Publication: Can the noninvasive morphokinetic analysis by time‑lapse imaging replace embryo biopsy for preimplantation diagnosis?

Thursday, April 30th, 2015

ABSTRACT

Embryo selection has moved from static observations to a dynamic evaluation. Time-lapse imaging can be utilized more effectively for improving in vitro fertilization outcome using morphokinetic parameters to choose chromosomally competent or euploid embryos. This noninvasive evaluation has the future potential to reduce the need for embryo biopsy for preimplantation genetic diagnosis wherever there are expertise related, ethical, legal or economic reasons for not doing it. Key Words: Embryo biopsy, in vitro fertilization, morphokinetics, preimplantation genetic diagnosis, time‑lapse imaging

INTRODUCTION

Success of in vitro fertilization (IVF) treatment has remained low despite advances in the field of reproductive medicine. It is now estimated that about 60–90% of all transferred embryos in IVF cycles do not implant. Hence, it can be stated that a large proportion of implantation failures are probably attributable to embryonic factors more than anything else.[1]
Selecting the best possible embryo for transfer is a major challenge in assisted conception. The primary methods for selection of gametes and embryos for transfer are subjective and static. They are based on punctual, discontinuous observations providing limited information.[2,3] Embryos which are deemed most suitable for transfer are the ones that display precise growth observed at fixed times of development, e.g., fertilization (observation of pronuclei at 16–18 h postinsemination), syngamy (at 23 h); early cleavage (at 26 h postintracytoplasmic sperm injection and 28 h post‑IVF); day 2 cleavage (at 44 h); day‑3 cleavage (at 68 h); morula (at 92 h) and blastocyst formation (at 116 h). However, these standard checkpoints are not informative of particular cellular events and precise kinetics of embryonic development occurring between any two observations. Recently published studies suggest that the selection of embryo/s or blastocyst/s for transfer with the best potential for implantation should not be based only on number of cells and morphological assessment on the day of transfer.[4,5]

There is a need for reliable noninvasive methods for embryonic assessments to aid in the selection of the best embryos from a cohort available. At present, time‑lapse imaging has emerged as a promising tool for selecting embryos. The integration of incubation with safe controlled culture environment, together with inspection of dynamic events with the opportunity to observe the fertilization process and early development has provided a new direction in the evaluation of embryos.[6]

It is well‑known that aneuploidy plays a major role in implantation failure, and early miscarriage thereby affecting the live birth rates of assisted reproduction. Even the best morphological embryos have been found to be chromosomally abnormal. The inherent imprecision of single embryo transfer has been highlighted in a recent publication where a 44.9% aneuploidy rate was detected for blastocysts from patients with a good prognosis.[7] Comparative methodology for aneuploidy screening is rapidly replacing conventional preimplantation genetic screening (PGS) using the fluorescence in situ hybridization (FISH) technique. PGS, now referred to as comprehensive chromosomal screening for all the 24 chromosomes using the new simple nucleotide polymorphism (SNP) or comparative genomic hybridization (CGH) arrays, quantitative polymerase chain reaction (QPCR) or next generation sequencing, is more informative and accurate compared to the prior FISH techniques.[8,9]

Preimplantation genetic screening is being used to improve embryo selection and assisted reproductive outcomes. The approach to using time‑lapse together with PGS using array CGH for aneuploidy detection was first presented at ESHRE meeting in 2012.[10] Embryo biopsy remains an essential procedure for PGS which is invasive, and the expertise may not be readily available at all places. Furthermore, the embryo biopsy skill learning curve is mandatory to not affect embryos adversely. In addition, PGS may not be always possible to perform due to legal restrictions, social or simply cost‑related issues. Morphokinetic selection could be used for all the IVf cycles where the couples dont opt for PGD/PGS for ethical, economical or legal reasons.

Campbell et al. described the use of time‑lapse imaging and morphokinetic analysis to identify embryos at risk for having single or multiple aneuploid chromosome constitution.[11,12] They used SNP array and array CGH from trophectoderm biopsies. The aneuploidy rate was quoted as 60% in this study. The aneuploid embryos showed delayed initiation of blastocyst formation and reached full blastocyst stage later compared to euploid embryos.

A recent prospective randomized controlled trial has shown statistically significant improvement in reproductive out‑comes with the use of multivariate morphokinetic model using time‑lapse imaging.[13] Other studies have linked patterns of embryonic divisions to blastocyst formation, ploidy, and implantation success.[8,14,15]

Time‑lapse imaging is currently being evaluated in various trials with concomitant use of CGH.[8,9,11] When both techniques are employed together, the total number of embryos to be biopsied and screened for aneuploidy would be expected to be much less and less expensive. Therefore, using risk models created by these studies could significantly reduce the financial burden of aneuploidy screening and enable more couples to afford this costly technique.

