Patent Application
20140087415
The present invention relates to a method and to a system for selecting embryos for in vitro fertilization based on the timing, and duration of observed cell cleavages and associated cell morphology.
Infertility affects more than 80 million people worldwide. It is estimated that 10% of all couples experience primary or secondary infertility (Vayena et al. 2001). In vitro fertilization (IVF) is an elective medical treatment that may provide a couple who has been otherwise unable to conceive a chance to establish a pregnancy. It is a process in which eggs (oocytes) are taken from a woman's ovaries and then fertilized with sperm in the laboratory. The embryos created in this process are then placed into the uterus for potential implantation. To avoid multiple pregnancies and multiple births, only a few embryos are transferred (normally less than four and ideally only one (Bhattacharya et al. 2004)). Selecting proper embryos for transfer is a critical step in any IVF-treatment. Current selection procedures are mostly entirely based on morphological evaluation of the embryo at different timepoints during development and particularly an evaluation at the time of transfer using a standard stereomicroscope. However, it is widely recognized that the evaluation procedure needs qualitative as well as quantitative improvements.
One approach is to use ‘early cleavage’ to the 2-cell stage, (i.e. before 25-27 h post insemination/injection), as a quality indicator. In this approach the embryos are visually inspected 25-27 hours after fertilization to determine if the first cell cleavage has been completed. However, although the early cleavage as well as other early criteria may be a quality indicator for development into an embryo there is still a need for quality indicators for implantation success and thereby success for having a baby as a result.
All patent and non-patent references cited in the application, or in the present application, are also hereby incorporated by reference in their entirety.
Previous studies have often focused on the embryo development before embryonic genome activation. However, the present inventors have found that monitoring the timing and duration of the subsequent cleavages, wherein embryonic genome activation takes place, may lead to additional quality criteria (sometimes referred to as “late phase criteria”), that are very useful in the selection of embryos in order to increase implantation success.
Accordingly, the present invention relates to a method and to a system to facilitate the selection of optimal embryos to be transferred for implantation after in vitro fertilization (IVF) based on the timing, and duration of observed cell cleavages.
In a first aspect the invention relates to a method for determining embryo quality comprising monitoring the embryo for a time period, and determining one or more quality criteria for said embryo, and based on said one or more quality criteria determining the embryo quality. In particular the invention may be applied to human embryos and the obtained embryo quality measure may be used for identifying and selecting embryos suitable of transplantation into the uterus of a female in order to provide a pregnancy and live-born baby.
Thus, in one embodiment the invention relates to a method for determining embryo quality comprising monitoring the embryo for a time period, and determining one or more quality criteria for said embryo, wherein said one or more quality criteria is based on the extent of irregularity of the timing of cell divisions when the embryo develops from four to eight blastomeres, and/or wherein said one or more quality criteria is based on determining the time of cleavage to a five blastomere embryo (t5) and wherein t5 is between 48.7 hours and 55.6 hours, and/or wherein said one or more quality criteria is based on the ratio of two time intervals, each of said two time intervals determined as the duration of a time period between two morphological events in the embryo development from fertilization to eight blastomeres, and based on said one or more quality criteria determining the embryo quality.
Thus, in a further aspect the invention relates to a method for selecting an embryo suitable for transplantation, said method comprising monitoring the embryo as defined above obtaining an embryo quality measure, and selecting the embryo having the highest embryo quality measure.
In a further aspect the invention relates to a system having means for carrying out the methods described above. Said system may be any suitable system, such as a computer comprising computer code portions constituting means for executing the methods as described above. The system may further comprise means for acquiring images of the embryo at different time intervals, such as the system described in pending PCT application entitled “Determination of a change in a cell population”, filed Oct. 16, 2006.
In a yet further aspect the invention relates to a data carrier comprising computer code portions constituting means for executing the methods as described above.
a Uneven blastomere size at the 2 cell stage (2nd cell cycle)—
Cleavage time is defined as the first observed timepoint when the newly formed blastomeres are completely separated by confluent cell membranes, the cleavage time is therefore the time of completion of a blastomere cleavage. In the present context the times are expressed as hours post IntraCytoplasmic Sperm Injection (ICSI) microinjection, i.e. the time of fertilization. Thereby the cleavage times are as follows:
Duration of cell cycles is defined as follows:
Synchronicities are defined as follows:
Cleavage period: The period of time from the first observation of indentations in the cell membrane (indicating onset of cytoplasmic cleavage) to the cytoplasmic cell cleavage is complete so that the blastomeres are completely separated by confluent cell membranes. Also termed as duration of cytokinesis.
Fertilization and cleavage are the primary morphological events of an embryo, at least until the 8 blastomere stage. Cleavage time, cell cycle, synchrony of division and cleavage period are examples of morphological embryo parameters that can be defined from these primary morphological events and each of these morphological embryo parameters are defined as the duration of a time period between two morphological events, e.g. measured in hours.
A normalized morphological embryo parameter is defined as the ratio of two morphological embryo parameters, e.g. cc2 divided by cc3 (cc2/cc3), or cc2/cc2—3 or cc3/t5 or s2/cc2.
The following discrete (binary) variables can be used
Rearrangement of cellular position=Cellular movement (see below)
Cellular movement: Movement of the center of the cell and the outer cell membrane. Internal movement of organelles within the cell is NOT cellular movement. The outer cell membrane is a dynamic structure, so the cell boundary will continually change position slightly. However, these slight fluctuations are not considered cellular movement. Cellular movement is when the center of gravity for the cell and its position with respect to other cells change as well as when cells divide. Cellular movement can be quantified by calculating the difference between two consecutive digital images of the moving cell. An example of such quantification is described in detail in the pending PCT application entitled “Determination of a change in a cell population”, filed Oct. 16, 2006. However, other methods to determine movement of the cellular center of gravity, and/or position of the cytoplasm membrane may be envisioned e.g. by using FertiMorph software (ImageHouse Medical, Copenhagen, Denmark) to semi-automatically outline the boundary of each blastomere in consecutive optical transects through an embryo.
Organelle movement: Movement of internal organelles and organelle membranes within the embryo which may be visible by microscopy. Organelle movement is not Cellular movement in the context of this application.
Movement: spatial rearrangement of objects. Movements are characterized and/or quantified and/or described by many different parameters including but restricted to: extent of movement, area and/or volume involved in movement, rotation, translation vectors, orientation of movement, speed of movement, resizing, inflation/deflation etc.
