Embryo aneuploidy screening with CGH arrays in the absence of implantation

Transcription

1 Mini-revue mt Médecine de la Reproduction, Gynécologie Endocrinologie 214 ; 16 (2) : Embryo aneuploidy screening with CGH arrays in the absence of implantation Dépistage d aneuploïdie embryonnaire avec microarrays en cas d échec d implantation Sandra Garcia-Herrero 1 Lorena Rodrigo 1,2 Emilia Mateu 1 Vanessa Peinado 1 Miguel Milán 1 Inmaculada Campos-Galindo 1,2 Maria Vera 1 Amparo Mercader 3 Fernando Bronet 4 Antonio Requena 4 Carlos Simón 1,2,5,6 Carmen Rubio 1,2 1 IVIOMICS, Paterna, Valencia, 2 Instituto Universitario IVI/INCLIVA Valencia, <carmen.rubio@iviomics.com> 3 IVI Valencia, Valencia, 4 IVI Madrid, Madrid, 5 Fundación Instituto Valenciano de Infertilidad (FIVI), Valencia, 6 Department of Obstetrics and Gynecology, School of Medicine, Stanford University, CA, USA médecine thérapeutique Reprints : C. Rubio Médecine de la Reproduction Gynécologie Endocrinologie Abstract. The most common method for embryo selection after In vitro fertilization (IVF) stands on morphologic and developmental characteristics. However, the implantation potential of human embryos produced in vitro remains low for some groups of patients. For this reason, the assessment of embryo viability by aneuploidy screening could improve IVF outcome in poor prognosis patients. Comprehensive chromosome screening (CCS) in combination with other selection techniques may help to improve the probability of selecting a chromosomally normal embryo for transfer by accurately identifying euploid embryos, and thus leading to improved ART outcomes. CCS is becoming a routine procedure in IVF cycles for many poorprognosis patient groups such as those suffering from implantation failure, increased maternal age, recurrent pregnancy loss, or carriers of structural or numerical chromosome abnormalities, that have predisposing factors for abnormal embryos. Key words: implantation failure, comprehensive chromosome screening, CGH arrays Résumé. La méthode de sélection embryonnaire la plus commune en fécondation in vitro (FIV) repose sur les caractéristiques morphologiques et de développement embryonnaire. Cependant, le potentiel implantatoire des embryons humains reste faible pour certains groupes de patients. Pour cette raison, l évaluation de la viabilité embryonnaire par dépistage d aneuploidie pourrait améliorer les résultats en FIV chez les patients de mauvais pronostic. Le Comprehensive chromosome screening (CGS) combiné à d autres techniques de sélection pourrait augmenter la probabilité de sélectionner un embryon chromosomiquement normal pour le transfert en identifiant avec précision les embryons euploïdes, et donc aboutir à de meilleurs résultats en AMP. CGS devient actuellement une procédure de routine dans les cycles de FIV dans de nombreux groupes de patients de mauvais pronostic, tels que les échecs répétés d implantation, l âge maternel avancé, les fausses couches à répétition ou chez les patients porteurs d anomalies chromosomiques de nombre ou de structure, tous ces facteurs prédisposant au développement d embryons anormaux. Mots-clés : échec d implantation, comprehensive chromosome screening, CGH arrays The most common method for embryo selection after In vitro fertilization (IVF) stands on morphologic and developmental characteristics. However, the implantation potential of human embryos produced in vitro remains low for some groups of patients one reason being that the majority are chromosomally abnormal [1]. For this reason, the assessment of embryo viability by aneuploidy screening could improve IVF outcome in poor prognosis patients. Most methods of embryo viability assessment involve morphological evaluation at different preimplantation developmental stages, including assessment of blastomere number, multinucleation, embryo fragmentation, and blastocyst formation [2] ; more recently time lapse systems such as Embryoscope have also been used as a routine IVF laboratory tool for better embryo selection by morph kinetic parameters [3]. The relationship of morphology with embryo aneuploidy was first evaluated with fluorescent in situ hybridization (FISH) studies for 7-8 chromosomes. In our experience, having analyzed almost 7, embryos, we observed that despite the significant effect that aneuploidy has on embryonic development, a remarkable percentage of chromosomally abnormal embryos still develop to the blastocyst stage and have good morphological features : in one study 42.8 % of chromosomally abnormal embryos reached the blastocyst stage doi:1.1684/mte To cite this article: Garcia-Herrero S, Rodrigo L, Mateu E, Peinado V, Milán M, Campos-Galindo I, Vera M, Mercader A, Bronet F, Requena A, Simón C, Rubio C. Embryo aneuploidy screening with CGH arrays in the absence of implantation. mt Médecine de la Reproduction, Gynécologie Endocrinologie 214; 16 (2): doi:1.1684/mte

