CN110688985A

CN110688985A - Automated monitoring of in vitro cultured embryos

Info

Publication number: CN110688985A
Application number: CN201910982050.7A
Authority: CN
Inventors: 尼尔斯·B·拉姆辛; 梅特·莱格德斯曼德; 延斯·K·冈德森; 索伦·伯斯戈德; 英吉·埃雷波·阿格霍姆
Original assignee: Unisense Fertilitech AS
Current assignee: Unisense Fertilitech AS
Priority date: 2012-08-30
Filing date: 2013-08-29
Publication date: 2020-01-14
Also published as: WO2014033210A1; CN104755608A; ES2831867T3

Abstract

A computer-implemented method for automated detection of changes and/or abnormalities in developmental conditions of in vitro cultured embryos, comprising the steps of: a) obtaining a first data set comprising morphokinetic parameters related to the development of a first group of embryos, b) obtaining a second data set comprising morphokinetic parameters related to the development of a second group of embryos, c) modifying the first and second data sets by removing morphokinetic parameter outliers from said first data set and or said second data set, d) calculating the difference between a specific morphokinetic parameter of the modified first data set and the corresponding morphokinetic parameter of the modified second data set, and monitoring said morphokinetic differences, thereby detecting a change in the development conditions of the first and second group of embryos.

Description

Automated monitoring of in vitro cultured embryos

The application is a divisional application of Chinese patent application 201380055588.4, having application date of 2013, 8, 29 and entitled "automated monitoring of in vitro cultured embryos".

Technical Field

The invention relates to a system and a method for automatically investigating development conditions of in vitro cultured embryos. The invention is applicable to quality control of embryo handling in connection with IVF to ensure that the quality of the transferred embryos is maintained. Thus, the present invention may be a tool for use in a fertility clinic to investigate and maintain the high implantation potential of in vitro cultured embryos.

Background

Infertility affects more than eighty million people worldwide. It is estimated that 10% of all couples experience primary or secondary infertility. In Vitro Fertilization (IVF) is a selective medical treatment that provides couples who otherwise would be unable to conceive an opportunity to conceive. This is a process in which eggs (oocytes) are removed from a female ovary and then fertilized by sperm in a laboratory. The embryos produced during this procedure are then placed into the uterus for potential implantation.

Quality Control (QC) is an important issue in IVF clinics to monitor the quality of the treatment and different processes of the clinic that affect the developmental conditions of the embryos. Quality control can be performed by monitoring the variation of a running average of the resulting variables, such as

Biochemical pregnancy rate (number of HCG positives per embryo transfer)

Fetal heart rate (number of fetal heartbeats per embryo transferred)

Miscarriage Rate (number of abortions per implantation of embryo)

KID ratio (known number of implantations per known result (defined as fetal heart beat or HCG positive))

The value of such monitoring is undoubted, but the monitoring response is slow due to the limited number of transferred embryos and even lower numbers of known implanted embryos in a particular clinic. Another problem is the time lag due to delays in waiting for HCG test results, scan results, and miscarriage information. Since this method has limited sensitivity to discrete binary results (pregnant/not pregnant, aborted/not aborted, implanted/not implanted), this means that a large population is required to assess whether significant changes have occurred. This causes the quality control monitoring to be delayed again.

Disclosure of Invention

Accurate, fast and sensitive reactions are required if quality control concerns are raised, such as failure to follow optimal operations (e.g. errors in embryo handling), toxic laboratory instruments or consumables (e.g. toxic oils or media) or environmental changes (contamination of laboratory air). Precision, so that only corrective measures are taken when something is really wrong, speediness, so that corrections are made as quickly as possible, and sensitivity, so that even small changes in operation can be detected and corrected. Therefore, there is a need for a sensitive and fast response tool for monitoring the operation of a clinic.

Accordingly, one embodiment of the present invention is directed to a computer-implemented method for automated detection of changes and/or abnormalities in developmental conditions of in vitro cultured embryos, comprising the steps of:

a) obtaining a first data set comprising morphokinetic parameters associated with the development of a first set of embryos,

b) obtaining a second data set comprising morphokinetic parameters associated with the development of a second group of embryos,

c) modifying the first and second data sets by removing morphokinetic parameter outliers from said data sets,

d) calculating a difference between a particular morphokinetic parameter from the modified first data set and a corresponding morphokinetic parameter from the modified second data set,

e) monitoring said morphokinetic differences to detect a change in a developmental condition of said first group of embryos and said second group of embryos.

Morphokinetic variables are known to be important indicators of embryo implantation success rate. In the present invention, morphokinetic parameters are used as quality control indicators, thereby forming a surrogate for the relevant variables that monitor pregnancy rate or are positively delayed. The great advantage of using the morphokinetic parameters is that embryos in clinics are about an order of magnitude higher than the number of embryos transferred and that variables are available at the end of the culture (2-5 days after fertilization). Moreover, the number of available morphokinetic parameters is about an order of magnitude higher than the number of embryos, since each embryo "produces" multiple morphokinetic parameters during different developmental stages. So by monitoring the morphokinetic parameters instead of the pregnancy rate, speed and accuracy can be increased. Because, for example, the time of occurrence of a cleavage is a continuous variable, rather than discrete, the sensitivity of the statistical test that can be used to test whether two groups differ significantly is greatly increased.

The invention is most naturally used for human embryos, but can also be used for monitoring of any mammalian embryo.

Definition and embryo quality parameters

The cleavage time is defined as the point in time when the first observed blastomeres are completely separated by confluent cell membranes, and thus the cleavage time is the time at which cleavage of the blastomere is complete. In the context of the present invention, time is expressed as hours after ICSI microinjection or after the time to mix semen and oocyte in IVF (i.e., the time of insemination). This is the time at which sperm cells are deliberately introduced into the egg cells. However, in this context, the term fertilization is also used to describe this point in time. Thus, the cleavage time was as follows:

t 2: time to cleavage to 2 blastomere embryo

T 3: time to cleavage to 3 blastomere embryos

T 4: time to cleavage of 4 blastomere embryos

T 5: time to cleavage to 5 blastomere embryos

T 6: time to cleavage to 6 blastomere embryos

T 7: time to cleavage to 7 blastomere embryos

T 8: time to cleavage of 8 blastomere embryos

The duration of the cell cycle is defined as follows:

cc1 ═ t 2: the first cell cycle.

Cc2 ═ t3-t 2: the second cell cycle, as the duration of the 2 blastomere embryo.

Cc2b ═ t4-t 2: the second cell cycle of both blastomeres was taken as the duration of the 2 blastomere embryo and the 3 blastomere embryo.

Cc3 ═ t5-t 3: the third cell cycle, the duration of the 3 blastomere embryos and 4 blastomere embryos.

Cc2 — 3 ═ t5-t 2: the second and third cell cycles are used as the duration of the 2-, 3-and 4-blastomere embryos.

Cc4 ═ t9-t 5: the fourth cell cycle, duration of 5, 6, 7 and 8 blastomere embryos.

Short and long cell cycle

In directly cracked embryos, the time between cell divisions appears to be insufficient for DNA replication throughout the genome. For the first three cell cycles:

cell cycle 1, cc1, is not a standard cell cycle, as it combines processes of fertilization, pronucleus formation and disappearance, until the first cell division occurs, at which point the maternal and paternal alleles combine in the embryonic genome. The combined genome may have replicated before cell division from zygote to 2 cells. cc 1-t 2.

Cell cycle 2, cc2, the time from completion of the first cell division to form two cells to the next cell division to form three cells, i.e., cc 2-t 3-t 2.

Cell cycle 3, cc3, is the time required for the third round of DNA replication. When a 4-cell embryo divides into 5 cells, there should be 5 complete copies of the genome and the two most recent cells in the 5-cell embryo should successfully complete their third round of DNA replication. Since the fastest dividing cells are most likely to be the first to appear after the first cleavage of a 2-cell embryo into 3 cells, the best estimate of the fastest cell cycle is cc3 ═ t5-t3, the time to cleave to 5 cells minus the time to 3 cells.

Short cell cycle embryos, SCC

In short cell cycle embryos, any of the following can be found:

cc1 ═ t2<15hr, or

Cc2 ═ t3-t2<5hr, or

·cc3＝t5-t3<5hr

Long cell cycle embryos, LCC

A similar concept that may be equally useful is "long cell cycle embryos". These embryos are very slow-developing embryos with an abnormally long duration of their cell cycle. Long cell cycle embryos have similar poor prognosis and are significantly less likely to be bedded compared to more normal embryos (as shown below). However, the category "long cell cycle embryos" is not as well defined as short cell cycles, since the high-end distribution is continuous and the implantation potential is gradually reduced. Therefore, it is not clear how to define the boundaries of this category. The limits of the long cell cycle embryo species were chosen to correlate LCC species with significantly reduced implantation potential, similar to that observed in short cell cycle embryos. Thus, long cell cycle species are defined using dimensions comparable to those of the short cell cycle species of the embryo:

long cell cycle embryos, LCC, meaning:

cc1 ═ t2>32hr, or

Cc2 ═ t3-t2>20hr, or

·cc3＝t5-t3>25hr

Middle cell cycle embryos, MCC

All embryos belonging to this group, i.e., wherein

Cc1 is 15 to 32 hours, and

cc2 is 5 to 20 hours, and

cc3 is a 5 to 25 hour embryo classified as a medium cell cycle embryo. These are "normal" embryos with a normal cleavage pattern. MCC embryos typically comprise about 60% of all embryos and are useful for comparing embryo development between clinics. For example, a first set of embryos can be selected from MCC embryos.

Synchronicity is defined as follows:

s2 ═ t4-t 3: synchronicity of cleavage from 2 blastomere embryos to 4 blastomere embryos.

S3 ═ t8-t 5: synchronization of cleavage from 4 blastomere embryos to 8 blastomere embryos.

S3a ═ t6-t 5; s3b ═ t7-t 6; s3c ═ t8-t 7: the duration of each cell division involved from the development of a 4 blastomere embryo to an 8 blastomere embryo.

