Authors: Javier del Río, Belén Gómez-Giménez & Iris Martínez
"The relationship between blastocyst morphology and implantation has been investigated mainly according to expansion, trophectoderm and inner cell mass"
The main goal of in vitro fertilization (IVF) is the birth of a single healthy child. However, the consequences and the effects of assisted reproductive techniques on children’s short- and long-term health have always been a source of discussion. Although IVF techniques and protocols have dramatically improved, the overall success rates are still relatively low, and assisted reproduction units still face the challenge of improving pregnancy rates (1). For this purpose, transfer of a single human embryo at blastocyst stage is becoming more common in the practice of assisted reproduction (2). It allows a better synchronization between the endometrium and the embryo and the possible selection of embryos with a higher implantation potential (3).
Several morphology- or kinetics-based approaches have been described to select the best blastocyst in order to increase pregnancy rates. However, the yielded results are conflicting and the outcome is a matter of never-ending and controversial debates, specially regarding blastocyst stage (4, 5).
BLASTOCYST ASSESSMENT SYSTEMS
The relationship between blastocyst morphology and subsequent blastocyst implantation has been investigated according to various criteria. Traditionally, morphology has been evaluated after embryo compaction (6). The significance of examining the embryo after compaction is the ability to examine it after embryonic genome activation. Furthermore, the obvious benefit of looking at the blastocyst is the possibility to examine both cell types. The extent to which the trophectoderm (TE) develops will reflect the embryo’s ability to attach and implant in the endometrium, whereas development of the inner-cell mass (ICM) is obviously crucial for the progress of the foetus (7).
There have been described several assessment systems to predict the success of blastocyst implantation. However, Gardner's grading system seems to be a better predictor of pregnancy rates (8, 4, 7). Following this method, blastocysts are initially scored from 1 to 6 based on their degree of expansion and hatching status, and ICM and TE grading is then assessed from A to D depending on their morphology (9).
It was felt that expansion was important for cavity formation. This process requires both extensive energy utilization through the Na+/K+ ATPases on the basolateral membrane of the TE and formation of effective tight junctions between TE cells to form a barrier. Therefore, expansion seems to be a reflection of embryo competence (7).
Recently, Richardson et al. proposed a simplified blastocyst grading system. These authors demonstrated both its prognostic potential and the inter- and intra-observer variability. This grading scheme was able to effectively predict clinical outcomes in terms of implantation, clinical pregnancy and live birth. Slight variation existed both between and within embryologists grading the embryos but, overall, consistency in their analyses was similar to, if not better than, those associated with more complex grading systems (10).
However, most of the grading systems that are currently used for assessing viability of IVF embryos are subjective, relying on visual inspection of morphological characteristics of the embryos that are qualitatively evaluated. Grading based on qualitative criteria is imprecise, and it inevitably results in inter-observer variability and in intra-observer to some extent, as well (10).
MORPHOLOGICAL ASSESSMENT AND OUTCOME RESULTS
As it has been exposed, there is a need for increased knowledge about the relative impact of each morphology parameter at the blastocyst stage (and their potential correlation) on predicting the probability of successful implantation and pregnancy (1, 2, 11).
Shapiro et al. compared up to 25 parameters in order to develop predictive models of clinical pregnancy within a set of blastocyst transfer cycles (12). Among these variables, blastocyst diameter seemed to be the most significant predictor of clinical pregnancy in the multivariate models. The authors concluded that embryos developing into expanded blastocyst stage on day 5 were approximately twice as likely to implant, compared to those for which expansion was delayed until day 6 (13, 3). This is supported by Van den Abbeel and coauthors, who found that high scores of blastocyst expansion and hatching stage, ICM and TE grade were all significantly associated with increased pregnancy and live birth rates after fresh transfers (11). The finding that the expansion and hatching stage is the most important parameter when selecting a blastocyst for transfer (11) is in contrast with some retrospective cohort studies that suggest TE grading to have the strongest predictive power for treatment outcome in fresh transfers (14, 15).
