Overview of the Embryo Selection Process and Indicators of Embryo Quality

Abstract: Overview of the Embryo Selection Process’ basic methods and its strong indicators of embryo quality. 

The present article focuses on representing the basic overview of peculiarities of the Embryo Selection process and comparison of different methods of Embryo Selection, used in Aassisted Reproduction technologies. Selection of the most viable embryo(s) for transfer has always been essential, as embryos that are cryopreserved are thought to have a reduced chance of implanting after thawing. Usually, one or two embryos that are considered to have the best chance of implanting are selected for transfer. Subsequently, supernumerary embryos with a good chance of implanting are selected for Cryopreservation and possible transfer in the future while remaining embryos are discarded. Embryo selection is performed using one or a combination of embryo characteristics. Available evidence suggests that, for the overall population, day 3 and day 5 selection yield similar results but better than zygote selection results. There has been revealed three basic methods [and their criterion] which are used for accurate selection of the most viable embryo(s) for transfer: 

1. Morphological evaluation; 
2. Preimplantation Genetic Screening; 
3. Time–lapse technology. Prospective studies correlating embryo characteristics with documented implantation potential, utilizing databases of individual embryos, are also needed.

Introduction

In–Vitro Fertilization therapy is closely associated with a high rate of multiple pregnancies, a consequence of the number of embryos transferred. In most In–Vitro Fertilization (IVF)Intracytoplasmic Sperm Injection (ICSI) programmes approximately one ongoing pregnancy in three is multiple. Sometimes, there is a great challenge in avoiding even twin pregnancies in Assisted Reproduction Technologies, and this can be accomplished with elective Single Embryo Transfer and a good Cryopreservation programme. The need to characterize embryos with optimal implantation potential is obvious.

One of the most important and unsolved problems in In–Vitro Fertilization is to decide which embryos are more suitable to implant and therefore should be transferred. In order to reduce the number of embryos transferred, it is vital to have very efficient selection criteria to identify those embryos that are more likely to implant and develop into pregnancy. Selection of the most viable embryo(s) for transfer has always been essential, as embryos that are cryopreserved are thought to have a reduced chance of implanting after thawing. This dilemma could be overcome, if it were possible to select embryos with a very high implantation potential. Culturing for a prolonged period of time until the blastocyst stage is a way of tackling this problem. However, as culture conditions are still imperfect, the longer culture lasts, the fewer embryos suitable for transfer are left. It would be more convenient if an equally effective selection could be performed, but at an earlier stage. In an attempt to establish better selection criteria, we decided to examine retrospectively the characteristics of embryos that all had resulted in an ongoing implantation. What is essential to emphasize is that the consequences of the application of these criteria should also be examined. Recent developments challenge this concept. Evidence is accumulating that all embryos can now be cryopreserved and transferred in subsequent cycles without impairing pregnancy rates. The only parameter that could possibly be improved by Embryo Selection would be time to pregnancy, if embryos with the highest implantation potential are transferred first.

In the majority of In–Vitro Fertilization cycles multiple embryos are created after Ovarian Hyperstimulation. The viability of these embryos, and as a consequence the chance for an embryo to successfully implant, is subject to biological variation. To achieve the best possible live birth rates after In–Vitro Fertilization while minimizing the risk for multiple pregnancy, one or two embryos that are considered to have the best chance of implanting are selected for transfer. The essence of the Embryo Selection procedure consists in ideal accuracy of the experts’ identification the high–quality embryos and in answering the integrative question: which of the identical embryos of good morphology, good cleavage rate and with adequate metabolic milieu should be selected for transfer? And which should be cryopreserved? Subsequently, supernumerary embryos with a good chance of implanting are selected for Cryopreservation and possible transfer in the future while remaining embryos are discarded.

Opinions vary as to whether single Embryo Transfer should or should not be accompanied by some strategy of optimized selection of the Embryo to Transfer. If Embryo Selection is pushed too far and embryos considered as having a high implantation potential are too strictly defined or if the proposed technique is too complicated or expensive, the number of Single Embryo Transfer cycles will remain low and the impact on the twinning rate limited. If on the other hand, only one embryo is transferred without using strict selection criteria, the pregnancy rates might drop below what is acceptable [50].

Documented ongoing implantation is the gold standard for a particular embryo’s competence. Published data show it to be a continuous biological variable, varying between 0% for the ‘worst’ and ,60% for the ‘best’ embryos. Labelling an embryo as a ‘top quality embryo’ or a ‘high implantation potential embryo’ or a ‘putative high competence embryo’ remains a clinically useful (when communicating with patients) but intrinsically oversimplified representation of this graduated implantation potential [50].

Efforts have been made to correlate embryo implantation potential with characteristics of follicles [122; 123; 124], the oocyte [6; 29; 86; 114; 115; 121; 123; 125; 133], the fertilized zygote [14; 70; 71; 72; 82; 99; 100; 101; 108; 135], the early cleaving embryo, either on day 2 [30; 51; 53; 67; 73; 74; 102; 103; 115; 117; 126; 138] or day 3 [21; 26; 27; 28; 47;48; 49; 61; 62; 66; 67; 92; 104; 110; 116; 119; 120; 127; 128; 129; 136], the morula [113] and the blastocyst [1; 21; 37; 39; 41; 45; 61;62; 80; 92; 93; 107; 119; 120; 126; 139].

Some studies are clinical, either randomized comparisons or cohort studies, which clearly stated the Embryo Selection Technique. Others are retrospective analyses correlating Pregnancy Rates (PR) and Implantation Rates (IR) with particular recorded embryo characteristics either used or not used as a selection method.

1. Morphological evaluation [method for Embryo Selection]

The ability to select the most viable embryo for transfer is an increasingly important aspect to an IVF Treatment cycle. Key considerations for selection of the most viable embryos include that the assessment be non–invasive and that it occurs in a timely fashion that can easily occur within the operations of the clinical laboratory. As such, methods to increase selection that are based on embryo morphology are an attractive option.

