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Time Lapse Technology

by: Natalia Basile, M.Sc.

TIME LAPSE TECHNOLOGY:

Conventional embryo evaluation based on standard microscopic observations has contributed significantly to the success of In Vitro Fertilization (IVF). However, success rates remain relatively low, with clinical pregnancy rates (PRs) of ~ 30% per transfer as reported by the Center for Disease Control and Prevention and the

European registers by ESHRE. (http://apps.nccd.cdc.gov/art/Apps/FertilityClinicReport.aspx ; Ferrareti et al., 2012).

In addition to this, and as consequence of the pressing need to reduce the number of multiple births, embryologists are required more and more to improve the selection methods in order to a) reduce the number of embryos transferred and b) to identify those with the highest implantation potential. This constitutes a great challenge specially considering that reducing the number of embryos transferred could jeopardize the overall success rates of IVF programs.  Therefore, new strategies have been proposed: culturing embryos up to the blastocyst stage to improve selection based on morphology; to perform genomic array hybridization (array-CGH) to improve selection based on chromosomal content and more recently, to study embryo kinetics through time lapse technology to improve selection based on kinetic markers.  In this review we will compare embryo evaluation with time-lapse monitoring systems (TMS) vs. standard micorscopy; we will go over the different equipment available for clinical use and we will review the most recent published articles based on this technology.

 

Embryo development and TMS:

Early embryo development is a dynamic process that begins with the fusion of the spermatozoa and the oocyte. The resulting zygote undergoes epigenetic reprograming followed by a series of embryo cleavages that are initially dependent on the maternal RNA and protein content. Later on, genome activation takes place and the pre-implantation embryo will most likely compact into a morula followed by differentiation to the blastocyst stage.  During the past 30 years we have described this “dynamic” process based on “static” observations and great knowledge has been acquired. However, it is inevitable to think of two major disadvantages:

  1. Static observations are linked to specific time-points during the day with the consequent loss of information and disturbance of optimal culture conditions
  2. They only allow qualitative interpretations that are highly subjective and prone to substantial inter-observer and moderate intra-observer variations (Baxter Bendus et al, 2006)

On the other hand TMS, a combination of camera + microscope + incubator + computer, offer the possibility of culturing embryos in a very controlled environment while capturing images at intervals between 5 and 20 minutes. This type of technology has been used for decades especially in the research area (Cole et al ,1967; Massip et al, 1980; Grissart et al, 1994) and recently, thanks to the great improvements in bioinformatics, time lapse systems have evolved from home made versions (Payne et al, 1997; Lemmen et al, 2008) to high tech commercial equipment more suitable for clinical use (Kirkegaard et al, 2012; Meseguer et al, 2011; Cruz et al 2011; Pribenszky et al, 2010; Wong et al, 2010). (Fig 1).

 

Figure 1: Home made technology vs. High technology.

TMS offers several advantages when compared to standard microscopy. It allows maintenance of optimal culture conditions during the entire period of embryo culture. It is known that pH, temperature and humidity are crucial for the correct development of embryos in vitro and it is inevitable to avoid alterations in these parameters when performing standard microscopy.  In addition to this, embryos are cultured either directly on an imaging device (Primo Vision, Vitrolife Eeva; Auxogyn) or placed on the imaging device at a set frequency (EmbryoScope, Unisense Fertilitech).  In contrast to standard microscopy (performed only once a day), TMS offers continuous monitoring of the embryos allowing us to perform precise determinations of cell divisions and a closer observation of morphological events such as the initiation of compaction and the appearance of the blastocoele cavity among others. Moreover, it allows us to detect abnormal events that would normally occur between observations like: irregular divisions, formation and reabsorption of fragments, appearance/disappearance of multinucleation, etc.

 

Culturing embryos in TMS has enlightened embryologists about the possible errors in embryo grading. It is well known that the morphological appearance of an embryo can change in a very short period of time (Fig 2) leading to different decisions regarding to the type and number of embryos transferred. Montag et al, (2011) in a very interesting article points out the fact that static observations may be misleading presenting data that compares time-lapse imaging with standard morphological observations. In this study embryos assessed within a time spam of 4 h (38 h, 40 h and 42 h) presented a different morphology or blastomere number in almost half of the embryos (49.1%) leading once again to a different score.

 

Figure 2: Different grading for the same embryo at different time points during a time frame of 6 hours.

