CIC Edizioni Internazionali. The power and the limits of morphokinetic. parameters in selecting embryos with full capacity. capacity to implant

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1 Mini-review The power and the limits of morphokinetic parameters in selecting embryos with full capacity to implant Sandrine Chamayou Antonino Guglielmino Unit of Reproduction Medicine, HERA Institute, Catania, Italy Address for correspondence: Sandrine Chamayou, PhD Unit of Reproduction Medicine, HERA Institute Via Barriera del Bosco, 51/ Sant Agata Li Battiati (Catania), Italy Summary Embryo morphokinetic analysis is an emerging tool for selecting embryo(s) for transfer. Each embryonic cellular event can be recorded, observed and dated without disturbing culture conditions. The morphokinetic parameter is the numerical data that links regular and irregular phenotypes with the exact moment of appearance. The duration of each embryonic process such as the time that takes a mother cell to divide in two sister cells, the synchronization of two sister-cells division or the time that takes a collapsed blastocyst to re-expand can be measured. From these numerical data a multitude of studies has been published relating embryo morphokinetics with patient characteristics, ovarian stimulation or laboratory protocols. Different Authors have created models to select the embryos with the highest probability to implant. The effectiveness of time-lapse technology to increase pregnancy rate remains to demonstrate. It is possible that instead of one model for embryo selection, different models should be applied for embryo selection or deselection according to medical and biological contests. Nowadays the complete in vitro development of each embryo is fully accessible. The new technology advances will help to further increase our knowledge on embryo development and improve in vitro culture conditions. KEY WORDS: embryo development, embryo selection, morphokinetic parameter, morphology, timelapse. Introduction Since the beginning of human in vitro fertilization treatments, embryos have been selected for in uterus transfer according to their morphology observed at fixed times (1). The disadvantages of this method are that embryo development events between two observations are missed and each observation needs the cells (embryo) to be removed from the incubator to be placed on the microscope plate, exposing them to temperature and ph variations. The recent introduction of time-lapse technology in the IVF practice has made accessible the continuous monitoring of embryo development without disturbing in vitro culture conditions. In this work, the potential and limits of morphokinetic parameters in selecting embryos with full capacity to implant are exposed. In a first time, the principle and history of time-lapse technology are described. All regular and atypical phenotypes of embryo development can be annotated and dated. Correlating each specific embryologic event observed with the precise time of occurring, the concept of morphokinetic parameter emerged. As a consequence the duration of each embryonic process can be calculated. We study which are the parameters due to couple characteristics (such as women s weight or response to ovarian stimulation) or those depending on the protocols used by the IVF laboratory (fertilization method, in vitro culture conditions, cryo - preservation, embryo biopsy) that modify embryokinetics. Many studies have been performed to determine the morphokinetic parameter(s) informative on the embryo competence to develop in in vitro conditions and to implant. Several models established by Authors have been tested by others, not always Current Trends in Clinical Embriology 2015; 2 (2):

2 S. Chamayou et al. with the expected success. The improvement of pregnancy rate due to embryo selection after timelapse monitoring remains to be demonstrated. Furthermore the study of embryo kinetic is unsuitable to select aneuploid embryos at least at cleavage stage. All these concepts are developed in the present review. The principle and history of time-lapse technology Time-lapse is a photography concept that consists in the recording of a slow process at regular intervals. The continuous projection of the frames gives an accelerated view of the process. In embryology, the time-lapse monitoring of human embryos is only possible if the embryo is maintained at 37 C, in an appropriated culture medium and controlled atmosphere. The light energy of the camera system necessary for the continuous observation must not be harmful to the embryo (2). One of the first reported time-lapse monitoring experiences was made by Cole in 1967 (3) when the escape of mouse blastocyst from the zona pellucida was precisely described and measured. In 1973, Kaufman (4) described the first cleavage events on mouse embryos and the effect of temperature on embryo development timing. On human, the timing of pronuclear formation, DNA synthesis and cleavage in human 1-cell embryos was detailed for the first time by Capmany et al. in 1996 (5). Gonzales et al. in 1996 (6) observed the cytoplasmic extensions of the trophectoderm cells during in vitro human embryo hatching. From the year 2008 and up today, the number of publications on embryo time-lapse monitoring constantly increased (7, 8). Nowadays, several devices that can be positioned inside conventional incubators or specific time-lapse incubators are equipped with sophisticated software analysis programs for manual annotation. Other time-lapse systems automatically annotate each embryological division up to 8-cell stage. The details of embryo development The fascinating aspect of time-lapse monitoring is that each embryonic cellular event can be recorded, observed and dated with relative precision. Conventionally, t0 is the time of sperm injection in the ooplasm (ICSI) or oocyte immersion in a sperm solution (conventional IVF). Each passage of fertilization is visible: second polar body extrusion, the first and second pronuclei appearance, the number and size of the nucleoli in each pronucleus, the time of nucleoli vanishing and complete pronuclei disappearance with complete pronucleus membrane fading. The female pronucleus formation and its migration from the site of the second polar body extrusion towards the male pronucleus can be observed. Each cell-stage is annotated when the cell division is completed with the two cells fully distinguishable. At each cleavage stage the number of nuclei and relative size of blastomere can be observed. Each passage from the beginning of morula compaction with the formation of tight-junction to the beginning of cavitation due to the accumulation of blastocoele fluid, the successive blastocyst expansions and collapses, the hatching and the morphologies of inner cell mass and trophectoderm cells can be detailed. One of the most interesting aspects of time-lapse recording is the possibility to observe the development of embryo irregularities. As in the conventional observations, in time-lapse monitoring it is possible to know (this time with certitude) the number of pronuclei at zygote stage and to detect a zygote with abnormal pronuclei number (1, 3 or more). The percentage and history of extra-cell fragmentation, the number of polynucleated cells (at cleavage stage) and the presence of vacuoles or atretic cells can be determined for each embryonic stage. Some cellular phenomena are appreciable only with a dynamic observation such as abnormal cell division (more than two sister cells are produced from one mother cell also called trichotomous mitosis), the number of successive blastocyst expansions and collapses. On the other hand, a limitation of time-lapse monitoring is that all systems record images on one or more predefined focus planes. It is not possible to observe the details of the cells on a continuous z-axe as it is with conventional microscopy. As a consequence, intermediate stages such as cell division before the complete cell detachment can be mis-interpreted as high fragmentation while the cells have an irregular shape before they become round again. Definition of morphokinetic parameters and calculation of durations The morphokinetic parameter is the chronometer parameter that links the time with a specific cellular event. In other words the morphokinetic param- 48 Current Trends in Clinical Embriology 2015; 2 (2): 47-55

3 01-Chamayou_. 22/07/15 15:14 Pagina 49 na zi on al i Predictability of morphokinetic parameters In te r Figure 1 - Embryo kinetic. time-lapse user group has proposed an universal guideline for the definition and annotation of dynamic human embryo monitoring (9). The conversion of morphological events in numbers (times) has led to a multitude of statistic studies linking embryo-kinetic with the embryo competence to develop and to implant. C IC Ed iz io ni eter dates each specific embryologic event such as the completion of second polar body detachment, the times of appearance and fading of each pronucleus, each cell stage (t2 for 2-cell stage, etc.), the beginning of cell compaction, blastulation, blastocyst expansion, blastocyst herniation, completed blastocyst hatching. The irregular events can be dated such as the appearance and disappearance of smooth endoplasmic reticulum, the time of trichotomous mitosis and cell-fusion, planar blastomere arrangement, the beginning and ending of embryo rolling. The term embryo rolling is used when blastomeres move on themselves without dividing. The duration of a specific embryonic process is the lapse of time between the first and the last frame in which the embryonic event is continuously observed. The duration can be defined for each or both pronucleus/i observation, the time that takes a mother cell to divide in two sister cells (cell cycle), the synchronization of two sister cells divisions, full morula compaction process, and for blastocyst events such as the time for full blastulation, expansion, collapse, re-expansion and herniation (9) (Figure 1). The irregular embryonic events such as embryo rolling, the duration of smooth endoplasmic reticulum observation can be