Preimplantation morphologic parameters are the standard criteria used in embryology laboratories as a means of selecting the assumed most viable embryos for transfer. Many different selection criteria and systems exist, all of which have been reported to have correlations with implantation and fetal development. However, none are totally definitive and none can offer anything greater than a 30–40% implantation rates in high–prognosis patients.

Successful outcomes after In–Vitro Fertilization (IVF) procedures depend on many factors, one of the most critical being embryo quality [94; 98]. Implementation of a good Embryo Scoring system has many potential benefits, such as:

1. Allowing proper selection of embryos for transfer, 
2. Standardizing embryo grading between embryologists, 
3. Reducing high order multiple births by adjustment of transfer number according to embryo quality, 
4. Permitting comparison of embryo quality between patient cycles, and 
5. Evaluation of different culture regimens [28].

Embryo quality is widely regarded as the single most important factor for the chance of a successful In–Vitro Fertilization Treatment (IVF Treatment) [54]. Scoring of embryo morphology [morphological evaluation] and cleavage rate is a clinically useful method in Embryo Selection and may serve as a proxy for embryo quality [10; 41; 45; 58)]. On the basis of multiple morphological characteristics at one or several stages of preimplantation development, embryos are selected for transfer [31; 50]. 

In the connection with the above, the key objective of morphological evaluation is to establish which Embryo Score variables are the most essential for high–quality Embryo Selection process for further Single Embryo Transfer (SET) at the early cleavage stage. That is why at the present stage, perfecting the existing systems of identifying high–quality embryo during Embryo Selection process and developing new higher–precision ones are among priorities in the theory and practice of improving expert systems for special purposes of morphological evaluation, which is a considerable challenge for embryologists.

This objective presupposes realizing the following tasks: 

1. To characterize the basic morphological characteristics [scoring] of the top–quality embryo which should be taken into consideration when selecting embryo for further transfer; 
2. To compare characteristics revealed through retrospective analysis of the scientific theories, based on scientific researches.

Analyzing criterions of morphological evaluation, established by scientists Holte J., Berglund L., Milton K., Garello C., Gennarelli G., Revelli A., Bergh T. there was investigated whether the five commonly used embryo variables, namely: 

1. Blastomere Number (BL)
2. The proportion of mononucleated blastomeres (or presence of Multinuclearity; NU), 
3. The degree of Fragmentation (FR), 
4. The size variation of the blastomeres (equality, EQ) and
5. The symmetry of the embryo (SY), were independent predictors of implantation potential of the embryo [58]. 

Of those five variables, only three were independent predictors in multivariate regression analysis: 

1. Blastomere Number (BL), 
2. The size variation of the blastomeres (equality, EQ) and 
3. The proportion of mononucleated blastomeres (or presence of multinuclearity; NU). 

Although also significant in univariate testing, the variables degree of Fragmentation (FR) and symmetry of the embryo (SY) did not retain significance in the multivariate model. The Holte et al. (2007) study was conducted on Double Embryo Transfers (DET), and thus the primary outcome was either the implantation of both or neither of the transferred embryos. The study resulted in a clinically useful algorithm, the Integrated Morphology Cleavage score (IMC), which enables evidence–based scoring of embryos on Day 2 after Oocyte Retrieval in a 10–point scale. The Integrated Morphology Cleavage score (IMC) forms an essential part of a validated prediction model as an aid in the decision between Single Embryo Transfer (SET) and Double Embryo Transfer (DET) [59], a model which has been in clinical use in several clinics for a number of years.

Further investigations of criterions of morphological evaluation, made by Rhenman A., Berglund L., Brodin T., Olovsson M., Milton K. Hadziosmanovic N., Holte J., on the basis of classic embryo morphological markers and their relation to chance of a successful In–Vitro Fertilization (IVF)/ Intracytoplasmic Sperm Injection (ICSI) Treatment, have led to revealing that the Embryo Selection model, derived from only Single Embryo Transfer (SET) in a much larger cohort and with live birth as main end–point, is very similar to the one presented previously [58]. In univariate analysis, the results were almost identical, confirming that treatment outcome is associated with cleavage rate in a non–linear way, and in a linear way with nuclear status, degree of fragmentation and variation in blastomere size. After regression modelling, the degree of fragmentation qualified in the final new model instead of the variation in blastomere size, which was incorporated in the previous model [58].

Thus, their new algorithm, the revised Integrated Morphology Cleavage score (rIMC), incorporates 

1. Cleavage rate, 
2. The percentage of blastomeres showing a normal nucleus, and 
3. The degree of fragmentation, whereas the variation in blastomere size and the symmetry of the cleavage do not qualify for the final model [91]. 

The conclusions of their large prospective study on visual Embryo Scoring variables in Single Embryo Transfer (SET) show that blastomere number (in a non–linear way), the proportion of mononucleated (including information on multinucleation) blastomeres and the degree of fragmentation (both linearly) have independent prognostic power to predict high–quality embryo with high implantation potential. Together, these variables are incorporated in an algorithm, which is presented as the revised Integrated Morphology Cleavage score (rIMC score). It is suggested that applying the revised Integrated Morphology Cleavage score (rIMC), gives an easy and structured clinical guide to the ranking and selection of embryo/s most suitable for transfer in the early cleavage stage [91].

The other significant study represents numerous morphological characteristics that should have been taken into consideration when selecting high–quality embryo, and comprises the following: 

1. Morphology of the oocyte; 
2. ATP content and mitochondrial distribution in oocytes; 
3. Pronuclear body breakdown; 
4. Number and symmetry of distribution of nucleolar bodies in zygote pronuclei; 
5. Early (25–27 hours after fertilization) or late first cleavage for day 2 embryos; 
6. Number and symmetry of blastomeres, fragmentation and presence or absence of multinucleation in early cleaving embryos; 
7. Number of blastomeres undergoing compaction and 
8. The morphology of the compaction process in day 4 morulas and 
9. Blastocyst morphology in day 5 or day 6 embryos [50]. 

