Dans les échecs de PMA, plusieurs facteurs peuvent affecter le développement embryonnaire jusqu'au stade blastocyste : la stimulation ovarienne inappropriée ; la maturation ovocytaire (Moore et al ; 1998), l'âge maternel, les facteurs paternels (Schoolcart et al 1999, Janny et Menezo 1994) ; le manque de facteurs de croissance (KYE 1997) ainsi que la présence d'anomalies chromosomiques ou nucléaires (Munne et al 2002, Kuliev et al 2003).
Au stade pré-implantatoire un grand nombre d'embryons présentent des mosaïques et des mixoploïdies chromosomiques (Benk et al, 1993 ; Bielenska et al 2002). La qualité ainsi que le potentiel de développement embryonnaire peuvent varier en relation du degré du désordre génomique, la morphologie, la fragmentation cellulaire et l'intégrité nucléaire. Il a été suggéré que l'apparition de fragmentation de l'ADN au stade pré-implantation puisse être associée à une apoptose qui peut causer la perte de blastomères ou la mort de l'embryon en totalité.
Dans cette étude, nous avons analysé la fragmentation de l'ADN nucléaire par apoptose avec la technique TUNEL fluorescente in situ et une estimation des aneuploïdies majeures par FISH multicouleur dans des embryons bloqués et/ou avec un retard de développement. Parallèlement, des morulas bloquées ont été analysées par des puces à ADN après amplification génomique totale par DOP PCR pour avoir une idée globale du profile génomique de ces embryons par l'analyse de 24 chromosomes.
L'analyse de 52 embryons par TUNEL et FISH a démontré un pourcentage très élevé d'anomalies chromosomiques et d'apoptose dans les embryons bloqués et/ou avec un retard de développement. Nous n'avons pas observé d'aneuploïdies avec une simple trisomie pour les chromosomes analysés (5 chromosomes).
La majorité des anomalies détectées sont des monosomies ou des aneuploïdies multiples. Mais d'autres facteurs peuvent expliquer l'arrêt embryonnaire parce qu'on a observé des embryons arrêtés avec un statut nucléaire et chromosomique normal. Après amplification génomique totale de l'ADN des morulas bloquées, l'utilisation d'un ADN de référence et l'hybridation sur une puce à ADN (1Mb), on a observé un mosaïcisme bas qui peut toucher tous les chromosomes.
On pense qu'avec la technique de FISH classique où l'on cherche les aneuploïdies les plus observées en diagnostique prénatal, on n'analyse peut être pas les meilleurs chromosomes candidats au blocage embryonnaire précoce (chromosomes 1, 9, 14 et 15, 16, 22).
La combinaison de la technique TUNEL avec la FISH explique bien le statut nucléaire des embryons aux premiers stades du développement mais l'inconvénient de ces deux techniques c'est qu'elles nécessitent la fixation du matériel analysé ; donc elles se présentent comme des outils de diagnostique et non un test de viabilité embryonnaire. Après biopsie embryonnaire, soit au stade précoce ou au stade tardif, l'amplification génomique et l'hybridation sur puce à ADN, nous donnera le profile génomique des 24 chromosomes sous 48 heures, ce qui représente une nouvelle alternative à l'hybridation in situ en séquentielle pour le diagnostic des aneuploïdies au stade pré-implantatoire.
In human IVF procedures. It has been proposed that during extended culture, good quality embryos with superior developmental potential are eliminated from the presumably "abnormal" ones showing poor embryo quality and development (Huisman et al., 1994).
There are number of factors that have been shown to affect the survival of the embryos beyond 8-cell or up to the blastocyst stage. Among them are inappropriate stimulation regimens, oocyte maturation and suboptimal culture conditions (Moor et al., 1998), maternal age and paternal factors (Janny and Menezo et al., 1994, Jones et al., 1998; Schoolcraft et al., 1999), lack of growth factors (Kaye, 1997) and the presence of chromosomal and/or nuclear abnormalities (Munne et al., 1995). There can also be some intrinsic factors that may trigger the elimination of embryos with low developmental potential (Hardy, 1999). A large population of human embryos are mosaics, that is, while some cells are diploid, there exist some blastomeres with chromosomal abnormality (Harper, 1995; Bielanska et al., 2002). Depending on the degree of abnormalities as well as the proportion of cells with normal/abnormal chromosomal content, embryo morphology and developmental competence as well as quality can vary.