Evaluations based on TMS add objectivity to the process of embryo selection. However, the automatic recognition of embryonic traits can be problematic depending on the quality of the image, the morphological differences depending on the developmental stage and the position and transparency of the embryo. Thanks to the development of bioinformatics, new programs that allow the automatic analysis of the images obtained under the microscope have been created. These programs also allow the quantitative assessment of key aspects of embryo development and morphology and the storage of the data related to these determinations. These data can then be used to improve our knowledge on early embryonic development and lead to morphological classification systems that are more objective and potentially more sophisticated.

Despite the many advantages of TMS a few disadvantages should also be mentioned. From an embryologist point of view one of the main limitations is the inability to rotate embryos under observation. We must remember that embryos are three dimensional, and that some of the structures that are clearly visible on one plane (for instance multinucleation) can disappear in the next one, especially in highly fragmented embryos.  Another concern is related to the positioning of the embryos within the field of view of the microscope. TMS that culture embryos individually do so by placing the embryos in a multiwell dish located in a motorized arm (Embryoscope).  The motorized arm rotates the dish at a set frequency in order to place each embryo within the field of view of the microscope.  A different type of system avoids rotations by culturing embryos in shared media drops very close to each other (Primo Vision, Eeva). These systems utilize dishes with microwells specially design to hold all the embryos constantly within the field of view of the microscope. Both settings are valid although concerns regarding possible displacement of embryos in group culture microwells may limit the utility of these systems in some situations; for instance in pre implantation genetic patients for whom individual culture is practically mandatory.

Another issue that has raised concerns is the potential detrimental effect of light exposure.  Although the safety of these systems has been evaluated from a clinical point of view (Cruz et al, 2011; Kirkegaard et al, 2012) a proper description of the amount of light exposure on TMS was necessary. Meseguer et al (2011) did so by placing a scalar irradiance microsensor inside the slide during the entire process of embryo culture. Similar measurements were made on standard microscopes used in fertility clinics. The total exposure time in the TMS during 3 day culture and acquisition of 1420 images was 57 s, which compares favourably with the 167 s microscope light exposure time reported for a standard IVF treatment (Ottosen et al, 2007).

 

TMS- Equipments

IVF laboratories currently using TMS on a clinical level are choosing one of these two options:

a)    To insert a microscope inside a commercially available incubator (Primo      Vision®, Vitrolife; Eeva, Auxogyn)

b)   To have all the items integrated in one single equipment (EmbryoScope, Unisense Fertilitech).

 

Table 1 summarizes the technical and clinical characteristics that of the different TMS currently available for clinical use. In regards to cost, we must acknowledge that TMS are expensive and, in the short term, we expect less expensive equipments due to the existing competition between different brands.

EMBRYOSCOPE-UNISENSE

PRIMO VISION- VITROLIFE

EEVA- AUXOGYN

Technical Characteristics
Integrated Incubator Yes No No
Optics Bright field Bright field Dark field
Frecquency of images Every 10 minutes Every 10 minutes Every 5 minutes
Focal Planes 7 1 1
Capacity of patients 6/system 1/camera 1/camera
N embryos/pt 12 individual culture 16 group culture 12 group culture
Clinical Characteristics
Automatic Diagnostic Tool No No Yes
Operator Dependant Yes Yes No
Time consuming analysis Yes Yes Automatic
Selection algorythm User defined User defined Yes
Blastocyst Prediction on day 3 No No Yes (Wong, 2010)
Implantation Prediction on day 3 Yes (Meseguer, 2011) No No

Table 1: Summary of technical and clinical characteristics: Embryoscope, PrimoVision and Eeva.

 

Time lapse studies

The first study in humans using TMS was published back in 1997 by Payne et al.  In this study, early parameters of development including timing of the extrusion of the second polar body, synchrony in appearance of pronuclei (PN) and abuttal of PN were observed in a small set of oocytes (n=50) and correlated with embryo quality on day 3. Almost ten years later, Mio and Maeda (2008) increased the number of embryos analyzed and the period of observation and described morphokinetic events in 286 embryos from fertilization and up to the blastocyst stage. That same year, Lemmen et al, analyzed 102 zygotes during the first day of development finding correlations between higher number of cells on day 2 and embryos presenting: i) early disappearance of PN and ii) early onset of the first cleavage.  Moreover the study analyzed implanted vs. non implanted embryos and interestingly implanted embryos presented better synchrony in the appearance of nuclei after the first cleavage although it was not proposed as a parameter to select embryos.