Dynamic characteristics such as pyruvate and glucose metabolism [42] or amino acid turnover [60] can also be studied but not in an immediate clinical context.

Morphological evaluation for day 2 embryos

Most IVF laboratories continue to grade embryos solely on the basis of cell number and percentage fragmentation as was traditionally done for day 2 embryos. Additional morphological features, some unique to day 3 embryos, may be essential in selecting embryos most likely to blastulate and implant. The following parameters should be used to grade the embryos: cell number, Fragmentation Pattern (FP), cytoplasmic pitting, compaction, equal sized blastomeres, blastomere expansion and absence of vacuoles [28].

The three main morphological features considered in embryo grading systems to date have been cell number, blastomere size and shape and degree of fragmentation [23; 87; 95; 111; 112; 138]. 

One such scoring system, the embryo quality or EQS score [23], was based on an assessment of blastomere symmetry, cytoplasmic appearance and amount of fragmentation. These same authors also proposed a formula for describing an Embryo’s Development Rate (EDR), based on the ratio of the time at which an embryo was observed to reach a particular stage and the expected time interval. Both the EQS and EDR were found to be interrelated and gave insight into pregnancy potential of individual embryos. Other scoring systems such as the Average Morphology Score (AMS) [95] and the cumulative embryos score (CES) [112; 130] incorporate number of embryos transferred into the final score. All of these scoring systems were designed for evaluation of embryos on day 2 of culture. A scoring system specific for day 3 embryos has not been extensively looked at.

Morphological evaluation for day 3 embryos

Culture and transfer of embryos on day 3 allows for additional assessment of embryonic development and improvement in selection criteria. A recent study on day 3 embryos [4] suggests that the pattern of fragmentation on day 3 may in fact give a better measure of implantation potential than the degree of fragmentation, which has always been a predominant feature in day 2 Embryo Scoring systems. In yet another study, assessing morphological attributes of day 3 embryos giving rise to pregnancies, investigators observed two features which they suggested might be early indicators of embryos likely to undergo embryonic compaction [132]. The first was an increase in cytoplasmic granularity and the appearance of tiny pits in the cytoplasm of blastomeres. The second was an increase in cell: cell adherence and loss of definition between individual blastomeres. Another feature that they scored was blastomere expansion. In high quality embryos, individual blastomeres were expanded and touched the zona, leaving very little perivitelline space [28].

Lynette Scott, Ruben Alvero, Mark Leondires and Bradley Miller in their article “The morphology of human pronuclear embryos is positively related to blastocyst development and implantation”, transparently described initial zygote scoring system: there are five pronuclear categories based on both the number and distribution of nucleoli in the pronuclei. They are graded 1–5 according to nuclear size, nuclear alignment, nucleoli alignment and distribution and the position of the nuclei within the zygote. Grade 1 zygotes have equal numbers of nucleoli aligned at the pronuclear junction. The absolute number is between three and seven. Grade 2 zygotes have equal numbers of nucleoli of equal sizes in the same nuclei but with one nucleus having alignment at the pronuclear junction and the other with scattered nucleoli. Grade 3 zygotes have equal numbers and sizes of nucleoli (between three and seven) which are equally scattered in the two nuclei. Grade 4 zygotes have unequal numbers (a difference of more than one nucleolus) and/or sizes of nucleoli. Grade 5 zygotes ae those with pronuclei that are not aligned, are of grossly different sizes or are not located in the central part of the zygote [104]. This zygote scoring system was experimentally revised and, consequently, following morphological parameters for Day 3 Embryo Scoring were established: on day 3, embryos were scored as grade 1–5 according to certain morphological criteria. Grade 1 = 8–cells, <10% fragmentation, good cell–cell contact, no multinucleated blastomeres; grade 2 = 8–cell, 10–20% fragmentation or lacking good cell–cell contact, no multinucleated blastomeres; grade 3 = 6–7–cells or 8–cells with 20% fragmentation or uneven blastomere size, no multinucleated blastomeres; grade 4 = >8–cells or 4–6–cells or 8–cells with >20% fragmentation or uneven blastomere size or multinucleated blastomeres; grade 5 = <4–cells or grossly fragmented or with half of the blastomeres being multinucleated [104].

Performing embryo transfer on day 3 after insemination instead of day 2 was proposed by Dawson et al. (1995) in order to allow good morphology embryos to develop for one more day and, consequently, avoid transferring embryos with the potential to arrest. The results showed significantly higher implantation rates following transfer on day 3, and a lower miscarriage rate [25].

Morphological evaluation for day 4 embryos

Single Embryo Transfers (SETs) require the most viable embryo from a cohort to be selected. Day 4 embryos may provide a selection advantage similar to blastocyst transfer. However, there was no adequate morphological system to assess Day 4 embryos. Therefore, the accurate morphological system to assess day 4 embryos was developed by scientists Deanne Feil, Richard C. Henshaw and Michelle Lane. Their study is unique to the current literature as it is the first to analyze pregnancy rates following the transfer of single embryos on Day 4 of development.

In clinical In–Vitro Fertilization therapy, the majority of embryos are traditionally transferred on Day 2 or Day 3 following insemination. However, transfer of cleavage stage embryos at this time involves transferring embryos prior to activation of the embryonic genome, and before compaction and blastulation, which are critical stages of development. One of the clear advantages of blastocyst transfer is that the embryo is returned to the uterus at a stage of development where it would normally reside [43]. Furthermore, there is a significant selection advantage, as embryos that are unable to activate the embryonic genome or complete compaction and cavitation can be easily identified and excluded for transfer [9; 13; 44].