Different measurements of cellular or organelle movement may thus be used for different purposes some of these reflect the extent or magnitude of movement, some the spatial distribution of moving objects, some the trajectories or volumes being afflicted by the movement.
Embryo quality is a measure of the ability of said embryo to successfully implant and develop in the uterus after transfer. Embryos of high quality will successfully implant and develop in the uterus after transfer whereas low quality embryos will not.
Embryo viability is a measure of the ability of said embryo to successfully implant and develop in the uterus after transfer. Embryos of high viability will successfully implant and develop in the uterus after transfer whereas low viability embryos will not. Viability and quality are used interchangeably in this document
Embryo quality (or viability) measurement is a parameter intended to reflect the quality (or viability) of an embryo such that embryos with high values of the quality parameter have a high probability of being of high quality (or viability), and low probability of being low quality (or viability). Whereas embryos with an associated low value for the quality (or viability) parameter only have a low probability of having a high quality (or viability) and a high probability of being low quality (or viability)
The search for prognostic factors that predict embryo development and the outcome of in vitro fertilization (IVF) treatment have attracted considerable research attention as it is anticipated that knowledge of such factors may improve future IVF treatments.
As discussed above one promising predictive factor is the precise timing of key events in early embryo development. Studies that involve imaging have been limited to measurements of early development, such as pronuclear formation and fusion, and time to first cleavage (Nagy, Z. P. 1994, Fenwick, J. 2002, Lundin, K. 2001, Lemmen, J. G. 2008). An important finding of the time-lapse analysis is a correlation between the early cleavage pattern to the 4-cell stage and subsequent development to the blastocyst stage. Morphokinetic analysis on the development of bovine embryos have also been published, where timing, duration and intervals between cell cleavages in early embryo development successfully predicted subsequent development to the expanded blastocyst stage (Ramsing 2006, Ramsing 2007).
The present inventors have performed a large clinical study involving many human embryos and monitoring the development, not only until formation of a blastocyst, but further until sign of implantation of the embryo. In this study important differences in the temporal patterns of development between the embryos that implanted (i.e. embryos that were transferred and subsequently led to successful implantation) and those that did not (i.e. embryos that were transferred but did not lead to successful implantation) were observed.
It has been found that there exists an optimal time range for parameters characterizing the embryonic cell divisions. Embryos which cleave at intermediate timepoints have significantly improved chance of ongoing implantation when compared with embryos that either developed faster or slower. The observations support the hypothesis that the viability of embryos is associated with a highly regulated sequence of cellular events that begin at the time of fertilization. In this large clinical study on exclusively good quality embryos, it has been confirmed that an embryo's capability to implant is correlated with numerous different cellular events, e.g. timing of cell divisions and time between divisions, as well as uneven blastomere size and multinucleation. The complexity, structure and parameters in the models must be adaptable to different clinical situations like incubation temperature, transfer times, culture media and other.
Timing of early events in embryonic development correlates with development into a blastocyst, however it has been found that the development into a blastocyst does not necessarily correlate with successful implantation of the embryo. By using implantation as the endpoint, not only embryo competence for blastocyst formation, but also subsequent highly essential processes such as hatching and successful implantation in the uterus is assessed.
Thus, the data allows the detection of later developmental criteria for implantation potential. The results in particular indicate that timing of later events such as the cleavage to the five cell stage are a consistently good indicator of implantation potential, and that the discrimination between implanting and non-implanting embryos is improved when using the later cell division events, e.g. t5 as opposed to the earlier events (t2, t3 and t4). The presented data indicate that incubating the embryos to day 3, which enables evaluation of timing for cell divisions from five to eight cells, after completion of the third cell cycle, can give additional important information that will improve the ability to select a viable embryo with high implantation potential.
Accordingly, in a first aspect the invention relates to a method for determining embryo quality comprising monitoring the embryo for a time period, and determining one or more quality criteria for said embryo, and based on said quality criteria determining the embryo quality. In the present context, the embryo quality is a quality relating to implantation success.
The selection criteria (quality criteria) can be based on single variables, composite variables (variables that can be calculated from other variables) and multiple variables (more variables at once).
The quality criteria used herein are preferably criteria relating to the phase from a 2 to 8 blastomere embryo, in particularly from 4 to 8 blastomere embryo, and accordingly, the present quality criteria may be determination of the time for cleavage into a 5 blastomere embryo, 6 blastomere embryo, 7 blastomere embryo, and/or 8 blastomere embryo.
The present quality criteria is a preferably criteria obtained within the time period of from 48 to 72 hours from fertilisation. As discussed above, the clock starts at the time of fertilisation which in the present context is meant to be the time of injection of the sperm, such as by ICSI microinjection. Preferably the embryo is monitored for a time period comprising at least three cell cycles, such as at least four cell cycles.
In particular it has been found that the time for cleavage into a 5 blastomere embryo has an important impact on the implantation success, and therefore the quality criteria is preferably determination of the time for cleavage into a 5 blastomere embryo, i.e. t5. As shown below t5 should preferably be in the range of from 47-58 hours from fertilisation, more preferably in the range of 48-57 hours from fertilisation, more preferably in the range of 48.7-55.6 hours from fertilisation.
The time for cleavage into a 2 blastomere embryo has an important impact on the implantation success and t2 should preferably be less than 32 hours from fertilisation, more preferably less than 27.9 hours from fertilisation. In a further embodiment of the invention t2≧24.3 hours.
The time for cleavage into a 3 blastomere embryo may have an impact on the implantation success and t3 should preferably be less than or equal to 40.3 hours from fertilisation. In a further embodiment of the invention t3≧35.4 hours.
The time for cleavage into a 6 blastomere embryo may have an impact on the implantation success and t6 should preferably be less than 60 hours from fertilisation.
The time for cleavage into a 7 blastomere embryo may have an impact on the implantation success and t7 should preferably be less than 60 hours from fertilisation.
The time for cleavage into an 8 blastomere embryo may have an impact on the implantation success and t8 should preferably be less than 60 hours from fertilisation more preferably less than 57.2 hours from fertilisation.
The duration of the period as a 2 blastomere embryo, i.e. the second cell cycle cc2=t3−t2, may have an impact on the implantation success and cc2 should preferably be less than 12.7 hours.
The duration of the period as a 2 and 3 blastomere embryo, i.e. the second cell cycle for both blastomeres cc2b=t4−t2, may have an impact on the implantation success and cc2 should preferably be less than 12.7 hours. In a further embodiment of the invention cc2>5 hours.