And (3) cleavage stage: the time period from the first observation of a cell membrane depression (indicating the onset of cytoplasmic cleavage) to the completion of cytoplasmic cell cleavage such that the blastomere is completely separated by confluent cell membranes. Also known as the duration of cytokinesis.

Fertilization and cleavage are the initial morphological events of an embryo, at least up to the 8 blastomere stage. Cleavage time, cell cycle, division synchronicity and cleavage phase are examples of morphological embryo parameters that may be defined by these initial morphological events, and each of these morphological embryo parameters is defined as the duration of the time period between two morphological events, e.g., measured in hours.

The 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/cc 2.

The durations of multiple cell cycles (e.g., CC1, CC2, CC3, and CC4) may be combined to form one common standardized parameter:

wherein CCi is selected from CC1 to CC4, for example. In one embodiment of the invention, the high CC_{Standardization}Values represent poor embryo quality because one or more of the variables CCi are far from the median, i.e., instead of using the absolute values of CCi, the interrelationships of the variables are used. The median may be calculated based on the entire population or a portion of the population (e.g., embryos with known and positive implantation). Another equivalent variable using logarithmic values (lCC)_{Standardization}) Can also be used for evaluating embryo quality.

Similarly, synchronicity of cell division Si (e.g., S2, S3, and S4) can combine to form a common normalization parameter:

in one embodiment of the invention, high S_{Standardization}Values represent poor embryo quality because one or more synchronizations are longer than the median. Using another equivalent variable (lS) to the logarithmic value_{Standardization}) Can also be used for evaluating embryo quality.

Variable CC_{Standardization}And S_{Standardization}This can be calculated based on the first, second, third or fourth cell cycle, depending on the duration of the culture.

In some embodiments, the morphological parameters of embryos used according to certain embodiments of the present invention may be established by combining a plurality of morphological features associated with the development of the respective embryo. For example, in some implementations, the morphological parameters may be derived by obtaining values for a plurality of morphological features associated with development of the embryo during the observation period, e.g., features such as cell cleavage time (t2, t3, t4 …) and/or the difference between subsequent cell cleavage times/divisions (t3-t2, t4-t3, t5-t4 …) and/or cycle duration (cc1, cc2, cc3 …, etc.). The value of the continuous variable is then determined by combining the differences between the obtained values of the plurality of features and the corresponding reference values in a predefined manner. The reference value can be determined, for example, from values for a plurality of characteristics obtained for one or more reference embryos (e.g., KID-positive embryos) having known developmental potential. Morphological parameters for embryos used according to certain embodiments of the present invention may thus be established by continuous variables. In this regard, the morphological parameters may correspond to embryo development potential determined according to the principles set forth in co-pending patent application PCT/EP2013/063240 filed on 25/6/2013, the entire contents of which are incorporated herein by reference.

According to some embodiment implementations, the step of combining the differences between the obtained values of the plurality of morphological features and the corresponding reference values may take into account a weighting value associated with each of said reference values. For example, the weighting values may be statistically determined from the values of a plurality of characteristics obtained for a plurality of reference embryos having known developmental potential. For example, the weighting value may be determined by the variance of the correlation values obtained for a plurality of reference embryos.

Some example implementations of the invention may also include obtaining values for other various characteristics associated with embryo development during the observation period; determining the value of another continuous variable by combining the differences between the obtained values and the corresponding reference values of the other plurality of features in another predefined manner; and establishing the developmental potential of the embryo also based on the determined value of the further continuous variable.

In some examples, the morphological characteristics of embryos can be determined by combining/aggregating the differences of multiple characteristics observed for each embryo with corresponding reference values (e.g., determined for a KID-positive embryo population) to produce what can be referred to as Generalized Irregular Variables (GIVs), which in some cases are defined as:

wherein cci is the series of cell cycle durations observed for embryos, cci_mIs the corresponding series of mean cell cycle durations observed in a reference group of embryos (e.g., a positive KID population from patients no older than 35 years), and cci_vIs the corresponding variance value associated with the reference population. The parameter n is the number of cell cycle durations that make up the series cci. Using variance value cci_vLet the difference (cci-cci)_m) Normalization as part of the combination. This means that differences in the specific cell cycles (i-values) that exhibit relatively high variation in the sample population contribute less to the GIV values than differences in cell cycles that exhibit relatively low variation in the sample population. In combination, let the difference (cci-cci)_m) Squaring, which means that, for a given difference, whether it is a positive or negative value (i.e., regardless of cci versus cci)_mLong or short) whose contribution to the GIV is the same. The series cci may comprise a plurality of different cell cycle durations in different implementations as discussed further below.

Generally, when the study embryo shows a regular cleavage pattern, the GIV as defined above is low, whereas when the embryo shows an irregular cleavage pattern, the value is high.

In some examples, the morphokinetic characteristics of an embryo may be referred to as a Generalized Time Variable (GTV), defined as:

where Δ tj is the time difference between a series of subsequent cell divisions observed for an embryo, Δ tj_mIs the average of the corresponding series observed in an embryonic reference group (e.g., a positive KID population from patients no older than 35 years) toAnd Δ tj_vIs the corresponding variance value associated with the reference population. The parameter k is the number of values that make up the series Δ tj. Using the variance value Δ tj_vLet the difference (Δ tj- Δ tj)_m) Normalization as part of the combination. This means that differences in specific cell cycles (j-values) that exhibit relatively high variation in the sample population contribute to GIV values less than those that exhibit relatively low variation in the sample population. In this example, the contribution of each time difference to the GTV depends on whether the time difference for subsequent cell division is faster or slower than the average observed in the positive KID population (i.e., whether the difference is a positive or negative value).

A co-pending application PCT/DK2012/050236 entitled "Adaptive embryo selection criteria by iterative customization and collaborative optimization", filed on 29/06/2012 by the same applicant, relates to the problem of adjusting embryo quality criteria between populations of embryo cultures under different culture conditions (e.g. in different clinics). This application is incorporated by reference herein in its entirety. However, a quality parameter (e.g., CC)_{Standardization}、ICC_{Standardization}、S_{Standardization}And IS_{Standardization}) Can help ensure that quality models can be applied directly between different populations of embryos cultured under different culture conditions, since they are based on variables that are insensitive to differences in operating conditions. Another example of a quality parameter is a quality parameter based on a relative time period (e.g., CC2/CC3), a variable (e.g., mean or median, e.g., CC2/CC2_ median) divided by a central estimate of this variable, where the center is scaled according to the central estimate and the cutoff is scaled according to variance estimates (e.g., variance, standard deviation, percentile), or a quality parameter utilizing a target interval.

The following discrete (binary) variables may be used

MN 2: multinuclear phenomena observed at the 2 blastomere stage; the value "true" or "false" may be taken.

MN2 val: the number of multinucleated cells at the 2-cell stage (0, 1, 2).

MN 4: multinuclear phenomena observed at the 4 blastomere stage; the value true or false may be used.

MN4 val: the number of multinucleated cells at the 4-cell stage (0, 1,2,3, 4).

EV 2: 2 homogeneity of blastomeres in blastomere embryos; the value "true" (i.e., uniform) or "false" (i.e., non-uniform) may be used.

Blastocyst-related parameters

Blastocyst quality standards are one example of embryo quality standards. The following blastocyst-related parameters may be used:

initial densification (IC)The first time at which densification between two or more blastomeres was observed is described. Densification is a process in which tight junctions and increased exposure of the desmosomes to the cleavage ball results in decreased intercellular spaces and blurred cell contours (see figure 3). Densification can be seen at the 6-8 cell stage, but few embryos are cleaved to 16 cells or more before densification occurs.

Densification/mulberry embryo (M)Is defined as the first time that no plasma membrane between any blastomeres is visible. When densification is complete, no plasma membranes are visible between any blastomeres that are densified, and the embryo can be defined as a morula. This stage is characterized by a process in which tight junctions and enhanced desmosomal contact with the cleavage ball results in decreased intercellular space and blurred cell contours. Densification/morula was sometimes seen at 6-8 cell stages of the third division (synchrony) stage (S3), but was most often seen near or just at the beginning of the fourth synchrony stage (S4) after S3. Few embryos had cracked to 16 cells or more before densification occurred.

Initial Differentiation of Trophoblasts (IDT)Defined as the first time during the densification phase that distinct trophoblast cells were identified. It describes the onset of trophoblast differentiation. The blastomeres gradually flatten and elongate, creating a barrier between the external environment and the inner cellular portion of the morula.

Onset of cavitation (cavitation)/early blastocyst/Blastocyst (BI)Is defined as the first time a fluid-filled cavity (blastocoel cavity) can be observed. Which describes the densification of blastocysts with embryosThe beginning of the transition period between periods. The embryo is usually held for a period of time at this transition stage before entering the true blastocyst stage. Air conditionerChamberThe onset of differentiation usually occurs immediately after the trophoblast cells differentiate. The outer layer of the morula contacting the external environment begins to actively pump salt and water into the intercellular spaces, thus beginning to form cavities (blastocoel cavities).

Initial differentiation of inner cell Mass (IDCIM)Defined as the first time that an inner cell mass can be identified after OC (onset of cavitation). IDCIM describes the initiation of inner cell mass development. Eccentrically located clusters of cells are connected by gap junctions, where the boundaries between cells do not seem to be well defined.

Initiation of blastocyst expansion(EB) is defined as the first time an embryo has filled the perivitelline space and started to move/expand the zona pellucida. EB describes the initiation of embryo expansion. The zona pellucida appears to be thinned as the blastocyst is expanded.

Blastocyst in Hatch (HB)Is defined as the first time the trophoblast cells escape/penetrate the zona pellucida.

Fully incubated blastocyst (FH)Is defined as when hatching is complete and the zona pellucida is detached.

Number of contractions (NC (X))Describe the spaceChamberNumber of contractions (X) the embryo undergoes after the start of transformation. In multiple embryos, shrinkage can be very large and result in a large reduction in embryo volume. Shrinkage 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})The degree of vacuolation of the embryos after undergoing densification is described. The vacuolation degree is classified into 0-3 grades (0: no vacuolation; 1: small vacuolation, in which small vacuoles appear after densification but embryo development does not seem to be affected; 2: medium vacuolation, in which large vacuoles appear after densification and embryo development is affected to some extent; 3: severe vacuolation, in which large vacuoles appear after densification and embryo development is severely affected). In this event, vacuoles can be mistaken for blastocyst vacuolesChamberAnd (4) transforming.