2 [4]. More recently, in a study using comparative genomic hybridization (CGH) with 24-chromosome screening, a weak association between blastocyst morphology and aneuploidy was described [5]. Therefore, pre-implantation genetic screening (PGS) in combination with other selection techniques may help to improve the probability of selecting a chromosomally normal embryo for transfer by accurately identifying euploid embryos, and thus leading to improved ART outcomes. PGS is becoming a routine procedure in IVF cycles for many poor-prognosis patient groups that have predisposing factors for abnormal embryos, such as increased maternal age, those with recurrent pregnancy loss, or carriers of structural or numerical chromosome abnormalities. Implantation failure : definition Couples are diagnosed with repetitive implantation failure (RIF) after two or three failed IVF attempts or failed IVF treatments after the cumulative transfer of more than 1 good-quality embryos. Nevertheless, criteria to define RIF are not homogenous and its exhaustive and comprehensive definition has not yet been agreed on [6, 7]. RIF remains a big challenge to the clinician because its causes can be multiple and are still poorly defined, and embryonic as well as endometrial factors can play an important role in this condition. The causes of RIF can be summarized as follows : defects in the embryo-endometrium relationship, the negative effect of stimulation protocols, uterine malformations, problems with implantation genes (more than 1 have been identified so far), immunological factors, infections, and problems related to the embryo itself. Among embryonic causes, genetic abnormalities, in particular embryonic aneuploidy or zona pellucida hardening, could adversely influence implantation [6]. Several approaches have been used in RIF patients to select for better quality embryos, such as blastocyst co-culture methods [8] and the analysis of the chromosomal constitution of embryos [9]. Other strategies have attempted to improve the implantation ability of the embryos, for example, through assisted hatching [1]. Pre-implantation genetic screening studies using fluorescence in situ hybridization technology in cases of implantation failure Until recently PGS using FISH technology for a selected panel of chromosomes has been the most widely applied methodology for aneuploidy screening. Only the chromosomal abnormalities which are most implicated in spontaneous miscarriages and those with live births were analyzed. Although the benefits of PGS with FISH have been controversial, with optimized embryo culture systems, appropriate patient inclusion criteria, and more comprehensive genetic tests, there could still be a place for it in an IVF setting. In this sense, our own experience differs from the previously published studies. Our group conducted a prospective randomized control trial (RCT) [7] to evaluate the usefulness of PGS in RIF patients younger than 4 years old ; we screened embryos for nine chromosomes (13, 15, 16, 17, 18, 21, 22, X, and Y) and performed day-5 blastocyst transfer which resulted in an increase in live birth rates in the PGS group compared to controls. This RCT also showed a trend towards improved live-birth rates when PGS was performed in a selected group of RIF patients for whom other potential causes had already been discarded. The live-birth rates per patient with and without PGS were 47.9 % and 27.9 % but unfortunately these did not reach statistical significance ; embryo development, blastocyst formation rates, and implantation ability were not detrimentally affected by embryo biopsy. Although we did not find a significant improvement as a result of PGS in this population, the higher live-birth rates observed with PGS in our study contrast with a previously published RCT also performed in RIF patients which found no significant differences in clinical pregnancy rates between PGS patients and control groups [11]. Nevertheless, this could be attributed to stricter inclusion criteria as well as to the number of biopsied cells and chromosomes analyzed in this study. We therefore