The duration of the period as a 3 and 4 blastomere embryo, i.e. cc3=t5−t3, may have an impact on the implantation success and cc3 should preferably be less than or equal to 16.3 hours. In a further embodiment of the invention cc3≧5 hours or cc3≧12.9 hours.
The duration of the period as a 2, 3 and 4 blastomere embryo, i.e. cc2—3=t5−t2, may have an impact on the implantation success and cc2—3 should preferably be less than or equal to 28.7 hours. In a further embodiment of the invention cc2—3≧24 hours.
The synchrony in division from a 2 blastomere embryo to a 4 blastomere embryo, i.e. s2=t4−t3, may have an impact on the implantation success and s2 should preferably be less than 1.33 hours or less than 0.33 hours.
The synchrony in division from a 4 blastomere embryo to an 8 blastomere embryo, i.e. s3=t8−t5, may have an impact on the implantation success and s3 should preferably be less than 2.7 hours.
Multiple variables may be used when choosing selection criteria. When using multiple variables it can be an advantage that the variables are selected progressively such that initially one or more of the variables that can be determined early with a high accuracy are chosen, e.g. t2, t3, t4 or t5. Later other variables that can be more difficult to determine and is associated with a higher uncertainty can be used (e.g. multinuclearity, evenness of cells and later timings (e.g. after t5)).
In addition to t5 other criteria may be added to determine the embryo quality. In one embodiment the present quality criteria is combined with determination of second cell cycle length in order to establish the embryo quality. In another embodiment the present quality criteria is combined with determination of synchrony in cleavage from 2 blastomere embryo to 4 blastomere embryo.
Accordingly, in one embodiment the embryo quality is determined from a combination of determination of time for cleavage to a 5 blastomere embryo and determination of the second cell cycle length.
Furthermore, three different criteria may be combined, for example so that determination of time for cleavage to a 5 blastomere embryo and determination of the second cell cycle length are combined with determination of synchrony in cleavage from 2 blastomere.
In one embodiment of the invention an embryo quality criterion is selected from the group of normalized morphological embryo parameters, in particular the group of normalized morphological parameters based on two, three, four, five or more parameters selected from the group of t2, t3, t4, t5, t6, t7 and t8. By normalizing the parameters the time of fertilization may be “removed” from the embryo quality assessment. Further, a normalized morphological embryo parameter may better describe the uniformity and/or regularity of the developmental rate of a specific embryo independent of the environmental conditions, because instead of comparing to “globally” determined absolute time intervals that may depend on the local environmental conditions, the use of normalized parameters ensure that specific ratios of time intervals can be compared to “globally” determined normalized parameters, thereby providing additional information of the embryo development. E.g. the ratio cc2/cc3 may indicate whether the duration of cell cycle 2 corresponds (relatively) to the duration of cell cycle 3, cc2/cc2—3 provides the duration of the period as a 2 blastomere embryo relative to the duration of the period as a 2, 3 and 4 blastomere embryo, s2/cc2 provides the synchronicity from 2 to 4 blastomere relative to the duration of the period as a 2 blastomere embryo and cc3/t5 provides the duration of cell cycle 3 relative to the time of cleavage to a 5 blastomere embryo.
In one embodiment of the invention the normalized morphological embryo parameter cc2/cc2—3=1−cc3/cc2—3=(t3−t2)/(t5−t2) should be between 0.41 and 0.47.
In one embodiment of the invention the normalized morphological embryo parameter cc3/t5=1−t3/t5 should be greater than 0.3 or between 0.26 and 0.28.
In one embodiment of the invention the normalized morphological embryo parameter s2/cc2=(t4−t3)/(t3−t2) should be less than 0.025.
In one embodiment of the invention the normalized morphological embryo parameter s3/cc3=(t8−t5)/(t5−t3) should be less than 0.18.
In one embodiment of the invention the normalized morphological embryo parameter cc2/cc3=(t3−t2)/(t5−t3) should be between 0.72 and 0.88.
Irregularity from 4 to 8 Blastomeres
The timing of the individual cell divisions when the embryo develops from 4 to 8 blastomeres (i.e. s3a=t6−t5), s3b=t7−t6 and s3c=t8−t7) may be associated with embryo quality and success of implantation. These timings may demonstrate the competence of each individual cell to perform a cell division. Possible irregularities or abnormalities in the mitosis may result in large differences between the value of s3a, s3b and/or s3c. Thus, in a further embodiment of the invention an embryo quality criterion is the extent of the irregularity of the timing of cell divisions, such as irregularity of the timing of cell divisions until the 8 blastomere embryo, such as irregularity of the timing of cell divisions when developing from 4 to 8 blastomere embryo. In a further embodiment of the invention an embryo quality criterion is the maximum of s3a, s3b and s3c. Preferably the maximum of s3a, s3b and s3c is less than 1.5 hours. In a further embodiment of the invention an embryo quality criterion is the maximum of s3a, s3b and s3c divided by s3, preferably max(s3a, s3b, s3c)/s3 is less than 0.5. Please note that max(s3a, s3b, s3c)/s3 is a normalized morphological embryo parameter based on t5, t6, t7 and t8.
Multi nucleation may be an embryo quality parameter, in particular multi nucleation observed at the 4 blastomere stage (MN4). Preferably no multi nucleation should be present at the 4 blastomere stage, thus preferably MN4=False.
Even size of the blastomeres may be an embryo quality parameter, in particular a two blastomere embryo should have blastomeres of even size, thus preferably EV2=True.
EV2: Evenness of the blastomeres in the 2 blastomere embryo; can take the values “True” (i.e. even) or “False” (i.e. uneven).
An embryo population may be subject to one or more exclusion criteria in order to exclude embryos from the population with a low probability of implantation success, i.e. the outliers. This may be embryos that fulfil many of the positive selection criteria but show unusual behaviour in just one or two selection criteria. Examples of exclusion criteria are the discrete criteria such as blastomere evenness at t2 and multi nuclearity at the four-blastomere stage. However, exclusion criteria may also be applied to the morphological embryo parameters. It has long been known that slowly developing embryos are an indication of poor quality, reflected in a very high value of t2 (>31.8 hours), but cleavage from one blastomere directly to three blastomeres may also be an indication of a poor quality embryo associated with low implantation rate. This may be reflected in very low values for cc2 and cc3.