Following this first group of observational studies and as a consequence of the great improvements in TMS the clinical application of this technology has dramatically increased, especially in Europe. Thus a great number of interesting and promising studies have been performed and publications are revolutionizing the field of ART.  Two articles are worth mentioning:  Wong et al (2010) and Meseguer et al (2011).  Both of these studies have proposed algorithms to predict embryo viability although with different end points; Wong´s study focuses on blastocyst formation and Meseguer´s study on implantation. Wong et al used 242 supernumerary frozen zygotes in a study that combined time lapse imaging and gene expression profile. Images were taken every 5 minutes using low-power LED light in a dark field illumination system. Gen expression analysis was performed every 24 hours for small groups of embryos. Three parameters were proposed to predict blastocyst formation: duration of the first cytokinesis (14.3 ±6.0 min); time between the first and second mitotic division (11.1 ±2.2h) and the time between the second and third mitosis (1.0 ±1.6h). In the second study, Meseguer et al analyzes 247 fresh embryos with known implantation data. Images were taken every 15 minutes using low intensity red illumination (635nm) in a bright field illumination system.  Three parameters were also proposed to predict implantation: t5 or time between intracitoplasmic sperm injection and the 5 cell stage (48.8-56.6 hours); cc2 or the time between the first and second mitotic division (≤ 11.9 hours) and s2 or the time between the second and third mitotic division (≤ 0.76 hours). Interestingly two of the three parameters coincide with Wong´s study. A set of negative predictive factors were also identified including: direct cleavage from 1-3 cells (or DC2-3 < 5 hours), uneven blastomere size at the 2 cell stage and multinucleation at the 4 cell stage. Finally, by combining standard morphology + exclusion criteria + inclusion criteria an algorithm for embryo selection was proposed to classify embryos into 10 categories. Another recent study also utilized blastocyst formation rate as an end point. In this case, Hashimoto et al (2012) reported that high quality blastocysts presented shorter times for completion of the second and third division when compared to low quality blastocysts (0.7 vs. 3.7 hours and 5.7 vs. 16.9 hours respectively). This suggests that embryos with potential to develop into high quality blastocysts can be selected as early as day 2-3 of culture.

These studies represent only the beginning of a long list of publications.  Many groups have gone a little further already while evaluating the potential intrinsic and extrinsic factors that could affect embryo kinetics including: the type and dose of gonadotropins utilized to stimulate patients (Muñoz et al, 2012); the type of culture media utilized for embryo culture (Basile et al, 2013; Ciray et al 2012); the effect of the patient´s weight (Bellver et al, 2013) and the effect of the chromosomal content among others (Basile et al, 2013; Campbell et al, 2013). Moreover a multicenter study (Rubio et al, 2012) confirmed the direct cleavage from 1 to 3 cells (DC2-3 < 5 hours) as a negative marker of implantation after reporting that only 1% of the embryos presenting this type of abnormal behaviour implanted. Also another study worth mentioning is the one by Meseguer et al (2012) where reproductive outcome from 10 different clinics was retrospectively compared for patients that had their embryos cultured in standard incubators (SI) vs. a TMS. The study concluded that culturing and selecting embryos with TMS significantly improved the relative probability of clinical pregnancy (+20.1% per oocyte retrieval, +15.7% per embryo transfer). The elevated clinical pregnancy rate was attributed to a combination of stable culture conditions and the use of morphokinetic parameters for embryo selection. Given the increasing number of studies based on TMS it is not surprising to find in the literature articles reviewing the use of TMS as a tool for embryo selection while at the same time summarizing published data (Kirkegaard et al, 2012; Meseguer et al 2012; Basile and Meseguer, 2012; Herrero and Meseguer, 2013; Wong et al, 2013).

In summary, great advances have occurred since the introduction of TMS. This new and exciting technology has led to the discovery of new and interesting data that can be applied on a daily basis in a clinical setting. We may now predict blastocyst formation as early on day 2 (Wong et al, 2010), it is possible to select embryos based on new algorithms (Meseguer et al, 2011; Wong et al, 2010) and we have learned that the morphological scoring of the embryos can change in a very short time spam (Montag et al, 2011).  In the future this technology will save time to the embryologist while offering at the same time 24-hours of continuous observation. It has the potential to improve the effectiveness of IVF cycles, reducing costs and increasing the ability to identify embryos with higher viability and implantation potential. Progress in this scientific field can become very important when implementing a policy of single embryo transfer considering that it offers the embryologist a system for decision making that is more powerful and that it is based not only on the real-time status of the embryo but in a more authentic and dynamic view of the entire process of the embryonic development. And last but not least, this technology will definitely aid in the transition from subjectivity to objectivity in IVF laboratories.