Cellular and nuclear fragmentation are among common observations during apoptosis in a variety of somatic cells, it has been suggested that appearance of these fragmentation patterns in preimplantation embryos could also be associated with apoptosis, leading to loss of certain blastomeres or a death of a whole embryo (Jurisicova et al., 1996; Levy et al., 1998).
In this study, we aimed to analyze the degree of DNA fragmentation and the extent of chromosomal abnormalities simultaneously on slow growing or arrested poor quality human embryos by applying TUNEL, FISH and micro Arrays techniques for genome profiling.
Materials and methods
This study was approved by Internal Review Board of Istanbul Memorial Hospital and informed consent was taken from each couple participated in this study. 60 embryos of 27 couples with male infertility were analyzed. Patients were selected in such a way that they either generated mostly (or all) embryos with poor quality hence donated their spare arrested or slow growing embryos that were eliminated before embryo transfer.
Embryo manipulation and fixation
Embryos were termed as arrested when no further development were observed after 24 hours of extended culture. For fixation, embryos were first transfered onto hypotonic solution droplet and treated with HCl-tween 20. After the total digestion, remaining cytoplasm was removed with methanol/acetic acid (3/1, v/v) solution and the nuclei were fixed onto the slide. The slides were then allowed to air dry at room temperature for 15 min.
Assesment of DNA fragmentation
DNA fragmentation was assessed by In Situ Death Detection Kit (1684795 - Roche Diagnostics) with the following protocol: Labeling solution is prepared by mixing 2.5 ul of solution 1 and 22.5 ul of solution 2 and added on each slide which is then covered with a large coverslip. Slides are then covered by aluminium folio and put into a humidity chamber for incubation at 37C for 45 minutes. After incubation, slides are washed with PBS for three times (1 minute for each). Slides are then left for air drying. Before the evaluation, 8 ul of DAPI is applied onto each slide and the slide is examined under a fluorescent microscope. Embryos having no fragmentation on their blastomeres were processed for fluorescent in situ hybridization. On the other hand, embryos having partial or complete fragmentation were excluded from the FISH part.
Fluorescent In Situ Hybridization
After the initial denaturation for 5 minutes at 73 ÞC the slides were hybridized with 3 ul of probe mix at 37ÞC. FISH analyses were performed using DNA probes specific for chromosomes 13, 18, 21, X and Y. Following hybridization step in a hybridization chamber (Hybrite, Vysis) at 370C nearly 8 hours, post-hybridization washes were performed in 0,4xSSC,0,3%NP40 for 2 min at 730C and 1 min in 2xSSC,0,1%NP40 at room temperature. After the drying of slides 10ml of DAPI was added onto each slide and slides were evaluated under a fluorescent microscope (Olympus BX 50, Japan), with recommended filters.
Genome profiling using BACs Micro Array
Embryos were treated for zona pellucida digestion. The total genome of each embryo was amplified by DOP PCR using Roche protocole with modification. After total genome DOP PCR of somatic reference DNA, both pCR products were labelled by Random priming ( Test Cy3 and control Cy5 and the reverse), as well as Cot –1 blocking DNA, are mixed and preciptated together. The pellets are dissolved in a hybridisation solution. After denaturation and pre-hybridisation, the labelled DNA solution were hybridised th the bAC arrays for 16 hours. A post hybridisation wash of the arrays was performed on 50% formamide solution and distilled water and the dried. The BAC arrays were scanned on a two color fluorescent scanner and the images were analysed using a commercial software.
Results and discussion
52 of 60 embryos ( 52 embyos for FISH/TUNEL and 8 for genome profiling ) were used for simultaneous apoptosis and aneuploidy assessment. After fixation, fluorescent in situ TdT-mediated dUTP nick end labelling (TUNEL) technique was applied and embryos which were negative for TUNEL were consecutively analysed by fluorescent in situ hybridisation (FISH) for chromosomes 13, 18, 21, X and Y. From 52 fixed embryos, 31 (54.3%) showed DNA fragmentation with TUNEL technique. In 26 embryos that were negative for TUNEL, interpretable FISH results were obtained for 25 embryos (96.2%). Nine were found to have normal chromosomal constitution for the chromosomes analysed (36.0%). However, 16 embryos were detected to be aneuploid (64.0%); 3 were found to be chaotic, 6 had complex aneuploidy, 6 had complete monosomy and 1 was polyploid. Our results show that high level of DNA fragmentation and aneuploidy are common on embryos with slow growth and/or low quality. These high rates can be due to early genetic or epigenetic events in preimplantation development leading to DNA fragmentation and/or developmental blocking. More detailed studies are needed to assess the effect of factors such as ovarian stimulation regimens and in vitro culture conditions. Moreover, application of simultaneous TUNEL and FISH techniques can be informative about the DNA integrity and aneuploidy.
