Patent Application
20150147770
The present invention relates to a method and to a system for selecting embryos for in vitro fertilization based on observed cell kinetics and cell morphology, in particular the embryo development in the later stages from the initiation of compaction and to the formation of the blastocyst.
Infertility affects more than 80 million people worldwide. It is estimated that 10% of all couples experience primary or secondary infertility. 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). 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.
A recent study (Wong et al. (2010)) focused on the embryo development before embryonic genome activation (EGA) indicating that success and failure in human embryo development is largely determined before EGA, thereby justifying an early implantation at day 2 after fertilization. A pending application from the present applicant PCT/DK2012/05018 (“Embryo quality assessment based on blastomere cleavage and morphology”) filed 31 May 2012 monitors the timing and duration of the subsequent cleavages, wherein embryonic genome activation takes place, and that led to additional embryo quality criteria. However, the present inventors have found that monitoring the embryo all the way to the blastocyst stages, during which the embryonic genome activation has taken over completely, may lead to a range of blastocyst quality criteria that are very useful in the selection of embryos that has transformed into blastocysts, in order to increase implantation success. Traditionally blastocysts have been evaluated based on the number of cells in the trophectoderm or in the inner cell mass, but these blastocyst parameters are difficult to quantify. A purpose of the present invention is therefore to develop new blastocyst quality criteria that are easier to quantify in order to evaluate embryo quality at the blastocyst stage.
Accordingly, the present invention relates to a method and to a system to facilitate the selection of optimal in vitro fertilized embryos to be transferred for implantation after their transformation into blastocysts based on morphological and/or kinetic parameters extracted during their development.
In a first aspect the invention relates to a method for determining embryo quality comprising monitoring the embryo for a time period, said time period comprising the transformation of the embryo from fertilization or initial compaction to blastocyst, i.e. one of the blastocyst stages, and determining one or more blastocyst quality criteria for said embryo, and based on said one or more blastocyst 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.
The blastocyst quality criteria may advantageously be combined with earlier embryo quality parameters, e.g. as listed in WO 2007/144001 and in pending PCT application PCT/DK2012/05018 entitled “Embryo quality assessment based on blastomere cleavage and morphology” filed at 31.05.2012 and thereby additional information of embryo quality and embryo viability. These applications are therefore also hereby incorporated by reference in their entirety.
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 ICSI microinjection or post time for mixing of semen and oocyte in IVF, i.e. the time of insemination. This is the time of the deliberate introduction of sperm into the ovum. However, herein the term fertilization is also used to describe this timepoint. 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 duration of a plurality of cell cycles (e.g. CC1, CC2, CC3 and CC4) can be combined to form a common normalized parameter:
where CCi e.g. is selected from CC1 to CC4. In one embodiment of the invention a high value of CCnorm indicates a poor embryo quality as one or more of the variables CCi is far from the median, i.e. it is not the absolute values of CCi that are used, but the mutual relation of the variables. The median may be calculated based on the whole population or parts of the population (e.g. embryos with known and positive implantation). Another equivalent variable using the logarithmic value instead (ICCnorm) may also be useful in assessing embryo quality.
Likewise the synchronicity Si of the cell divisions (e.g. S2, S3 and S4) may be combined to form a common normalized parameter:
In one embodiment of the invention a high value of Snorm indicates a poor embryo quality as one or more of the synchronicities is long compared to the. Another equivalent variable using the logarithmic value instead (ISnorm) may also be useful in assessing embryo quality.
The variables CCnorm and Snorm may be calculated based on the first, second, third or fourth cell cycle, depending on the duration of the incubation.