measured. In a view to compare the morphokinetic parameters and durations of specific embryo events with a cohort of embryos or between groups of patients inside the laboratory or with other IVF centers, and to relate the kinetic of embryo development with outcomes such as implantation and live birth rates or any other embryonic characteristic, it is fundamental to apply the same protocol of morphokinetic parameter annotation. An experienced Current Trends in Clinical Embriology 2015; 2 (2): What make the morphokinetic parameters vary The parameters that make the embryo kinetic vary depend on couple characteristics, ovarian stimulation, gamete morphology and IVF protocols. Couple characteristics and ovarian stimulation As previously found in studies based on embryo morphology only, there is no modification of the cleavage morphokinetic parameters according to the women weight (10). However, embryos from overweight and obese women reach the morula and blastocyst stages with a lower number of cells and faster than normo-weight women. This acceleration of embryo kinetic would be due to metabolic abnormalities such as diminished glucose consumption, altered profile of amino acid metabolism and increased endogenous triglyceride content (11). Female smoking impairs embryo development with a delay of embryo cleavage which could be responsible for lower pregnancy rates in smoking women (12). 49

4 S. Chamayou et al. On a group of young oocyte donors, it was demonstrated that embryo kinetic is independent of the type of gonadotrophin preparation (rfsh, HMG or rfsh+hmg). Nevertheless there is an anticipation of cell divisions and shorter cell cycles at cleavage stage in embryos produced from the women with the highest ovarian response after the lowest doses of gonadotrophin somministration (13) and when GnRH antagonist + GnRH agonist combination is used instead of GnRH agonist + hcg (14). Gamete morphology Until now no study related oocyte morphology (ooplasma texture, perivitelline space largeness, granulation under the perivitelline space, zona pellucida thickness, shape of the first polar body, meiotic spindle presence and position) with embryo kinetic. Looking at the spermatozoa morphology, Knez et al. in 2013 (15) showed how the pattern of vacuolization in the sperm nucleus can compromise the timing of embryo development until blastocyst stage. This would demonstrate that there is a paternal effect on embryo development. The sperm-derived genome is not completely silent in the period between fertilization and the first cleavage. If the level of DNA damage introduced by the sperm nucleus is too high to be repaired by the oocyte, the embryo slowly cleaves and finally arrests. IVF protocols: the fertilization method The procedures applied in the IVF laboratory modify the embryo kinetic. On mouse embryos, Wale and Gardner in 2010 (16) showed that embryos cultured in 20% oxygen are delayed at the first cleavage compared to embryos cultured in 5% oxygen, and this delay increases progressively and in an irreversible way as the embryos reach the further cleavages. The over oxygenation during the first embryo stages is toxic and can impact on DNA replication fidelity increasing the amount of DNA damages. The Authors conclude that based on kinetic analysis, the embryo culture in a reduced oxygen concentration should be taken in consideration in all clinical IVF laboratories. The embryos produced after IVF reach the 2- and 3-cell stages later than those produced after ICSI but both embryo kinetic timings become similar from the 4-cell stage (17). The fertilization method modifies the hatching process of in vitro fertilized human embryos (18). IVF embryos hatch from a large breach of the zona pellucida, while ICSI embryos get out displaying trophectoderm cells projection in combination with more gradual expulsion of the blastocyst. This change of hatching pattern according to fertilization protocol shows that the micro-injection pipette penetration during ICSI modifies the zona pellucida resistance to embryo pressure. IVF embryos hatch earlier than ICSI embryos. IVF protocols: in vitro culture conditions It is easy to study the effect of two culture media on embryo kinetic splitting an oocyte cohort in two groups and comparing the morphokinetics and frequencies of embryo irregularities (abnormal fertilization, multinucleated cells, trichotomous divisions...) in embryos produced from sibling oocytes. Using this approach, two groups found controversial results of the effect of culture media on embryo kinetic. According to Ciray et al. in 2012 (19), embryos cultured in single media were advanced from the first mitosis cycle and reached 2- to 5-cell stages earlier compared to those cultured in sequential media from the same manufacturer (Irvine). Nevertheless, the durations t3-t2 and t4-t3 were comparable. Embryo-kinetic differences would illustrate the differences in quantity for essential and non-essential amino-acids in each culture-media according to manufacturer data. Basile et al. in 2013 (20) compared a single step