos with mosaic forms of autosomal monosomies. The differences were found between the cells with monosomies for different autosomes in the mechanisms of origin, intertissue localization, and phenotypic effects. It was shown that monosomies for chromosomes 7, 15, 21, and 22 in a mosaic state with the normal cell line can be compatible with the early stages of postimplantation differentiation of the cytotrophoblast. Predominant compartmentalization of the cells with monosomies for chromosomes 21 and 22 in the extraembryonic mesoderm, a derivative of epiblast, can be a critical factor, which makes the normal morphogenesis of embryonic structures impossible.

Molecular cytogenetic characteristics of chromosome imbalance in cells of spontaneous human abortion fetuses with low proliferative activity in vitro

Karyotyping of noncultivated cells of 60 first-trimester spontaneous abortions (blighted ova and missed abortions) was carried out using fluorescence in situ hybridization (FISH) with centromere-specific DNA probes for all chromosomes of the karyotype. Conventional cytogenetic study of these abortions was impossible because of cell culture failures. The algorithm is proposed for molecular cytogenetic FISH analysis of interphase karyotypes. Chromosome abnormalities were found in 32 fetuses (53.3%). In groups of missed abortions and blighted ova, the frequency of numerical chromosome abnormalities was 50 and 60%, respectively. Both the numerical chromosome abnormalities typical of spontaneous human abortions (autosomal trisomies, sex chromosome aneuploidy, and polyploidy) and a relatively rare type of genomic imbalance unidentifiable by standard cytogenetic analysis (autosomal monosomies 7, 15, 21, and 22 in mosaic state) were observed. The frequency of these type of chromosome abnormalities comprised 19% of all known karyotype abnormalities determined in spontaneously perished embryos. Note that the level of confined placental mosaicism in embryos with low cell proliferative activity was 25%, which is substantially higher than the corresponding parameter (1-2%) determined by prenatal diagnosis of chromosome abnormalities in developing embryos. The results of interphase FISH analysis of cells with low proliferative activity in vitro suggest that the pathology of early fetal development and missed abortion in humans are associated with a wider spectrum of chromosome abnormalities.

Impact of parental gonosomal mosaicism detected in peripheral blood on preimplantation embryos

This study evaluated the chromosomal condition of embryos generated by patients with an altered karyotype due to gonosomal mosaicism and the clinical outcome after preimplantation genetic diagnosis (PGD) for aneuploidy. Thirty-six patients aged 34.6 ± 3.6 years performed 54 treatment cycles and had 295 embryos diagnosed by fluorescence in-situ hybridization (FISH). Thirty-seven per cent of the embryos were chromosomally normal and generated 19 clinical pregnancies after replacement in 39 cycles. Only one pregnancy miscarried, yielding a take-home baby rate of 33.3%. Autosomal monosomy and trisomy contributed 36.1% of total abnormalities and gonosomal aneuploidy 5.9%, similar to the results detected in patients who undergo PGD for increased maternal age. Reanalysis was performed on 114 non-transferrable embryos: 41 were found to be mosaics, which were grouped in three different types, chaotic mosaics (56%), aneuploid mosaics (29%) and diploid/haploid/polyploid mosaics (15%). The incidence of aneuploid mosaics was higher than expected compared with PGD patients of the same age and resembled the condition observed in patients of advanced maternal age. These findings suggest that constitutional carriers of sex chromosome mosaicism are predisposed to autosomal mosaicism of embryos, possibly due to errors of cell division. There is an indication that this tendency is higher in female than male carriers.