Densifying-depdensifying-densifying (CD)C)A phenomenon is described in which the densification of the embryo has already begun but the cleavage is disturbed. Again, the cell boundaries of the blastomeres became visible, but after a while the embryos returned to a densified composition.

Partial densification (PC)A non-uniform densification is described in which one or more blastomeres are not included in the densification.

In humans, Embryonic Gene Activation (EGA) usually occurs on the third day, around the 8-cell stage. Prior to EGA, it was observed that the embryo translated only maternally inherited mRNA, i.e., mRNA present in the oocyte when fertilized. The mRNA is located in different parts of the oocyte, so that when the oocyte/zygote divides, it segregates into different blastomeres. This separation is believed to be the basis for most of the cell differentiation that occurs prior to EGA. After EGA, the embryo begins to transcribe its own DNA, the cell becomes mobile and cell division becomes asynchronous. Since the cells are now transcribing their own DNA, differential expression of the paternal genes is observed for the first time at this stage. The transition before and after EGA is also called mesocyst or mesocyst transition.

Rearrangement of cell position ═ cell migration (see below)

Cell migration: movement of cell central and outer cell membranes. The internal movement of organelles within a cell is not a cellular movement. The outer cell membrane is a dynamic structure, so the cell boundary will constantly change position slightly. However, these slight fluctuations are not considered as cell movements. Cell movement occurs when the center of gravity of a cell and its position changes relative to other cells and when a cell divides. Cell movement can be quantified by calculating the difference between two successive digital images of the moving cell. An example of such quantification is described in detail in a pending PCT application entitled "Determination of a change in a cell population", filed on 16.10.2006. However, other methods of determining the movement of the center of gravity of the cell and/or the position of the cytoplasmic membrane are conceivable, for example by using fertimorphh software (ImageHouse Medical, Copenhagen, Denmark) to delineate, semi-automatically, the boundaries of each blastomere that extends through the embryo in the form of successive optical sections.

Other parameters:

organelle movement: the movement of internal organelles and membranes within the embryo can be visualized by microscopy. In the context of the present application, organelle movement is not cell movement.

Moving: spatial rearrangement of objects. The movement is characterized and/or quantified and/or described by a number of different parameters, including but not limited to: degree of movement, area and/or volume involved in the movement, rotation, translation vector, direction of movement, speed of movement, change in size, expansion/contraction, etc. Thus, different measurements of the movement of a cell or organelle can be used for different purposes, some reflecting the degree or amplitude of movement, some reflecting the spatial distribution of moving objects, some reflecting the trajectory or volume affected by the movement.

Embryo quality is a measure of the ability of the embryo to successfully colonize and develop in the uterus after implantation. High quality embryos have a higher probability of successfully implanting and developing in the uterus after transplantation than low quality embryos. However, even high quality embryos are not a guarantee of implantation, as the actual transfer and acceptance of the woman is highly influencing the final result.

Viability and mass are used interchangeably herein. An embryo quality (or viability) measurement is a parameter that reflects the quality (or viability) of an embryo, such that an embryo with a high quality parameter value has a high probability of being of high quality (or viability) and a low probability of being of low quality (or viability). Embryos with an associated low value of the quality (or viability) parameter have only a low probability of being of high quality (or viability) and a high probability of being of low quality (or viability).

Drawings

FIG. 1: nomenclature of cleavage patterns, showing the cleavage time (t2-t5), duration of the cell cycle (cc1-cc3), and synchronicity (s1-s3) associated with the resulting images.

FIG. 2: embryo development up to the blastocyst stage. The number refers to the number of blastomeres in each time period. The letters a to e refer to the following motive forcesParameters are as follows: a: densified/morula (M), b: initial Differentiation of Trophoblasts (IDT), c: air conditionerChamberOnset of chemosis/early blastocyst/Blastocyst (BI), d: initiation of blastocyst (EB); e: hatching Blastocyst (HB). Initial densification (IC), if present, can be observed between t5 and densification/morula (a), IC typically preceding densification/morula for several minutes to several hours. Partial densification (PC), if present, can be observed between periods a and c. Cavitation (vc (x)) and shrinkage (nc (x)), if any, can be observed between periods a and d +.

Namely:

dynamics of

a) densified/Mulberry embryos

b) Initial Differentiation of Trophoblasts (IDT)

c) Onset of cavitation/early blastocyst/blastocyst (B)

d) Initiation of dilation (OB)

e) Blastocyst in Hatch (HB)

Morphology of

t5-a) if there is initial densification (IC), intervals of IC can be observed, typically IC a few minutes to hours before "a".

a-c) if there is "partial densification" (PC), PC spacing is observed.

a-d +) if "degree of vacuolation (VC (X))" and number of contractions (NC (X)) "are present, a separation between VC (X) and NC (X)) is observed.

FIG. 3 shows the morphokinetic parameters (in this case, t2, t3 and t5) as a function of the medium in the breeding clinics. The whole cycle is from 2 months 2011 to 6 months 2011. Of the three media used (A, B, C), medium a gave the worst embryonic development (cell division occurred at the latest for medium a and t2, t3 and t5 were all higher). The culture medium A also gave a worse implantation rate and pregnancy rate. Medium B and medium C gave normal embryonic development and high implantation and pregnancy rates. The application of the present invention to the supervision of the morphokinetic parameters of embryos developing in different media reveals these problems in real time as they progress.

Figure 4a. series of images show that time t2 (the time to cleavage of an embryo producing a 2 blastomere, i.e., the time to resolution of cell division) occurred at 22.9 hours.

Figure 4b. series of images show cleavage directly into a 3 blastomere embryo. Cleavage from 1 cell to 3 cells occurs within one frame, therefore t3 ═ t2 and CC2 are therefore less than 5 hours, i.e., an example of SCC embryos.

FIG. 5 mouse embryo development with media at different temperatures (see example 1).

FIG. 6 duration between multiple cell divisions of mouse embryos for media at different temperatures (see example 1).

7a-c direct cleavage, involving a total of 32,382 recovered oocytes inseminated and placed in an Embryoscope incubator, was analyzed in a large data set containing 4,181 IVF treatments from eight IVF clinics. 18,024 annotated embryos were evaluated, 5491 of which were transplanted. As shown in fig. 7a, short cell cycles (26.1%) were seen in 4709 of the 18024 annotated embryos and long cell cycles (13.7%) were seen in 2464 of the 18024 embryos. The remaining 10851 embryos (60.2%) were MCC. In the transplanted embryos, the abundance of SCC and LCC decreased: SCC 14.1% (776 in 5491) and 9.2% (503/5491), as shown in fig. 7 b. The remaining 76.7% are "normally" dividing MCC embryos. The KID ratio is shown in fig. 7 c. Two abnormal classes, SCC and LCC, showed significantly reduced KID ratios. The relative adherence ratio of SCC embryos decreased to 47% for the MCC ratio, and the relative adherence ratio of LCC embryos decreased to 41% for 7.2%/17.7% for the MCC ratio. In one embodiment of the invention, therefore, SCC and/or LCC embryos are defined as embryos with morphokinetic parameter outliers. Also, changes in the relative values of SCC and/or LCC embryos can be an indicator of changes in developmental conditions, and by monitoring this change, early warning can be provided.

FIGS. 8 and 9 show the changes in the mean values of the morphokinetic parameters t2, t3, t4, t5, t8, s2, cc3 and t8-t5 of embryo cultures in different batches of oil. It is clearly seen that the differences and especially the oil of batch "L1" are clearly different, possibly resulting in a significantly lower embryo quality due to the small amount of toxic substances. It was seen that the embryos cultured in L1 developed more slowly and the cell cycle was longer (cc 3). The latest morphokinetic parameters t5 and t8 differed more significantly for cleavage time. Thus, monitoring morphokinetic parameters is a sensitive and fast-reacting tool for detecting the quality of cultured embryos.

FIGS. 10-14 embryos from cross-bred mice were tested using controlled amounts of TX-100(Triton X-100) in culture medium. The leftmost column is a control group without TX-100, and the amount of TX-100 in the other five groups gradually increases to 0.002%. FIG. 11 shows the blastocyst rate at 96 hours, that is, the rate at which the blastocyst stage is reached at 96 hours. Only group 5 using up to TX-100 differs significantly. FIG. 12 shows the variation of t2, t3, t4, t5 and t8 among the groups, and the trend of developmental slowing was observed with increasing amounts of TX-100, with the greatest difference at later cleavage times. FIG. 13 shows the change in s2, cc3, and t8-t5 between groups, with no other significant trend except that most groups had higher values than the control. FIG. 14 shows the variation of the time of occurrence of the different blastomeres and a clear trend of developmental slowing down with increasing amounts of TX-100 was observed.

Detailed Description

One way to identify viable embryos in a batch of embryos from IVF treatment would be to compare the recorded temporal patterns of cell division (expressed using morphokinetic parameters) with the recorded temporal patterns of cell division of embryos of past treatment cycles. Viable embryos are characterized by morphokinetic parameters that match those recorded in the past from embryos that have been implanted and produced live birth. In selecting a transferred embryo that exhibits morphokinetic parameters similar to positive embryos (i.e., from embryos that are undergoing pregnancy or successfully completing pregnancy) and, where possible, different from most negative embryos (i.e., those that fail to engraft or cause clinical abortion), it would be possible to increase the likelihood of pregnancy occurring and to obtain the desired outcome of fertility treatment. The present invention subverts this known principle: by monitoring the morphokinetic parameters, undesired differences or trends can be detected more quickly, and this can lead to early warnings directed to undesired differences in, for example, embryo processing. In addition, morphokinetic parameter supervision can also be used to alleviate fear after multiple implantation failures, as morphokinetic parameter analysis can show that embryo development is indeed normal.