Furthermore, analyzed embryos were classified according to their developmental stages. Among 8 prezygotes that were found to be arrested on day 1 of embryo development, 5 contained fragmented DNA (62.5%) and only 3 or them could be analyzed by FISH. Two of the 3 prezygotes analyzed contained normal chromosomal content for the chromsomes analyzed and in one both trisomy 18 and nonosomy X were detected. In 20 embryos, which were found to be either arrested on day 2 or slow growing on day 3, DNA fragmentation was observed in 12 of them (60.0%). For 8 embryos on which FISH were applied, only one of them was found to be normal. All embryos analyzed by FISH contained cellular fragmentation ranging from 10 to 20%. As a third group, 21 embryos, either arrested on day 3 or slow growing on day 4 were documented and 9 of them contained fragmented DNA (42.8%). Out of 12 embryos selected for FISH, 7 embryos were found to be chromsomally abnormal. Interestingly, almost all abnormal embryos contained variable degree of cytoplasmic fragmentations, which was hardly observed in embryos with normal chromosomal content. Only 8 embryos were analyzed for day 4-arrested/day 5-slow growth pattern and for them, 5 were positive for TUNEL assay (from the same patient). For three TUNEL negative embryos, however, in one of them FISH was not successful, one embryo was found to be normal, the other contained monosomy 22
Our results and previous observations in such a way that majority of the slow growing or arrested embryos were found to contain high percentage of DNA fragmentation and chromosomal abnormalities. Furthermore, we did not observe any aneuploidies containing a single trisomy of the chromosomes analyzed. In fact, majority of the abnormalities detected were monosomies or contain multiple abnormalities containing monosomies and double trisomies. In combination with the results of Bielanska et al., these data suggest that two forms of chromosomal abnormalities are very common among arrested embryos and they are eliminated during the first and the third cleavage (Bielanska et al., 2002). It is also consistent with the rarity of mutiple chromosome abnormalities, and the very low rates of double trisomies found among spontaneous abortions (Reddy, 1997).
There are also several retrospective studies performed on embryos in PGD cycles showing that some embryos of abnormal chromosomal structure (mostly trisomies) can efficiently reach blastocyst stage whereas some chromosomally normal embryos, although they are in good morphology, may fail to undergo differentiation in extended culture (Magli et al., 2000, Sandalinas et al., 2001). In this study, we have also observed several arrested embryos that had intact DNA structure and normal chromosome content for the five chromosomes analyzed. A common observation on these embryos was that they did not show significant cellular fragmentation and all of them were arrested between 3-8 cell stages. However whether they contained abnormalities on other chromosomes were not determined hence not known.
It has been suggested that most of the chromosomal abnormalities can arise during oogenesis and cell cycle checkpoints are not operational at this stage as well as during early cleavage divisions (Handyside and Delhany, 1997). Chromosome specific meiotic or mitotic errors may be a consequence of misalignment on a disorganized spindle and a consequent non-functional metaphase-anaphase checkpoint control (LeMarie-Adkins et al., 1997; Harrison et al., 2000).
Generally embryo culture media, conditions and culture systems are usually the first ones to be questioned when abnormal embryo development is observed. However, current literature showed that, most of the developmentally blocked or poor quality embryos have in fact intrinsic problems that can probably be traced back to gamete cell environment.
We have found that arrested or slow growing embryos of poor quality have in fact higher rate of DNA fragmentation and chromosomal abnormalities. We have also observed that the first technique was very advantegous for detecting nuclear fragmentation since it allows both localization and the quantification of the degree of nuclei with DNA fragmentation. Additionally, embryos having intact nuclear DNA could be processed for multicolour FISH analysis without further procedures or special treatments.
The combination of TUNEL and FISH analysis can be more effective in explaining the relative nuclear status of the developing embryos as well as blastomeres and spermatozoa analyzed. However, since both techniques necessitates the fixation of the samples analyzed, they can only be used as diagnostic purposes and not on viable human embryos whereas they can efficiently be used as predictors of sperm genome quality. The application of total genome application by DOP PCR from single or few cell and the microarrays CGH using 1 MB chip can be a new alternative to aneuploidy testing by FISH on preimplantation genetic diagnosis.