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Morphokinetics and embryo aneuploidy: has time come or not yet?

Published:March 28, 2013DOI:https://doi.org/10.1016/j.rbmo.2013.03.011

      Abstract

      Morphokinetic analysis of early human embryos in combination with trophectoderm biopsy and chromosomal enumeration by array technology has been shown to identify embryos with single and/or multiple aneuploidies. In a time-lapse imaging study, aneuploid embryos showed a delayed initiation of blastocyst formation and also reached the full blastocyst stage later compared with euploid embryos. Based on these findings, a model for a risk factor classification for aneuploidy of human embryos has been established. However, time values that allow differentiation between different risk classes have to be considered carefully as timings that are prognostic for one clinic may not apply directly to another clinic. Thus, risk models may need to be verified in specific IVF settings. Further studies are needed to verify that time-lapse imaging does allow subselection of embryos with a high aneuploidy risk and thus does hold the chance to restrict invasive biopsy procedures to those embryos where there is uncertainty regarding the chromosomal status.

      Keywords

      Time-lapse imaging of early human embryo development is increasingly used in the embryology laboratory as part of assisted reproduction treatment. It enables the change of embryo morphology to be followed over time and to document these changes in a database for detailed evaluation, which is also known as morphokinetic analysis. An increasing number of publications report on the variation of the time points for specific developmental stages as well as on the duration of cell-cycle length as prognostic markers for the potential of an embryo to implant (
      • Herrero J.
      • Meseguer M.
      Selection of high potential embryos using time-lapse imaging: the era of morphokinetics.
      ,
      • Wong C.
      • Chen A.A.
      • Behr B.
      • Shen S.
      Time-lapse microscopy and image analysis in basic and clinical embryo development research.
      ).
      In the May issue of Reproductive BioMedicine Online,
      • Campbell A.
      • Fishel S.
      • Bowmann N.
      • Duffy S.
      • Sedler M.
      • Hickmann C.F.L.
      Modelling a risk classification of aneuploidy in human embryos using non-invasive morphokinetics.
      go one step further and describe the use of time-lapse imaging and morphokinetic analysis to identify the risk of embryos for having a single- or multiple-aneuploid chromosome constitution. For the detection of the ploidy status, they used a single-nucleotide polymorphic array and array comparative genomic hybridization from trophectoderm biopsies. Due to biopsy on day 5, there was no effect of blastomere removal and zona opening on embryo morphokinetics, as would have likely been the case with biopsy on day 3. Among all embryos that were diagnosed, the rate of aneuploidy was 60%. The study revealed for embryos with single or multiple aneuploidies a delayed initiation of blastocyst formation. Furthermore, aneuploid embryos reached the full blastocyst stage later compared with euploid embryos. Based on this, the authors proposed a risk model with three categories. For the patients included in this study, 20% (12/59) of all aneuploid embryos fell into the high-risk group with morphokinetic characteristics that enabled a 100% cut off without overlap. For the medium-risk group, the chance of having a single- or multiple-aneuploid embryo was 69% (34/49) and for the low-risk group it was 36% (13/36). The authors concluded that using their risk model would allow the restriction of biopsy to less embryos – mostly those in the low- or medium-risk groups – depending on the perception of the couple.
      The idea of using time-lapse imaging and morphokinetic analysis is intriguing, because having available a completely non-invasive procedure to predict which embryo is euploid or aneuploid would allow the application of this technique for virtually every assisted reproduction cycle. The potential benefit of such an approach is obvious in view of published data on the incidence of aneuploidy even in oocytes from younger women (
      • Munnè S.
      • Ary J.
      • Zouves C.
      • Barnes F.
      • Cinioglu C.
      • Ary B.
      • Cohen J.
      Wide range of chromosome abnormalities in the embryos of young egg donors.
      ,
      • Yang Z.
      • Liu J.
      • Collins G.C.
      • Salem S.A.
      • Liu X.
      • Peck A.C.
      • Sills E.S.
      • Saleem R.D.
      Selection of single blastocyst for fresh transfer via standardized morphology assessment alone and with array-CGH for good prognosis IVF patients: results from a randomized pilot study.
      ).
      The first approach to using time-lapse imaging in combination with array-comparative genomic hybridization for aneuploidy detection was presented at ESHRE 2012 (
      • Davies S.
      • Christopikou D.
      • Tsorva E.
      • Karagianni A.
      • Handyside A.H.
      • Mastrominas M.
      Delayed cleavage division and a prolonged transition between 2- and 4-cell stages in embryos identified as aneuploidy at the 8-cell stage by array-CGH.
      ). It was based on biopsy on day 3 and reported for complex aneuploidies a later division to the 2-cell stage (t2 = time from insemination to division to 2 cells) and a longer time spend in the 3-cell stage (s2 = time period of the synchrony of the second cell cycle). Similarly another time-lapse imaging study reported that up to the 4-cell stage, euploid embryos exhibit precise cell-cycle variables, whereas only 30% of the aneuploid embryos show variables that fall into normal timing windows if assessed by automatic detection software (