media (Global) and sequential media (Sage) and found no statistical difference in t2, t3, the morphokinetic parameters t2, t3, t4 and t5, the durations t3-t2 and t4-t3. The interpretation of both studies results is limited because the two groups worked in different conditions (reduced oxygen for the first group and 20% oxygen for the second) and the manufacturers gave limited information on media composition. In 2010, Meseguer et al. (21) partially showed that the stable culture conditions in time-lapse incubator lead to increased pregnancy rate of relative 20% compared to conventional incubator. IVF protocols: oocyte vitrification modifies nucleoli activity and zygote kinetic In one of our studies (22), a 44-hour time-lapse analysis was made comparing the embryo-kinetic of 179 fertilized fresh oocytes to those of 168 fer- 50 Current Trends in Clinical Embriology 2015; 2 (2): 47-55

5 Predictability of morphokinetic parameters tilized sibling vitrified/warmed oocytes. From the results it appeared that oocyte vitrification anticipated pronuclei fading, one-cell stage timing and modified nucleoli activity by increasing their number and decreasing their diameter at the zygote stage. Conversely, the embryo kinetic at the cleavage stage was similar in length with the embryo kinetic of their sibling fresh oocytes. We suggested that oocyte vitrification induces changes in pronuclei stability, probably due to pronuclei envelop instability as well as modifications in nucleoli functionality. Therefore, the predictive morphokinetic parameters on embryo competence found from fresh oocytes must be revised when applied on embryos from vitrified/warmed oocytes. IVF protocols: embryo biopsy In the study of Kirkegaard et al. (23), 56 human embryos had one blastomere removed at 8-cell stage and their embryo kinetic until blastocyst stage were compared to 53 non-biopsied embryos. It resulted that the removal of a blastomere prolonged the cell-stage of embryo biopsy and delayed the compaction and the reaching of blastocyst stage. On the other hand, the duration of embryo-compaction and the time of hatching were similar to non-biopsied embryos. The blastocyst stage was shorter in the biopsied group. The conclusion was that the prolongation of compaction stage in biopsied embryo could be due to the use of Ca2+ free embryo-biopsy medium in a view to remove inter-blastomere junctions and making each blastomere easily removable. After the biopsy, the embryo is immersed again in a medium containing Ca2+ (and Mg2+). Due to Ca2+ reintegration, the embryo re-organizes inter-cellular boundaries such as focal gap junctions, tight junctions and cytoskeletal adherence between cells. Ca2+acts also on intra-cellular spatial organization through the cytoskeleton. In addition, the blastomere removal per se would contribute to post-biopsy stage embryo kinetic alterations. How to select the competent embryo to transfer Relationship between morphokinetics and embryo competence Several studies linked embryo morphokinetics within cleavage stage to the embryo competence to develop until blastocyst stage and implant. These analyses were made for both normal and abnormal phenotypes. Two studies linked the morphokinetic parameters at zygote stage with clinical outcomes for embryos produced from fresh oocytes after ICSI and transferred at cleavage stage. According to Aguilar et al. (24), the timing of the second polar body extrusion, pronuclei fading and duration of both pronuclei observation are the only morphokinetic parameters predictive of successful embryo implantation. In Azzarello et al. study (25), the pronuclei fading was also defined as predictive morphokinetic parameter because no embryo with pronuclei breakdown earlier than 20h 45min resulted in live birth. In Chamayou et al. study (26), for an embryo to develop in a competent blastocyst the two pronuclei must be visible for duration between 7.7 and 22.9h (Figure 2). An abnormal syngamy resulting in a prolonged duration of pronuclei observation would be due to a dysfunctional sperm aster (27). Studying the morphokinetics at cleavage stage on a larger number of embryos, Cruz et al. (28) found the timings t2, t3, t4 and the durations t3-t2 and t4- t3 to be linked to blastocyst formation and quality. Based on the analysis of 19 embryos, Dal Canto et al. (17) found that t7 and t8 were shorter in blastocyst able to implant. On the opposite, the cleavage kinetic of a competent embryo must not be too fast. In Rubio et al. (29) study, the embryos with fast cleavage from two to three cells (at 2-cell stage one cell divide within 5 hours, defined as direct cleavage by the Authors) had an implantation rate of 1.2% compared to 20.2% for embryos with a second cell cycle longer than 5 hours. Normally a cell cycle takes hours during which two consecutive phases of cytokinesis and replication of whole cell genome must occur. A too short cell-cycle would result in incomplete DNA replication and might be associated to an unequal distribution of DNA in each blastomere. Wirka et al. (27) studied the cellular development in embryos after direct cleavage, that is to say trichotomous mitosis. The potential to develop in competent blastocyst resulted superior in embryos that directly divided from 3-cell to 5-cell stage compared to those that directly divided from 1-cell to 3-cell stage. Their conclusion was that according to the embryo-stage of abnormal cleavage, embryos with fewer chromosomally abnormalities could express a mosaicism compatible with embryo development potential and implantation. Current Trends in Clinical Embriology 2015; 2 (2):