A specific exclusion criterion pointing out a group of embryos in a population with a low probability of implantation does not imply that the rest of the population has a high probability of implantation. An exclusion criterion only indicates poor quality embryos. Thus, in one embodiment of the invention said one or more quality criteria are combined with one or more exclusion criteria. An example of applying exclusion criteria to a population of embryos (based on KID data, see example 2) is shown in
The embryo is monitored regularly to obtain the relevant information, preferably at least once per hour, such as at least twice per hour, such as at least three times per hour. The monitoring is preferably conducted while the embryo is situated in the incubator used for culturing the embryo. This is preferably carried out through image acquisition of the embryo, such as discussed below in relation to time-lapse methods.
Determination of selection criteria's can be done for example by visual inspection of the images of the embryo and/or by automated methods such as described in detail in the pending PCT application entitled “Determination of a change in a cell population” filed Oct. 16, 2006. Furthermore, other methods to determine selection criteria's can be done by determining the position of the cytoplasm membrane by envisioned e.g. by using FertiMorph software (ImageHouse Medical) Copenhagen, Denmark). The described methods can be used alone or in combination with visual inspection of the images of the embryo and/or with automated methods as described above.
In particularly, the criteria may be combined in a hierarchical form, as shown in
The decision tree depicted in
The decision tree subdivided embryos into 6 categories from A to F. Four of these categories (A to D) were further subdivided into two sub-categories (+) or (−) as shown in
If the value of s2 falls inside the optimal range (≦1.76 hrs) the embryo is categorized as A or C depending on t5 and similarly if the value of s2 falls outside the optimal range the embryo is categorized as B or D depending on t5.
Finally, the embryo is categorized with the extra plus (+) if the value for cc2 is inside the optimal range (≦11.9 hrs) (A+/B+/C+/D+) and is categorized as A,B,C,D if the value for cc2 is outside the optimal range.
In the study discussed in example 1, the decision procedure divides all the 247 evaluated embryos in ten different categories containing approx. the same number of transferred embryos but with largely decreasing implantation potential (i.e. from 68% for A+ to 8% for E).
Decision tree models have also been constructed based on KID data from 1598 human embryos (see example 2). The two decision trees are based on quality and exclusion criteria from t2 onwards (
Combination with Measurements of Movement
The quality criteria discussed above may also be combined with determinations of movement of the embryo, such as i) determining the extent and/or spatial distribution of cellular or organelle movement during the cell cleavage period; and/or ii) determining the extent and/or spatial distribution of cellular or organelle movement during the inter-cleavage period thereby obtaining an embryo quality measure.
Volumes within the zona pelucida that are devoid of movement (or similarly areas in a projected 2D image of the embryo that remain stationary) are an indication of “dead” zones within the embryo. The more and larger these immotile “dead” zones the lower the probability of successful embryo development. Large areas within a time-lapse series of embryo images without any type of movement (i.e. neither cellular nor organelle movement) indicates low viability. Organelle movement should generally be detectable in the entire embryo even when only comparing two or a few consecutive frames. Cellular movement may be more localized especially in the later phases of embryo development.
The cell positions are usually relatively stationary between cell cleavages (i.e. little cellular movement), except for a short time interval around each cell cleavage, where the cleavage of one cell into two leads to brief but considerable rearrangement of the dividing cells as well as the surrounding cells (i.e. pronounced cellular movement). The lesser movement between cleavages is preferred.
In one embodiment, in order to determine movement relating to either cleavage and inter-cleavage periods, the length of each cleavage period may be determined as well as the length of each inter-cleavage period. Preferably the period of cellular movement in at least two inter-cleavage periods is determined as well as the extent of cellular movement in at least two inter-cleavage periods. Furthermore, it has been found that rapid cleavage seems to increase quality of the embryo, where rapid normally means less than 2 hours.
In relation to movement during cleavage and inter-cleavage periods we also refer to PCT application WO 2007/144001.
A neural network or other quantitative pattern recognition algorithms may be used to evaluate the complex cell motility patterns described above, for example using different mathematical models (linear, Princepal component analysis, Markov models etc.)
A particular use of the invention is to evaluate image series of developing embryos (time-lapse images). These time-lapse images may be analyzed by difference imaging equipment (see for example WO 2007/042044 entitled “Determination of a change in a cell population”). The resulting difference images can be used to quantify the amount of change occurring between consecutive frames in an image series.
The invention may be applied to analysis of difference image data, where the changing positions of the cell boundaries (i.e. cell membranes) as a consequence of cellular movement causes a range parameters derived from the difference image to rise temporarily (see WO 2007/042044). These parameters include (but are not restricted to) a rise in the mean absolute intensity or variance. Cell cleavages and their duration and related cellular re-arrangement can thus be detected by temporary change, an increase or a decrease, in standard deviation for all pixels in the difference image or any other of the derived parameters for “blastomere activity” listed in WO 2007/042044. However the selection criteria may also be applied to visual observations and analysis of time-lapse images and other temporally resolved data (e.g. excretion or uptake of metabolites, changes in physical or chemical appearance, diffraction, scatter, absorption etc.) related to embryo.
Of particular interest are the onset, magnitude and duration of cell cleavages that may be quantified as peaks or valleys, in derived parameter values. These extremes, peaks or valleys, frequently denote cell cleavage events. The shape of each peak also provides additional information as may the size of the peak in general. A peak may also denote an abrupt collapse of a blastomer and concurrent cell death. However, it may be possible to separate cell cleavage events and cell death events by the peak shape and change in base values before and after the event. The baseline of most parameters are usually not affected by cell cleavage whereas cell lysis is frequently accompanied by a marked change in the baseline value (for most parameters in a decrease following lysis.)
In summary, the present invention demonstrates that routine time-lapse monitoring of embryo development in a clinical setting (i.e. automatic image acquisition in an undisturbed controlled incubation environment) provide novel information about developmental parameters that differ between implanting and non-implanting embryos.
In some cases the term “embryo” is used to describe a fertilized oocyte after implantation in the uterus until 8 weeks after fertilization at which stage it becomes a foetus. According to this definition the fertilized oocyte is often called a pre-embryo until implantation occurs. However, throughout this patent application we will use a broader definition of the term embryo, which includes the pre-embryo phase. It thus encompasses all developmental stages from the fertilization of the oocyte through morula, blastocyst stages hatching and implantation.
An embryo is approximately spherical and is composed of one or more cells (blastomeres) surrounded by a gelatine-like shell, the acellular matrix known as the zona pellucida. The zona pellucida performs a variety of functions until the embryo hatches, and is a good landmark for embryo evaluation. The zona pellucida is spherical and translucent, and should be clearly distinguishable from cellular debris.
