WO2014033210A1 - Surveillance automatique d'embryons incubés in vitro - Google Patents

Surveillance automatique d'embryons incubés in vitro Download PDF

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WO2014033210A1
WO2014033210A1 PCT/EP2013/067888 EP2013067888W WO2014033210A1 WO 2014033210 A1 WO2014033210 A1 WO 2014033210A1 EP 2013067888 W EP2013067888 W EP 2013067888W WO 2014033210 A1 WO2014033210 A1 WO 2014033210A1
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embryos
embryo
morphokinetic
values
group
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PCT/EP2013/067888
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English (en)
Inventor
Niels B. Ramsing
Mette LÆGDSMAND
Jens K. Gundersen
Søren PORSGAARD
Inge Errebo AGERHOLM
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Unisense Fertilitech A/S
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Priority claimed from PCT/EP2013/063240 external-priority patent/WO2014001312A1/fr
Application filed by Unisense Fertilitech A/S filed Critical Unisense Fertilitech A/S
Priority to CN201380055588.4A priority Critical patent/CN104755608A/zh
Priority to EP13756438.1A priority patent/EP2890781B1/fr
Priority to ES13756438T priority patent/ES2831867T3/es
Publication of WO2014033210A1 publication Critical patent/WO2014033210A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/06—Bioreactors or fermenters specially adapted for specific uses for in vitro fertilization
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/46—Means for regulation, monitoring, measurement or control, e.g. flow regulation of cellular or enzymatic activity or functionality, e.g. cell viability
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/48—Automatic or computerized control
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06V—IMAGE OR VIDEO RECOGNITION OR UNDERSTANDING
    • G06V20/00—Scenes; Scene-specific elements
    • G06V20/60—Type of objects
    • G06V20/69—Microscopic objects, e.g. biological cells or cellular parts
    • G06V20/698—Matching; Classification

Definitions

  • the present invention relates to a system and a method for automatically surveying the developmental conditions of in vitro incubating embryos.
  • the present invention may be applied within quality control of embryo handling in relation to IVF to ensure that the quality of transferred embryos is maintained.
  • the present invention may be a tool for fertility clinics to survey and maintain a high implantation potential of in vitro incubating embryos.
  • Infertility affects more than 80 million people worldwide. It is estimated that 10% of all couples experience primary or secondary infertility.
  • In vitro fertilization (IVF) is an elective medical treatment that may provide a couple who has been otherwise unable to conceive a chance to establish a pregnancy. It is a process in which eggs (oocytes) are taken from a woman's ovaries and then fertilized with sperm in the laboratory. The embryos created in this process are then placed into the uterus for potential implantation.
  • Quality Control is an important issue in IVF clinics to monitor the quality of the treatment and the different processes in the clinics that has an effect on the
  • Quality control can be conducted by monitoring the development of running averages in outcome variables like
  • Fetal heats beat rates (number of fetal heart beats per transferred embryos)
  • KID ratio the number of known implantations (defined either as fetal heart beat or HCG positive) per known outcome
  • the quality control shall pick up on e.g. failure to comply to best practice (e.g. errors in the handling of the embryos), toxicity of lab utensils or consumables (e.g. toxic oil or media) or environmental changes (contamination of laboratory air) then the response needs to be accurate, fast and sensitive. Accurate so that correcting measures are only set in place if something is really wrong, fast so that modifications can be implemented as quickly as possible and sensitive so that even small changes in the performance can be detected and corrected. Therefore, a sensitive and fast responding tool for monitoring the clinic procedures is needed.
  • One embodiment of the invention therefore relates to a computer implemented method for automatically detecting variations and/or abnormalities in the developmental conditions of in vitro incubating embryos, the method comprising the steps of:
  • morphokinetic variables are important indicators for embryo implantation success rates.
  • morphokinetic parameters are utilized as quality control indicators and thereby form an alternative to monitoring the pregnancy rates or related variables which are delayed by definition.
  • the big advantage of using morphokinetic parameters is that the number of embryos in a clinic is around one magnitude higher than the number of embryos transferred and the variables are available as soon as the incubation is ended (2-5 days after fertilization). Furthermore, the number of available morphokinetic parameters is around one magnitude higher than the number of embryos, because each embryo "produces" a plurality of morphokinetic parameters during the different developmental stages. So by monitoring the morphokinetic parameters instead of the pregnancy rates both speed and accuracy may be increased. Since e.g. the timings of cleavages are continuous variables instead of discrete the sensitivity of the statistical tests that can be used to test if two groups are significantly different are greatly increased.
  • the present invention is most naturally applied to human embryos, but may also be applied within monitoring of any mammal embryos.
  • Cleavage time is defined as the first observed timepoint when the newly formed blastomeres are completely separated by confluent cell membranes, the cleavage time is therefore the time of completion of a blastomere cleavage.
  • the times are expressed as hours post ICSI microinjection or post time for mixing of semen and oocyte in IVF, i.e. the time of insemination. This is the time of the deliberate introduction of sperm into the ovum.
  • fertilization is also used to describe this timepoint.
  • the cleavage times are as follows: t2: Time of cleavage to 2 blastomere embryo
  • Duration of cell cycles is defined as follows:
  • cc3 is the time required for the third round of DNA replication.
  • Long cell cycle embryos are embryos that develop very slowly with unusual long durations for their cell cycles.
  • the long cell cycle embryos have a similar poor prognosis and are significantly less likely to implant than more normal embryos (as is shown below).
  • the class of "long cell cycle embryos” is less clearly defined than the short cell cycles as the distribution at the high end is continuous with a gradually decreasing implantation potential. It is therefore less clear how to define the boundaries for this class.
  • the boundaries for the long cell cycle embryo class has been chosen such that the LCC class correlate with a markedly reduced implantation potential similar to the one found for short cell cycle embryos.
  • a long cell cycle class is thus defined with a comparable size to the short cell cycle class of embryos:
  • MCC embryos are classified as medium cell cycle embryo. These are the "normal" embryos with a normal cleavage pattern.
  • the MCC embryos typically constitute about 60% of all embryos, which can be used to compare embryo development from clinic to clinic. E.g. the first group of embryos may be selected among MCC embryos.