In vivo, the blastocyst moves from the oviduct into the uterus on Day 4 of development, and at this stage, the embryo has compacted and is therefore developing its transporting epithelium [16].

Thoroughly investigating Day 4 embryos’ morphology, the scientists decided to assess on Day 4 of culture in a descriptive manner to establish the range of morphologies observed in the most accurate details. 

Scoring system developed for Day 4 embryos: 

Grade 1: early blastocyst, visible signs of cavitation occurring or completely compacted embryo, lacking negative morphological anomalies, e.g. vacuolation, excessive fragmentation, large number of excluded cells and self–cavitation of cells. 

Grade 2: Grade 1 compacted morula, with some morphological anomalies, e.g. one of the following present: vacuolation, excessive fragmentation, excluded cells, self–cavitation of cells or at least an eight–cell embryo, with partial compaction present or the majority of cells at least showing signs of compaction. 

Grade 3: partially compacted embryo with vacuoles or excessive fragmentation present or eight–cell embryo or greater, with no signs of compaction evident.

Grade 4: embryos with eight cells or greater, with no signs of compaction and vacuoles or excess fragments or embryos with less than eight cells, and no signs of compaction [35]. Embryo Selection for transfer is based on assessment of morphology, and in all cases the single highest quality embryo, as determined by the scoring system applicable to the stage of embryo development, dependent on the day of transfer (Day 4 or Day 5 of embryo culture), is transferred.

One of the key advantages of a Day 4 embryo assessment appears to be in the wide range of observed embryo development on Day 4, from cleavage stages through to early blastocysts. Therefore, it appears that this is a dynamic time period of development and scientists suggest that the increase in pregnancy rate observed may result from the assessment at this time assisting in selection of an “on–time” developing embryo. A further advantage of Day 4 morphology is that the Embryo Scoring system is an extension of a cleavage stage scoring system. Therefore, an embryo can be selected for transfer more quickly with the added benefit that there is no requirement for extra training that is necessary with blastocyst Embryo Selection for transfer [35].

A demonstrated benefit of blastocyst culture and transfer is the ability to determine which embryos have not yet activated the embryonic genome, which occurs at the 4–8 cell stage embryos [15]. Thus, those embryos with limited developmental potential in a cohort can be identified and excluded. One potential benefit of the use of Day 4 embryo transfers is the reduction in the time that an embryo exists ex vivo. It has been suggested that extending the time that an embryo is in culture may increase the susceptibility to disruption of epigenetic regulatory processes. The use of Day 4 selection and transfer may reduce this susceptibility [35].

The transfer of Day 4 embryos is beneficial in numerous ways; the embryo is returned to the uterus, to an environment where it would normally reside. This occurs post–genome activation to allow the embryo with the highest developmental potential to be selected from a cohort. An added advantage is being exposed to the uterine environment for the maximum time period, and an in vitro environment for a minimal time period, before implantation. In addition, uterine contractility is reduced at this time, all of which maximizes the potential for implantation [33; 69]. The ability to transfer the embryo to the uterus on Day 4 enables the benefits of Embryo Selection for transfer post–genome activation to be obtained, and may offer a viable alternative to Day 5 transfers [35].

Morphological evaluation for day 5 embryos

It has been suggested that transferring embryos at the blastocyst stage instead of selection at an earlier stage without knowing their developmental capability might enhance the implantation rate by a better embryo selection, thereby reducing the need to transfer more embryos [40]. The most competent embryos reaching the blastocyst stage are selected for transfer and the embryos that have arrested their development are identified and hence not transferred.

It is believed that gene expression of human embryos is switched on around the 8–cell stage immediately before compaction and that the blastocysts have a greater implantation potential compared with a cleavage–stage embryo, as it is characterized by an activated embryonic genome [15]. Therefore, nutrient requirements of embryos are different after this stage. Early embryos can grow in a simple salt solution, whereas they require more complex media after they reach the 8–cell stage. These changes also correspond to environmental changes in vivo since the embryo reaches the uterus from the Fallopian tube at the stage when compaction begins.

The possibility cannot be excluded that although the sequential media currently used for blastocyst culture allow embryos to develop to the blastocyst stage, they might compromise their implantation potential compared with blastocysts developed in vivo [5; 88].

Interestingly, that earlier Lynette Scott, Ruben Alvero, Mark Leondires and Bradley Miller in their article “The morphology of human pronuclear embryos is positively related to blastocyst development and implantation”, have represented blastocyst scoring system as: a good grade blastocyst needed to have: progression from 16–cell to compacted morula on day 4; and on day 5 the presence of a blastocoele or the signs of one beginning; defined trophectoderm with enough cells to form a continuous layer without a single cell stretching or flattening on the surface, a well–defined and organized inner cell mass; >60 cells [104], which after further scientific investigations of blastocyst morphological peculiarities was turned into more inclusive scoring model.

At present time, Day 5 blastocysts are selected for transfer or for Cryopreservation if they have an adequate number of even–sized cells, visible inner cell mass, an even trophectoderm with sufficient cells to form a continuous cell layer, no atretic areas and no excluded cells. In other words, the blastocysts are considered available for transfer or Cryopreservation when a big blastocoele is present (at least half the volume of the embryo), when the inner cell mass is identifiable and when the trophectoderm is formed by many cells forming a cohesive epithelium [106].

Scott L., Finn A., O’Leary T., McLellan S., Hill J. in their study “Morphologic parameters of early cleavage-stage embryos that correlate with fetal development and delivery: prospective and applied data for increased pregnancy rates” firstly brought together all the current scoring techniques in a single prospective trial as a means of testing their efficacy to the final outcome, delivery [106]. According to their scoring technique, the embryos are prospectively selected for transfer using the six most significant early parameters in a sequential/gated system: Pronuclear (PN) score, f Nuclear Precursor Bodies (NPB) ratio in the nuclei, day 2 size, nucleation and cell number and then by day 3 or 5 morphology. All data should be computed per embryo, and the clinical data should follow for the embryos, which would be used in transfer [106].