6 S. Chamayou et al. Embryo morphokinetics and chromosomal content Several Authors challenged to link embryo kinetic with chromosomal content. The embryo gender cannot be determined on the base of morphokinetic data analysis because there is no statistical difference between male and female embryos (30). Chavez et al. (31) studied the behaviour of the two cleavage divisions according to ploidy and led out that while normal cell cycle parameter timing was observed in all euploid embryos to the 4-cell stage, only 30% of aneuploid embryos exhibited parameter values within normal timing windows. The Authors supposed there may exist processes encompassing the formation of embryonic micronuclei, cellular fragmentation and resorption that would compensate blastomere aneuploidies and mosaicim. In Basile et al. (32) study, the existing correlation between the morphokinetic timings and durations at cleavage stage and chromosomal abnormalities were confirmed after day 3 embryo biopsy. Nevertheless, the choice to perform day 3 embryo biopsy is surprising because it has been previously established that embryo biopsy on cleavage stage to assess chromosomal content was not an adequate strategy because of the very high levels of chromosomal mosaicism (90%) (33). On the later embryo stages, Campbell et al. (34) found that the times of initiation of compaction and full blastocyst stage reaching were significantly delayed in multiple aneuploid embryos compared to euploid embryos. On the opposite Rienzi et al. (35) failed to correlate chromosomal abnormalities with morphokinetic parameters from cleavage to blastocyst stage. Flowcharts and embryo selection Meseguer et al. group (36) proposed the first model for embryo selection based on morphokinetic parameters. They published a hierarchical classification of embryos based on morphological screening and morphokinetics within the cleavage stages. This work had a big success in the IVF world and several Authors tested this model but failed to recover the same correlations. Meseguer et al. worked with 5% CO2 in air and on year-old donor eggs. These culture conditions and the tested population may be different than those met in the common IVF laboratories. Campbell et al. (37) tested the correlation previously found (34) between the peri-blastulation timings and embryos outcomes in terms of foetal heart beats and live birth. They confirmed their model because of a statistically significance in terms of foetal heart beats and live birth rates between cases in which the aneuploidy risk was low and medium. In 2013 we proposed a method for the routine use of morphokinetic parameters to identify the competent embryos in developing from day 3 to day 5 of in vitro culture and implanting while transferred on day 5 (26). In a retrospective time-lapse monitoring analysis of morphokinetic parameters for 72 fully implanted embryos and leading to pregnancies with chromosomally normal fetuses were compared to 106 non-implanted embryos and 66 embryos with arrested development from the same pool of implanted embryos. The morphokinetic parameters t1, t2, t4, t7, t8, the duration of both pronuclei observation and the synchronization t8- t5 resulted informative of embryo competence to develop to blastocyst stage; the cell cycle t5-t3 was informative on the embryo competence to implant when transferred on day 5 (Figure 2). Any embryo out of these ranges failed to implant. Our results were partially discordant with other studies results because embryo kinetic is sensitive to culture conditions (see Table 3 in Chamayou et al.) (26). Each laboratory should determine the values of the potentially informative morphokinetic parameter(s) in its own conditions. Conclusion The recent introduction and diffusion of embryo time-lapse monitoring devices has led to a multitude of studies witnessing the large scientific curiosity among embryologists. The complete development of all embryos produced in the IVF lab is now fully accessible. Every change in morphological aspect, from the second polar body extrusion to the complete embryo hatching can be recorded, observed and dated. Every embryonic process, from the duration of the second polar body extrusion after ICSI to each cycle of blastocyst collapse and re-expansion can be measured. All irregularities of embryo development can be annotated. Some of them such as trichotomous mitosis or cell fusion can be appreciated only in time-lapse monitoring. Furthermore, all the embryo development can be observed without disturbing in anyway the in vitro culture conditions. The first consequence of time-lapse introduction in an IVF lab is the increasing knowledge of the em- 52 Current Trends in Clinical Embriology 2015; 2 (2): 47-55