  • Cleavage period The period of time from the first observation of indentations in the cell membrane (indicating onset of cytoplasmic cleavage) to the cytoplasmic cell cleavage is complete so that the blastomeres are completely separated by confluent cell membranes. Also termed as duration of cytokinesis.
  • Fertilization and cleavage are the primary morphological events of an embryo, at least until the 8 blastomere stage.
  • Cleavage time, cell cycle, synchrony of division and cleavage period are examples of morphological embryo parameters that can be defined from these primary morphological events and each of these morphological embryo parameters are defined as the duration of a time period between two morphological events, e.g. measured in hours.
  • a normalized morphological embryo parameter is defined as the ratio of two
  • morphological embryo parameters e.g. cc2 divided by cc3 (cc2/cc3), or cc2/cc2_3 or cc3/t5 or s2/cc2.
  • the duration of a plurality of cell cycles (e.g. CC1 , CC2, CC3 and CC4) can be combined to r:
  • CCi e.g. is selected from CC1 to CC4.
  • a high value of CC nor m indicates a poor embryo quality as one or more of the variables CCi is far from the median, i.e. it is not the absolute values of CCi that are used, but the mutual relation of the variables.
  • the median may be calculated based on the whole population or parts of the population (e.g. embryos with known and positive
  • ICC norm logarithmic value
  • synchronicity Si of the cell divisions may be combined to form a common normalized parameter:
  • a high value of S norm indicates a poor embryo quality as one or more of the synchronicities is long compared to the.
  • Another equivalent variable using the logarithmic value instead (IS n0 rm) may also be useful assessing embryo quality.
  • the variables CC norm and S n0 rm may be calculated based on the first, second, third or fourth cell cycle, depending on the duration of the incubation.
  • a morphokinetic parameter for embryos which is used in accordance with certain embodiments of the invention may be established by combining a plurality of morphokinetic characteristics relating to the development of the respective embryos.
  • the morphokinetic parameter may be derived by obtaining values for a plurality of morphokinetic characteristics relating to the development of an embryo during an observation period, for example characteristics such as cell cleavage times (t2, t3, t4%) and / or differences between subsequent cell cleavage times / divisions (t3-t2, t4-t3, t5-t4...) and / or cycle durations (cc1 , cc2, cc3... etc).
  • a value for a continuous variable may then be determined by combining differences between the obtained values for the plurality of characteristics and corresponding reference values in a pre-defined manner.
  • the reference values may, for example, be determined from values for the plurality of characteristics obtained for one or more reference embryos of known development potential (e.g. KID positive embryos).
  • a morphokinetic parameter for an embryo which is to be used in accordance with certain embodiments of the invention may thus be established from the continuous variable.
  • the morphokinetic parameter may correspond with the development potential for an embryo determined in accordance with the principles set out in co-pending patent application PCT/EP2013/063240 filed 25 June 2013, the entire contents of which are incorporated herein by reference.
  • the step of combining differences between the obtained values for the plurality of morphokinetic characteristics and the corresponding reference values may take account of weighting values associated with each of the reference values.
  • the weighting values may, for example, be statistically determined from values for the plurality of characteristics obtained for a plurality of reference embryos of known development potential. For example, the weighting values may be determined from a variance of the relevant values obtained for the plurality of reference embryos.
  • Some example implementations of the present invention may further comprise obtaining values for a further plurality of characteristics relating to the development of the embryo during the observation period; determining a value for a further continuous variable by combining differences between the obtained values and corresponding reference values for the further plurality of characteristics in a further pre-defined manner; and establishing the development potential for the embryo based also on the determined value for the further continuous variable.
  • a morphokinetic characteristic for embryos may be determined by combining / aggregating differences for various characteristics seen for each embryo and corresponding reference values (e.g. determined for a KID positive population of embryos) to generate what might be referred to as a generalized irregularity variable (GIV), which in some cases may be defined as:
  • cci is a series of cell cycle durations observed for an embryo
  • cci m is the corresponding series of average cell cycle durations seen in a reference group of embryos (e.g. a positive KID population from patients under the age 35)
  • cci v are the corresponding variance values associated with the reference population.
  • the parameter n is the number of cell cycle durations comprising the series cci.
  • the differences (cci - cci m ) are normalized by the variance values cci v as part of the combining. This means that differences for particular cell cycles (values of i) which exhibit relatively high variance in the sample population contribute less to the value of GIV than differences for cell cycle which exhibit relatively low variance in the sample population.
  • the differences (cci - cci m ) are squared in the combining, and this means the contribution to GIV is the same for a given difference, regardless of whether it is positive or negative (i.e. whether cci is longer or shorter than cci m ).
  • the series cci may comprises durations for various different cell cycles in different implementations as discussed further below.
  • GIV as defined above is low when the study embryo exhibits a regular cleavage pattern and is high when the embryo exhibits an irregular cleavage pattern.
  • GTV generalized time variable
  • Atj is a series of differences in times for subsequent cell divisions observed for an embryo
  • Atj m is the corresponding series of average values seen in a reference group of embryos (e.g. the positive KID population from patients under the age 35)
  • Atj v are the corresponding variance values associated with the reference population.
  • the parameter k is the number of values comprising the series Atj.
  • the differences (Atj - Atj m ) are normalized by the variance values Atj v as part of the combining. This means that differences for particular cell divisions (values of j) which exhibit relatively high variance in the sample population contribute less to the value of GTV than for those which exhibit relatively low variance in the sample population. In this example the contribution to GTV for each time difference depends on whether the difference in times for a particular pair of subsequent cell divisions is faster or slower than the average seen in the positive KID population (i.e. whether the difference is positive or negative).
  • cc2/cc2_median or using target intervals where the center is scaled according to a central estimate and the boundaries are scaled according to a variance estimate (e.g. variance, standard deviation, percentiles).
  • a variance estimate e.g. variance, standard deviation, percentiles
  • MN2 Multi nucleation observed at the 2 blastomere stage; can take the values "True” or False”.
  • MN2val the number of multinuclear cells at the 2 cell stage (0, 1 ,2).
  • MN4 Multi nucleation observed at the 4 blastomere stage; can take the values "True” or False”.
  • MN4val the number of multinuclear cells at the 4 cell stage (0, 1 ,2,3,4).
  • a blastocyst quality criterion is an example of an embryo quality criterion.