Preimplantation genetic screening identifies euploid embryos to be transferred. However, the expertise is not available everywhere and the there is definitely a learning curve to acquire the skill. Hence, it is prudent to search for noninvasive alternatives for identification of normal embryos to improve the success in assisted reproduction.
Time‑lapse imaging provides continuous visual observation of embryo morphology and kinetics which may help in distinguishing euploid from abnormal embryos. Studies have been attempted to create risk models utilizing morphokinetics to select chromosomally normal embryos.[11,12] Based on the dynamic events, embryos can be classified as high risk or low risk with respect to chromosomal abnormalities. Hence, recognition of embryos at risk may aid in the selection of embryos for transfer that are more likely to be chromosomally normal. On the other hand, this approach may also help to reduce the number of embryos to be biopsied, thereby reducing the total cost of the procedure.

Chawla et al. [16] analyzed cleavage‑stage embryos morphokinetically, and day‑3 biopsy was performed for comprehensive chromosomal screening by CGH microarray. Some studies which have evaluated morphokinetic events and timings in correlation to embryonic aneuploidy have either used day‑3 or trophoectoderm biopsy and subsequently used FISH, CGH microarray or sequential nucleotide peptide (SNP) as a method of genetic analysis.

The different morphokinetic parameters are discussed below:

PRONUCLEI FADING

The time of pronuclei fading showed a significant difference between normal and abnormal embryos with array CGH analysis in the study by Chawla et al. [16] Similarly, Azzarello et al. [17] found that pronuclei breakdown occurred later in embryos resulting in a live birth compared to those that resulted in lower live birth rate. Lemmen et al. [18] showed early pronuclei breakdown as a good marker of embryo potential. However, Stevens et al. reported that time of pronuclei appearance, resolution, or size had no correlation to embryo aneuploidy utilizing trophectoderm biopsy and 24 chromosomes analysis with QPCR.[19] Some more recent publications also found no co-relation between time of pronuclei fading and aneuploidy utilizing trophectoderm biopsy and 24‑chromosome microarray.[9,10,20]

CYTOKINESIS

The results of the analysis by Chawla et al. [16] have shown that mean durations of t2, t5, CC2, CC3, and t5‑t2 differences are significantly different between normal and abnormal embryos suggesting that early cleavage divisions are delayed in aneuploid embryos compared to euploid embryos. They analyzed 460 embryos. The study by Davies et al. [10] and Basile et al. [21] supports these findings. Davies et al. reported a delayed first and second cleavage and prolonged transition between 2 and 4 cells in embryos with complex aneuploidies. Basile et al. [21] studied a total of 504 embryos and found a difference in kinetic behavior. Logistic regression analysis of their data revealed t5‑t2, followed by CC3 as the most relevant variables related to normal chromosomal content. On the basis of these results, the authors proposed an algorithm for embryo selection.

In contrast, Campbell et al.,[11] found that aneuploid and euploid embryos developed similarly up to the 8‑cell stage (day‑3) but had a significant delay in development in the periblastulation stage. They compared development parameters from 38 euploid embryos with 60 aneuploid embryos(total of 98 blastocysts) and created an aneuploidy risk model based on these observations. Multiple aneuploid embryos were found to be delayed at the initiation of compaction and time to reach the full blastocyst stage. In a study including 76 blastocysts observed with the EmbryoScope, Semeniuk et al. [20] showed that the timings of the first mitotic division, division from 2 to 3 cells, appearance of 4th blastomere and third mitotic division were not different between aneuploid and euploid embryos. In another study by Bayram et al. [22] utilizing day‑3 biopsy and FISH, 122 embryos were analyzed and no difference was found in cleavage time to the 8‑cell stage, duration or cleavage from 2‑ to 3‑cell or 3‑ to 4‑cell. However, they showed that euploid embryos had a shorter time to cleavage from 5‑ to 8‑cell. In the study by Friedman et al.,[23] 62.5% of all embryos exhibited normal timings for first cytokinesis, time of second mitosis, and synchrony of the third and fourth cell appearance. Using CGH microarray for chromosomal analysis, they showed that only 40% of the embryos with abnormal timings were euploid.

CONCLUSIONS

Substantial percentage of in vitro generated embryos tends to be chromosomally abnormal. Morphological parameters of embryos do not correlate well with chromosomal content. Time‑lapse imaging is a noninvasive technique and has become an integral part of the many IVF laboratories. Various studies have suggested dynamic morphokinetic analysis and risk models to identify normal embryos. Preliminary data suggest that this technique may assist in the selection of chromosomally competent embryos. This approach may enhance the success of assisted reproduction and promote single embryo transfer. Larger age‑adjusted data set or randomized controlled trials are necessary to draw clear conclusions. The important thing to note is that the data and risk models cannot be extrapolated from one lab to another. Specific risk models will need to be created for each laboratory and the parameters need to be validated with retrospective and prospective analysis over some time as the embryos can be affected by both external and internal factors specific to each laboratory.

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23. Friedman B, Chavez S, Behr B. Noninvasive imaging for the detection of human embryonic aneuploidy at the balstocyst stage. Fertil Steril 2012;98 Suppl 3:s38. Source of Support: Nil, Conflict of Interest: None declared.