In terms of identifying, morphological evaluation method has a number of considerable advantages. However, morphological evaluation also has certain disadvantages, such as the hyper–sensitivity of the systems of integrated morphology cleavage score to possible scoring constituents’ changes and various methodological modifications of systems’ representation formats of morphological criterion, a noticeable growth in the quantities of calculations when the precision of identifying increases, which causes difficulties while observing real time mode.

Despite many studies on embryo morphology have focused on a single factor, such as cleavage rate, degree of fragmentation, uneven cleavage and multinuclearity, all factors which are associated with implantation, which is now so essential in the theory and practice of embryology has hardly ever been inclusively described and analyzed comprehensively using multi–dimensional approach, which better reflects the common clinical situation, when ranking embryos with various degrees and types of morphological deviation is needed [3; 34; 52; 58; 89; 90; 105; 109]. A multivariate model is necessary to find out which variables have independent, and thus true predictive, impact on the outcome. Some variables may lack biological significance, but show an association with other variables of real importance and thus falsely give the impression of true predictors for embryo euploidy. The best way to avoid misclassification of embryos due to univariate or subjective Embryo Scoring is to perform prospective, large–scale examinations (studies) with several embryo variables, resulting in a scoring algorithm that is based on multivariate statistical analyses, in which all relevant variables ‘compete’. Such an algorithm thus includes only the factors that show independent impact on treatment outcome and it also gives the correct power balance between those variables [91].

Morphology alone cannot disclose whether a particular embryo will implant or not because embryologists are looking at a statistical correlation, not at an individual measurement, and because morphology alone cannot disclose all the information contained in the embryo. No single static observation gives all the information contained in an embryo’s morphology [50]. It is more logical to consider that a combination of different observations, preferably reflecting different aspects of implantation potential, should be used; for instance, it is likely that blastomere asymmetry and multinucleation both reflect aneuploidy [53] etc.

This the most important scientific conclusion we have made, underlines the great importance of an accurate Embryo Scoring Protocol for ranking and selecting embryos to transfer, and suggests that such precise and standardized scoring should be the cornerstone of full prediction models.

2. Preimplantation Genetic Screening (PGS) method of Embryo Selection

The best studied alternative selection method is Preimplantation Genetic Screening (PGS). The classical form of Preimplantation Genetic Screening (PGS) involves the biopsy at Day 3 of embryo development of a single cell of each of the embryos available in an In–Vitro Fertilization (IVF) cycle and analysis of this cell by Fluorescence In–Situ Hybridization (FISH) for aneuploidies, for a limited number of chromosomes. Only embryos for which the analyzed blastomere is euploid for the chromosomes tested are transferred [134]. Although this method of PGS has been increasingly used in the last decade, recent trials show that it actually decreases ongoing pregnancy rates compared with standard IVF with morphological selection of embryos [75].

In an effort to overcome some of the drawbacks of Preimplantation Genetic Screening (PGS) using cleavage stage biopsy and Fluorescence In–Situ Hybridization (FISH) new methods to determine the ploidy status of a single cell are developed, such as Comparative Genomic Hybridization arrays or single nucleotide polymorphism arrays [131]. Furthermore, in an attempt to avoid the confounding effects of chromosomal mosaicism, embryos are now biopsied at either the zygote or blastocyst stage [46]. In addition, increasing time and money are invested in the development of high–tech, non–invasive methods to select the best embryo for transfer in In–Vitro Fertilization (IVF) cycle. This includes metabolomic profiling, amino acid profiling, respiration–rate measurement and birefringence imaging [85]. In metabolomic profiling, spectrophotometric tests are used to measure metabolomic changes in the culture medium of embryos; in proteomic profiling, proteins produced by the embryo and released into the culture medium are identified; in amino acid profiling, amino acid depletion and production by the embryo is assessed using the culture medium; in respiration-rate measurement, the respiration rate of embryos is assessed; and in birefringence imaging, polarization light microscopy is used to assess the meiotic spindle or the Zona Pellucida [85].

3. Time–Lapse Technology (TLM) as the innovative method of Embryo Selection, which can be used for investigation of the embryo’s implantation capacities based on embryo development and embryo viability

Time–lapse observation presents an opportunity for optimizing Embryo Selection based on morphological grading as well as providing novel kinetic parameters, which may further improve accurate selection of viable embryos. In other words, time–lapse observation presents an opportunity for identification of the morphokinetic parameters specific to embryos that are capable of implanting [78].

Evaluation of embryos in vitro has improved greatly over the past 25 years. Classical embryo assessment has been supplemented by the evaluation of several additional morphological characteristics that allow prediction of the developmental potential of an embryo and the probability of achieving pregnancy for an infertile couple [8].

Several publications have proposed additional morphological evaluations to assess the timing of embryonic cell divisions that appear to be related to embryo viability [19; 68; 72; 81; 99; 108]. Many of these studies have investigated the relationship between the timing of the first embryonic division and the embryo quality [56]. The underlying reason for variation in the time of the first cell division is not clear; it could be related to culture conditions as well as intrinsic factors of the oocyte and sperm, maturity, genetic competence and metabolism [72]. Early cleavage in first embryonic division, operationally defined as an early cell division resulting in a 2–cell embryo at a time of inspection 25–27 hours post Intracytoplasmic Sperm Injection (ICSI), and its impact on pregnancy rate was first published by the Edwards group [32].