7 Predictability of morphokinetic parameters Figure 2 - Morphokinetic parameters informative on embryo ability to develop a viable blastocyst and implant while transferred on day 5 (26). bryologists on human embryo morphology and development. The biological meaning of specific regular and irregular phenotypes is discussed and the embryologists are more aware of the consequences of culture conditions on embryo development. In each IVF laboratory, the morphokinetic data of developed and implanted embryos can be compared to that of non-implanted ones. An inhome model of embryo selection (dis-selection) can be created, applied and tested for its efficacy. From the visualization of embryo development it is surprising that an embryo with several abnormalities such as one poly-nucleated blastomere at 2-cell stage followed by a trichotomous mitosis division can implant and lay to a normal baby. A vacuole present in the ooplasm at ICSI time can be expelled among the trophectoderm cells in a viable embryo. The embryo seems able to repair itself and to survive to (ambient) conditions in which it is placed even if these conditions are not always the best ones (e.i. high oxygen tension). The morphokinetic parameters vary according to a lot of variables such as couple characteristics, ovarian stimulation, sperm morphology, fertilization method, in vitro culture conditions, gamete cryopreservation or embryo biopsy. From this long list and taking in consideration that embryos with irregular phenotype(s) or altered kinetic can be viable, it appears clearly that the construction of a model for embryo selection has to be made with much care in a view to not eliminate the embryo with atypical phenotype and able to implant. As a definition, a model with the endpoint of embryo implantation gives the probability of an embryo to implant comparing its characteristics to a database of previously implanted embryos. Usually it is given a probability as high, medium or low to implant. As a consequence an embryo with a high probability can fail to implant while an embryo with low probability can implant. With this caution in mind we constructed our model without probability of implantation (or development) but taking in consideration the REAL morphokinetic parameters of day 3 embryos able to develop and implant when transferred on day 5, even if the data of a specific parameter for a competent embryo is far from the mean of the common competent embryo group (26). Several articles tried to link the chromosomal content with morphokinetic parameters at cleavage stage or later. The strategy of embryo biopsy on 8- cell stage to establish chromosomal status should be taken with caution because it has been previously established as inappropriate due to high proportion of blastomeres with mosaicism in viable embryos. Furthermore and until the 8-cell stage the embryo genome is silent, the embryonic processes rely on the molecular construction on the oocyte prior to the first meiotic division (38), that is to say when the female gamete is diploid. In fact, Wong et Current Trends in Clinical Embriology 2015; 2 (2):

8 S. Chamayou et al. al. (39) demonstrated that embryo development depends on mrna content. As a consequence the time-lapse monitoring cannot substitute preimplantation genetic screening for the cleavage stage or could substitute it for those chromosome abnormalities that would alter the embryo development after 8-cell stage. The time-lapse monitoring can predict the presence of aneuploidy only if the presence of aneuploidy is due to a non chromosomal cellular process that impairs embryo development and is also responsible of aneuploidy occurring (e.i. level of mitochondrial DNA). A systematic analysis concluded that the efficacy of time-lapse technology to increase pregnancy rates remains to establish but the in vitro culture conditions and patients profile of the studies were non-homogeneous (8). Instead of creating a single hierarchical model for embryo selection, it would be more efficient to construct different models according to patient selection, ovarian stimulation or laboratory protocols such as the choice of culture medium. In conclusion, the time-lapse monitoring technology in the IVF field has the big advantage to be a non-invasive technology, makes the embryos grow in very stable in vitro conditions and the embryologists more aware in the decision on embryo selection for transfer, freezing, prolonged embryo culture for biopsy on day 5, etc. A lot of work remains to be done to correlate embryo morphokinetics with gene expression and embryo physiology. The new technology advances will help us to further increase our knowledge on embryo development and improve in vitro culture conditions. References 1. Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology. 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