The pending application PCT/DK2012/050236 filed 29 Jun. 2012 and entitled “Adaptive embryo selection criteria optimized through iterative customization and collaboration” from the same applicant relates to the issue of adapting embryo quality criteria across populations of embryos cultures under different incubation conditions, e.g. in different clinics. This application is hereby incorporated by reference in its entirety. However, quality parameters like CCnorm, ICCnorm, Snorm and ISnorm may help to ensure that quality models will be directly applicable across different populations of embryos cultured under different incubation conditions, because they are based on variables that are insensitive to differences in running conditions. Another example of that is quality parameters based on relative time periods (e.g. CC2/CC3), variables divided with a central estimate of that variable (e.g. mean or median, e.g. cc2/cc2_median) or using target intervals where the center is scaled according to a central estimate and the boundaries are scaled according to a variance estimate (e.g. variance, standard deviation, percentiles).
The following discrete (binary) variables can be used
A blastocyst quality criterion is an example of an embryo quality criterion. The blastocyst quality criteria relate to the development of the embryo from compaction, i.e. initial compaction, to the hatched blastocyst. Compaction is a process wherein an intensification of the contacts between the blastomeres with tight junction and desmosomes result in reduction of the intercellular space and a blurring of the cell contours (see
Initial compaction (IC) describes the first time a compaction between 2 or more blastomeres is observed. Thus, IC marks the initiation of the compaction process.
Morula (M) is defined as the first time where no plasma-membranes between any blastomeres are visible. When the compaction process is complete no plasma-membranes between any of the blastomeres forming the compaction are visible and the embryo can be defined as a morula. Most often Morula is seen after S3 close to or right in the beginning of the fourth synchrony period (S4). Rarely do the embryos cleave to 16 cell or more before compaction is initiated.
Initial differentiation of trophectoderm (IDT) is defined as the first time during the morula stage where distinct trophectoderm cells are recognized. It describes the onset of differentiation of the trophectoderm cells. The blastomeres gradually become flattened and elongate creating a barrier between the outside environment and the inner cell part of the morula.
Early blastocyst (ERB) is defined as the first time a fluid-filled cavity, the blastocoel, can be observed. It is also referred to as “Onset of cavitation”. It describes the initiation of the transition period between the morula stage and the blastocyst stage of the embryo. Embryos often remain in this transition stage for a period of time before entering the actual blastocyst stage. The onset of cavitation usually appears immediately after differentiation of the trophectoderm cells. The outer layer of the morula with contact to the outside environment begins to actively pump salt and water into the intercellular space, as a result of which a cavity (the blastocoel) begins to form.
Blastocyst (Bl) is defined as where the fluid filled cavity is finally formed, i.e. the cavity does not increase significantly anymore before the blastocyst starts to expand (tEB)
Initial differentiation of inner cell mass (IDCIM) defined as the first time the inner cell mass can be recognized. IDCIM describes the initiation of inner cell mass development. An eccentrically placed cluster of cell connected of gab junction where the boundaries between the cells seem not well defined.
Onset of expansion of the blastocyst (EB) is defined as the first time the embryo has filled out the periviteline space and starts moving/expanding Zona Pelucidae. EB describes the initiation of the embryos expansion. As the blastocyst expands the zona pellucida becomes visibly thinner.
Hatching blastocyst (HB) is defined as the first time a trophectoderm cell has escaped/penetrated the zona pellucida.
Fully hatched blastocyst (FH) is defined as when hatching is completed with shedding zona pellucida.
Number of Contractions (NC (X)) describes the number of contractions (X) the embryo undergoes after the onset of cavitation. In many embryos the contractions can be quite large and lead to a large reduction of the embryonic volume. A contraction is defined as a reduction in the cross sectional surface area of the embryo of more than 15%.
Degree of vacuolization (VC (X); X={0, 1, 2, 3}) describes the extent of vacuolization after initiation of the morula stage. The degree of the vacuoles is rated by a 0-3 scale (0=no vacuolization; 1=small degree of vacuolization where small vacuoles appear but the embryonic development does not appear to be affected; 2=moderate degree of vacuolization where large vacuoles appear and embryonic development is affected to some extent; 3=severe vacuolization where very large vacuoles appear and embryonic development is severely affected. In this incidence the vacuoles can be mistaken for the blastocyst cavitation.