concluded that PGS with classic FISH is beneficial for this indication if proper blastomere biopsy procedures and good laboratory conditions are applied. Despite these results, there is still a clear need for a technique which can analyze all the chromosomes in a short period of time while still producing reliable and faithful results [7]. Pre-implantation genetic screening studies using comprehensive chromosome screening technology in implantation failure patients Methodology Several approaches toward 24-chromosome analysis have been developed under the general denomination of comprehensive chromosome screening (CCS). The first studies published used CGH protocols for metaphase II oocytes, cleavage-stage embryos, and blastocysts, while different technologies were later developed for 24- chromosome aneuploidy analysis in a shorter time frame. CGH-based microarrays, SNP arrays, and quantitative polymerase chain reaction-based techniques appeared to 113

3 Mini-revue be the most suitable. The first successful clinical application of array-cgh in single cells suggested that this is the most suitable technique owing to its efficiency, depth of detection, and the time required for analysis. A recently published review by Simpson et al. proposed that array- CGH is the preferred diagnostic approach for assessing 24-chromosome aneuploidy [12] and hence many IVF programs are now moving toward performing PGS analysis using this technology. In the majority of our work we performed day- 3 acgh analysis on a single cell from each embryo (figure 1) ; patients underwent ovarian stimulation using standardized protocols, and when at least two follicles reached 18 mm in diameter recombinant human chorionic gonadotropin (hcg, Ovitrelle, 25 mg, Merck- Serono, Geneva, Switzerland) was administered, and oocyte retrieval was scheduled 36 hours later. ICSI was performed in all cases [13], fertilization was assessed 17-2 hours after microinjection, and embryo cleavage was recorded every 24 hours. Embryo biopsy was performed on day-3 and can be summarized as follows : embryos were placed in a droplet containing Ca2 + /Mg2 + -free medium (G-PGD, Vitrolife, Göteborg, Sweden/LifeGlobal Guilford, CT), the zona pellucida was perforated using laser technology (OCTAX, Herbron, Germany), and one blastomere was withdrawn from each embryo. Only embryos with five or more nucleated blastomeres and less than 25 % fragmentation were biopsied. Individual blastomeres were placed in.2 ml PCR tubes containing 2 L PBS, and 1 % polyvinylpyrrolidone (PVP) was used for blastomere washing and handling. Properly developed euploid embryos were transferred on day-5, and surplus euploid embryos were vitrified either on day-5 or day-6. To perform day-3 acgh analysis, a single cell from each embryo was amplified using the Sureplex TM DNA amplification system (BlueGnome, Cambridge, UK) and the amplification quality was ensured by gel electrophoresis (Lonza, Rockland, USA). Sample and control DNA were labelled with Cy3 and Cy5 fluorophores following the manufacturer s instructions and labelling mixes were combined and hybridized on 24sure arrays (V2 and V3, BlueGnome, Cambridge, UK) for 6-12 hours. Each probe is specific to a different chromosomal region and occupies a discrete spot on the slide ; the technique involves the competitive hybridization of differentially-labeled test and reference DNA samples and chromosomal loss or gain is revealed by the color taken on by each spot after hybridization. Fluorescence intensity was detected using a laser scanner (Powerscanner, TECAN, Männedorf, Switzerland) and BlueFuse Multi software was used for data processing (BlueGnome, Cambridge, UK). As specified by the manufacturer (24sure microarray product description, eigth February 212, version 2.3, for model number ) the 24sure has an effective 1Mb resolution when using this software, which corresponds to the minimum size requirement specified by BlueGnome for detection of segmental aneuploidies. The entire protocol can be completed in less than 24 hours and therefore embryo transfer and vitrification of surplus euploid embryos can be scheduled for day-5 (figures 2 and 3). Results To date we have performed a total of 491 a-cgh PGS cycles, for couples who have had at least two previous IVF failures even with the transfer of good morphology embryos. The age of the women ranged from 29 to 44 years, and the difference in the incidence of chromosomal abnormalities correlated to maternal age : 69.4 % and 88.1 % of embryos