  • the following blastocyst related parameters may be used:
  • Initial compaction describes the first time a compaction between 2 or more blastomeres is observed.
  • Compaction is a process wherein an intensification of the contacts between the blastomeres with tight junction and desmosomes result in reduction of the intercellular space and a blurring of the cell contours (see fig. 3).
  • Compaction can be seen at the 6-8 cell stage but rarely do the embryos cleave to 16 cell or more before compaction occurs.
  • Compaction/Morula is defined as the first time where no plasma-membrane between any blastomeres are visible. When compaction is complete no plasma- membranes between any of the blastomeres forming the compaction are visible and the embryo can be defined as a morula.
  • the stage is characterized by a process with an intensification of the contacts between the blastomeres with tight junction and desmosomes resulting in reduction of the intercellular space and a blurring of the cell contours.
  • Compaction/Morula can sometimes be seen at the 6-8 cell stage during the 3 ' rd division (synchrony) period (S3) but most often compaction/Morula is seen after S3 close to or right in the beginning of the fourth synchrony period (S4). Rarely do the embryos cleave to 16 cell or more before compaction occurs.
  • IDT initial differentiation of trophectoderm
  • Onset of cavitation/early blastocyst/blastocyst is defined as the first time a fluid- filled cavity, the blastocoel, can be observed. It describes the initiation of the transition period between the compaction and the blastocyst stage of the embryo. Embryos often remain in this transition stage for a period of time before entering the actual blastocyst stage. Onset of cavitation usually appears immediately after differentiation of the trophectoderm cells. The outer layer of the morula with contact to the outside environment begins to actively pump salt and water into the intercellular space, as a result of which a cavity (the blastocoel) begins to form.
  • IDCIM Inner cell mass
  • Onset of expansion of the blastocyst is defined as the first time the embryo has filled out the periviteline space and starts moving/expanding Zona Pelucidae.
  • EB describes the initiation of the embryos expansion. As the blastocyst expands the zona pellucida becomes visibly thinner.
  • Hatching blastocyst is defined as the first time a trophectoderm cell has escaped / penetrated the zona pellucida.
  • Fully hatched blastocyst is defined as when hatching is completed with shedding zona pellucida.
  • NC (X) Number of Contractions (NC (X)) describes the number of contractions (X) the embryo undergoes after the onset of cavitation. In many embryos the contractions can be quite large and lead to a large reduction of the embryonic volume. A contraction is defined as a reduction in the cross sectional surface area of the embryo of more than 15%.
  • Compaction - de-compaction-compaction describes a phenomenon where an onset compaction of the embryo had started but becomes disrupted of cleavage. The cell boundaries of the blastomeres become visible again but after a while the embryo returns into a compacted composition.
  • Partial Compaction describes an uneven compaction where one or more of the blastomeres are not included in the compaction.
  • EGA embryonic gene activation
  • Cellular movement Movement of the center of the cell and the outer cell membrane. Internal movement of organelles within the cell is NOT cellular movement. The outer cell membrane is a dynamic structure, so the cell boundary will continually change position slightly. However, these slight fluctuations are not considered cellular movement. Cellular movement is when the center of gravity for the cell and its position with respect to other cells change as well as when cells divide. Cellular movement can be quantified by calculating the difference between two consecutive digital images of the moving cell. An example of such quantification is described in detail in the pending PCT application entitled “Determination of a change in a cell population", filed Oct 16 2006. However, other methods to determine movement of the cellular center of gravity, and/or position of the cytoplasm membrane may be envisioned e.g. by using
  • FertiMorph software (ImageHouse Medical, Copenhagen, Denmark) to semi- automatically outline the boundary of each blastomere in consecutive optical transects through an embryo. Other parameters
  • Organelle movement Movement of internal organelles and organelle membranes within the embryo which may be visible by microscopy. Organelle movement is not Cellular movement in the context of this application. Movement: spatial rearrangement of objects. Movements are characterized and/or quantified and/or described by many different parameters including but restricted to: extent of movement, area and/or volume involved in movement, rotation, translation vectors, orientation of movement, speed of movement, resizing, inflation/deflation etc. Different measurements of cellular or organelle movement may thus be used for different purposes some of these reflect the extent or magnitude of movement, some the spatial distribution of moving objects, some the trajectories or volumes being afflicted by the movement.
  • Embryo quality is a measure of the ability of said embryo to successfully implant and develop in the uterus after transfer. Embryos of high quality have a higher probability of successfully implant and develop in the uterus after transfer than low quality embryos. However, even a high quality embryo is not a guarantee for implantation as the actual transfer and the woman's receptivity highly influences the final result. Viability and quality are used interchangeably in this document.
  • Embryo quality (or viability) measurement is a parameter intended to reflect the quality (or viability) of an embryo such that embryos with high values of the quality parameter have a high probability of being of high quality (or viability), and low probability of being low quality (or viability). Whereas embryos with an associated low value for the quality (or viability) parameter only have a low probability of having a high quality (or viability) and a high probability of being low quality (or viability)
  • Fig. 2 The development of the embryo until the blastocyst stage.
  • the number refers to the number of blastomeres in each stage.
  • the letters a to e refer to the following kinetic parameters, a: Compaction/Morula (M), b: Initial differentiation of trophectoderm (IDT), c: Onset of cavitation/early blastocyst/blastocyst (Bl), d: Onset of expansion of the blastocyst (EB), e: Hatching Blastocyst (HB).
  • IDT Onset of trophectoderm
  • c Onset of cavitation/early blastocyst/blastocyst
  • Bl Onset of expansion of the blastocyst
  • HB Hatching Blastocyst
  • Initial Compaction (IC) can be observed between t5 and compaction/morula (a), if present usually IC precedes
  • Partial compaction can be observed between stages a and c if present.
  • Vacuolization VC(X)
  • NC(X) contractions
  • Fig. 3 shows the variation of morphokinetic parameters (in this case t2, t3 and t5) as a function of the culture medium in a fertility clinic. The total period runs from February 2011 to June 201 1.
  • media A provided the worst embryo development (latest cell division timing and t2, t3 and t5 are all higher for media A).
  • Media A also provided worse implantation rates and pregnancy rates.
  • Media B and Media C both provided normal embryo development and high implantation and pregnancy rates.