On the contrary, Basak Balaban et al. exposed that quantitative measurement of blastocysts and ICM is not a practical way to assess blastocyst quality, arguing that two-dimensional measurements of three-dimensional global structures can be misleading. The reasoning is that the size of a blastocyst may vary depending on the time the blastocyst is assessed under the microscope, and this may easily confuse grading (9). For this purpose, Almagor et al. tried to provide an easily measurable assessment of the ICM and evaluate its correlation with pregnancy rates in a series of single blastocyst transfers. They found a high ICM/blastocyst ratio associated with significantly increased pregnancy rates. Thus, they proposed this measure to be used as an additional strongly predictive parameter of successful implantation (16). Recently, Bouillon et al. have confirmed that clinical pregnancy and live birth rates were significantly higher for blastocysts with good TE and ICM quality, and so it was concluded that both rates decreased with morphology (4). Even though some blastocysts with non-optimal morphology are able to implant, it has been suggested that when selection is made among suboptimal blastocysts, preference should be given to those with a normal ICM (6).
However, the current goal for researchers is to establish the optimal perinatal outcome of singletons according to blastocyst morphology. This has been recently analyzed by Bouillon et al., who found no increased rates of adverse obstetric and perinatal outcomes after transfer of blastocysts with poor morphological features (4).
Figure 2. Examples of blastocyst grading: (a) 3AA blastocyst; (b) 3AB blastocyst; (c) 3BA blastocyst; (d) 4AA blastocyst; (e) 4AB blastocyst; (f) 4BA blastocyst; (g) 4CC blastocyst; (h) 5AA blastocyst; (i) 5CA blastocyst. For details of the EH stages and ICM and TE grades, see Materials and methods from Van den Abbeel (11).
BLASTOCYST ASSESSMENT BY TIME-LAPSE TECHNOLOGY
As previously explained, the most accepted blastocyst grading system is Gardner’s (17), based on the degree of blastocyst expansion and the morphological appearance of both the ICM and TE. However, since embryo development is a dynamic process, conventional grading practices may not detect subtle differences in morphology, which changes significantly over a time span of only a few hours (18). In order to obtain a complete picture of morpho-kinetic events occurring during embryo development a time-lapse system is needed. This technology offers continuous monitoring of embryos rather than just a limited number of discrete observations annotated through conventional assessment. Besides, time-lapse allows embryos to be cultured uninterruptedly, thus getting rid of embryo trafficking from and into the incubator (19). Nevertheless, the actual new and unique contribution of morpho-kinetics is the ability to predict how likely is for a zygote to reach the blastocyst stage in vitro. Several algorithms based on parameters detected by time-lapse, such as early divisions of cleavage-stage embryo, have recently been developed in IVF laboratories to predict blastocyst formation (20). In addition, some authors have made an effort to take time-lapse usefulness further, for instance, to predict the ploidy status of pre-implantation embryos (21, 22).
Implantation potential of blastocysts can be evaluated by means of time-lapse during its development. In this regard, three main events are currently being investigated: duration of both compaction and blastulation plus number of blastocyst collapse events (19, 23, 24).
Duration of compaction
After several cell divisions during the initial stages of embryonic development, the intercellular boundaries become obscured in a process called compaction, which maximizes the intercellular contact and gives rise to the morula (25). Although the compaction of embryos has not received sufficient attention in the IVF field, some studies have focused on the relationships between compaction patterns and embryo developmental potential. Embryos that begin to compact before the eight-cell stage exhibit aberrant in vitro development. Conversely, embryos that complete compaction on day 5 have a lower ability to develop into high-quality blastocysts than those that compact on day 4 (26). These results suggest that the compaction patterns of embryos can facilitate the prediction of their ability to develop both in vitro and in vivo.