An embryo is formed when an oocyte is fertilized by fusion or injection of a sperm cell (spermatozoa). The term is traditionally used also after hatching (i.e. rupture of zona pelucida) and the ensuing implantation. For humans the fertilized oocyte is traditionally called an embryo for the first 8 weeks. After that (i.e. after eight weeks and when all major organs have been formed) it is called a foetus. However the distinction between embryo and foetus is not generally well defined.
During embryonic development, blastomere numbers increase geometrically (1-2-4-8-16-etc.). Synchronous cell cleavage is generally maintained to the 16-cell stage in embryos. After that, cell cleavage becomes asynchronous and finally individual cells possess their own cell cycle. Human embryos produced during infertility treatment are usually transferred to the recipient before 16-blastomere stage. In some cases human embryos are also cultivated to the blastocyst stage before transfer. This is preferably done when many good quality embryos are available or prolonged incubation is necessary to await the result of a pre-implantation genetic diagnosis (PGD).
Accordingly, the term embryo is used in the following to denote each of the stages fertilized oocyte, zygote, 2-cell, 4-cell, 8-cell, 16-cell, morula, blastocyst, expanded blastocyst and hatched blastocyst, as well as all stages in between (e.g. 3-cell or 5-cell)
A final analysis step could include a comparison of the made observations with similar observations of embryos of different quality and development competence, as well as comparing parameter values for a given embryo with other quantitative measurements made on the same embryo. This may include a comparison with online measurements such as blastomere motility, respiration rate, amino acid uptake etc. A combined dataset of blastomere motility analysis, respiration rates and other quantitative parameters are likely to improve embryo selection and reliably enable embryologist to choose the best embryos for transfer.
Thus, in one embodiment the method according to the invention may be combined with other measurements in order to evaluate the embryo in question, and may be used for selection of competent embryos for transfer to the recipient.
Such other measurements may be selected from the group of respiration rate, amino acid uptake, motility analysis, blastomere motility, morphology, blastomere size, blastomere granulation, fragmentation, blastomere colour, polar body orientation, nucleation, spindle formation and integrity, and numerous other qualitative measurements. The respiration measurement may be conducted as described in PCT publication no. WO 2004/056265.
In a preferred embodiment the observations are conducted during cultivation of the cell population, such as wherein the cell population is positioned in a culture medium. Means for culturing cell population are known in the art. An example of culturing an embryo is described in PCT publication no. WO 2004/056265.
The invention further relates to a data carrier comprising a computer program directly loadable in the memory of a digital processing device and comprising computer code portions constituting means for executing the method of the invention as described above.
The data carrier may be a magnetic or optical disk or in the shape of an electronic card as for example the type EEPROM or Flash, and designed to be loaded into existing digital processing means.
The present invention further provides a method for selecting an embryo for transplantation. The method implies that the embryo has been monitored as discussed above to determine when cell cleavages have occurred.
The selection or identifying method may be combined with other measurements as described above in order to evaluate the quality of the embryo. The important criteria in a morphological evaluation of embryos are: (1) shape of the embryo including number of blastomers and degree of fragmentation; (2) presence and quality of a zona pellu-cida; (3) size; (4) colour and texture; (5) knowledge of the age of the embryo in relation to its developmental stage, and (6) blastomere membrane integrity.
The transplantation may then be conducted by any suitable method known to the skilled person.
The research project was conducted at the Instituto Valenciano de Infertilidad-IVI, Valencia. The procedure and protocol was approved by an Institutional Review Board, (IRB), which regulates and approves database analysis and clinical IVF procedures for research at IVI. The project complies with the Spanish Law governing Assisted Reproductive Technologies (14/2006). The present study included a total of 2903 oocytes from which 2120 embryos were generated in 285 IVF treatment cycles between September 2009 and September 2010. All embryos were obtained after fertilization by Intra Cytoplasmic Sperm Injection (ICSI) and were part of the clinic's standard (n=188) and ovum donation program (n=97). Time-lapse images were acquired of all embryos, but only transferred embryos with known implantation (i.e. either 0% implantation or 100% implantation) were investigated by detailed time-lapse analysis measuring the exact timing of the developmental events in hours-post-fertilization by ICSI.
The exclusion criteria for standard patients and recipients with respect to this study were: low response (less than 5 MII oocytes), endometriosis, Polycystic Ovarian Syndrome (PCOS), hydrosalpynx, BMI>30 kg/m2, uterine pathology (myomas, adenomyiosis, endocrinopaties, trombophylia, chronic pathologies, acquired or congenital uterine abnormalities), recurrent pregnancy loss, maternal age over 39 years old for standard patients and 45 for oocyte donation recipients (aging uterus), or severe masculine factor (presenting less than 5 million motile sperm cells in total in the ejaculate).
All donors were from the clinic's egg donation program. Only patients having fulfilled the inclusion criteria were included in the study. Briefly, donors were between 18 and 35 years old without current or past exposure to radiation or hazardous chemical substances, drug use, no family history of hereditary or chromosomal diseases, a normal karyotype, and tested negative for fragile X Syndrome and sexually transmitted diseases as stated by Spanish law (Garrido, N. 2002). The mean age of the male patients of the study population was 37.9 years (SD=5.2). The mean age of the female population was 36.9 years (SD=4.9). All donors had normal menstrual cycles of 26-34 days duration, normal weight (BMI of 18-28 kg/m2), no endocrine treatment (including gonadotrophins and oral contraception) in the 3 months preceding the study, normal uterus and ovaries at transvaginal ultrasound (no signs of polycystic ovary syndrome), and antral follicle count (AFC)>20 on the first day of gonadotrophin administration, after down-regulation with GnRH agonist (Meseguer, M. 2010).
Prior to controlled ovarian stimulation (COS), cycles with GnRH agonist protocols were used. In GnRH agonist protocols, patients started with administration of 0.5 mg leuprolide acetate (Procrin®; Abbott, Madrid, Spain) in the midluteal phase of the previous cycle, until negative vaginal ultrasound defined ovarian quiescence. Patients with adequate pituitary desensitization started their stimulation, and the dose of GnRH-agonist was reduced to 0.25 mg per day until the day of hCG administration (Melo, M. 2009).