Partial Compaction (PC) describes an uneven compaction where one or more of the blastomeres are not included in the compaction process.
In humans embryonic gene activation (EGA) typically occurs on day 3, around the 8-cell stage. Before EGA embryos are observed to translate only maternally inherited mRNA, i.e. that mRNA which is present in the oocyte when it is fertilized. The mRNA is localized in different parts of the oocyte, so that as the oozyte/zygote divides it is segregated into different blastomeres. This segregation is thought to underlie much of the differentiation of cells that occurs before EGA. After EGA the embryo begins to transcribe its own DNA, cells become motile and cell division becomes asynchronous. Since the cells are now transcribing their own DNA, this stage is where differential expression of paternal genes is first observed. The transition around EGA is also referred to as midblastula or midblastula transition.
Chromosomal content Aneuploidy is an abnormal number of chromosomes and is a type of chromosome abnormality. Aneuploid embryos can have one or more missing chromosomes and/or one or more extra chromosomes. Aneuploidy occurs during cell division when the chromosomes do not separate properly between the two cells. An aneuploid embryo is an embryo which contains an aneuploidy. Correspondingly a euploid embryo is an embryo that is characterized as being chromosomally normal. Euploid (i.e. normal) embryos have the proper number of chromosome pairs. E.g. a euploid human embryo has 23 pairs of chromosomes for a total of 46 chromosomes.
Most cases of aneuploidy result in termination of the developing fetus, but there can be cases of live birth. An extra or missing chromosome is a common cause of genetic disorders (birth defects). It is therefore of particular interest to be able to detect aneuploid IVF embryos before transfer, because some of these embryos have the ability to develop into live births, however possibly with unwanted genetic disorders.
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 have a higher probability of successfully implant and develop in the uterus after transfer than low quality embryos. However, even a high quality embryo is not a guarantee for implantation as the actual transfer and the woman's receptivity highly influences the final result.
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)
a: A picture of an embryo immediately prior to initial compaction.
b: A picture of an embryo at the time of initial compaction. Compaction is a process wherein an intensification of the contacts between the blastomeres with tight junction and desmosomes result in reduction of the intercellular space and a blurring of the cell contours as seen in
a: A picture of an embryo before full Morula.
b: Same embryo as in
a: A picture of an embryo immediately prior to initial differentiation of trophectoderm.
b: Same embryo as in
a: A picture of an embryo immediately prior to onset of cavitation (early blastocyst).
b: Same embryo as in
a: A picture of a blastocyst prior to onset of expansion of the blastocyst (EB).
b: Same embryo as in
a: A picture of an expanded blastocyst immediately prior to hatching.
b: Same embryo as in
a and 9b are pictures of embryos with only partial compaction which is an uneven compaction process where one or more of the blastomeres are not included in the compaction, as illustrated by the markings in the figures.
a, 10b and 10c are pictures of embryos illustrating different degrees (1, 2 and 3) of vacuolization, which is the extent of vacuolization after the morula stage.
a and 11b are pictures of the same embryo with twenty minutes difference. In
a and 13b: Known Implantation data (see example 1) divided into quartiles with respect to t2 and with the expected value for each quartile (
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. 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.
It has been found that there exists an optimal time range for parameters characterizing the embryonic cell divisions. 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 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. 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, and the development into a blastocyst is a necessity for a successful implantation and thus the formation of a blastocyst is a quality parameter in itself. However it has been found that the development into a blastocyst does not necessarily correlate with successful implantation of the embryo.
Supporters of early implantation at day 2 have argued that the extended culture of embryos to the blastocyst stage at around day 5 give rise to potential risks because the culture period is significantly prolonged which may disrupt embryo integrity. However, an extended culture period to the blastocyst stage has several advantages. Cultured human embryos have an average blastocyst formation rate of only approx. 30-50%, and by extending the culture period a large part of the low quality embryos have automatically been excluded, by not forming the blastocyst. Furthermore, after EGA at around the 5-8 blastomere stage the embryos own DNA controls the development. By evaluating the embryos at the blastocyst stage high quality embryos can be identified with a higher degree of certainty.