  • Fig. 4a A series of images showing where the time of t2 (time of cleavage where a 2 blastomere embryo is created, i.e. the time of resolution of the cell division) is seen to happen at 22.9 hours.
  • Fig. 4b A series of images showing direct cleavage to a 3 blastomere embryo.
  • Fig. 5 Mouse embryo development with varying temperature of the incubation medium (see example 1).
  • Figs. 7a-c Direct cleavage has been analysed in a large data material containing 4, 181 IVF treatments from eight IVF clinics which involve a total of 32,382 retrieved oocytes that were inseminated and placed in EmbryoScope incubators. 18,024 annotated embryos have been evaluated of which 5491 were transferred. As shown in fig. 7a short cell cycles were found in 4709 of the 18024 annotated embryos (26.1 %) and long cell cycles in 2464 of 18024 embryos (13.7%). The remaining 10851 embryos (60.2%) were MCC.
  • the KID ratio is shown in fig. 7c.
  • the two aberrant classes SCC and LCC display a significantly reduced KID ratio.
  • SCC and/or LCC embryos are therefore defined as embryos with morphokinetic parameter outliers.
  • variation in the relative amount of SCC and/or LCC embryos may be an indicator of change in developmental conditions and by monitoring this variation early warnings may be provided.
  • Figs. 8 and 9 show the variation in the average of the morphokinetic parameters t2, t3, t4, t5, t8, s2, cc3 and tS-t5 for embryos cultures in different lots of oil.
  • the differences are clearly seen and especially oil lot "L1 " is significantly different, possibly due to a small amount of a toxic substance that leads to markedly lower embryo quality.
  • the embryos cultured in L1 are seen to develop more slowly and with a longer cell cycle (cc3).
  • cc3 cell cycle
  • monitoring the morphokinetic parameters is a sensitive and fast responding tool to survey the quality of incubating embryos.
  • Figs. 10-14 Experiments were conducted with controlled amounts of TX-100 (Triton X- 100) in culture media for embryos from hybrid mice. The leftmost columns are the control group with no TX-100 and the five other groups have increasing amounts of TX- 100, up to 0.002%.
  • Fig. 1 1 shows the blastocyst rate at 96 hours, i.e. the rate of embryos having reached the blastocyst stage at 96 hours. Only group 5 with most TX- 100 is significantly different.
  • Fig. 12 shows the variation of t2, t3, t4, t5 and t8 across the groups and a trend towards a slower development with increasing amounts of TX- 100 is seen with the most pronounce differences at the later cleavage times.
  • Fig. 13 shows the variation of s2, cc3 and t8-t5 across the groups with no clear trends other than most groups have higher values than the control group.
  • Fig. 14 shows the variation for the timings of different blastocyst stages and a clear trend towards a slower development with increasing amounts of TX-100 is seen.
  • a way to identify a viable embryo in a cohort of embryos from an IVF treatment would be to compare the recorded temporal pattern of cell division, represented by the morphokinetic parameters, to the recorded temporal patterns of cell division from embryos in past treatment cycles.
  • a viable embryo would be characterized by having morphokinetic parameters that match the recorded morphokinetic parameters from embryos that implanted and resulted in a live birth in the past.
  • the embryo for transfer that display morphokinetic parameters resembling those of positive embryos (i.e. embryos from ongoing or successfully completed pregnancies) and differ where possible from the majority of negative embryos (i.e.
  • the present invention turns this known principle upside down: By monitoring the morphokinetic parameters undesirable differences or trends may be detected much sooner and this may give rise to early warnings pointing to e.g. unintended differences in embryo handling. On the other hand morphokinetic parameter surveillance may also be used to alleviate fears after multiple implantation failures, because morphokinetic parameter analysis can show that embryo development is indeed normal.
  • the factors that have been shown to influence embryo development, and consequently the derived morphokinetic parameters include: Temperature, media composition, pH, C0 2 and oxygen, growth factors, cultivation vessel etc.
  • Other factors such as patient age, etiology, BMI, stimulation protocol (agonist/antagonist, type of hormone rFSH/hMG), embryo handling (pipettes, fertilization method, assisted hatching, removal of blastomeres, polar bodies or trophectoderm cells by biopsy) and even experience and competence of the person handling the embryos, have been proposed by various scientists to influence embryo development and in particular the timing of cellular events such as cell cleavage.
  • one embodiment of the invention applies within quality control in a fertility clinic by comparing average cleavage patterns of embryos in recent treatment cycles with cleavage patterns from past cycles.
  • Temporal changes in general morphokinetic parameters for good quality embryos may indicate an unintended change in protocol, such as bad lot of media, problems with incubators, pipette tips, etc.
  • the steps of the claimed method can be repeated and the second dataset can thus be continuously updated with the most recent embryo data, such as the most recent embryo data selected from a specific time period or selected from a predefined number of the most recent embryos, such as the most recent embryos from a predefined time period, such as number of hours, days, weeks or months.
  • Said predefined number of embryos or predefined time period may advantageously be determined as a user input. The developmental conditions can thereby be continuously surveyed.
  • the method is computer implemented and thus the functionality of issuing a warning when the morphokinetic difference is above a predefined level may advantageously be implemented.
  • the type of the warning may be dependent of the severity of the detected difference, e.g. green light for normal, yellow light for possible problems and red light for a serious quality issue.
  • the first group of embryos will typically be a reference group or control group and the second group of embryos may be the group of embryos that are monitored and compared to the control group.
  • the detected morphokinetic difference between the groups of embryos can be determined by standard statistical methods known in the art, e.g. the predefined level for issuing a warning may be determined as a predefined level above the standard deviation of one or more of the morphokinetic parameters from the first dataset.
  • Morphokinetic parameter outliers may be defined as relative outliers, i.e. the outermost 3% of the population. However, it may be an advantage to define morphokinetic parameter outliers as absolute outliers, i.e. a morphokinetic parameter is defined as an outlier if it outside a predefined absolute number. This is the case for SCC embryos and LCC embryos, as defined herein, that are examples of absolute outliers. The lower limit of 5 hours for the SCC embryos comes from the fact that 5 hours is not enough time for DNA replication of the whole genome. The morphokinetic parameter outliers may be excluded from the first datasets and/or from the second dataset.