An interesting work on this issue has been recently published by Mizobe and collaborators (23). The study retrospectively examined the outcome of 299 embryos from 243 patients, which were transferred at blastocyst stage. The whole early development was analysed by comparing morpho-kinetic parameters between implanted and non-implanted embryos, and measuring the time length of specific events, particularly of embryo compaction. Compaction length was calculated by using values of beginning and end of compaction. Beginning of compaction was considered as the time point when the intercellular boundaries became diffuse somewhere in the embryo, while fully compaction was defined as the point when blastomeres were finally unified into one cluster. Compaction length was significantly shorter in blastocysts resulting in pregnancies compared to those that failed to do so. These results indicate a correlation between the length of compaction and implantation potential. This finding is in agreement with the results from previous studies, which observed that the compaction patterns of embryos affected the rates of good-quality blastocyst formation and implantation (26, 27, 28). By contrast, some studies have reported that compaction time of embryos does not affect clinical pregnancy rates (29, 30).
Duration of blastulation
Blastulation is the process through which a morula becomes a blastocyst. Two different structures will arise to form the blastocyst out of the compacted blastomeres of the morula. The first sign of blastulation is compaction and differentiation of the outer blastomeres, forming the TE. This compaction gives the structure a watertight condition, allowing the fluid later secreted to be contained (31). Then, a different group of blastomeres normally located at the centre of the morula start to get closely attached to each other by the formation of Gap junctions, thus facilitating cell communication. It is these cells that differentiate into the ICM (the future embryoblast) and acquire a polarized location at one edge of the embryo. Such polarization creates a cavity, the blastocoel, and gives rise to the structure termed blastocyst. The trophoblasts (TE cells), in turn, continuously pump fluid into the blastocoel, which results in an enhanced size of the blastocyst. This increased volume leads the embryo to hatch through the zona pellucida (32).
A recent study conducted by Mumusoglu analysed whether time-lapse morpho-kinetic variables differ among those euploid blastocysts that result in ongoing pregnancy after single embryo transfer (24). For that purpose, 129 patients who had been transferred a single embryo after an ICSI cycle with PGS were considered. Embryos were cultured in a time-lapse incubator up to the moment of TE biopsy, and 23 time-lapse morpho-kinetic parameters were annotated. After biopsy, blastocysts were vitrified and transferred within the next cycle. When comparing all time-lapse parameters, only blastulation time was statistically different: it had lasted shorter in successfully implanted blastocysts than in those that had not implanted. Blastulation time was calculated as the interval from initiation of blastulation up to full blastocyst formation (33, 34). Even though only a few studies have genetically tested euploid blastocysts (21, 22), all of them have pointed out that faster-developing euploid blastocysts might exhibit higher implantation potential. Even so, further large-scale studies are needed in order to confirm such an association (24).
Blastocyst collapse events
The phenomenon of blastocyst collapse is actually the shrinkage caused by the efflux of the blastocoel fluid due to the loss of cell bindings along the TE. When blastocysts expand, fluid gradually accumulates in the blastocoel -mediated by the sodium pump (Na+/K+-ATPase) (35), resulting in an increased pressure on both the TE and the zona. In parallel, TE cells produce lysins that are involved in the zona weakening and hatching. Formerly to implantation, the embryo needs to leave the zona behind, place adjacent to the endometrial epithelium and then make first contact with the uterus (36). Thus, embryo hatching from the zona is thought to be related to collapse-expansion cycles.
By using a time-lapse monitoring system, it has been observed that many of the human blastocysts that reach stage 5 of expansion experience one or more collapse events of the blastocoel cavity, producing a separation of part (if not all) of the TE cells from the zona (19). In a study conducted by IVI Valencia and IVI Murcia clinics (19), blastocyst collapse was analysed to determine its potential influence on reproductive outcomes and whether it may serve for prognostic purposes. 460 patients and data from over 500 blastocysts known to have implanted were included in the study. Blastocyst collapse was considered to have occurred if the separation between TE and the zona pellucida was higher than 50% of the volume. Blastocysts that had experienced just one collapse event were found to present a significantly reduced implantation potential when compared to those transferred after having experienced none. The authors proposed that the molecular mechanisms underlying this association could be related to the mechanical stress suffered from by the embryo, which could result in an excessive energy consumption that would adversely affect the consequent development (19).