Thus, the data allows the detection of blastocyst related developmental criteria for implantation potential. The results in particular indicate that timing of late events, such as the onset of cavitation, are a consistently good indicator of implantation potential, and that the discrimination between implanting and non-implanting embryos is improved when using blastocyst quality criteria, e.g. tBl as opposed to the earlier events (t2, t3 and t4). The presented data indicate that incubating the embryos to the blastocyst stage can give additional important information that will improve the ability to select a viable embryo with high implantation potential. The claims list a number of embryo quality criteria and blastocyst quality criteria that may be applied singly or combined in groups to assess embryo quality.
One embodiment of the invention relates to a method for determining embryo quality comprising monitoring the embryo for a time period and determining one or more blastocyst quality criteria for said embryo, wherein said time period comprises the time from fertilization to a blastocyst stage, and wherein 1) the duration of a first time period from fertilization until translation of maternally inherited mRNA in the blastomeres is completed and 2) the duration of a second time period from initiation of transcription of the blastomeres own DNA to said blastocyst stage are determined, and wherein a blastocyst quality criterion is the ratio of said first and second time periods, and based on said one or more blastocyst quality criteria determining the embryo quality.
A further embodiment of the invention relates to a method for determining embryo quality comprising monitoring the embryo for a time period, said time period comprises the time from fertilization to a blastocyst stage, wherein 1) the duration of a first time period from fertilization to a 5 blastomere embryo and 2) the duration of a second time period from the 5 blastomere embryo to said blastocyst stage are determined, and wherein a blastocyst quality criterion is the ratio of said first and second time periods, and based on said blastocyst quality criterion determining the embryo quality.
The time from fertilization to the blastocyst stage is thereby divided into two time periods and the ratio between these time periods is a blastocyst quality criterion. The reason for dividing at the 5 blastomere stage is that this is approx. the time of embryonic gene activation. Thus, in a further embodiment of the invention the time period comprises the time from fertilization to a blastocyst stage, wherein 1) the duration of a first time period from fertilization until translation of maternally inherited mRNA in the blastomeres is completed and 2) the duration of a second time period from initiation of transcription of the blastomeres own DNA to said blastocyst stage are determined, and wherein a blastocyst quality criterion is the ratio of said first and second time periods.
In a further embodiment of the invention the ratio of the second time period divided by the first time period is an indicator of high embryo quality if said ratio is greater than a predefined value.
A corresponding blastocyst quality criterion can be provided by determining 1) the duration of a first time period from fertilization to blastocyst, and 2) the duration of a second time period from initiation of transcription of the blastomeres own DNA to said blastocyst stage and taking the ratio of these time periods. This ratio provides information on how much of the total time period from fertilization to blastocyst the embryo's own DNA is in control. Again, the ratio of the second time period divided by the first time period is an indicator of high embryo quality if said ratio is greater than a predefined value. This ratio can be seen as a measure for the relative development speed in a certain period relative to the overall development speed until that stage. Thus, it seems that the embryos that take more time to develop from the time where the embryos own genome takes over at EGA relative to the overall development time has a higher probability to implant. Or similarly: The embryos that take more time to develop from the time where the embryos own genome takes over at EGA relative to the time before EGA when the maternally inherited mRNA is in control, has a higher probability to implant.
Early cleavage (i.e. low t2) has long been known as an embryo quality indicator. Low t4 and t5 are also quality indicators, thereby showing that fast development before EGA is an indicator of high quality. Herein it is also demonstrated that embryos that reach the blastocyst stage (Blastocyst, tBl) before approx. 96 hours have a higher probability for implantation, thereby showing that fast development in general all the way to the blastocyst stage is a quality indicator. It is therefore surprising that a slow phase in this fast development, i.e. the phase after EGA, is a quality indicator. Typically there is no clear-cut determination of the time of EGA and it may be defined at e.g. t4, t5, or t8. It may also be defined that “before EGA” is the development until the 4 blastomere stage, i.e. t4, and “after EGA” may be defined as after the 8 blastomere stage.