  • Monitoring the variation in the morphokinetic parameters may be a good indicator for quality issues as many of the morphokinetic parameters are quality parameters for the embryo quality. However, it may be an advantage to monitor the number or frequency or distribution of morphokinetic outliers, in particular in the second dataset, as any variation in the outliers may be an indicator of quality issues.
  • the second group of embryos is a subset of the first group of embryos such that the second dataset is a subset of the first dataset.
  • morphokinetic parameters are selected from the group of:
  • n ⁇ 1 , ... , 8 ⁇ , duration of cell cycles cc1 , cc2, cc2b, cc3, cc2_3 and cc4,
  • the following may be selected as a morphokinetic parameter:
  • cc/ m is the mean value or median value of cci and cc/ v is the variance of cci.
  • the first and/or second group of embryos are embryos that has been fertilized, preserved and/or incubated under a specific set of conditions.
  • the first group of embryos may have been fertilized under a different set of conditions than the second group of embryos.
  • These conditions may be selected from the group of: type of fertilization treatment, preservation such as cryopreservation, incubating temperature, type of media, specific incubator, specific treatment such as hormonal treatment, hormonal treatment, aspiration, male factor, specific incubator, incubation temperature, type of media, type of oil, PGD Treatment, transfer, cryopreservation, thawing, time outside incubator, number of fertilization treatments.
  • the second group of embryos are embryos that originate from predefined embryo donors.
  • the predefined embryo donors may be selected from the group 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 diseases, Hiv, Hep, PCO's, individuals with current or past exposure to radiation or hazardous chemical substances, individuals with current or past drug use, individuals with a BMI higher or lower than a predefined level, smokers, non-smokers, individuals with normal or abnormal menstrual cycle.
  • the second group of embryos may be selected as embryos from specific patient groups (e.g. younger patients), patients with specific diagnosis (e.g. endometriosis), specific fertilization treatments (e.g. ICSI), embryos that has received special pre- treatment (e.g. cryopreserved).
  • specific patient groups e.g. younger patients
  • patients with specific diagnosis e.g. endometriosis
  • specific fertilization treatments e.g. ICSI
  • embryos that has received special pre- treatment e.g. cryopreserved.
  • the lengths of the cell cycles cci are all important variables in determining the quality of the embryo. Combinations of the morphokinetic parameters may be used to construct a new morphokinetic parameter that is resilient to the rarely occurring extremes in timing of the specific cleavage events
  • CCi m is the median (average) value of cci and cci v is the variance of cci.
  • CC n0 rm e.g. 3
  • x is set for CC n0 rm (e.g. 3) it means that if the CCi's from the second group of embryos on average fall more than x standard deviations (of the KID positive, transferred or all) from the average value of the first group of embryos (e.g. KID positive, transferred or all) that data will be excluded.
  • the second group of embryos is selected among embryos with this specific treatment.
  • groups of embryos that have been handled by a specific person of the laboratory or clinic staff can be selected as the second group of embryos to evaluate if changes has occurred over time or if there is a general difference between staff performance.
  • the first group of embryos comprise preimplantation data from implanted embryos that have resulted in ongoing
  • the first group is selected to reflect high quality embryos with proven track record.
  • an embryo dataset (e.g. a first or second embryo dataset) comprise morphokinetic parameters for
  • predefined hours after insemination e.g. less than 20% fragmentation 68 hours after insemination
  • GQE Good quality embryos
  • Embryos selected by excluding poorly developing embryos, e.g. by excluding Sec and/or Lcc embryos or by employing other exclusion criteria as e.g. described in pending applications PCT/DK2012/05018 or EP 12174432.0, the latter filed at
  • the morphokinetic parameters are selected from the group of; - the timing and/or duration cell-division periods and inter-division periods,
  • said second group of embryos dataset is substantially smaller than the first group of embryos, such as 2 times smaller, such as 5 times smaller, such as 10 times smaller, such as 50 times smaller, such as 100 times smaller, such as 200 times smaller, such as 500 times smaller, such as 1000 times smaller.
  • the embryos are cultured and/or monitored in an incubator.
  • the embryos are monitored through image acquisition, e.g. by means of time-lapse microscopy equipment, such as image acquisition at least once per hour, preferably image acquisition at least once per half hour such as image acquisition at least once per twenty minutes, such as image acquisition at least once per fifteen minutes, such as image acquisition at least once per ten minutes, such as image acquisition at least once per five minutes, such as image acquisition at least once per two minutes, such as image acquisition at least once per minute.
  • the method according to the invention may be computer implemented or at least partly computer implemented thereby providing an efficient customizable tool for fertility clinics. I.e.
  • the method according to the invention may be implemented in automated incubator systems for culturing and monitoring embryos, such as human embryos.
  • the selection processes and the quality control of e.g. culture media and other culturing conditions may be more or less automated, i.e. fully manual with the software assisting the users with proposed decisions, semi-automatic or fully automatic with the incubator system making all the decisions, including early warnings, based on data analysis.
  • the invention relates to a system having means for carrying out the methods described above.
  • Said system may be any suitable system, such as a computer comprising computer code portions constituting means for executing the methods as described above.
  • the system may further comprise means for acquiring images of the embryo at different time intervals, such as the system described in WO 2007/042044.
  • the invention relates to a data carrier comprising computer code portions constituting means for executing the methods as described above.
  • the present inventors have performed a large clinical study involving many human embryos and monitoring the development, not only until formation of a blastocyst, but further until sign of implantation of the embryo.
  • important differences in the temporal patterns of development between the embryos that implanted i.e. embryos that were transferred and subsequently led to successful implantation
  • those that did not i.e. embryos that were transferred but did not lead to successful implantation
  • By using implantation as the endpoint not only embryo competence for blastocyst formation, but also subsequent highly essential processes such as hatching and successful implantation in the uterus is assessed.
  • Timing of early events in embryonic development correlates with development into a blastocyst, and the development into a blastocyst is a necessity for a successful implantation and thus the formation of a blastocyst is a quality parameter in itself.
  • the data allows the detection of blastocyst related developmental criteria for implantation potential.
  • the results in particular indicate that timing of late events, such as the onset of cavitation, are a consistently good indicator of implantation potential, and that the discrimination between implanting and non-implanting embryos is improved when using blastocyst quality criteria, e.g. tBI as opposed to the earlier events (t2, t3 and t4).