were abnormal in women younger than 4 years (n = 274) or older than 4 years (n = 217) respectively. Complex abnormalities most frequently contributed to the increase in the total number of abnormalities with age (12.3 % vs % in the two age groups) as shown in Table 1. As expected, the increase in the incidence of chromosome abnormalities with maternal age is reflected in a decrease in the percentage of cycles with at least one normal embryo for transfer. In the youngest group of patients, 75.5 % of the cycles resulted in embryos suitable for transfer whereas this number decreased to only 37.8 % for women over 4 years of age. Pregnancy rates were also higher in the group of patients under 4 years compared to the oldest group (52.6 % and 41.5 % respectively), although they had similar miscarriage rates (Table 2). Most of these cycles were performed on day-3 embryo biopsies, and in only 24 of the cycles blastocyst biopsy with deferred embryo transfer was carried out. In this subset of patients, pregnancy rates per transfer were 73.3 % with an Figure 1. Day-3 embryo biopsy scheme. 114

4 Results <24hours Sample 1 Sample 2 Biopsy Cell (s) loading Amplification (~ 3 hrs) (98.3%) Cy3 Cy5 Labelling (2 hrs) DNA precipitation (~ 1 hrs) 24sure BlueGnome 2684 clones IMb coverage BlueFuse Multi software Hybridisation (5-12 hrs) Washing (~ 1/2 hr) Scanning Figure 2. CGH array flowchart. implantation rate of 58.3 % and the percentage of abnormal embryos using this approach was 57.8 % which is significantly lower than when day-3 biopsies were used (77. %;p<.5). These clinical outcomes were also evaluated according to the number of previous implantation failures, in this case only considering cycles from patients younger than 4 years of age. We found that the percentage of abnormal embryos was not affected by the number of previous implantation failures (71.9 % with two failures and 69.5 % with six or more previous failures) but that there was a decrease in the percentage of embryos with a chaotic division pattern (from 18.6 % with two failures to 8.5 % with six or more failures). We concluded that the clinical outcome or the chances of a successful pregnancy are not affected by the number of previous implantation failures (figure 4). The contribution of sperm concentration to the outcome of RIF cycles is shown in figure 5 : again, only cycles from patients younger than 4 years of age were considered. There were higher pregnancy and implantation rates in cycles in which sperm counts were less than 1 million sperm/ml, however, the percentage of chromosomally abnormal embryos did not differ for different sperm concentrations. These results could indicate that when a sperm factor is identified as a potential cause of poor implantation in regular IVF cycles, the selection of chromosomally normal embryos for transfer may be able to improve pregnancy rates per transfer to more than 6. %, with the previous failures in the remaining cases likely resulting from other undefined causes. The number of MII-stage oocytes may also contribute to the outcome of patients with RIF. In patients younger than 4 years of age the main limitation of the reduced number of oocytes was that there were possibilities for fewer embryo transfer cycles ; in patients with less than 5 MII oocytes embryos were transferred in 6 % of cycles whereas this was over 9 % when more than 16 MII oocytes were retrieved. However, the percentage of abnormal embryos, as well as pregnancy and miscarriage rates, were similar independently of the number of MII-stage oocytes obtained. The parameter mostly affected by the number of MII oocytes is the percentage of cycles with transfer and therefore the pregnancy rate per cycle, with a significant decrease 1 MII oocytes (33.6) compared to the group of cycles performed with more than 1 MII (46.8 ; p=.3) (figure 6). Conclusions Different therapy options have been offered to RIF patients with unknown etiology in recent years. The data on the positive effect of assisted zona pellucida hatching 115