Several factors have been shown to affect embryonic development, e.g., the timing of cell division. Factors that have been shown to affect embryo development and therefore the resulting morphokinetic parameters include: temperature, Medium composition, pH, CO₂And oxygen, growth factors, culture vessels, and the like. Other factors such as patient age, etiology, BMI, stimulation regimen (type of agonist/antagonist, hormone rFSH/hMG), embryo handling (pipette, fertilization method, assisted incubation, blastomere removal by biopsy, polar or trophoblast cells), and even the experience and skill of the person handling the embryo, are suggested by many scientists to affect the timing of embryonic development, particularly cellular events such as cellular cleavage.

Thus, one embodiment of the invention finds application in quality control in fertility clinics by comparing the average cleavage pattern of embryos from the most recent treatment cycle with the cleavage pattern of past cycles. Temporal changes in general morphokinetic parameters of good quality embryos may indicate undesirable changes in protocols, such as bad batch media, problems with incubators, pipette tips, and the like.

For continuous monitoring, the steps of the claimed method may be repeated and thus the second data set may be continuously updated with up-to-date embryo data, e.g. up-to-date embryo data selected from a certain time period or from a predefined number of up-to-date embryos, e.g. up-to-date embryos from a predefined time period (e.g. hours, days, weeks or months). Preferably, said predefined number of embryos or predefined time period is determined as user input. And development conditions can be continuously checked.

The method is computer-implemented, so that the function of issuing a warning is advantageously performed when the morphological kinetic differences are above a predefined level. The type of warning may depend on the severity of the detected difference, e.g. a normal green light, a possibly problematic yellow light and a serious quality problem red light.

The first group of embryos will typically be the reference or control group, while the second group of embryos will be the group of embryos that is monitored and compared to the control group. The detected morphokinetic differences between the groups of embryos may be determined by standard statistical methods known in the art, e.g., the predefined level for issuing the warning may be determined as a predefined level that exceeds the standard deviation of one or more morphokinetic parameters in the first data set.

The morphokinetic parameter outliers can be defined as relative outliers, i.e., the outermost 3% of the population. However, it may also be advantageous to define a morphokinetic parameter outlier as an absolute outlier, i.e. if a certain morphokinetic parameter is outside a predefined range of absolute numbers, it is defined as an outlier. This is the case for SCC embryos and LCC embryos as defined herein as examples of absolute outliers. The lower 5 hour limit for SCC embryos comes from the fact that 5 hours are an insufficient time for DNA replication of the entire genome.

Morphological kinetic parameter outliers may be excluded from the first data set and/or the second data set. Monitoring the change in the morphokinetic parameters can be a good indicator of quality problems, since multiple morphokinetic parameters are quality parameters of embryo quality. However, it is also advantageous to monitor the number or frequency or distribution of morphokinetic outliers, especially in the second data set, since any change in outliers can be an indicator of quality problems.

Only specific morphological kinetic parameters that are outliers may be excluded from the data set. It may also be advantageous to exclude all data from a particular embryo that has one or more morphokinetic outliers in development.

The second set of embryos may be a subset of the first set of embryos such that the second data set is a subset of the first data set.

The method according to any one of the preceding claims, wherein the morphokinetic parameters are selected from the group consisting of:

time to cleavage of an n blastomere embryo, tn, where n ═ 1, …,8,

cell cycle durations cc1, cc2, cc2b, cc3, cc2_3, and cc4,

synchronicity s2, s3, s3a, s3b and s3c,

the number of blastomeres at a particular predefined point in time,

the time, extent or duration of occurrence of cellular and/or organelle movement,

any combination of these morphokinetic parameters.

The following may be selected as morphokinetic parameters:

where i ═ {1,2,3,4}, cci_mIs the mean or median of cci and cci_vIs the variance of cci.

In one embodiment of the invention, the first and/or second group of embryos are embryos that have been fertilized, stored and/or cultured under a specific set of conditions. The first group of embryos may be fertilized under a different set of conditions than the second group of embryos. These conditions may be selected from the group: type of fertilization therapy, preservation (e.g., cryopreservation), culture temperature, culture media type, specific incubator, specific therapy (e.g., hormone therapy), hormone therapy, suction, male factor, specific incubator, culture temperature, culture media type, oil type, PGD therapy, transplant, cryopreservation, thawing, time outside incubator, number of fertilization treatments.

In one embodiment of the invention, the second group of embryos are embryos derived from a predefined embryo donor. The predefined embryo donors may be selected from the group consisting of: individuals younger or older than a predefined age, embryo donors in a specific treatment or stimulation protocol, embryo donors in a specific fertilization treatment, embryo donors with a specific diagnosis (e.g. hereditary chromosomal disease, Hiv, Hep, PCO), individuals currently or previously exposed to radiation or dangerous chemicals, individuals currently or previously used with drugs, individuals with a BMI higher or lower than a predefined level, smokers, non-smokers, individuals with a normal or abnormal menstrual cycle.

The second group of embryos may be selected as embryos from a specific patient population (e.g., younger patients), embryos from patients with a specific diagnosis (e.g., endometriosis), embryos for a specific fertilization therapy (e.g., ICSI), embryos that have received a specific pretreatment (e.g., cryopreservation).

A particular problem arises if the clinic occasionally has patients with very deviating cleavage patterns. These patient numbers then severely affect the mean of the time of occurrence. In this case, monitoring the above variables for selected groups of embryos is a solution.

The duration of the cell cycle cci (cc1, cc2, cc3, and cc4) is an important variable in determining embryo quality. The combination of morphokinetic parameters can be used to construct a new morphokinetic parameter suitable for the extreme case that rarely occurs during the time of occurrence of a specific cleavage event

Wherein CCi_mIs the median (mean) of CCi and CCi_vIs the variance of cci. These can be calculated, for example, for a set of KID-positive embryos, from all transferred embryos, or from all embryos. If CC is set_{Standardization}By a maximum limit x (e.g., 3), it is meant that if the average CCi from the second group of embryos exceeds the average x-fold standard deviation (e.g., the standard deviation of a KID-positive embryo, a transferred embryo, or all embryos) from the first group of embryos (KID-positive embryos, transferred embryos, or all embryos), that data will be excluded.

To monitor the best laboratory procedure being followed, embryos that have undergone a particular treatment can be isolated to monitor the variables of those embryos for changes over time, i.e., a second group of embryos selected from embryos that have been treated with that particular treatment. The group of embryos handled by a particular laboratory worker or clinic staff may also be selected as the second group of embryos to assess whether there is a general difference in time or staff handling.

In another embodiment of the invention, the first group of embryos contains pre-implantation data for implanted embryos that result in pregnancy continuation (ingoing pregnancy), live babies, Fetal Heart Beats (FHB), and/or fetal cysts. That is, the first set is selected to reflect high quality embryos with verified trace records.

In yet another embodiment, the embryo data set (e.g., the first or second embryo data set) comprises the morphokinetic parameters of an embryo:

1) all embryos in the group of embryos monitored, or

2) Functionally defined subgroups from the group of embryos.

That is, all embryos in the set of monitored embryos (i.e., all embryos that were monitored at a particular clinic) may be selected as a frame of reference for statistical calculations.

Or only a subset is selected, wherein the subset is defined by function. Examples of sub-groups defined by function:

-all the fertilized embryos in the group,

embryos that have divided to at least a predefined number of cells a predefined number of hours after insemination, e.g. embryos that have divided to at least 7 cells 68 hours after insemination,

embryos with less than a predefined percentage of fragmentation for a predefined number of hours after insemination, e.g. embryos with less than 20% fragmentation 68 hours after insemination,

an embryo that is not multinucleated (multinucleated) at a certain cell stage (e.g. at the four-cell stage),

embryos that have been classified as "good quality embryos" (GQE) by a qualified embryologist,

-embryos that have been selected for freezing or transplantation,

embryos that have been selected for transfer, and/or

-an implanted embryo.

Embryos selected by exclusion of dysplastic embryos, for example by exclusion of Scc and/or Lcc embryos or by using other exclusion criteria, as described, for example, in pending applications PCT/DK2012/05018 or EP12174432.0, EP12174432.0 filed at 29.06.2012, entitled "Embryo quality assessment based on blastocyst development" (Embryo quality assessment based on blastocyst development).

In another embodiment of the invention, the morphokinetic parameters are selected from the group consisting of:

-the time and/or duration of occurrence of cell division periods and inter-division periods,

-the time of occurrence and/or duration of the cleavage time, cleavage phase and cell cycle time;

the occurrence and/or duration of the fragmentation and static phases,

-synchronicity of cell division;

the occurrence, extent or duration of cellular and/or organelle movement,

-the time of occurrence, extent or duration of a quality criterion (such as the quality criterion described in PCT/DK 2012/05018),

blastocyst quality standards as described in EP 12174432.0.

-the time of occurrence and/or duration of cell-division periods and inter-division periods determined for the first, second, third, fourth, fifth and/or sixth cell division;

-the occurrence times and/or durations of cleavage time, cleavage phase and cell cycle time determined for the first, second, third, fourth, fifth and/or sixth cell division;

-synchronicity of the second and third cell divisions;

-the occurrence, extent or duration of cellular and/or organelle movement determined for the first, second, third, fourth, fifth and/or sixth cell division;

-the time of occurrence, extent or duration of cellular and/or organelle movement determined between the first, second, third, fourth, fifth and/or sixth cell divisions;

in another embodiment of the invention, the second set of embryo data sets is significantly smaller than the first set of embryos, e.g., as small as 1/2, e.g., as small as 1/5, e.g., as small as 1/10, e.g., as small as 1/50, e.g., as small as 1/100, e.g., as small as 1/200, e.g., as small as 1/500, e.g., as small as 1/1000.

In another embodiment of the invention, embryos are cultured and/or monitored in an incubator. Preferably, the embryo is monitored by image acquisition, e.g. by means of a time-lapse (time-lapse) microscope device, e.g. at least once per hour of image acquisition, preferably at least once every half hour of image acquisition, e.g. at least once every twenty minutes of image acquisition, e.g. at least once every fifteen minutes of image acquisition, e.g. at least once every ten minutes of image acquisition, e.g. at least once every five minutes of image acquisition, e.g. at least once every two minutes of image acquisition, e.g. at least once every minute of image acquisition.