Figure 4. Drawing tools used with Embryovieverw for blastocyst collapse evaluation. First, a line was drawn across the embryo diameter (A). Then, the two circumferences that define the contracted blastocyst and the inner surface of the zona pellucida were outlined (B) [for more details, go to Materials and methods from Marcos (19)].
In spite of the data discussed above, the negative association between blastocyst collapse and implantation potential is not yet clear. In a report by Bodri and colleagues (37), blastocysts were classified according to the number of collapses: embryos with no collapses represented 54% of the total, 22% of the embryos had experienced one single collapse, and multiple collapse events occurred in 24% of the blastocysts. Whereas the live birth rate was observed to decrease as the number of embryo collapse increased, multivariate analyses suggested blastocyst collapse not to be a significant predictor. Rather, it was found to be a confounding factor, along with other morpho-kinetic variables such as time up to two-cell division completion and female age. Therefore, it was concluded that blastocyst collapse patterns should not be evaluated alone without stronger predictors of reproductive outcomes being taken into account (37).
TIME-LAPSE AS A MEANS TO EVALUATE EMBRYO QUALITY
As previously exposed, the use of time-lapse technology is recently common in embryology laboratories because of its noticeable potential for enhancing embryo selection. Using these technologies, Desai et al. analysed possible kinetic differences between embryos with limited potential and those that accomplished in vitro blastocyst formation and/or implantation (38). Certain parameters such as time of pronuclear formation and cleavage stage were found to be different in embryos reaching blastocyst stage vs. poor-quality embryos. Moreover, a large number of embryos were found to present multinucleation and reverse cleavage, but they were able to form a blastocyst with optimal criteria for freezing (38), which resembles previous reports on the dynamic nuclear formation of blastocysts by Ergin and coauthors (39).
With respect to blastocyst formation, Motato et al. (2016) proposed two models to classify embryos based on their probability of reaching blastocyst stage and implantation (40). However, the study was limited by parameters such as subjective criteria from different clinics with different culture media (40). Consequently, it would be reasonable to keep on research on this subject in order to achieve a consensus regarding embryo classification and implantation potential (40).
CONSIDERING DAY-2 AND DAY-3 EMBRYO MORPHOLOGY BEFORE DAY-5 TRANSFER
Even though blastocyst stage is currently widely accepted as the optimal moment for embryo transfer, cleavage stage has been traditionally regarded as the right moment in global practice. In fact, it still continues to be so in some laboratories, and early transfer into the uterus has been proposed to be advantageous to the embryo due to the limited time exposed to the in vitro environment (41). However, there exist two main arguments supported by extensive scientific literature to explain why blastocyst transfer after extended culture has advantages over the traditional cleavage-stage transfer:
First of all, when the embryo arrives to the uterus in natural conditions it has already reached morula stage, which corresponds to, at least, day 4 of in vitro culture (42). This means blastocyst stage is the most physiologically compatible stage for transfer, since it allows a better synchronization between embryonic stage and endometrial receptivity (43) [you can read more about the optimal day for embryo transfer in our previous article here].
Secondly, several studies have reported higher implantation potential for blastocysts compared to cleavage-stage embryos (6, 41), the first transferred blastocyst being reported in 1995 (44). Furthermore, some authors have postulated that a large proportion of morphologically normal day-3 embryos are actually chromosomally abnormal or mosaic, which may contribute to the 80-90% rate of implantation failure observed after cleavage-stage embryo transfer (45).
EXTENDING EMBRYO CULTURE UP TO BLASTOCYST STAGE
Considering the need for further studies on the subject, and the fact that day-3 embryos can actually implant and develop successfully, does it really make sense to extend embryo culture up to blastocyst stage?