The abovementioned blastocyst stage may be selected from the group of: initial compaction (IC), Morula (M), initial differentiation of trophectoderm cells (IDT), early blastocyst (ERB), blastocyst (Bl), expansion of blastocyst (EB), first contraction (CPS(1)), second contraction (CPS(2)), third contraction (CPS(3)), fourth contraction (CPS(4)), fifth contraction (CPS(5)), sixth contraction (CPS(6)), seventh contraction (CPS(7)), hatching blastocyst (HB), and fully hatched blastocyst (FH). Thus, tIC is the time from fertilization to initial compaction, tM is the time from fertilization to Morula, etc.
A further blastocyst quality criterion may be the determination of the absolute or relative 2D and/or 3D expansion of the blastocyst, e.g. the speed of the blastocoel expansion, where e.g. a quick expansion may be a quality indicator. A further blastocyst quality criterion may be the largest degree of expansion of the blastocyst, e.g. the diameter prior to expansion relative to the largest embryo diameter for the expanded blastocyst. Thus, a blastocyst quality criterion may be the determination of the diameter and/or the volume of the embryo at the onset of expansion. Further, a blastocyst quality criterion may be the determination of the maximum diameter and/or the maximum volume of the blastocyst before hatching.
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.
In one embodiment of the invention an embryo quality criterion is selected from the group of normalized morphological embryo parameters, e.g. 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.
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 number of contractions of the blastocyst, the degree of vacuolization and uneven compaction. 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 despite of a fast t3.
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 blastocyst quality criteria are combined with one or more exclusion criteria.
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
In the decision tree depicted in
If the value of s2 falls inside the optimal range 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 (A+/B+/C+/D+) and is categorized as A, B, C, D if the value for cc2 is outside the optimal range.
The decision tree models can be evaluated using receiver operator characteristic (ROC) methods evaluated by multi-class AUC. Multiclass AUC expresses how well the model sorts the embryos with respect to probability for implanting. AUC lies between 0.5 and 1 where 0.5 is the sorting power of a random model (no effect of the model) and a higher AUC indicate a better sorting compared to the random model.
Decision tree models have been constructed based on KID data from 407 human embryos (see example 1) and the multiclass AUC have been determined. The two decision trees in
The probability of implantation of a specific embryo from a specific woman depends on many other parameters. However, this dataset provides a unique opportunity to test the quality and exclusion criteria presented herein in order to optimize the classification of IVF embryos. E.g. to classify (in terms of quality) a number of embryos taken from a single woman in order to select the best embryo(s) for transfer. Possibly none of the embryos from a single woman fulfils all optimal quality criteria because all embryos are mediocre or poor quality. However, a transfer must be performed and a classification of the embryos is therefore important to select the best of the embryos. Thus, the highest possible AUC is naturally preferred but within the field of embryo selection any improvement in sorting compared to the random model is good and can be considered to improve the selection of good embryos.
The criteria may also be combined in form of a logistic regression model that predicts the odds of implantation success of the embryo (see example 2). The model can be affected by both discrete and continuous variables. The continuous variables used shall have a monotone effect on the odds (either increasing with increasing value of the variable or decreasing with the value of the variable).
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 pellucida 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 blastomere 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 is 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.
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 blastomeres and degree of fragmentation; (2) presence and quality of a zona pellucida; (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.
This analysis is based on known implantation data (KID) of 407 embryos incubated under different conditions (patient characteristics, clinical practices and rules and regulations). The KID embryos are all transferred embryos with known implantation. With multiple embryo transfers 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. t5) 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
Odds ratio (OR) of two groups with associated probabilities (pi and pj) is calculated as
The odds of the first group is
and the odds of the second group is
The odds ratio for implantation of two groups can be tested using the Fishers test providing the p-value. The odds ratio provides the “odds” for being inside the target group. A high odds ratio for a target group for a specific parameter is better. With H0 odds ratio is 1.