The method according to the invention may be computer implemented or at least partly computer implemented, thereby providing an effective customizable tool for a birth clinic. That is, the method according to the present invention may be performed in an automated incubator system for culturing and monitoring embryos (e.g., human embryos). By implementing the present invention in such an automated incubator system, the selection process and quality control of, for example, media and other culture conditions can be almost automated, i.e., fully manual in terms of software that assists the user in making decisions, semi-automated or fully automated in terms of the incubator system that makes all decisions (including pre-alarms) based on data analysis.

In another aspect, the invention relates to a system having means for carrying out the above method. The system may be any suitable system, such as a computer comprising computer code portions constituting means for performing the method described above.

The system may in turn comprise means for acquiring images of the embryo at different time intervals, such as the system described in WO 2007/042044.

In yet another aspect, the invention relates to a data carrier comprising computer code portions constituting means for implementing the above method.

Late stage monitoring

The search for prognostic factors that predict the outcome of embryo development and In Vitro Fertilization (IVF) treatment has attracted considerable research attention, as knowledge of these factors is expected to improve future IVF treatments. One promising predictor is the precise time of occurrence of key events in early embryonic development. Studies involving imaging are limited to measuring early development, such as prokaryotic 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 for delayed analysis is the correlation between early cleavage pattern, cleavage to the 4-cell stage, and subsequent development to the blastocyst stage. Also disclosed is a morphokinetic analysis of bovine embryonic development in which the time of occurrence, duration and interval between cell cleavage in early embryonic development successfully predicted the development of the blastocyst stage followed by expansion of the blastocyst (Ramsing 2006, Ramsing 2007).

The present inventors have conducted a large clinical study involving multiple human embryos and monitored development that persists not only until blastocyst formation, but further until there is evidence of embryo implantation. In this study, implanted embryos (i.e., embryos that were transplanted and subsequently successfully implanted) and those that were not implanted (i.e., embryos that were transplanted but were not successfully implanted) were observedIs not provided withSuccessfully implanted embryos) are used. By taking implantation as an endpoint, not only the blastocyst formation ability of the embryo, but also subsequent highly necessary processes, such as hatching and successful implantation in the uterus, were evaluated.

It has been found that there is an optimal time frame for the parameters characterizing embryonic cell division. Observations support this assumption: the viability of the embryo is associated with a highly regulated sequence of cellular events that begins at fertilization. This clinical study of only good quality embryos confirmed that the implantation ability of the embryos was associated with a number of different cellular events. The complexity, structure and parameters of the model must be adapted to different clinical situations, such as culture temperature, transplantation time, culture medium, etc.

The timing of early events in embryo development is related to the development into a blastocyst that is essential for successful implantation, and thus, blastocyst formation is itself a quality parameter. However, studies have found that there is no necessary correlation between the development into blastocysts and the successful implantation of the embryos.

Supporters of early implantation at day 2 believe that prolonged culture of embryos to the blastocyst stage around day 5 poses a potential risk because the culture period is significantly prolonged, which may destroy embryo integrity. However, the extended culture period to the blastocyst stage has several benefits. The cultured human embryos have only an average blastocyst formation rate of about 30-50%, and by extending the culture period, most of the low quality embryos are automatically excluded because no blastocysts are formed. Furthermore, the embryo's own DNA controls development after EGA before and after the 5-8 blastomere stage. By evaluating embryos at the blastocyst stage, high quality embryos can be identified with greater certainty.

Thus, the data allow the detection of blastocyst-associated developmental criteria for implantation potential. The results are particularly indicative of late events (e.g., null)ChamberOnset of chemogenesis) is an indicator of consistently good implantation potential, and improved discrimination between implanted and non-implanted embryos when blastocyst quality criteria (e.g., tBI) are used, as opposed to earlier events (t2, t3 and t 4). The data indicate that culturing embryos to blastocyst stage can yield additional important information that will improve the ability to select for viable embryos with high implantation potential. Furthermore, monitoring the late stage embryos up to the blastocyst stage may better show quality problems, as shown in FIGS. 10-14, where the effects of (for example) toxic oils are more pronounced in the later stages. Therefore, it is advantageous to include blastocyst quality criteria in the morphokinetic parameters selected herein (i.e., the morphokinetic parameters selected for quality supervision).

A number of embryo quality (selection) criteria are listed below, as well as blastocyst quality criteria, which can be applied individually or in combination in groups to evaluate embryo quality and thereby evaluate quality issues in monitoring and automating the study of developmental conditions.

Multiple variables

When selecting the selection criteria, a number of variables may be used. When multiple variables are used, the variables are selected step by step to first select one or more variables, e.g., t2, t3, t4, or t5, that are determined early with high accuracy. Other variables that are more difficult to determine and have higher uncertainty are then reused.

Normalized or relative parameters

The selection criteria derived from the morphokinetic parameters cannot be applied universally, since the developmental conditions may vary from clinic to clinic. Thus, it is advantageous to define normalized morphological kinetic parameters, e.g. based on two, three, four, five or more parameters selected from the group of t2, t3, t4, t5, t6, t7 and t 8. By normalizing the parameters, the fertilization time can be "removed" from the embryo quality assessment. Furthermore, the normalized morphological embryo parameters may better describe the uniformity and/or regularity of the development rate of a particular embryo independent of environmental conditions, as the use of normalized parameters ensures that the ratio of time intervals can be compared to "globally" determined normalized parameters, which may not be compared to "globally" determined absolute time intervals, which may depend on local environmental conditions, thereby providing additional information on embryo development. In addition, normalizing morphokinetic parameters may provide additional related quality issues in embryo development.

Thus, a blastocyst quality criterion may be used to determine 1) the duration of a first time period from fertilization to completion of translation of maternally inherited mRNA in a blastomere, and 2) the duration of a second time period from initiation of transcription of the blastomere's own DNA to the determination of the blastocyst stage, wherein the blastocyst quality criterion is the ratio of the first and second time periods.

Thus, a blastocyst quality criterion may be used to determine 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 the determination of the blastocyst stage, wherein the blastocyst quality criterion is the ratio of the first and second time periods.

The time from fertilization to blastocyst stage is thus divided into two time segments and the ratio between these time segments is the blastocyst quality criterion. The reason for the division at the 5 blastomere stage is presumably the time of embryonic gene activation. Thus, the duration of 1) a first time period from fertilization to completion of translation of maternally inherited mRNA in the blastomere, and 2) a second time period from the start of transcription of the blastomere's own DNA to the determination of the blastocyst stage is determined, wherein the blastocyst quality criterion is the ratio of the first and second time periods.

The corresponding blastocyst quality criterion can be provided by determining 1) the duration of the first time period from fertilization to blastocyst, and 2) the duration of the second time period from the start of transcription of blastomere self-DNA to the blastocyst stage and taking the ratio of these time periods. This ratio provides information on how much time the embryo's own DNA is in control during the entire time period from fertilization to blastocyst.

These ratios can be considered as a measure of the relative rate of development over a period of time relative to the overall rate of development up to that period. Any change in these ratios may indicate that the development of the embryo is accelerating or decelerating, which may be indicative of quality problems in the development conditions.

The blastocyst stage can be selected from the group consisting of: initial densification (IC), densification/morula (M), initial differentiation of trophoblast cells (IDT), early blastocyst (ERB), empty blastocystChamberOnset of chemomorphism/Blastocyst (BI), blastocyst Expansion (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 (HB), and complete hatching (FH). Thus, tIC is the time from fertilization to initial densification. tM is the time from fertilization to densification/morula, etc.

Examples of embryo quality criteria

One blastocyst quality criterion was the determination of tB_i-t_iWherein tB_iSelected from the group of { tM, tBL, tEB } and t_iSelected from the group { t5, t6, t7, and t8 }.

One blastocyst quality criterion was determination (tB)_i-t_i)/t_iWhich isMiddle tB_iSelected from the group of { tM, tBL, tEB } and t_iSelected from the group { t5, t6, t7, and t8 }.

One blastocyst quality criterion was determination (tB)_i-t_i)/tB_iWherein tB_iSelected from the group of { tM, tBL, tEB } and t_iSelected from the group { t5, t6, t7, and t8 }.

One blastocyst quality criterion is the determination of the time from fertilization to the blastocyst stage.

One blastocyst quality criterion is the determination of one or more of the following blastocyst morphological parameters:

tIC-the time from fertilization to initial densification,

tM-time from fertilization to densification/morula,

ttit is the time from fertilization to initial differentiation of trophoblast cells,

tERB-the time from fertilization to early blastocyst,

tBI from fertilization to emptyChamberThe time at which the formation is to begin,

time from fertilization to blastocyst expansion,

tCPS (1) is the time from fertilization to first contraction,

tCPS (2) — the time from fertilization to the second contraction,

tCPS (3) is the time from fertilization to third contraction,

tHB is the time from fertilization to hatch, and

tFH-time from fertilization to complete hatching.

tBI-tM，

tEB-tB，

tHE-tEB，

tEB-tM，

tBI-tCPS(1)，

tBI-tCPS(2)，

tEB-tCPS(1)，

tEB-tCPS(2)，

tCPS(2)-tCPS(1)，

tCPS(3)-tCPS(2)。

one blastocyst quality criterion is the determination of absolute or relative 2D and/or 3D expansion of the blastocyst.

One blastocyst quality criterion is to determine the diameter and/or volume of the embryo at the beginning of expansion.

One blastocyst quality criterion is to determine the maximum diameter and/or maximum volume of the blastocyst before hatching.

The normalized embryo morphology parameters may be selected from the group consisting of:

cc2/cc2_3＝(t3-t2)/(t5-t2)，

cc3/cc2_3＝(t5-t3)/(t5-t2)，

cc3/t5＝1-t3/t5，

s2/cc2＝(t4-t3)/(t3-t2)，

s3/cc3 ═ t8-t5)/(t5-t3, and

cc2/cc3＝(t3-t2)/(t5-t3)。

one quality criterion is to determine the degree of irregularity in the timing of cell division when an embryo develops from 4 to 8 blastomeres.