For COS the treatments proceeded as previously described (Melo, M. 2010). Briefly, donors and patients treated with 150 IU of rFSH (Gonal-f; Merck Serono) plus 75 IU HP-hMG (Menopur; Ferring). The fixed starting dose of 225 IU gonadotropins per day was initiated on day 3 of menstruation and sustained for the first 5 days of controlled ovarian stimulation, during which serum E2 was assessed. The gonadotropin dose was adjusted if necessary. Serial transvaginal ultrasound examinations were initiated on day 5 of controlled ovarian stimulation and were performed every 48 hours to monitor the follicular growth. Human chorionic gonadotropin (hCG) (Ovitrelle, Serono Laboratories, Madrid, Spain) was administered subcutaneously when at least eight leading follicles reached a mean diameter ≧18 mm. Daily administration of gonadotrophins and the GnRH agonist was discontinued on the day of hCG administration. Transvaginal oocyte retrieval was scheduled 36 hours later. Serum E2 and P levels were measured on the morning of hCG administration. Samples were tested with a microparticle enzyme immunoassay Axsym System (Abbott Cientifico S.A., Madrid, Spain). The serum E2 kit had a sensitivity of 28 pg/mL and intraobserver and interobserver variation coefficients of 6.6% and 7.7%, respectively.
Protocol for Endometrial Preparation of Recipients:
can be found in (Meseguer, M. 2008; Meseguer, M. 2010). Briefly, patients with ovarian function were down-regulated with a single dose of 3.75 mg of Triptorelin (Decapeptyl 3.75, Ipsen Pharma S.A., Madrid, Spain) administered IM in the secretory phase of the previous cycle. Hormonal replacement started on day 1 of the cycle after ovarian downregulation was confirmed with vaginal ultrasound. Patients started oral administration of 2 mg/day of E2 valerate (Progynova, Schering Spain, Madrid, Spain) from days 1 to 8; 4 mg/day from days 9 to 11; and 6 mg/day from day 12 on. Patients without ovarian function started hormonal replacement directly. After 14 days of E2 valerate administration, vaginal ultrasound was performed and serum E2 determined. If recipients were ready to receive oocytes, they waited having 6 mg/day of E2 valerate until an adequate donation was available. After embryo transfer for luteal phase support all patients received a daily dose of 200 mg for standard patients and 400 mg for oocyte recipients of vaginal micronized progesterone (Progeffik Effik, Madrid Spain) every 12 hours.
Follicles were aspirated and the oocytes were washed in Quinn's Advantage medium (QAM) (SAGE, Rome, Italy). After washing, oocytes were cultured in Quinn's Advantage Fertilization medium (QAFM) (Sage Rome, Italy) at 5.2% CO2 and 37° C. for 4 hours before oocyte denudation. Oocyte stripping was carried out by mechanical pipetting in 401 U/mL of hyaluronidase in the same medium (QAFM). After this ICSI was performed in a medium containing HEPES (QAM) (Garcia-Herrero, S. 2010). ICSI was performed at 400× magnification using an Olympus IX7 microscope. Finally the oocytes was placed in pre-equilibrated slides (EmbryoSlide® Unisense FertiliTech, Aarhus Denmark).
The slides are constructed with a central depression containing 12 straight-sided cylindrical wells each containing a culture media droplet of 20 μL Quinn's Advantage Cleavage medium (QACM). The depression containing the 12 wells was filled with an overlay of 1.4 mL mineral oil to prevent evaporation. The slides were prepared at least 4 hrs in advance and left in an incubator to pre-equilibrate at 37° C. in the 5.0% CO2 atmosphere. After pre-equilibration all air bubbles are meticulously removed before the oocytes are placed individually in dropplets and incubated in the time-lapse monitoring system until embryo transfer 72 hour later approximately. The time-lapse instrument, EmbryoScope®, (ES), (Unisense FertiliTech, Aarhus, Denmark) is a tri gas oocyte/embryo incubator with a built in microscope to automatically acquire images of up to 72 individual embryos during development.
The imaging system in the ES uses low intensity red light (635 nm) from a single LED with short illumination times of 30 ms per image to minimize embryo exposure to light and to avoid damaging short wavelength light. The optics comprise of a modified Hoffmann contrast with a 20× speciality objective (Leica Place) to provide optimal light sensitivity and resolution for the red wavelength. The digital images are collected by a highly sensitive CCD camera (1280×1024 pixels) with a resolution of 3 pixels per μm. Image stacks were acquired at 5 equidistant focal planes every 15 minutes during embryo development inside the ES (i.e. from about 1 hr after fertilization to transfer on day 3 about 72 hrs after fertilization). Embryo exposure to light during incubation was measured with a scalar irradiance microsensor with a tip diameter of 100 μm placed within the EmbryoScope at the position of the embryo in the EmbryoSlide. Similar measurements were made on standard microscopes used in fertility clinics. The total exposure time in the time-lapse system during 3 day culture and acquisition of 1420 images were 57 seconds, which compares favourably with the 167s microscope light exposure time reported for a standard IVF treatment (Ottosen et al, 2007). As the light intensity measured with the within the ES with the scalar irradiance microsensor was much lower than the light intensity in microscopes used in IVF clinics, the total light dose during 3 day incubation in the time-lapse system was found to be 20 J/m2 (i.e. 0.24 μJ/embryo) as opposed to an exposure of 394 J/m2 during microscopy in normal IVF treatments (i.e. 4.8 μJ/embryo) based on average illumination times from (Ottosen et al, 2007) and measured average intensities with the scalar irradiance microsensor. Furthermore, the spectral composition of the light in the ES was confined to a narrow range centred around 635 nm, and thus devoid of low wavelength light below 550 nm, and comprise about 15% of the light encountered in a normal IVF microscope.
Successful fertilization was assessed at 16-19 h post-ICSI based on digital images acquired with the time-lapse monitoring system. Embryo morphology was evaluated on days 2 (48 h post ICSI) and 3 (72 h post ICSI) 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 (Alikani, M. 2000). Embryo selection were performed exclusively by morphology based on: i) absence of multinucleated cells; ii) between 2-5 cells on day-2; iii) between 6-10 cells on day 3; iv) total fragment volume of less than 15% of the embryo and; v) the embryo must appear symmetric with only slightly asymmetric blastomeres (Meseguer, M. 2006; Muriel, L. 2006; Meseguer, M. 2008). A total of 522 embryos were transferred to 285 patients.