Subsequently, many studies have used this concept as the basis for their publications [14; 19; 36; 72; 99; 100; 101; 108; 118] and all have found that the transfer of early cleavage embryos results in higher implantation and pregnancy rates compared with Embryo Transfers with delayed division. However, many transfers in these studies involved more than one embryo and many included a mix of early and late cleaving embryos; thus, it is difficult to obtain conclusive evidence that the implantation can be attributed to the early cleavage [14; 19; 36; 99; 100; 101; 108; 118]. In one fascinating study, Van Montfoort et al. (2004) investigated this, by comparing Embryo Transfers that composed entirely of early cleaving embryos with transfers that composed entirely of late cleaving embryos in 253 double transfers and 165 single transfers. They found that there were significantly higher pregnancy rates in the early cleavage group in both single and double transfers. The blastocyst formation rate for early cleaving embryos also increased, and the miscarriage rate decreased compared with the late cleaving group [128].

It is important to highlight that it is unclear if early cleavage is an independent predictor of pregnancy or if it is correlated with other variables such as embryo morphology and cell number. Several studies have shown that early cleavage embryos have significantly higher numbers of cells and better viability compared with late–cleaving embryos [36; 72; 99; 100; 101; 108].

A key consideration in many studies of timing of first cleavage is the limited number of observations, which restricts temporal assessment of a given phenomenon to a determination if an event occurred before or after a particular time point. Knowledge of the exact time a given event occurred cannot be obtained with a limited number of discrete observations. Indeed, one of the fundamental problems of embryo quality assessment is the static evaluation of a dynamic developing entity. Current classification scores analyze the morphology at a few predefined time points during embryo development preimplantation, with the consequent lack of information about what happened between the analyzed time points. Thus, continual monitoring might provide one strategy to collect a complete picture of embryo developmental kinetics [78].

Using a time–lapse photography system, Lemmen et al. (2008) found that embryos that implant have an earlier disappearance of pronuclei and first division and an increased cell number on Day 2 of embryonic development. They also found a correlation between a higher pregnancy rate and synchronicity in re–appearance of nuclei in the two blastomeres formed after the first division [68]. In a more recent study, Wong et al. (2010) found that development of human embryos to the blastocyst stage was correlated with: 

1. The duration of the first cytoplasmic cleavage from 1 cell to 2 cells; 
2. Time between division to 2 cells and subsequent division to 3 cells and 
3. Time between division to 3 cells and subsequent division to 4 cells. 

However, none of the embryos in that study were transferred. It is thus unclear if embryos with the suggested morphokinetic cleavage pattern would have implanted [137].

All the above determines formulating a deeply actual embryological mission of our time, consisting in modelling [creating] the perspective model for embryo development investigation, which presupposes the most accurate Embryo Selection process for further transfer of high–quality embryo with high implantation potential. Consequently, all the above–listed scientific theories and their basic concepts were implemented in scientific investigations of identifying the innovative methods of Embryo Selection and the corresponding integrity of activities directed at creating the most accurate Embryo Scoring systems, based on embryo development.

In the past few years Time–Lapse Technology (TLM) has been adopted for the study of embryo development [2; 22; 55; 63; 65]. The Time–Lapse System (TMS) utilized, EmbryoScope™, consists of an incubator with a built–in microscope able to acquire images automatically every 10–20 minutes and in up to seven focal planes. One of the main benefits of these systems resides in the use of different models or algorithms known to improve clinical outcomes by predicting embryo viability. Time–lapse monitoring of embryo development may represent a superior way to culture and select embryos in vitro. Authors that have contributed with different predictive markers, models or algorithms include: Wong et al. (2010), Meseguer et al. (2011), Azzarello et al. (2012), Dal Canto et al. (2012), Campbell et al. (2013), Chamayou et al. (2013) and Basile et al. (2014).

The first inclusive algorithm proposed for time–lapse technology’s adoption for the study of embryo development was published in 2011 [78].

This algorithm classified embryos through a combination of 

1. Morphological assessment, 
2. Exclusion criteria and 
3. Inclusion criteria. 

This algorithm introduced a new way of classifying and selecting embryos, a more complete one, based not only on morphological assessment but also on kinetic markers (nowadays measurable). 

This improvement can be attributed not only to more stable culture conditions but also to the use of morphokinetic parameters to select embryos destined for transfer [12].

The use of morphokinetic parameters to select embryos destined for transfer was previously investigated, analyzed, discussed and described in article “The use of morphokinetics as a predictor of embryo implantation”, written by scientists Meseguer M., Herrero J., Tejera A., Hilligsoe K.M., Ramsing N., Remohi J. This study presents to our knowledge the largest set of transferred embryos after time–lapse analysis, and thus, a novel opportunity to correlate morphokinetic parameters to implantation and ongoing pregnancy. The purpose of this study was to generate and evaluate a tool for the selection of viable embryos based on the exact timing of embryo development events together with morphological patterns by using an automatic time–lapse system to monitor embryo development. The Spanish scientific group, in 2011 presented a clinical study with time–lapse imaging of embryo development for 247 transferred embryos, using a tri–gas IVF incubator with a built–in camera designed to automatically acquire images at defined time points, has simultaneously monitored up to 72 individual embryos without removing the embryos from the controlled environment. Images were acquired every 15 minutes in five different focal planes for at least 64 hours for each embryo. As a result of embryo–development monitoring, the scientists have found that several parameters were significantly correlated with subsequent embryo implantation (for example, time of first and subsequent cleavages as well as the time between cleavages) [78].

It was scientifically established that the following parameters should be considered for the most accurate Embryo Selection process as the most predictive ones: 

1. Time of division to 5 cells, t5 (48.8–56.6 hours after Intracytoplasmic Sperm Injection – ICSI); 
2. Time between division to 3 cells and subsequent division to 4 cells, s2 (≤0.76 hour) and 
3. Duration of cell cycle two, i.e. time between division to 2 cells and division to 3 cells, cc2 (≤11.9 hours). 