One quality criterion is to determine the maximum cleavage time per blastomere when the embryo develops from 4 to 8 blastomeres.

One quality criterion is to determine the ratio between the longest cleavage time per blastomere when the embryo develops from 4 to 8 blastomeres and the duration of the total period from 4 to 8 blastomeres; max (s3a, s3b, s3c)/s 3.

In combination with movement measurement

The above quality criteria may also be combined with determinations of embryo movement, such as i) determining the extent and/or spatial distribution of movement of a cell or organelle during the cleavage phase of the cell; and/or ii) determining the extent and/or spatial distribution of movement of cells or organelles during the inter-cleavage period, thereby obtaining an embryo quality measure.

The volume within the zona pellucida absent movement (or similar area in the projected 2D image of the embryo that remains stationary) represents a "dead" region within the embryo. The more and larger these immobile "dead" regions, the lower the likelihood of successful development of the embryo. Large areas within a series of time-lapse embryo images that do not have any type of movement (i.e., neither cell nor organelle movement) indicate low viability. Organelle movement should generally be detectable throughout the embryo, even when only two or a few consecutive frames are compared. Cell movement can be more localized, especially at later stages of embryonic development.

The cell location is usually relatively quiescent (i.e., little cell movement) between cell cleavages, except for short time intervals before and after each cell cleavages, when one cell cleavages into two resulting in a brief but extensive rearrangement (i.e., significant cell movement) of the dividing cells and surrounding cells. The less movement between cleavage is better. Thus, any unintended change in cell or organelle movement can be an indicator of quality issues related to developmental conditions.

For the cleavage phase and movement during the cleavage phase, we also refer to PCT application WO 2007/144001.

Other examples of morphokinetic parameters

As described above, the morphokinetic parameters used in accordance with certain embodiments of the invention may be established by combining a plurality of morphokinetic features associated with the development of an embryo. For example, in some implementations, the morphokinetic parameters can be derived by obtaining values for a plurality of morphokinetic characteristics associated with the development of the embryo during the observation period, such as characteristics associated with any of cell cleavage time, time difference between paired cell divisions, and cell cycle duration. The value of the continuous variable may be determined by combining the differences between the obtained values and the corresponding reference values. The reference value can be determined, for example, from values for a plurality of features obtained for one or more reference embryos (e.g., KID-positive embryos) having known developmental potential. The morphological kinetic parameters may then be based on the determined values of the continuous variables.

Thus, in accordance with practice of one embodiment of the present invention, a method for determining a morphokinetic parameter of an embryo (referred to herein as a study embryo) by generating a value of a continuous variable from a plurality of characteristics associated with the development of the embryo may comprise the following steps:

in a first step S1, a plurality of characteristics relating to the development of the study embryo during the observation period are obtained. These characteristics may be based substantially on cleavage times determined using conventional time-lapse embryo imaging. One or more features may be based on the time of occurrence of prokaryotic decay/disappearance (tPN decay (or tPNf)).

In one example, the features may comprise a series of cell cycle durations cci of the cell cycle sequence. For example, the plurality of features may include a series of values: cc2a (t3-t2), cc2b (t4-t2), cc3a (t5-t3), cc3b (t6-t 3), cc3c (t7-t 4), and cc3d (t8-t 4). That is, for this example, the sequence encompasses all cell cycle durations from cc2a to cc3d (i.e., all cell cycle durations up to the 8-blastomere embryo excluding cc 1). If there is an unmeasured duration of a particular cell cycle for a given embryo (e.g., because the time to occurrence of the associated cleavage event cannot be properly determined due to insufficient measurement or because no cleavage event has occurred until the end of the culture time (tEnd)), the missing cell cycle may not be included in the sequence.

In a second step S2, the mean and variance of features observed in a population of one or more reference embryos of known developmental potential for those corresponding to those obtained for the study embryo in step S1 are obtained. These means and variances can be read, for example, from a memory or other storage of the apparatus implementing the method. The mean and variance can be obtained by retrospective analysis of images of embryos that were advanced to successful implantation. Embryos that have obtained mean and variance for a given study embryo may be referred to as reference embryos. The reference embryo may in some cases comprise an embryo that is expected to be in the same clinic as the study embryo, e.g., to assist in accounting for differences between clinics associated with different culture conditions. That is, step S2 may also include selecting an appropriate grouping of reference embryos to derive the mean and variance of the reference embryos based on the characteristics of the study embryos. The mean and variance may be determined according to conventional statistical analysis techniques, such as may involve the discarding of outlier data, and the like. It should be understood that the term "mean" is used broadly herein to refer to typical/representative/indicative values of parameters observed in a population of samples. In this regard, the mean may, for example, correspond to the mean, mode (modeval), or median of the relevant features of the reference population (positive KID population).

In a third step S3, the difference between the value of each feature observed for the study embryo and the corresponding mean feature associated with a population of embryos with known developmental potential (e.g., KID positive) is determined.

In a fourth step S4, morphokinetic parameters (corresponding to continuous variables) of the study embryo are determined by combining/aggregating the differences determined for each feature in a manner weighted by the respective variances. Thus, in one particular example, the morphokinetic parameters (GIV) are defined as:

where cci is the series of cell cycle durations observed for studying embryos, cci_mIs the corresponding series of mean cell cycle durations observed in a reference set of embryos (e.g., a positive KID population from patients no older than 35 years), and cci_vIs the corresponding variance associated with the reference population. The parameter n is the number of cell cycle durations that make up the series cci. Difference (cci-cci)_m) Using variance value cci_vNormalization as part of the combination. This means that differences in the specific cell cycles (values of i) that exhibit relatively high variation in the sample population contribute less to the value of GIV than differences in cell cycles that exhibit relatively low variation in the sample population. In combination, let the difference (cci-cci)_m) Squaring, which means that, for a given difference, whether it is a positive or negative value (i.e., regardless of cci versus cci)_mLonger or shorter) that contribute equally to the GIV.

Generally, when the study embryo shows a regular cleavage pattern, the GIV is low, whereas when the embryo shows an irregular cleavage pattern, the value is high.

The particular morphological kinetic parameters based on the particular sequence of cell cycle durations cci ═ cc2a, cc2b, cc3a, cc3b, cc3c, and cc3d in this example may be referred to herein as the first generalized irregular variable GIV 1.

Thus, the above steps S1-S4 represent a method of establishing morphokinetic parameters of an embryo according to one embodiment of the present invention. It will be appreciated that similar methods may be used to establish morphokinetic parameters of an embryo, using different features associated with studying the development of an embryo and/or by combining the features in different ways to produce different morphokinetic parameters.

For example, while the first generalized irregular variable GIV1 described above is based on the durations of the cell cycles cc2a, cc2b, cc3a, cc3b, cc3c, and cc3d (or at least the durations of the measured/non-deleted cell cycles), other generalized irregular variables may be based on the durations of other cell cycles. For example, the following variables are defined to provide different morphokinetic parameters for use in accordance with embodiments of the present invention:

GIV2 (second generalized irregular variable): similar to GIV1, but also including cc1, i.e., GIV2 can be calculated in a similar manner as GIV1, but based on cci-cc 1, cc2a, cc2b, cc3a, cc3b, cc3c, and cc3 d.

GIV3 (third generalized irregular variable): including only the second generation cell cycle (cc2a and cc2b), i.e., GIV3 can be calculated in a similar manner to GIV1, but based on cci ═ cc2a and cc2 b.

GIV4 (fourth generalized irregular variable): including only the second and third generation shortest cell cycles (cc2a and cc3a), i.e., GIV4 can be calculated in a similar manner to GIV1, but based on cci ═ cc2a and cc3 a.

However, the generalized irregular variables of the above examples are based on cell cycle duration as defined in terms of cell cleavage time, it being understood that other generalized irregular variables may be based on other times of occurrence (and/or durations between times of occurrence) associated with other embryonic development events. For example, according to some embodiments of the present invention, generalized irregular variables used as morphokinetic parameters can be established using time of occurrence (tPNf) definitions relative to time of prokaryotic decay. One example, which may be referred to as GIV5, may be defined as follows:

GIV5 (fifth generalized irregular variable): comprises (t3-tPNf) and cc3 a.

In each case, if any of the characteristics of the embryo constituting the generalized irregular variable are missing (e.g. because they are not properly measured or do not occur before the end of the culture time), the corresponding characteristics may not be counted in the calculation of the continuous variable (morphokinetic parameter), with a corresponding decrease in the value of n.

Other morphokinetic parameters may be determined by features other than the duration of the cell cycle that are relevant to studying the development of an embryo.

For example, the method for changing the above method with respect to steps S1 to S4 may include the following steps:

in the first step T1, the characteristic relating to the development of the study embryo may instead comprise the time difference Δ tj between a series of subsequent cell divisions (or morphological phases). For example, the plurality of features may include a series of values: Δ t1(═ t2), Δ t2(═ t3-t2), Δ t3(═ t4-t3), Δ t4(═ t5-t4), Δ t5(═ t6-t5), Δ t6(═ t7-t6), Δ t7(═ t8-t7) — that is, the time difference between subsequent cell divisions up to 8 blastomere stages. That is, for this example, the sequence includes all of the time between a single cell and subsequent cell divisions of the 8-blastomere embryo, or at least all of these times considered to be appropriately measured (i.e., no deletions).

In a second step T2, the mean and variance observed in a population of one or more reference embryos of known developmental potential (e.g., KID-positive embryos) for the features obtained for the study embryo in step T1 are obtained.

In a third step T3, the difference between the value of each feature observed for the study embryo and the corresponding mean feature associated with a population of embryos with known developmental potential (e.g. KID positive) is determined.

In a fourth step T4, the morphokinetic parameters (corresponding to continuous variables) of the studied embryo are determined by combining/aggregating the differences determined for each feature in a manner weighted by the respective variances. Thus, in one particular example, the morphokinetic parameters (GTV) are defined as:

where Δ tj is the time difference observed for a series of subsequent cell divisions for the study embryo, Δ tj_mIs the average of the corresponding series, Δ tj, observed in a reference group of embryos (e.g., a positive KID population from patients no older than 35 years)_vIs the corresponding variance associated with the reference population. The parameter k is the number of values that make up the series Δ tj. The difference (Δ tj- Δ tj)_m) Using variance Δ tj_vNormalization as part of the combination. This means that the difference in the specific cell divisions exhibiting relatively high variation in the sample population (value of j) contributes less to the value of GTV than those exhibiting relatively low variation in the sample population. In this example, the contribution of each time difference to the GTV depends on whether the time difference for subsequent cell divisions of a particular pair is faster or slower than the average in the positive KID population (i.e., whether the difference is a positive or negative value).