Retrospective analysis of the acquired images of each embryo was made with an external computer, EmbryoViewer workstation (EV), (Unisense FertiliTech, Aarhus, Denmark) using image analysis software in which all the considered embryo developmental events were annotated together with the corresponding timing of the events in hrs after ICSI microinjection. Subsequently the EV was used to identify the precise timing of the 1st cell division. This division was the division to 2 cells and a shorthand notation of t2 is used in the following. Annotation of the 2nd (i.e. to 3 cells, t3), 3rd (4 cells, t4) and 4th (5 cells, t5) cell division were done likewise. For the purpose of this study, time of cleavage was defined as the first observed timepoint when the newly formed blastomeres are completely separated by confluent cell membranes. All events are expressed as hours post ICSI microinjection.
The duration of the second cell cycle was defined as the time from division to a two blastomere embryo until division to a 3 blastomere embryo. cc2=t3−t2, i.e. the second cell cycle is the duration of the period as 2 blastomere embryo.
The second synchrony s2 was defined as the duration of the division from a 2 blastomere embryo to a 4 blastomere embryo (s2=t4−t3) which corresponds to the duration of the period as 3 blastomere embryo.
The detailed analysis was performed on transferred embryos with 100% implantation (i.e. where the number of gestational sacs confirmed by ultrasound matched the number of transferred embryos) (N=61); and on embryos with 0% implantation, (where no biochemical pregnancy was achieved) (N=186).
The number of embryos transferred was normally two, but in some cases 1 or 3 embryos were transferred because of embryo quality or patient wishes. Supernumerary embryos were frozen for potential future transfers using IVI standard vitrification technique (Cobo et al. 2008). The β-hCG value was determined 13 days after embryo transfer and the clinical pregnancy was confirmed when a gestational sac with foetal heartbeat was visible after 7 weeks of pregnancy.
The exact timings of embryo events in hrs after ICSI microinjection largely followed normal distributions for the implanted embryos, but that was typically not the case for the not implanted embryos (Shapiro-Wilk test). The distributions of the not implanted embryos typically had long tails extending to later timing values. To investigate whether the variances in the exact timings of embryo events were different between the implanted and not implanted embryos the Brown-Forsythe's test for homogeneity of variances was used, since it does not demand normality of the tested distributions. The Mann-Whitney U-test was used to test whether the median values in the exact timings of embryo events were significantly different between the implanted and not implanted embryos.
To describe the distribution of the probabilities of implantation, timings were converted from continuous variables into a categorical variable using quartiles for all observations of each of the measured parameters. A system based on ordinations giving four categories (timing quartiles) with equal number of observations in each of them was used to obtain these categories. By this procedure, bias due to differences in the total number of embryos in each category was avoided. Hereafter the percentage of embryos that implanted for each timing quartile was calculated to assess the distribution of implantation in the different categories.
The derived embryo timings were analyzed using Student's T-test when comparing two groups, and Analysis of Variance (ANOVA) followed by Bonferroni's and Scheffe's post hoc analysis when multiple groups were considered. Chi square tests were used to compare between categorical data. For each timing variable an optimal range was defined as the combined range spanned by the two quartiles with the highest implantation rates. Additionally, a binary variable was defined with the value inside (outside) if the value of the timing variable was inside (outside) the optimal range.
The odds ratio (OR) of the effect of all binary variables generated on implantation were expressed in terms of 95% confidence interval (CI95) and significance. By performing the logistic regression analysis, the effect of optimal ranges and other binary variables on implantation were quantified. Significance was calculated using the omnibus test (likehood ratio), and the uncertainties uncovered by the model were evaluated by Negelkerke R2, a coefficient that is analogous to the R2 index of the linear regression analysis. ROC curves were employed to test the predictive value of all the variables included in the model with respect to implantation. ROC curve analysis provides AUC values (area under the curve) that are comprised between 0.5 and 1 and can be interpreted as a measurement of the global classification ability of the model.
Statistical analysis was performed using the Statistical Package for the Social Sciences 17 (SPSS Inc., Chicago, Ill.) and MedCalc Software (Ghent, Belgium).
The primary etiology of female infertility was: poor oocyte quality 34.7% (n=99); advanced maternal age 24.6% (n=70); premature ovary failure 6.0% (n=17); normal 23.8% (n=68), tubal obstruction 2.5% (n=7); low ovary response 8.4% (n=24). Average E2 levels prior to hCG injection were 1701 (SD=991) pg/ml. A total of 201 embryos gave successful implantation (gestational sac) out of the total 522 transferred, giving rise to a 38.5% implantation rate. The biochemical pregnancy rate per transfer was 55.1% (n=157) and ongoing pregnancy rate per transfer were 49.8% (n=142).
A single gestational sac was frequently observed after dual embryo transfer. As it was not possible to ascertain with certainty, which of the two transferred embryos that implanted, these embryos were excluded from further analysis. All embryos with known implantation were selected for further retrospective analysis. This analysis comprise 247 embryos; 61 with 100% implantation (number of gestational sacs matched with number of transferred embryos) and 186 with 0% implantation (no biochemical pregnancy was achieved).
The correlation between morphokinetic parameters analyzed with the EV time lapse tool and embryo implantation was investigated. For 51 embryos of the total 247 (20.6%) morphological events were observed that were apparently related to poor embryo development. These three events were; A) Direct cleavage from zygote to 3 blastomere embryo, defined as: cc2=t3−t2<5 hours. (N=9). B) Uneven blastomere size at the 2 cell stage during the interphase where the nuclei are visible (N=26). Blastomeres are considered uneven sized if the average diameter of the large blastomere is more than 25% larger than the average diameter of the small blastomere. This definition implies that the volume of the large blastomere should be at least twice the volume of the small blastomere. C) Multinucleation at the 4 cell stage during the interphase where the nuclei are visible (N=28) The embryo is considered multinucleated if more than one distinct nucleus is observed in one (or more) blastomeres. From those 51 embryos only 4 implanted (8%) (two with uneven blastomere size and two that were multinucleated). Given the low implantation rate observed in the embryos showing these events it was suggested to use the listed observations as exclusion criteria for embryo selection as the frequency of implanting was very low (4 out of 51 i.e. 8%).
Cleavage times for the first four divisions are shown in
More detailed evaluation of the distribution of all divisional timings was performed. An example, the timing for cell division to five cells, t5, is shown in
The average timing of t2, t3, t4 and t5, together with cc2 and s2 for the analysed transferred embryos with known outcome are presented in Table 1, values for those implanted and not implanted were also calculated. The standard deviation for each of the variables is also included in the table. Additionally, the results of the Shapiro-Wilk test for normal distribution are included in Table I. Exact timings of embryo events follow normal distributions for the implanted embryos for all parameters (except s2). On the other hand the exact timings of embryo events for the not implanted embryos don't follow normal distributions, but exhibit tails at the later timings. Only the parameter t5 follows a normal distribution for the not implanted embryos (see also
As expected from the distributions of cleavage times shown in
This supports the hypothesis that viable embryos follow a predefined developmental schedule with greater fidelity than non-implanting embryos.