5 Mini-revue Fused Chart (Sample vs Male References) (GRCh37) Log2 Ratio Ch1/Ch2 Chromosomal Position Euploid embryo X Y Fused Chart (Sample vs Male References) (GRCh37) Log2 Ratio Ch1/Ch2 Chromosomal Position Loss of chromosome X Y Figure 3. Examples of CGH arrays profiles. Table 1. Chromosomal abnormalities according to maternal age < 4 years 4 years Total Mean MII Mean analyzed No. informative embryos % abnormal embryos 69.4 a 88.1 a 77. % chaotic pattern 13.5 b 16.3 b 14.6 % complex abnormal 12.3 c 25.2 c 17.6 % segmental abnormalities Chi-square test a,b p<.5 on clinical outcomes in RIF patients were controversial [1, 14]. Similarly, some centers, including ours, have chosen to culture embryos to the blastocyst stage to improve the outcome for these patients. In one study published by our group [15] there was a favorable outcome when Table 2. Clinical results according to maternal age < 4 years 4 years Total No. of cycles Mean Age No. of transfer 75.5 a 37.8 b 58.8 Mean embryos transferred Pregnancy rate Implantation rate Miscarriage rate Chi-square test a p<.5 embryos were co-cultured to the blastocyst stage before embryo transfer compared with transferring them on day- 2, although oocytes from patients with RIF showed less favorable results, indicating that RIF should not have been caused by embryonic causes in ovum donation. In another study, pregnancy and implantation rates were shown to 116

6 failures 3 failures 4 failures 5 failures 6 failures % transfer Pregnancy rate Implantation rate Miscarriage rate Figure 4. Clinical Outcome according to the number of previous failures < 5 mill/ml (n = 42 cycles) 5-1 mill/ml (n = 33 cycles) 11-2 mill/ml (n = 42 cycles) 5 > 2 mill/ml (n = 157 cycles) % transfer Pregnancy rate Implantation rate Miscarriage rate Figure 5. Clinical outcome according to the sperm concentration. 117

7 Mini-revue <=5 MII (n = 25 cycles) 6-1 MII (n = 19 cycles) MII (n = 89 cycles) 16-2 MII (n = 33 cycles) > 2 MII (n = 17 cycles) % transfer Pregnancy rate Implantation rate Miscarriage rate Figure 6. Clinical outcome according to the number of MII oocytes retrieved. decline dramatically in repeated IVF cycles with blastocyst transfer [16]. Both of these studies imply that blastocyst culture alone may not improve the clinical outcome of repeated IVF attempts and that there may be intrinsic problems with the embryos from these patients. Indeed, a recent study using CCS found a moderate relationship between blastocyst morphology and euploidy [17], and the ability of these embryos to implant seemed to be determined mainly by the chromosomal complement of preimplantation embryos rather than the developmental and morphological parameters conventionally used for blastocyst evaluation. RCTs with RIF patients which used FISH showed a trend towards better live birth rates with PGS in a selected group of these patients for whom other potential causes had already been discarded [7]. Until now, FISH has been the most widely used technique for aneuploidy screening, but there is clearly a need for a technique capable of analyzing all the chromosomes, while producing reliable and faithful results in a short period of time so that embryo cryopreservation can be avoided. Several approaches towards 24-chromosome analysis have already been developed, and preliminary CGH studies have suggested a promising future for its application in PGS [18-21]. Despite this, to date there have been no RCTs which address the use of CCS with acgh in RIF patients. Our group performed a retrospective study compiling data from more than 188 cycles of couples suffering RIF, which supported the application of conventional aneuploidy screening for this group of patients [22]. However, the clinical outcome following analysis of all 24 chromosomes did improve pregnancy and implantation rates for different indications (including RIF) to a higher degree than the previously available technology (FISH) in which only a limited number of chromosomes could be analyzed. Keltz et al. also found that CCS led to a reduction in the number of the embryos transferred, reduced multiple pregnancy rates, and resulted in a trend towards lower miscarriage rates [23]. In summary, our results from 467 couples with repetitive implantation failure whose embryos were screened for aneuploidy in all 24 chromosomes showed that there are different factors that affect the clinical outcomes. The best prognosis can be expected in couples younger 4 years of age, with a sperm concentration lower than 1 million sperm/ml, and there is a higher probability of successful implantation when more than 15 MII-stage oocytes are retrieved before embryo transfer. The number of previous failures only increases the probability of couples producing embryos with a complex division pattern but 118