This morphokinetic parameter, GTV, may be referred to herein as the total time variable, GTV. Generally, GTV is low when the study embryo exhibits relatively fast development, and high when the embryo exhibits relatively slow development.

Specific morphokinetic parameters based on specific sequences Δ t1(═ t2), Δ t2(═ t3-t2), Δ t3(═ t4-t3), Δ t4(═ t5-t4), Δ t5(═ t6-t5), Δ t6(═ t7-t6), Δ t7(═ t8-t7) as in this example may be referred to herein as the third generalized time variable GTV 3.

It will also be appreciated that similar methods may be used to establish morphokinetic parameters of embryos using different characteristics associated with studying the development of the embryo.

For example, while the third generalized time variable, GTV3, as described above, is based on all times between subsequent cell divisions from a single cell to an 8-blastomere embryo, other generalized irregular variables may be based on other sequences of time differences. For example, the following variables are defined to provide different morphokinetic parameters for use in accordance with embodiments of the present invention:

GTV1 (first generalized time variable): similar to GTV3 but using only the time difference between the last two cell divisions observed up to the 8 blastomere stage. That is, if t7 and t8 are annotated, Δ ti is (t8-t 7); or if t8 is missing and t6 and t7 are annotated, Δ ti ═ (t7-t 6); or if t8 and t7 are missing and t5 and t6 are annotated, Δ ti ═ t6-t 5; or if t8, t7, and t6 are missing and t4 and t5 are annotated, Δ ti ═ t5-t 4; or if t5-t 8 are missing and t3 and t4 are annotated, Δ ti ═ t4-t 3; or if t4-t 8 are missing and t2 and t3 are annotated, Δ ti ═ t3-t 2; if t3-t 8 are missing and t2 is annotated, Δ ti — t 2. In each case, the parameter k is 1.

GTV2 (second generalized time variable): similar to GTV1 but in the absence of time, the later cleavage time in the pair (cleavage time) was replaced by tEnd (end of culture time). That is, if t7 and t8 are annotated, Δ ti is (t8-t 7); or if t8 is missing and t7 is annotated, Δ ti ═ (tEnd-t 7); or if t7 and t8 are missing and t6 is annotated, Δ ti ═ (tEnd-t 6); or if t6-t 8 are missing and t5 is annotated, Δ ti ═ (tEnd-t 5); or if t5-t 8 are missing and t4 is annotated, Δ ti ═ (tEnd-t 4); or if t4-t 8 are missing and t3 is annotated, Δ ti ═ (tEnd-t 3); or if t3-t 8 are missing and t2 is annotated, Δ ti ═ t (tEnd-t 2). The mean and variance of the reference population may be calculated based on Δ ti without replacement. For GTV2, parameter k is always 1.

GTV3 (third generalized time variable): as described above, this morphokinetic parameter uses the time of occurrence between all consecutive divisions that were not missing from the data up to the 8 blastomere phase, i.e., Δ ti ═ ((t8-t7), (t7-t6), (t6-t5), (t5-t4), (t4-t3), (t3-t2), t 2). If Δ ti from this sequence is missing for a particular embryo, it is ignored for the calculation. For a particular embryo, the parameter k is the number of Δ ti used in the calculation.

GTV4 (fourth generalized time variable): similar to GTV3, but using all sequential divisions, if deleted, the final division time was replaced with the culture termination time. Δ ti ═ ((t8-t7), (t7-t6), (t6-t5), (t5-t4), (t4-t3), (t3-t2), t 2). If ti is missing, it is replaced with tEnd. The mean and variance of the reference population can be calculated based on Δ ti without substitution. For this particular embryo, k is the number of Δ ti used in the calculation.

GTV5 (fifth generalized time variable): similar to GTV3, but using the time of occurrence of the whole cell cycle. That is, Δ ti ═ ((t8-t4), (t4-t2), t 2). If Δ ti is missing, it is ignored. For a particular embryo, k is the number of Δ ti used in the calculation.

GTV6 (sixth generalized time variable): similar to GTV5, but using all whole cell cycles, if deleted, the last division time was replaced with tEnd. Δ ti ═ ((t8-t4), (t4-t2), t 2). If ti is missing, it is replaced with tEnd. The mean and variance are calculated based on Δ ti without substitution. For a particular embryo, k is the number of Δ ti used in the calculation.

GTV7 (seventh generalized time variable): similar to GTV2, but only the time period from fertilization to the time of occurrence of the last annotation was used. That is, Δ ti is t8 if t8 is annotated, t7 if t8 is missing and t7 is annotated, t6 if t7 and t8 are missing and t6 is annotated, and so on. For GTV7, parameter k is always 1.

GTV8 (eighth generalized time variable): similar to GTV3, but if t8 is annotated, then t8 is used, and in other cases, if t8 is missing, then tEnd is used. For GTV8, parameter k is always 1.

GTV9 (ninth broad time variable): similar to GTV2, but using the period up to the blastocyst stage to evaluate on day 5 post-insemination. If tSB and tB are annotated, Δ ti is (tB-tSB); or if tB is missing and tSB is annotated, Δ ti ═ (tEnd-tSB); or if tSB and tB are missing and tM is annotated, Δ ti ═ t (tEnd-tM); or if tM to tB is missing and t8 is annotated, Δ ti ═ t 8; or if t 8-tB is missing and t7 is annotated, Δ ti ═ t 7; or if t 7-tB is missing and t6 is annotated, Δ ti ═ t 6; or if t 6-tB is missing and t5 is annotated, Δ ti ═ t 5; or if t 5-tB is missing and t4 is annotated, Δ ti ═ t 4; or if t 4-tB is missing and t3 is annotated, Δ ti ═ t 3; or if t3 to tB are missing and t2 is annotated, Δ ti ═ t 2. The mean and variance may be calculated based on Δ ti without substitution. For GTV9, parameter k is always 1.

GTV10 (tenth generalized time variable): similar to GTV4, but used for all periods up to the blastocyst stage. For evaluation on day 5 after insemination, Δ ti ═ ((tB-tSB), (tSB-tM), (tM-t8), (t8-t7), (t7-t6), (t6-t5), (t5-t4), (t4-t3), (t3-t2), t 2). If a certain occurrence is missing, it is replaced by a tEnd. The mean and variance are calculated based on Δ ti without substitution. For GTV10, the parameter k is the number of Δ ti used in the calculation for that particular embryo.

Embryo

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 time the embryo becomes a fetus. According to this definition, a fertilized oocyte is generally referred to as a pre-embryo until implantation occurs. However, in this patent application we will use the broader definition of the term embryo, which encompasses the pre-embryonic stage. Thus, it encompasses all developmental stages from fertilization of an oocyte to morula, blastocyst stage, hatching and implantation.

The embryo is approximately spherical and consists of one or more cells (blastomeres) surrounded by a gelatinous shell (an acellular matrix called zona pellucida). Zona pellucida performs multiple functions until the embryo hatches, which is a good landmark for embryo evaluation. The zona pellucida is spherical and translucent and should be clearly distinguishable from cell debris.

When an oocyte is fertilized by fusion or injection of sperm cells (spermatozoa), an embryo is formed. The term is also traditionally used after hatching (i.e., rupture of zona pellucida) and subsequent implantation. In humans, fertilized oocytes are traditionally referred to as embryos at the first 8 weeks. After that (i.e. after eight weeks and when all major organs have formed) it is called a fetus. However, the distinction between embryo and fetus is generally not strictly defined.

During embryonic development, blastomere numbers increase in a geometric manner (1-2-4-8-16-etc.). Synchronized cell cleavage is generally maintained until the 16-cell stage of the embryo. Thereafter, cell cleavage becomes asynchronous and eventually, each cell has its own cell cycle. Human embryos produced during infertility treatment are typically transferred to a recipient prior to the 16-blastomere stage. In some cases, human embryos are also cultured to the blastocyst stage prior to transplantation. This is preferably done in the following cases: when many good quality embryos are available or require prolonged culture to await the results of pre-implantation genetic diagnosis (PGD).

Thus, the term embryo is used hereinafter to denote each of the following stages: fertilized oocytes, zygotes, 2-cells, 4-cells, 8-cells, 16-cells, morulae, blastocysts, expanded blastocysts and hatched blastocysts, and all stages in between (e.g., 3-cells or 5-cells).

Examples

Example 1

Under similar conditions, the development of three different groups of mouse embryos cultured in three media at different temperatures was studied, i.e., only the temperature differed between the three different groups. The temperature of the medium was evaluated by measuring the slide holder temperature using a YSI precision thermometer.

The three different temperatures were 36.5 ℃ (33 embryos), 37.5 ℃ (63 embryos), and 38.5 ℃ (35 embryos), respectively. As shown in the table below, almost all mouse embryos reached the blastocyst stage.

Temperature (. degree. C.) of slide holder N blastocyst fraction (%)

The following table shows the mean time to occurrence measured for different cell divisions, morula and blastocyst stages.

These data are plotted in three graphs as shown in fig. 5. The differences between the different cell divisions are shown in figure 6. The data and graphs show that an increase in medium temperature significantly accelerated development.

To evaluate developmental differences, a relative rate coefficient k may be defined. If k is at the reference temperature (T)_b) When 1 is set, the following relationship can be determined:

k(T)＝1+α*(T_b-36.5)

where T is the temperature ℃ and α is the temperature-dependent coefficient.

Expected time T for a given temperature T, and T (T)_b) Related, inversely proportional to k (T):

t(T)＝t(T_b)/k(T)

the above linear simplification provides the advantage that only a single parameter needs to be evaluated. On the other hand, it may only be effective within a narrow temperature range. However, in the case of human embryo culture, the expected maximum temperature span is slightly below ± 1 ℃ so that the actual effect of the non-linearity is considered negligible.