As above-stated, morphologically normal embryos may actually present chromosome abnormalities, which proves the insufficiency of morphological criteria to evaluate implantation rates (46). Because of embryo plasticity, the proportion of chromosomally abnormal cells varies within the culture; corrupted cells can be eliminated, thus resulting in a good-quality blastocyst developing from a poor-quality cleavage-stage embryo (47). Some studies have evaluated pregnancy rates derived from transfers of blastocysts with previous poor quality as cleavage-stage embryos, finding an approximate success rate of 45% after culture and freezing of embryos at an early stage for another cycle. The conclusion of this being a valid practice to avoid the repetition of IVF-ICSI treatments (48) agrees with recent findings showing that low-scoring day-2 or 3 embryos, which are not considered transferable, can still result in successful blastulation and end up in a live birth (49, 50).
All this said, the right question now would be: should day-2 and day-3 embryo morphology be considered before transfer at day 5 when blastocysts reach a similar good quality?
A recent retrospective study by Herbemont has suggested that only the quality of the transferred blastocyst may be predictive of the subsequent clinical outcome, whereas morphological aspects at day 2 or day 3 have limited interest (51). These same results had previously been observed by Guerif; even though early morphological parameters were relatively helpful to predict blastocyst development, their value to predict blastocyst morphology was limited, and so they provided no significant additional information that could prognosticate blastocyst implantation and live birth rates (6). A few years earlier and with the same goal in mind, Zech and coauthors carried out a prospective randomized study in which they compared ongoing pregnancy rates per single embryo transfer between day 3 and day 5. When good-quality embryos were available, pregnancy rates were found to be higher after blastocyst transfer. Therefore, the authors concluded that morphological criteria-based seleccion at day 3 may not be a suitable procedure when just one embryo is to be transferred out of a cohort of all morphologically good ones. Thus, and as stated by the authors, extending embryo culture up to day 5 may result in a better strategy in order to correctly identify and select those embryos with higher implantation potential, provided there is a sufficient number of top-quality eight-cell embryos available (52).
On the contrary, a study performed by Silber (2014) found that blastocysts arising from poor-quality embryos displayed lower implantation and pregnancy rates compared to good-quality embryos. These discrepancies could be due to different criteria used to score embryo quality (53). So, in order to minimise discrepancies between studies, the use of time-lapse is currently established as a common approach to evaluate embryo morpho-kinetics. In fact, reduction in the time of embryo exposure to the environment outside the incubator has been demonstrated to enhance both embryo quality and blastulation rates (54).
Nevertheless, to answer the question previously postulated, a prospective randomized study would be needed that compare at least two similar good-quality blastocysts, one arising from a good-quality day-2/3 embryo, and to the other from a poor-quality one (51).
It is important to take into account that the main population features of different patients, such as paternal age, maternal BMI, parental smoking or cause of infertility may influence clinical outcomes. Moreover, certain methodological aspects also need to be considered, like blastocyst evaluation by the same personal (in order to minimize variation) or the consistent use of the same type of culture media (to avoid potential effects on birth-weight and other traits), just as previously suggested (4).
A universal embryo grading system needs to be validated, before widespread implementation in IVF laboratories. Also, it has not yet been clearly established which morphological feature of blastocysts (expansion, TE or ICM) is the most reliable as a predictive factor for post-transfer implantation success. Consequently, there is still a debate between authors about the true outcomes of single transfer of low-quality blastocysts (4).
Morpho-kinetics assessment, along with chromosomal screening, may ultimately help identify euploid embryos with the highest developmental potential (55). Since these features are susceptible of being affected by in vitro culture conditions, each embryology laboratory should define their own cut-off points in order to standardise time-lapse variables (24).
Finally, it should be taken into account the fact that embryo quality is not the only parameter with influence on implantation rates; endometrial receptivity is also involved, and it may be greatly determined by a variety of factors (56) [to learn more about endometrium status and receptivity, read our previous post here].
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