  • tBI e.g. tBI
  • incubating the embryos to the blastocyst stage can give additional important information that will improve the ability to select a viable embryo with high implantation potential.
  • monitoring the embryos at the later stages up to the blastocyst stages may give better indication of quality issues and as shown in figs. 10-14 the implications of e.g. a toxic oil are more pronounced at the later stages. It may therefore be an advantage to include blastocyst quality criteria in the herein selected morphokinetic parameters, i.e. selected for quality surveillance
  • Multiple variables may be used when choosing selection criteria.
  • the variables are selected progressively such that initially one or more of the variables that can be determined early with a high accuracy are chosen, e.g. t2, t3, t4 or t5. Later other variables that can be more difficult to determine and is associated with a higher uncertainty can be used.
  • normalized morphokinetic parameters e.g. normalized morphological parameters based on two, three, four, five or more parameters selected from the group of t2, t3, t4, t5, t6, t7 and t8.
  • normalized morphological embryo parameter may better describe the uniformity and/or regularity of the developmental rate of a specific embryo independent of the environmental conditions, because instead of comparing to "globally" determined absolute time intervals that may depend on the local
  • a blastocyst quality criterion may be to determine 1 ) the duration of a first time period from fertilization until translation of maternally inherited imRNA in the
  • blastomeres is completed and 2) the duration of a second time period from initiation of transcription of the blastomeres own DNA to said blastocyst stage are determined, and wherein a blastocyst quality criterion is the ratio of said first and second time periods.
  • a blastocyst quality criterion may be 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 said blastocyst stage are determined, and wherein a blastocyst quality criterion is the ratio of said first and second time periods.
  • the time from fertilization to the blastocyst stage is thereby divided into two time periods and the ratio between these time periods is a blastocyst quality criterion.
  • the reason for dividing at the 5 blastomere stage is that this is approx. the time of embryonic gene activation.
  • a corresponding blastocyst quality criterion can be provided by determining 1 ) the duration of a first time period from fertilization to blastocyst, and 2) the duration of a second time period from initiation of transcription of the blastomeres own DNA to said blastocyst stage and taking the ratio of these time periods. This ratio provides information on how much of the total time period from fertilization to blastocyst the embryo's own DNA is in control.
  • ratios can be seen as measures of the relative development speed in a certain period relative to the overall development speed until that stage. Any variation in these ratios may indicate that the embryos are accelerating or decelerating in their development and this may be an indicator of quality issues in the developmental conditions.
  • the abovementioned blastocyst stage may be selected from the group of: initial compaction (IC), compaction/morula (M), initial differentiation of trophectoderm cells (IDT), early blastocyst (ERB), onset of cavitation/blastocyst (Bl), expansion of blastocyst (EB), first contraction (CPS(1)), second contraction (CPS(2)), third contraction (CPS(3)), fourth contraction (CPS(4)), fifth contraction (CPS(5)), sixth contraction (CPS(6)), seventh contraction (CPS(7)), hatching (HB), and fully hatched (FH).
  • IC initial compaction
  • M initial differentiation of trophectoderm cells
  • IDTT initial differentiation of trophectoderm cells
  • ERP early blastocyst
  • Bl onset of cavitation/blastocyst
  • EB blastocyst
  • first contraction CS(1)
  • second contraction CPS(2)
  • CCS(3) third contraction
  • CPS(4) fourth contraction
  • a blastocyst quality criterion is determination of tB, - 1, wherein tB, is selected from the group of ⁇ tM, tBI, tEB ⁇ and ti is selected from the group of ⁇ t5, t6, t7 and t8 ⁇ .
  • a blastocyst quality criterion is determination of (tB, - 1,) / 1, wherein tBj is selected from the group of ⁇ tM, tBI, tEB ⁇ and tj is selected from the group of ⁇ t5, t6, t7 and t8 ⁇ .
  • a blastocyst quality criterion is determination of (tB, - tj) / tB, wherein tB, is selected from the group of ⁇ tM, tBI, tEB ⁇ and t, is selected from the group of ⁇ t5, t6, t7 and t8 ⁇ .
  • a blastocyst quality criterion is determination of the time from fertilization to a blastocyst stage.
  • a blastocyst quality criterion is determination of one or more of the following morphological blastocyst parameters:
  • tM time from fertilization to compaction/morula
  • tIDT time from fertilization to initial differentiation of trophectoderm cells
  • tERB time from fertilization to early blastocyst
  • tBI time from fertilization to onset of cavitation
  • tEB time from fertilization to expansion of blastocyst
  • tCPS(2) time from fertilization to second contraction
  • tCPS(3) time from fertilization to third contraction
  • tHB time from fertilization to hatching
  • a blastocyst quality criterion is determination of one or more of the following morphological blastocyst parameters:
  • a blastocyst quality criterion is determination of the absolute or relative 2D and/or 3D expansion of the blastocyst.
  • a blastocyst quality criterion is determination of the diameter and/or the volume of the embryo at the onset of expansion.
  • a blastocyst quality criterion is determination of the maximum diameter and/or the maximum volume of the blastocyst before hatching.
  • cc3/cc2_3 (t5-t3)/(t5-t2)
  • cc2/cc3 (t3-t2)/(t5-t3).
  • a quality criterion is determination of the extent of irregularity of the timing of cell divisions when the embryo develops from 4 to 8 blastomeres.
  • a quality criterion is determination of the maximum cleavage time for each blastomere when the embryo develops from 4 to 8 blastomeres.
  • a quality criterion is determination of the ratio between the maximum cleavage time for each blastomere when the embryo develops from 4 to 8 blastomeres and the duration of the total time period from 4 to 8 blastomeres; max(s3a,s3b,s3c)/s3.
  • the quality criteria discussed above may also be combined with determinations of movement of the embryo, such as i) determining the extent and/or spatial distribution of cellular or organelle movement during the cell cleavage period; and/or ii) determining the extent and/or spatial distribution of cellular or organelle movement during the inter- cleavage period thereby obtaining an embryo quality measure.