By using the above mouse embryo data, k (T) and t (T) were optimized using the time to divide into 5 cells (t5), and α was estimated to be 0.080 ± 0.015 (95% CI).

Q₁₀The values are calculated as:

where R is the ratio and T is the temperature.

Using the above parameters, mouse embryo data and test + -1 deg.C span, the equation gives Q₁₀This is 2.22, which is within the normal expected range of 2-3 for biological systems (Reyes et al, 2008, Mammalian peripheral circadian oscillators are temperature compensated).

The same calculations were performed on a set of data from 1397 individual embryos obtained from different clinics. The culture conditions for these human embryos are therefore not similar to the mouse embryos described above. However, the clinics all belong to the same chain of IVF clinics using the same instrument. In addition to temperature, all embryos were transferred using the same procedure. Here again using t5, the estimated value for α becomes 0.058. + -. 0.028 (95% CI) based on k (T) and t (T) optimizations.

Unlike mouse embryos, these human embryos were cultured under slightly different conditions. The resulting human embryo data cannot therefore be compared to the same extent as the mouse embryo data. However, data from human embryos again indicate that higher temperature media accelerates development. By continuously monitoring the morphokinetic parameters, an increase in development speed can be detected and provide an early warning of problems.

This problem can be associated with an undesirable increase in temperature, which can be corrected prior to actually transferring any embryos.

Claims

1. A computer-implemented method for automated detection of changes and/or abnormalities in developmental conditions of in vitro cultured embryos, comprising the steps of:

c) modifying the first data set and the second data set by removing morphokinetic parameter outliers from the first data set and or the second data set,

e) monitoring said morphokinetic differences to detect a change in a development condition of said first group of embryos and said second group of embryos.

2. The method of claim 1, wherein said step c) is optional.

3. The method of claim 1, wherein said step c) is non-optional.

4. The method according to any of the preceding claims, wherein the second data set comprises up-to-date embryo data.

5. The method according to any of the preceding claims, wherein the steps a) -d) are repeated and the second data set is continuously updated with the latest embryo data.

6. The method according to any of the preceding claims, wherein the second data set comprises latest embryo data selected from a specific time period or from a predefined number of latest embryos.

7. The method according to any of the preceding claims, further comprising the step of issuing a warning when the difference in morphological kinetics is above a predefined level.

8. The method of claim 7, wherein the predefined level is determined to be a predefined level above a standard deviation of one or more morphokinetic parameters from the first data set.

9. The method according to any of the preceding claims, wherein the steps a) -d) are repeated, thereby continuously measuring developmental conditions.

10. The method according to any of the preceding claims, wherein at least one of the morphokinetic parameter outliers is defined as a relative outlier.

11. The method according to any of the preceding claims, wherein at least one of the morphokinetic parameter outliers is defined as an absolute outlier.

12. The method according to any of the preceding claims, further comprising the step of monitoring the number of outliers rejected.

13. The method according to any of the preceding claims, wherein the morphokinetic parameter outliers are excluded from the dataset.

14. The method according to any of the preceding claims, wherein all morphokinetic parameters of embryos with one or more morphokinetic parameter outliers are excluded from the dataset.

15. The method of any of the preceding claims, wherein the second group of embryos is a subset of the first group of embryos such that the second data set is a subset of the first data set.

16. The method according to any of the preceding claims, wherein the second group of embryos is selected as the latest embryo, e.g. a predefined number of embryos as the latest embryo, e.g. the latest embryos from a predefined time period, e.g. a predefined number of hours, a predefined number of days, a predefined number of weeks or a predefined number of months.

17. The method of claim 16, wherein the predefined number of embryos or predefined time period is determined as a user input.

18. The method according to any one of the preceding claims, wherein the morphokinetic parameters are selected from the group of:

-time to cleavage to n blastomere embryos, tn, where n ═ 1, …,8,

the duration of the cell cycle cc1, cc2, cc2b, cc3, cc2_3 and cc4,

synchronicity s2, s3, s3a, s3b and s3c,

-the number of blastomeres at a specific predefined point in time,

the occurrence, extent or duration of cellular and/or organelle movement,

-any combination of these morphokinetic parameters.

19. The method according to any one of the preceding claims, further comprising the step of calculating a running average or a running median of the morphokinetic parameter.

20. The method according to any of the preceding claims, further comprising the step of calculating the mean (mean) value, median value, variance value and/or standard deviation value of the morphokinetic parameter.

21. The method according to any of the preceding claims, wherein the following is a morphokinetic parameter:

where i ═ {1,2,3,4}, cci_mIs the mean or median of cci, and cci_vIs the variance of cci.

22. The method according to any of the preceding claims, wherein at least one morphokinetic parameter of an embryo is established by: obtaining values of a plurality of features related to embryo development during an observation period and determining a value of the at least one morphokinetic parameter by combining differences between the obtained values and corresponding reference values of the plurality of features in a predefined manner.

23. The method of claim 22, wherein the reference value is determined from values of the plurality of features obtained for at least one reference embryo.

24. The method of claim 22 or 23, wherein the step of combining differences between the obtained values and the reference values takes into account a weighting value associated with each of the reference values.

25. The method of claim 24, wherein the weighting values are statistically determined from values of the plurality of features obtained for a plurality of reference embryos having known developmental potential.

26. The method of claim 25, wherein the weighting values are determined from a variance of values obtained for the plurality of reference embryos.

27. The method of any one of claims 22 to 26, wherein the morphokinetic parameter represents a measure of regularity in morphological development of the embryo.

28. The method of any one of claims 22 to 27, wherein the plurality of characteristics comprises a plurality of cell cycle durations, cci, of the embryo.

29. The method according to any one of claims 22 to 28, wherein said plurality of characteristics comprises a plurality of time differences, Δ tj, between subsequent cell divisions of the embryo.

30. The method according to any of the preceding claims, wherein at least one of the morphokinetic parameters is based on a generalized irregular variable, GIV, determined according to:

wherein cci is the series of cell cycle durations of the embryo, cci_mIs the average cell cycle duration of the corresponding series observed in the embryos of the reference population, cci_vIs the variance value of the respective cell cycle durations of the corresponding series observed in the reference population, and n is the number of cell cycle durations comprising the cell cycle duration series.

31. The method according to any of the preceding claims, wherein at least one of the morphokinetic parameters is based on a generalized time variable, GTV, which is determined according to:

where Δ tj is the time difference between a series of subsequent cell divisions of the embryo, Δ tj_mIs the average of the corresponding series, Δ tj, observed in embryos of the reference population_vIs the variance of the respective time differences observed in the reference population for the corresponding series of subsequent cell divisions, and k is the number of time differences comprising the series.

32. The method according to any of the preceding claims, wherein the morphokinetic parameter outliers are selected from the group consisting of: embryos that are cleaved directly from 1 cell to 3 cells and cc2<5 hours, embryos that are cleaved directly from 2 cells to 5 cells, short cell cycle embryos, long cell cycle embryos, and embryos that undergo multinucleation.

33. The method according to any of the preceding claims, wherein short cycle embryos (SCC) and/or long cycle embryos (LCC) are defined as embryos with morphokinetic parameter outliers.

34. The method according to any of the preceding claims, wherein the first and/or second group of embryos are embryos that have been fertilized, stored and/or cultured under a specific set of conditions.

35. The method of claim 34, wherein the first group of embryos have been fertilized under a different set of conditions than the second group of embryos.

36. The method of any one of claims 34 to 35, wherein the conditions are selected from the group consisting of: type of fertilization treatment, preservation such as cryopreservation, culture temperature, culture media type, specific incubator, specific treatment such as hormone treatment, suction, male factor, specific incubator, culture temperature, culture media type, oil type, PGD treatment, transplantation, cryopreservation, thawing, time outside incubator, number of fertilization treatments.

37. The method of any one of the above claims, wherein the second group of embryos are embryos processed by a specific person.

38. The method according to any of the preceding claims, wherein the second group of embryos are embryos derived from a predefined embryo donor.

39. The method of claim 38, wherein the predefined embryo donors are selected from the group consisting of: individuals younger or older than a predefined age, embryo donors in a specific treatment or stimulation protocol, embryo donors in a specific fertilization treatment, embryo donors with a specific diagnosis such as hereditary chromosomal disorders, Hiv, Hep, PCO, individuals currently or previously exposed to radiation or harmful chemicals, individuals currently or previously administered drugs, individuals with a BMI higher or lower than a predefined level, smokers, non-smokers, individuals with a normal or abnormal menstrual cycle.

40. The method according to any of the preceding claims, wherein the first group of embryos is selected from all implanted embryos, implanted embryos of known fate, implanted embryos that result in a continuation of pregnancy, live babies, Fetal Heart Beats (FHB), and/or fetal capsules.

41. The method according to any of the preceding claims, wherein the second group of embryos is selected from the group consisting of:

a) all of the fertilized embryos had been,

b) embryos that have been selected for freezing or transplantation,

c) the embryos that have been selected for transfer,

d) embryos that have been classified as "good quality embryos" (GQE) by a qualified embryologist,

e) an implanted embryo.

42. The method according to any of the preceding claims, wherein the embryo is monitored by image acquisition, such as at least once per hour image acquisition, such as at least once per half hour image acquisition, such as at least once per twenty minute image acquisition, such as at least once per fifteen minute image acquisition, such as at least once per ten minute image acquisition, such as at least once per five minute image acquisition, such as at least once per two minutes image acquisition, such as at least once per minute image acquisition.

43. The method according to any of the preceding claims, wherein the embryo is monitored by means of a time-lapse microscopy device.

44. The method according to any of the preceding claims, wherein the embryo has been fertilized in vitro.

45. The method according to any of the preceding claims, wherein the embryo is cultured and/or monitored in an incubator.

46. The method of any one of the above claims, wherein the embryo is a human embryo.

47. A system for determining embryo quality comprising means for carrying out the steps of any one of claims 1 to 46.

48. A computer comprising computer code portions constituting means for carrying out the method of claims 1 to 46.