The scientists have also observed aberrant behavior such as multinucleation at the 4–cell stage, uneven blastomere size at the 2–cell stage and abrupt cell division to three or more cells, which appeared to largely preclude implantation [78].

In their scientific conclusions, scientists established that the image acquisition and time-lapse analysis system makes it possible to determine exact timing of embryo cleavages in a clinical setting. They proposed a multivariable model based on their findings to classify embryos according to their probability of implantation. The efficacy of this classification should be evaluated in a prospective randomized study that ultimately will determine if implantation rates can be improved by time–lapse analysis [78].

How Time–Lapse Technology (TLM) is used for Embryo Selection? 

The time–lapse instrument EmbryoScope^TM is a tri–gas incubator with a built–in microscope to automatically acquire images of up to 72 individual embryos during development. Embryos are investigated by detailed time–lapse analysis measuring the exact timing of the developmental events in hours post insemination by Intracytoplasmic Sperm Injection (ICSI).

Exemplifying how time–lapse technology is used for Embryo Selection, we should look through the experimental part of Meseguer et al. (2011) investigation, where Embryo Score and culture conditions are described. At the first stage, embryos are monitored: successful fertilization can be assessed at 16–19 hours post Intracytoplasmic Sperm Injection (ICSI) based on digital images acquired with the time–lapse monitoring system. Embryo morphology is evaluated on Days 2 (44–48 hours post ICSI) and 3 (64–72 hours post ICSI) [78], based on the acquired digital images, taking into account the number, symmetry and granularity of the blastomeres, type and percentage of fragmentation, presence of multinucleated blastomeres and degree of compaction as previously described [5]. 

Embryo Selection should be performed exclusively by morphology based on: 

1. The absence of multinucleated cells; 
2. Between 2 and 5 cells on Day 2; 
3. Between 6 and 10 cells on Day 3; 
4. Total fragment volume <15% of the embryo and 
5. The embryo must appear symmetric with only slightly asymmetric blastomeres [76; 77; 84].

At the second stage, the acquired images of each embryo are analyzed for further time–lapse evaluation of morphokinetic parameters. 

Retrospective analysis of the acquired images of each embryo is made with an external computer, EmbryoViewer® workstation, using an image analysis software in which all the considered embryo developmental events were annotated together with the corresponding timing of the events in hours after ICSI microinjection. Subsequently, the EmbryoViewer® workstation is used to identify the precise timing of the first cell division. This division is the division to 2 cells and a shorthand notation of t2 is used in the following. The second (i.e. to 3 cells, t3), third (4 cells, t4) and fourth (5 cells, t5) cell division should be also annotated. For the purpose of this study, scientists define time of cleavage as the first observed time point when the newly formed blastomeres are completely separated by confluent cell membranes. The time of all events is expressed as hours post ICSI microinjection [78].

How morphology categories can be compared with time-lapse categories?

Embryos with defective morphology by conventional criteria should be excluded from further examination and not replaced. To make a comparison with the time–lapse classification categories, scientists retrospectively evaluated the morphology of the transferred embryos using the following categories:

Category 1: The two Pronuclei (2PN) embryo consists of 2 cells at 27 hours post insemination, 4 cells at Day 2 and 8 cells at Day 3. Even blastomere size at the 2, 4 and 8 cell stage, no multinucleation is observed at any time and the fragmentation is <10% [78].

Category 2: The two Pronuclei (2PN) embryo consists of 1–2 cells at 27 hours, 3–4 cells at Day 2 and 6–8 cells at Day 3. Only one mismatch is allowed, i.e. either 1 cell at 27 hours, 3 cells at Day 2 or 6–7 cells at Day 3. Blastomeres are even sized at the 2, 4 and 8 cell stage; no multinucleation is observed at any time and the fragmentation is <20% [78].

Category 3: The two Pronuclei (2PN) embryo consists of 1–2 cells at 27 hours, 2–4 cells at Day 2 and 6–8 cells (or morula) at Day 3. The embryo can have asymmetric blastomeres and multinucleation can be observed in maximally one blastomere at each stage. The degree of fragmentation is <20% [78].

Category 4: The 1PN or two Pronuclei (2PN) embryo embryo consists of 1–2 cells at 27 hours, 2–6 cells at Day 2 and 4 to more than 8 cells or morula at Day 3. The embryo can have asymmetric blastomeres and be multinucleated. The degree of fragmentation is <50% [78].

Category 5: The embryo consists of any number of cells at 27 hours, Day 2 and Day 3. Asymmetric blastomere size, multinucleation and any degree of fragmentation is allowed. Atretic embryos and embryos with arrested development belong to this category [78].

To describe the distribution of the probabilities of implantation, timings should be converted from continuous variables into categorical variables by dividing them into groups based on their quartiles. By this procedure, the scientists avoided bias due to differences in the total number of embryos in each category. After that it is recommended to calculate the percentage of embryos that implanted for each timing quartile to assess the distribution of implantation in the different categories [78].

Scientific experts Basile N., Vime P., Florensa M., Aparicio Ruiz B., García Velasco J.A. and J. Remohí M. Meseguer in their study “The use of morphokinetics as a predictor of implantation: a multicentric study to define and validate an algorithm for Embryo Selection”, proposed a different experimental design from the one previously published by their group. Their objective was to identify kinetic markers associated with a higher implantation rate and to develop an improved version of their previous algorithm that was suitable for different IVF clinics. For this reason, they have designed a two–phase study based on a larger data set from four different IVF clinics. Phase 1: Development of the algorithm: this phase included cases where Embryo Selection was based purely on standard morphological assessment. Phase 2: Validation of the algorithm: in this phase, Embryo Selection was based on standard morphological assessment plus our relevant morphokinetic criteria [12].