  • volumes within the zona pellucida that are devoid of movement are an indication of "dead” zones within the embryo. The more and larger these immotile "dead” zones the lower the probability of successful embryo development. Large areas within a time-lapse series of embryo images without any type of movement (i.e. neither cellular nor organelle movement) indicates low viability. Organelle movement should generally be detectable in the entire embryo even when only comparing two or a few consecutive frames. Cellular movement may be more localized especially in the later phases of embryo development.
  • the cell positions are usually relatively stationary between cell cleavages (i.e. little cellular movement), except for a short time interval around each cell cleavage, where the cleavage of one cell into two leads to brief but considerable rearrangement of the dividing cells as well as the surrounding cells (i.e. pronounced cellular movement). The lesser movement between cleavages is preferred. Thus, any unexpected variation in cellular or organelle movement may be an indicator of quality issues in relation to the developmental conditions.
  • morphokinetic parameters to be used in accordance with certain embodiments of the invention may be established by combining a plurality of morphokinetic characteristics relating to the development of embryos.
  • a morphokinetic parameter may be derived by obtaining values for a plurality of morphokinetic characteristics relating to the development of an embryo during an observation period, for example characteristics relating to any of cell cleavage times, time differences between pairs of cell divisions, and cell cycle durations.
  • a value for a continuous variable may be determined by combining differences between the obtained values and corresponding reference values.
  • the reference values may, for example, be determined from values for the plurality of characteristics obtained for one or more reference embryos of known development potential (e.g. KID positive embryos).
  • a morphokinetic parameter may then be based on the determined value for the continuous variable.
  • a method for determining a morphokinetic parameter for an embryo by generating a value for a continuous variable from a plurality of characteristics relating to the development of the embryo in accordance with an implementation of an embodiment of the invention may comprise various steps as follows: In a first step S1 a plurality of characteristics relating to the development of the study embryo during an observation period are obtained. These characteristics may fundamentally be based on cleavage times determined using conventional time-lapse embryonic imaging. One or more characteristics may be based on the timing of pronuclei fading / disappearance (tPNfading (or tPNf)).
  • the characteristics may comprise a series of cell cycle durations cci for a sequence of cell cycles.
  • the missing cell cycle(s) may be left out of the sequence.
  • a second step S2 average and variance values seen in a population of one or more reference embryos of known development potential (e.g. KID positive embryos) for characteristics corresponding to those obtained for the study embryo in step S1 are obtained. These may, for example, be read from a memory or other storage of an apparatus executing the method.
  • the average and variance values may be obtained through retrospective analysis of images of embryos that proceeded to successful implantation.
  • the embryos for which the average and variance values are obtained for a given study embryo may be referred to as reference embryos.
  • the reference embryos may in some cases comprise embryos that have been expected at the same clinic as the study embryo, for example to help take account of inter-clinic variations associated with different incubation conditions.
  • step S2 may also comprise selecting an appropriate grouping of reference embryos for which to obtain the average and variance values based on characteristics of the study embryo.
  • the average and variance values may be determined in accordance with conventional statistical analysis techniques, for example potentially involving the discarding of outlier data, and so forth.
  • the term "average” is used broadly herein to refer to a typical / representative / indicative value for a parameter seen in a sample population.
  • the average may, for example, correspond to a mean, mode or median value of the relevant characteristic for the reference population (positive KID population).
  • a difference between the value of each characteristic seen for the study embryo and the corresponding average characteristic associated with the population of known development potential (e.g. KID positive) embryos is determined.
  • a morphokinetic parameter for the study embryo (corresponding to a continuous variable) is determined by combining / aggregating the differences determined for each characteristic in a way which is weighted by the respective variance values.
  • a morphokinetic parameter (GIV) is defined as:
  • cci is the series of cell cycle durations observed for the study embryo
  • cci m is the corresponding series of average cell cycle durations seen in a reference group of embryos (e.g. the positive KID population from patients under the age 35)
  • cci v are the corresponding variance values associated with the reference population.
  • the parameter n is the number of cell cycle durations comprising the series cci.
  • the differences (cci - cci m ) are normalized by the variance values cci v as part of the combining. This means that differences for particular cell cycles (values of i) which exhibit relatively high variance in the sample population contribute less to the value of GIV than differences for cell cycle which exhibit relatively low variance in the sample population.
  • steps S1 to S4 discussed above represents a process for establishing a morphokinetic parameter for an embryo in accordance with an embodiment of the invention. It will be appreciated that similar methods may be used to establish a morphokinetic parameter for an embryo using different characteristics relating to the development of the study embryo and / or by combining the characteristics in a different way to generate a different morphokinetic parameter.
  • the first generalized irregularity variable GIV1 as described above is based on durations of cell cycles cc2a, cc2b, cc3a, cc3b, cc3c and cc3d (or at least the durations of the ones which are measured / not missing)
  • other generalized irregularity variables may be based on durations of other cell cycles.
  • the following variations may be defined to provide different morphokinetic parameters for use in accordance with embodiments of the invention:
  • a generalized irregularity variable used as a morphokinetic parameter in accordance with some embodiments of the invention may be established using timings defined relative to the time of pronuclei fading (tPNf).
  • GIV5 can be defined as follows: GIV5 (fifth generalized irregularity variable): comprising (t3 - tPNf) and cc3a.
  • the corresponding characteristic may be left out of the calculation of the continuous variable (morphokinetic parameter), with the value of n being correspondingly reduced.
  • morphokinetic parameters may be determined from characteristics relating to the development of the study embryo other than cell cycle durations.
  • a variation of the method discussed above with reference to steps S1 to S4 may comprise the following steps:
  • the characteristics relating to the development of the study embryo may instead comprise a series of time differences Atj between subsequent cell divisions (or morphological stages).
  • step T2 average and variance values seen in a population of one or more reference embryos of known development potential (e.g. KID positive embryos) for the characteristics obtained for the study embryo in step T1 are obtained.
  • reference embryos of known development potential e.g. KID positive embryos
  • a difference between the value of each characteristic seen for the study embryo and the corresponding average characteristic associated with the population of known development potential (e.g. KID positive) embryos is determined.
  • a morphokinetic parameter for the study embryo (corresponding to a continuous variable) is determined by combining / aggregating the differences determined for each characteristic in a way which is weighted by the respective variance values.
  • GTV morphokinetic parameter
  • Atj is the series of differences in times for subsequent cell divisions observed for the study embryo
  • Atj m is the corresponding series of average values seen in a reference group of embryos (e.g. the positive KID population from patients under the age 35)
  • Atj v are the corresponding variance values associated with the reference population.