Some Chromosomal Abnormalities in Early Embryos Preclude Blastocyst Formation in Vitro

Objectives: Embryo selection is an important contributor to success after IVF. A number of studies have identified correlations between early embryo morphology, chromosomal integrity, and developmental competence. Our goal was to extend those studies to blastocysts.Design: This is a prospective study to determine which chromosomal abnormalities in cleavage stage embryos are compatible with development to the blastocyst stage and which are selected against in extended culture.Materials and Methods: Embryos used for this project were donated for research in accordance with guidelines approved by our Internal Review Board, including informed written consent in each case. Embryos were obtained from 27 patients (29 cycles) undergoing preimplantation genetic diagnosis (PGD) for aneuploidy using FISH with probes for chromosomes X, Y, 1, 13, 15, 16, 18, 21 and 22. A total of 179 embryos were diagnosed as chromosomally abnormal or were unsuitable for embryo replacement (male embryos with X-linked disease or embryos with no FISH result during PGD). After blastomere biopsy on day 3, embryos were cultured in microdrops of G2 medium and their development was monitored daily until day 5. Arrested embryos were fixed using Tarkowsky’s method (1966); blastocyst fixation was according to the protocol of Clouston et al. (1997). All embryos were analyzed by two rounds of FISH with the same probes used in the original PGD analysis.Results: Of the 179 embryos cultured to day five, 48 were classified as blastocysts. The morphology of those embryos was optimal in six, sub-optimal in 19, and abnormal in 19; four embryos were not classified. The following table shows the chromosome abnormalities developing to blastocyst. If blastocyst with abnormal morphology were not transferred, 52% of the abnormalities arriving to blastocyst detected would be eliminated. Tabled 1Mono- somicTrisomicHaploid>40% mosaic<40% mosaicNormalDay 333384612513Blastocyst3∗1109166(∗two monosomies 21, one monosomy X) Open table in a new tab Discussion: By extending in vitro culture period to five days, embryos with haploidy and autosomal monosomy (with the exception of monosomy 21) are eliminated as these anomalies lead to early embryonic arrest. However, other abnormalities such as extensive (>40%) mosaicism, trisomy and polyploidy cannot be completely screened out by extended culture. The only option for exclusion of such embryos from transfer is PGD. Objectives: Embryo selection is an important contributor to success after IVF. A number of studies have identified correlations between early embryo morphology, chromosomal integrity, and developmental competence. Our goal was to extend those studies to blastocysts. Design: This is a prospective study to determine which chromosomal abnormalities in cleavage stage embryos are compatible with development to the blastocyst stage and which are selected against in extended culture. Materials and Methods: Embryos used for this project were donated for research in accordance with guidelines approved by our Internal Review Board, including informed written consent in each case. Embryos were obtained from 27 patients (29 cycles) undergoing preimplantation genetic diagnosis (PGD) for aneuploidy using FISH with probes for chromosomes X, Y, 1, 13, 15, 16, 18, 21 and 22. A total of 179 embryos were diagnosed as chromosomally abnormal or were unsuitable for embryo replacement (male embryos with X-linked disease or embryos with no FISH result during PGD). After blastomere biopsy on day 3, embryos were cultured in microdrops of G2 medium and their development was monitored daily until day 5. Arrested embryos were fixed using Tarkowsky’s method (1966); blastocyst fixation was according to the protocol of Clouston et al. (1997). All embryos were analyzed by two rounds of FISH with the same probes used in the original PGD analysis. Results: Of the 179 embryos cultured to day five, 48 were classified as blastocysts. The morphology of those embryos was optimal in six, sub-optimal in 19, and abnormal in 19; four embryos were not classified. The following table shows the chromosome abnormalities developing to blastocyst. If blastocyst with abnormal morphology were not transferred, 52% of the abnormalities arriving to blastocyst detected would be eliminated. Tabled 1Mono- somicTrisomicHaploid>40% mosaic<40% mosaicNormalDay 333384612513Blastocyst3∗1109166(∗two monosomies 21, one monosomy X) Open table in a new tab Discussion: By extending in vitro culture period to five days, embryos with haploidy and autosomal monosomy (with the exception of monosomy 21) are eliminated as these anomalies lead to early embryonic arrest. However, other abnormalities such as extensive (>40%) mosaicism, trisomy and polyploidy cannot be completely screened out by extended culture. The only option for exclusion of such embryos from transfer is PGD.