To demonstrate Embryo Scoring and Embryo Selection, based on time–lapse observation, the scientists propose to evaluate embryo morphological parameters. All the embryos included in that study were transferred on Day 3 after Oocyte Retrieval (D3). This decision was taken by the scientists before Oocyte Retrieval based on previous medical criteria. Embryos with defective morphology by conventional criteria were excluded from further examination and not replaced. Embryo morphology was evaluated at 48 hours and 72 hours post Intracytoplasmic Sperm Injection (ICSI). Evaluated parameters included: cell number, symmetry and granularity of the blastomeres, the type and percentage of fragmentation (fragment defined as an anuclear, membrane–bound extracellular cytoplasmic structure and calculating the % of the total volume of the embryo constituted by fragments), presence of multinucleated blastomeres and degree of compaction [78].

Following the scientific investigation of embryo morphological features, what is essential to emphasize is that embryos with acceptable morphology were subjected to analysis of time–lapse images of each embryo on an external computer with software developed for time–lapse image analysis (EmbryoViewer workstation). 

The morphological features scored were: 

1. Direct Cleavage (DC) from zygote to three blastomere embryo; 
2. Uneven blastomere size (UBS) at the 2–cell stage during the interphase where the nuclei are visible. Blastomeres were considered uneven sized if the average diameter of the large blastomere was 25% larger than the average diameter of the small blastomere; 
3. Multinucleation (MN) at the 4–cell stage during the interphase where the nuclei are visible [12].

Kinetic parameters measured were: 

1. Time of cleavage to 2–blastomere embryo (t2); 
2. Time of cleavage to 3–blastomere embryo (t3); 
3. Time of cleavage to 4-blastomere embryo (t4); 
4. Time of cleavage to 5-blastomere embryo (t5). 

Additionally, duration of the second cell cycle (cc2), i.e. the duration of the 2–blastomere embryo phase (t3 − t2), and synchrony (s2) in divisions from a 2–blastomere embryo to a 4–blastomere embryo (t4 − t3) were calculated [12].

Furthermore, the scientists constructed a hierarchical classification model based on that described by Meseguer et al. (2011): 

1. Morphological screening; 
2. The new morphological criteria; 
3. Timing of cell division to three cells (t3); 
4. Duration of second cell cycle, cc2, i.e. the time from division to a two blastomere until division to a three blastomere embryo; 
5. Timing of cell division to five cells [78].

Experimentally, using morphological embryo evaluation and relevant time–lapse criteria (t3, cc2 and t5), and correlating it with Implantation Rates (IR), there was established new algorithm. In order to validate the new algorithm, scientists studied 885 cycles of Intracytoplasmic Sperm Injection (ICSI) and transferred 1620 embryos using morphological embryo evaluation and our relevant time–lapse criteria (t3, cc2 and t5), and correlated it with IR. Their morphological criteria were DC, UBS in the second cell cycle and MN in the third cell cycle.

For each developmental event, scientists observed the time ranges with the higher density of implanting embryos and defined optimal ranges. A new algorithm was then built based on the information from a logistic regression model. The kinetic variables were included in the new algorithm: t3, cc2 and t5 in combination with the new morphology criteria (Direct Cleavage (DC), Uneven Blastomere Size (UBS) and Multinucleation (MN)) [12].

It is evident that kinetic markers represent one of the most potent non–invasive tools to improve embryo selection nowadays. Several independent groups have focused on discovering these markers with somehow similar results [7; 22; 57; 78; 96; 137]. However, it is undeniable that the majority of studies are based on small data sets and therefore may not be valid for all embryo cohorts as several factors have shown to impact morphokinetics [20; 38; 64; 83]. Embryo selection remains the focus of many improvements in In–Vitro Fertilization (IVF) Technology. Many of the non–invasive approaches such as metabolomics, proteomics, secretomics, still remain as poorly applicable on a daily basis in the IVF laboratory. On the other hand, several authors have proposed the use of early [78; 137] or late [17; 24] kinetic markers of embryo development to elaborate different algorithms to improve embryo selection. Some have validated these algorithms with retrospective studies [79] and some have performed prospective randomized trials [65; 97] with contradictory results. Kirkegaard et al. (2013) did not find significant differences in the morphokinetic behaviour of implanting versus non–implanting embryos suggesting that kinetic markers may not be universally applicable [12].

Conclusion 

The present article was focused on representing the basic overview of peculiarities of the Embryo Selection process and comparison of different methods of Embryo Selection, used in Assisted Reproduction technologies. There has been revealed three basic methods [and their criterion] which are used for accurate selection of the most viable embryo(s) for transfer: 

1. Morphological evaluation; 
2. Preimplantation Genetic Screening; 
3. Time–lapse technology.

Embryo quality is widely regarded as the single most important factor for the chance of a successful IVF Treatment. Scoring of embryo morphology and cleavage rate is a clinically useful method in Embryo Selection and may serve as a proxy for embryo quality. Selection of the best embryo for transfer is crucial for the elective Single–Embryo Transfer (eSET). During the past twenty–five years, various approaches were suggested, including morphological classification, proteomic and metabolomic investigations and time lapse follow–up. There is still a lack of evidence–based standardized morphological and cleavage variables for ranking and selection of the embryo with the greatest chance of resulting in a live baby. The variation in Embryo Scoring models makes comparisons between different clinics and Treatment strategies difficult and many of the non–invasive approaches such as metabolomics, proteomics, secretomics, still remain as poorly applicable on a daily basis in the IVF laboratory. Solid data on how to rank and select individual embryos with different types and degrees of deviations from the “ideal” morphologic and cleavage appearance are still scarce. Thus, in clinical practice, it is not clear if, for example, an embryo with ‘too fast’ or ‘too slow’ cleavage and no fragmentation should be selected for transfer before an embryo with normal cleavage rate and moderate fragmentation, or if a slight variation in blastomere size is a worse prognostic sign than the absence of a visible normal nucleus in a proportion of the blastomeres.

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