For nearly all time variables the first quartile has the highest implantation rate, indicating that fast embryo development to the blastocyst stage is indicative for high implantation rate. Only with morula this is different since there is no significant difference between the first and the second quartile with regard to tM. OR is highest with tBl and also with the most significant rejection of the H0 hypothesis. This may be related both to the fact that this is an important morphological characteristic, but it may also reflect that the stage is easy to determine from time-lapse images.
From table 4 it is seen that the number of contractions should not be more than two. And that the degree of vacuolization should not be more than one. The uneven compaction is also significant. These three criteria are suitable for excluding blastocysts that have a relatively low implantation rate (OR>3), i.e. they are suitable exclusion criteria.
There is a slightly significantly lower implantation rate with a low time period between the 8 cell stage and the morula stage. This might be related to media change. The time from blastocyst to the second contraction (if this is occurring) needs to be long. Also the difference between the first two contractions should be long. This supports that fewer and later contractions may be used as quality indicator.
The relative variables that include variables from both the early part of the embryo development and blastocyst variables all have a tendency towards a higher probability in the groups with a high value of the relative variables. The relative variables with only differences between blastocysts stages have less clear indications. However, please note that these variables are only based on data from 407 KID embryos.
Logistic regression is commonly used to establish models that describe the effect of continuous variables (e.g. tBl) and discrete variables (e.g. UC) on a binomial outcome (e.g. KID_value (implantation/no implantation)). The model fits the log transformed odds (p/(1−pi)) to a linear combination of continuous and discrete variables Xj.
Where pi is the probability of observation i, Xi,j is the value of the jth variable on the ith observation, β0 is the intercept parameter, βj is the slope parameter and ε is a random error. The model is multiplicative and exponential. A negative value of βj means that a continuous variable has a decreasing effect on the model output with increasing values of the variable. With discrete variables the different values of the variable has different associated βj and if the variable takes that value the value of Xj is 1.
Two examples of logistic regression models describing the data are presented herein. The first model includes only variables that can be observed in the blastocysts stage and the second includes also earlier characteristics
The Akaike information criterion (AIC) was used to evaluate the relative goodness of fit of the model and to choose which variables to include in the model. AIC is a measure of both model accuracy and model complexity. If AIC increased when including an effect of a variable in the model this effect was not included.
AIC=2k−2 ln(L)
Where k is the number of estimated parameters and L is the likelihood of the model.
The LRmodel A has effect of tBl (significant), the number of contractions (three contractions significantly different from no contractions) and uneven compaction (almost significant). Including also variables from the earlier stages result in LRmodel B with an effect of tBl (significant), the number of contractions (2 and 3 significantly different from no contractions) and there is a slightly significant effect of multinuclearity at the four cell stage and of uneven compaction.
As example 1 this analysis is based on known implantation data (KID) of 407 embryos incubated under different conditions (patient characteristics, clinical practices and rules and regulations). In this example 351 embryos are selected because they had annotations for tBl and tBE. The KID embryos are all transferred embryos with known implantation. With multiple embryo transfers 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. Implantation successes (squares) and failures (triangles) have been plotted in
The chart in
The outcome of the embryos of these three sections is very different. The implantation ratio of embryos falling within section “1” is 0.59, the implantation ratio of embryos falling within section “2” is 0.37, whereas the implantation ratio of embryos falling within section “3” is 0.14. This is also visible in
The invention will now be described in further detail with reference to the following items:
where CCi is the duration of a cell cycle.
where Si is the synchrony of a division.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/DK2012/050188 | May 2012 | DK | national |
| 12174432.0 | Jun 2012 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2013/061260 | 5/31/2013 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61663856 | Jun 2012 | US | |
| 61707321 | Sep 2012 | US |