      • Chavez S.L.
      • Loewke K.E.
      • Han J.
      • Moussavi F.
      • Colls P.
      • Munné S.
      • Behr B.
      • Reijo Pera R.A.
      Dynamic blastomere behaviour reflects human embryo ploidy by the four-cell stage.
      ). These results implied that the majority of aneuploid embryos had aberrant timings up to the 4-cell stage. However, this study only relied on reference timings from eight euploid embryos, which is probably too small a number to establish normal cell-cycle variables.
      In contrast, the study presented here by Campbell and colleagues for 38 euploid embryos versus 60 aneuploid embryos showed no significant difference with regard to the timing of early events in development (t2; t3 = time from insemination to division to 3 cells; cc2 = time period of the second cell cycle (t3 − t2) from 2 to 3 cells; s2). Whether this is due to aneuploidy testing on day 5 using trophectoderm cells instead of day-2–3 blastomeres, as in the previously mentioned studies, remains unclear at present.
      Campbell and colleagues established a risk classification model. Although this by itself is a major step forward, there is still a long way to go before having an absolute classification as to which embryo is 100% euploid or 100% aneuploid.
      Further, like most studies publishing absolute values for timing events, one should be careful to transfer the time values presented in this study to another clinical setting. Timing of embryo development is a process, which is influenced by numerous variables from the laboratory side and presumably also from the patient side. Temperature and pH are the most obvious denominators, but culture medium does play a role too (
      • Ciray H.N.
      • Aksoy T.
      • Goktas C.
      • Ozturk B.
      • Bahceci M.
      Time-lapse evaluation of human embryo development in single versus sequential culture media – a sibling oocyte study.
      ). So defining the exact boundaries for the timing of the different risk classification groups in the proposed model is important, as variations should be expected for individual laboratories.
      Time-lapse imaging should not be considered as a competitive methodology replacing preimplantation genetic screening (PGS). Nevertheless, the recent findings may influence how PGS is performed in several ways. Time-lapse imaging in a closed incubator does improve blastocyst formation rates and hence allows for having more blastocysts on day 5 from which to select. Independently of the number of blastocysts that are available for biopsy on day 5, it would be beneficial to restrict the diagnosis to those blastocysts where the risk classification model does not allow for a clear discrimination. An economic approach would be to biopsy all embryos but to diagnose initially only those that are considered at low risk while vitrifying the remaining blastocysts scored with a medium-to-high aneuploidy risk for eventual later diagnosis and a potential transfer. This in itself would cut the cost for array-based diagnosis of PGS by at least one-third.
      Last but not least, one should not forget that there are couples having difficulties in accepting an invasive procedure like trophectoderm biopsy but still wish to reduce their risk for failed implantation or early pregnancy loss due to aneuploidy. For these patients, time-lapse imaging may offer an alternate to PGS – knowing that in the low-risk group there is still a chance for having an aneuploid embryo – and it could be applicable to young women who have an overall risk of 30–50% for aneuploidy, according to recent publications (
      • Munnè S.
      • Ary J.
      • Zouves C.
      • Barnes F.
      • Cinioglu C.
      • Ary B.
      • Cohen J.
      Wide range of chromosome abnormalities in the embryos of young egg donors.
      ) and where one can expect a reasonable number of oocytes enabling an elective single-embryo transfer at the blastocyst stage on day 5.
      In view of the increasing usage of time-lapse imaging, it should be possible to get further results on the clinical value of the proposed risk model from other clinics within a reasonable time period. Further information is also needed in order to elucidate if the time values underlying the model can be affected by certain variables and. in particular, if maternal age has an influence too. In the study by
      • Campbell A.
      • Fishel S.
      • Bowmann N.
      • Duffy S.
      • Sedler M.
      • Hickmann C.F.L.
      Modelling a risk classification of aneuploidy in human embryos using non-invasive morphokinetics.
      , the indication for PGS was manifold: recurrent miscarriage, repeated implantation failure and severe male factor infertility as well as previous aneuploidy or advanced female age. Based on this heterogeneity, one could assume that the risk classification model in aneuploid embryos is not affected by the underlying patient characteristics. However, it remains the case that, for the overall timing of development, values for the time points of tSB (time from insemination to the start of blastulation) and tB (time from insemination to formation of a full blastocyst) can be different in other laboratory settings.
      This study by
      • Campbell A.
      • Fishel S.
      • Bowmann N.
      • Duffy S.
      • Sedler M.
      • Hickmann C.F.L.
      Modelling a risk classification of aneuploidy in human embryos using non-invasive morphokinetics.
      is the first to open a new field of applied clinical research that may ultimately lead to new concepts and strategies for PGS. The overall applicability needs to be shown by follow-up studies – and there is no doubt that these will come.

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