Creation of monosomic derivatives of human cultured cell lines.

Monosomic mammalian cell lines would be ideal for studying gene dosage effects, including gene imprinting, and for systematic isolation of recessive somatic mutants parallel to the invaluable mutants derived from haploid yeast. But autosomal monosomies are lethal in early development; although monosomies appear in tumors, deriving cell lines from these tumors is difficult and cannot provide several syngenic lines. We have developed a strategy for generating stable monosomic human cells, based on random autosomal integration of the gpt plasmid, partial inhibition of DNA topoisomerase II during mitosis to promote chromatid nondisjunction, and selection against retention of gpt. These are likely to be valuable as a source of otherwise inaccessible mutants. The strategy can also be used to generate partial mammalian monosomies, which are desirable as a source of information on recessive genes and gene imprinting.

The early lethality of autosomal monosomy in the mouse.

Using male mice doubly heterozygous for pairs of Robertsonian translocation chromosomes that have one arm in common, mouse embryos monosomic for 11 of the 19 autosomes have been generated. All of these monosomies result in death prior to or during the implantation period, with only rare survivors being detected 6 days after fertilization. For some of the monosomies the onset of lethality can be detected during the third or fourth day of development, but others do not begin to die until sometime after the late blastocyst stage on day 4. Retardation of development, as revealed by decreased cell numbers, is often detectable prior to or after the onset of the lethal period. The period during which death occurs may spread over several days and does not coincide with any of the developmental landmarks of the pre- or peri-implantation period. Genetic factors that may affect the rate of cellular proliferation or other aspects of embryonic development appear to play an important role in determining exactly when individual monosomies result in death. The universal early lethality of the autosomal monosomies leads to the conclusion that a large number of loci scattered over all of the autosomes are involved in processes that are so concentration dependent that a 50% reduction is sufficient to produce very serious consequences.

Ethanol-induced late fetal death in mice exposed around the time of fertilization

In the mouse, all autosomal monosomies and trisomies are lethal by the time of birth (Searle, 1981). To test whether ethanol ingested by females shortly after mating induces nondisjuction, as reported by Kaufman (1983) on the basis of cytological evidence, we attempted to determine whether the incidence of intrauterine death was affected by this treatment. The incidence of late death (day 11 postconception or later) was found to be significantly increased when ethanol was administered 2 h following a 30-min mating period, but not when the interval was shorter. Measurements of early death were not sensitive enough (because of the high control frequency) to show an effect of ethanol treatment. Limited cytological data showed an induced incidence of trisomy in line with the excess frequency of late death, but the trisomy incidence by itself was not significantly different from control. The overall level of effect in the present experiment was lower than that reported by Kaufman.

Chromosomal control of early embryonic development in mice I. Experiments on embryos with autosomal monosomy

The effects of autosomal monosomy on early embryonic development were studied in mice with Robertsonian and reciprocal (non-Robertsonian) translocations. It was found that monosomy for autosomes 1, 2, 3, 5, 6, 16, 17 and 19 had substantially different effects on preimplantation development and survival of mouse embryos. Monosomy for autosomes 1, 3, 6, 16 and 19 does not affect cleavage, compaction and blastulation and in some cases is compatible with implantation. Most of these embryos, however, die as early blastocysts (Ms 3, 6 or 19) and some of them are eliminated at early postimplantation stages (Ms 1 or 16). The embryos with monosomy for autosomes 2, 5 or 17 can be identified during cleavage owing to the reduced blastomere number and pathological changes in the nuclei. Most of these monosomies do not survive beyond the morula stage. The results indicate that differential genetic activity of autosomes in mice becomes already evident in very early embryonic development. A hypothetical mechanism for homologous autosome activation at the onset of embryonic development in mice is suggested.