  • the parameter k is the number of values comprising the series Atj.
  • the differences (Atj - Atj m ) are normalized by the variance values Atj v as part of the combining. This means that differences for particular cell divisions (values of j) which exhibit relatively high variance in the sample population contribute less to the value of GTV than for those which exhibit relatively low variance in the sample population. In this example the contribution to GTV for each time difference depends on whether the difference in times for a particular pair of subsequent cell divisions is faster or slower than the average seen in the positive KID population (i.e. whether the difference is positive or negative).
  • This morphokinetic parameter GTV may be referred to herein as a generalized time variable, GTV.
  • GTV is low when the study embryo exhibits a relatively fast development and is high when the embryo exhibits a relatively slow development.
  • morphokinetic parameter for an embryo using different characteristics relating to the development of the study embryo.
  • GTV3 as described above is based on all times between subsequent cell divisions from a single cell to an eight- blastomere embryo
  • other generalized irregularity variables may be based on other sequences of time differences.
  • the following variations may be defined to provide different morphokinetic parameters for use in accordance with embodiments of the invention:
  • GTV1 (first generalized time variable): similar to GTV3 but using only the time difference of the last two cell divisions observed up to the 8 blastomere state. I.e.
  • the parameter k is 1.
  • GTV2 (second generalized time variable): similar to GTV1 but with the later division time (cleavage time) in a pair replaced with tEnd (the end of the incubation time) where timings are missing.
  • the parameter k is the number of Ati used in the calculation for a particular embryo.
  • the parameter k is always 1 .
  • GTV8 (eighth generalized time variable): similar to GTV3 but using t8 if it is annotated and otherwise using tEnd if t8 is missing.
  • the parameter k is always 1.
  • Mean and variance may be calculated based on the Ati without substitution.
  • the parameter k is always 1.
  • Mean and variance is calculated based on the Ati without substitution.
  • the parameter k is the number of Ati 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 stage it becomes a foetus. According to this definition the fertilized oocyte is often called a pre-embryo until implantation occurs. However, throughout this patent application we will use a broader definition of the term embryo, which includes the pre-embryo phase. It thus
  • An embryo is approximately spherical and is composed of one or more cells
  • the zona pellucida performs a variety of functions until the embryo hatches, and is a good landmark for embryo evaluation.
  • the zona pellucida is spherical and translucent, and should be clearly distinguishable from cellular debris.
  • An embryo is formed when an oocyte is fertilized by fusion or injection of a sperm cell (spermatozoa).
  • spermatozoa sperm cell
  • the term is traditionally used also after hatching (i.e. rupture of zona pelucida) and the ensuing implantation.
  • the fertilized oocyte is traditionally called an embryo for the first 8 weeks. After that (i.e. after eight weeks and when all major organs have been formed) it is called a foetus. However the distinction between embryo and foetus is not generally well defined.
  • blastomere numbers increase geometrically (1-2-4-8- 16- etc.). Synchronous cell cleavage is generally maintained to the 16-cell stage in embryos. After that, cell cleavage becomes asynchronous and finally individual cells possess their own cell cycle.
  • embryos produced during infertility treatment are usually transferred to the recipient before 16-blastomere stage.
  • human embryos are also cultivated to the blastocyst stage before transfer. This is preferably done when many good quality embryos are available or prolonged incubation is necessary to await the result of a pre-implantation genetic diagnosis (PGD).
  • PGD pre-implantation genetic diagnosis
  • the term embryo is used in the following to denote each of the stages fertilized oocyte, zygote, 2-cell, 4-cell, 8-cell, 16-cell, morula, blastocyst, expanded blastocyst and hatched blastocyst, as well as all stages in between (e.g. 3 -cell or 5- cell)
  • the table below shows the measured average timing for different cell divisions, the morula and blastocyst stage. Temp. 2 cells 3 cells 4 cells 5 cells 6 cells 7 cells 8 cells 9+ cells
  • k 1 at base temperature (T b ) the following relationship can be assumed:
  • T is the temperature in °C and a is the temperature dependency coefficient.
  • the expected time f for a given temperature T, relative to t ⁇ T b ), is inversely proportional to k(T):
  • the Q 10 value is calculated as
  • the clinics belong to the same chain of IVF clinics using the same instrumentation. All embryos have been transferred with homogenised procedures, besides temperature.
  • t5 here again, and optimising according to k(T) and t(T), the estimate for a becomes 0.058 ⁇ 0.028 (95 % CI).

Abstract

La présente invention concerne un procédé informatique permettant de détecter automatiquement des variations et/ou des anomalies dans les conditions de développement d'embryons incubés in vitro, le procédé comprenant les étapes consistant à : a) obtenir un premier ensemble de données comprenant des paramètres morphocinétiques liés au développement d'un premier groupe d'embryons, b) obtenir un second ensemble de données comprenant des paramètres morphocinétiques liés au développement d'un second groupe d'embryons, c) modifier le premier et le second ensemble de données par extraction des paramètres morphocinétiques aberrants du premier ensemble de données et du second ensemble de données, d) calculer la différence entre les paramètres morphocinétiques spécifiques du premier ensemble de données modifié et les paramètres morphocinétiques correspondant du second ensemble de paramètres modifié, et surveiller ladite différence morphocinétique, ce qui permet de détecter des variations dans les conditions de développement du premier et du second groupe d'embryons.
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WO2015143350A1 (fr) 2014-03-20 2015-09-24 Auxogyn, Inc. Mesure quantitative de blastocystes humains et cinétique de croissance de la morphologie de la morula
WO2016050964A1 (fr) * 2014-10-03 2016-04-07 Unisense Fertilitech A/S Évaluation d'embryons
GB2531699A (en) * 2014-10-03 2016-05-04 Unisense Fertilitech As Embryo Assessment
CN106795474A (zh) * 2014-10-03 2017-05-31 尤尼森斯繁殖技术公司 胚胎评定
JP2017529844A (ja) * 2014-10-03 2017-10-12 ウニセンス フェルティリテック アー／エス 胚の評価
CN106795474B (zh) * 2014-10-03 2020-05-26 尤尼森斯繁殖技术公司 胚胎评定
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