8 does not impact their overall clinical implications. Nevertheless, new RCTs should be conducted in the near future to assess the feasibility of different platforms and biopsy approaches for clinical practice and to test their potential to increase live birth rates, while also performing a more comprehensive aneuploidy screening for different indications, including RIF [2, 23]. Disclosures: References The authors have nothing to disclose. 1. Munne S, Cohen J. Chromosome abnormalities in human embryos. Hum Reprod Update 1998;4: Munne S, Wells D, Cohen J. Technology requirements for preimplantation genetic diagnosis to improve assisted reproduction outcomes. Fertil Steril 21 ; 94 : Meseguer M, Herrero J, Tejera A, et al. The use of morphokinetics as a predictor of embryo implantation. Hum Reprod 211 ; 26 : Rubio C, Rodrigo L, Mercader A, et al. Impact of chromosomal abnormalities on preimplantation embryo development. Prenat Diagn 27 ; 27 : Alfarawati S, Fragouli E, Colls P, et al. The relationship between blastocyst morphology, chromosomal abnormality, and embryo gender. Fertil Steril 211 ; 95 : Pehlivan T, Rubio C, Rodrigo L, et al. Impact of preimplantation genetic diagnosis on IVF outcome in implantation failure patients. Reprod Biomed Online 23;6: Rubio C, Bellver J, Rodrigo L, et al. Preimplantation genetic screening using fluorescence in situ hybridization in patients with repetitive implantation failure and advanced maternal age : two randomized trials. Fertil Steril 213 ; 99 : Simon C, Mercader A, Garcia-Velasco J, et al. Coculture of human embryos with autologous human endometrial epithelial cells in patients with implantation failure. J Clin Endocrinol Metab 1999 ; 84 : Gianaroli L, Magli MC, Ferraretti AP, Munne S. Preimplantation diagnosis for aneuploidies in patients undergoing in vitro fertilization with a poor prognosis : identification of the categories for which it should be proposed. Fertil Steril 1999 ; 72 : Magli MC, Gianaroli L, Ferraretti AP, Fortini D, Aicardi G, Montanaro N. Rescue of implantation potential in embryos with poor prognosis by assisted zona hatching. Hum Reprod 1998;13: Blockeel C, Schutyser V, De Vos A, et al. Prospectively randomized controlled trial of PGS in IVF/ICSI patients with poor implantation. Reprod Biomed Online 28 ; 17 : Simpson JL. Preimplantation genetic diagnosis to improve pregnancy outcomes in subfertility. Best Pract Res Clin Obstet Gynaecol 212 ; 26 : Rubio C, Mercader A, Alama P, et al. Prospective cohort study in high responder oocyte donors using two hormonal stimulation protocols : impact on embryo aneuploidy and development. Hum Reprod 21 ; 25 : Bider D, Livshits A, Yonish M, Yemini Z, Mashiach S, Dor J. Assisted hatching by zona drilling of human embryos in women of advanced age. Hum Reprod 1997 ; 12 : Rubio C, Rodrigo L, Mercader A, et al. Impact of chromosomal abnormalities on preimplantation embryo development. Prenat Diagn 27 ; 27 : Shapiro BS, Richter KS, Harris DC, Daneshmand ST. Dramatic declines in implantation and pregnancy rates in patients who undergo repeated cycles of in vitro fertilization with blastocyst transfer after one or more failed attempts. Fertil Steril 21 ; 76 : Capalbo A, Rienzi L, Cimadomo D, et al. Correlation between standard blastocyst morphology, euploidy and implantation : an observational study in two centers involving 956 screened blastocysts. Hum Reprod 214 (Epub ahead of print). 18. Voullaire L, Collins V, Callaghan T, McBain J, Williamson R, Wilton L. High incidence of complex chromosome abnormality in cleavage embryos from patients with repeated implantation failure. Fertil Steril 27 ; 87 : Schoolcraft WB, Fragouli E, Stevens J, Munne S, Katz-Jaffe MG, Wells D. Clinical application of comprehensive chromosomal screening at the blastocyst stage. Fertil Steril 21 ; 94 : Rubio C, Rodrigo L, Mir P, et al. Use of array comparative genomic hybridization (array-cgh) for embryo assessment : clinical results. Fertil Steril 213 ; 99 : Fragouli E, Wells D, Whalley KM, Mills JA, Faed MJ, Delhanty JD. Increased susceptibility to maternal aneuploidy demonstrated by comparative genomic hybridization analysis of human MII oocytes and first polar bodies. Cytogenet Genome Res 26 ; 114 : Rodrigo L, Mateu E, Mercader A et al. New tools for embryo selection : comprehensive chromosome screening by array comparative genomic hybridization. BioMed Research International Volume 214, Article ID Keltz MD, Vega M, Sirota I, et al. Preimplantation genetic screening (PGS) with Comparative genomic hybridization (CGH) following day 3 single cell blastomere biopsy markedly improves IVF outcomes while lowering multiple pregnancies and miscarriages. J Assist Reprod Genet 213 ; 3 :