Since all parameters show significantly different variances the non-parametric Mann-Whitney U-test was used for comparison of the medians. The median values were not significantly different between the implanting and not implanting embryos for any of the parameters except for s2. The s2, synchrony of second and third cell division were significantly different between implanted embryos with median value 0.50h and not-implanted embryos with median value 1.00h, (p=0.0040).
24.3-25.8
32
25.8-27.9
30
35.4-37.8
39
37.8-40.3
32
36.4-38.9
36
38.9-41.6
31
48.8-52.3
37
52.3-56.6
40
<11.0
23
11.0-11.9
39
<0.30
36
0.30-0.76
28
The four quartiles for the timing of each of the investigated parameters are presented in Table 2 together with percentages of implanting embryos in each quartile. The categories defined by these quartiles were used to establish optimal ranges based on the two consecutive quartiles with highest implantation probabilities (entries in bold typeface in Table 2). Observed parameters with significantly higher implantation rate for parameters inside the optimal range as compared to outside the range are presented in
For all cleavage times assessed (t2, t3, t4 and t5), embryos whose cleavage was completed in the two central quartiles displayed the highest implantation rates, and were consequently combined in an optimal range for each parameter (
For all cleavage times there was a significant difference in implantation rate between embryos within the optimal range as opposed to those outside the range (
For both the duration of the second cell cycle, cc2, and the synchrony of cell cleavages in the transition from 2-cell stage to 4-cell stage, s2 (i.e. the duration of the three cell stage), the embryos that cleaved in the two first quartiles was found to have significantly higher implantation rate that those falling in the last two quartiles (
A logistic regression analysis were used to select and organize which observed timing events, expressed as binary variables inside or outside the optimal range as defined above, should be used together with the morphological exclusion criteria. The model identified the time of division to five cells, t5 OR=3.31 (CI95% 1.65-6.66) followed by synchrony of divisions after the two cell stage, s2 OR=2.04 (CI95% 1.07-4.07) and the duration of the second cell cycle, cc2 OR=1.84 (CI95% 0.95-3.58) as the most promising variables characterizing implanting embryos.
A logistic regression model was defined by using exclusion variables plus t5, s2 and cc2. A ROC curve analysis to determine the predictive properties of this model with respect to probability of implantation gave an area under the curve AUC value of 0.720 (CI95% 0.645-0.795).
These data was used to generate the hierarchical selection model described herein (
This analysis is based on known implantation data (KID) of 1598 embryos from 10 different clinics. The KID embryos are all transferred embryos with known implantation. With multiple embryos were transferred only total failure of implantation or total success is used. All multiple transfers with implantation that have less implanted embryos than transferred were discarded to enable the implantation success for the specific embryo. The implantation success takes the value 1 if the transferred embryo led to successful implantation implanted and 0 if not. The number of embryos (N) used for calculating the expected value (probability of success) of the target and non-target groups is different for different variables.
The data were divided into quartiles with respect to a single continuous variable (e.g. t2) and the expected value (probability of getting a success with one trial) of each quartile was calculated. From these quartile groups a new group was formed (the target group) either by the quartile with the highest expected value or by two or three neighboring quartiles having similar probability (see example in
All the variables tested in table 4 can be used to exclude embryos with very low implantation rate since the implantation success rates of the embryos outside the target groups are 0.23 and below. The two criteria cc2<5 h and cc3<5 h are associated with a low implantation success rate. This may be due to direct cleavage from 1 to 3 blastomeres and 2 to 5 blastomeres indicating a mismatch in DNA replication or in the cell division in general. The embryos with these irregular division patterns will have asynchronous time-lapse data and may disturb any statistical calculation if they are included in the data. The embryos with cc1 (t2) longer than 32 h are also associated with a low implantation success rate and are embryos that develop slowly, possibly due to immaturity of the oocytes.
Another option is to use composite variables calculated using the primary morpho-kinetic variables (timings and time periods). Especially interesting are variables that express the ratio between two morphological time periods. These types of normalized variables may hold information that is better for predictive models since they may take out some of the variability that may arise due to differences in temperature and other environmental variables and since they may be less sensitive to the definition of fertilization time. This could for example be cc2/cc2—3 and cc3/cc2—3 (the fraction of the first and second cell cycle out of the first two cell cycles) or s2/cc2 and s3/cc3 (the synchronicity of the first cell or second cell cycle relative to the time of the first or second cell cycle). The timing of the individual cell divisions in s3 (t8−t5), i.e. s3a (t6−t5), s3b (t7−t6), s3c (t8−t7) is believed to be of interest since they may demonstrate the competence of each individual cell to perform a cell division. Possible irregularities or abnormalities in the mitosis may result in large differences between the value of s3a, s3b and/or s3c (i.e. one high max value).
The invention will now be described in further details with reference to the following items:
27. The method according to item 26, wherein said quality criterion is an indicator of high embryo quality if cc2/cc3=(t3−t2)/(t5−t3) is between 0.7 and 0.9, or between 0.71 and 0.89, or between 0.72 and 0.88.
54. The method according to item 53, wherein said quality criterion is an indicator of high embryo quality if s3=t8−t3 is less than 5 hours, or less than 4.5 hours, or less than 4.3 hours, or less than 4.2 hours, or less than 4.1 hours, or less than 4 hours, or less than 3.9 hours, or less than 3.8 hours, or less than 3.7 hours, or less than 3.6 hours, or less than 3.5 hours, or less than 3.4 hours, or less than 3.3 hours, or less than 3.2 hours, or less than 3.1 hours, or less than 3 hours, or less than 2.9 hours, or less than 2.8 hours, or less than 2.7 hours, or less than 2.6 hours, or less than 2.55 hours, or less than 2.53 hours, or less than 2.51 hours, or less than 2.5 hours, or less than 2.4 hours, or less than 2.3 hours, or less than 2.2 hours, or less than 2.1 hours, or less than 2 hours, or less than 1.8 hours, or less than 1.6 hours, or less than 1.4 hours, or less than 1.2 hours, or less than 1 hour.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/DK2012/050188 | 5/31/2012 | WO | 00 | 11/26/2013 |
| Number | Date | Country | |
|---|---|---|---|
| 61491483 | May 2011 | US |
