WO2015001458A1 - Procédé d'évaluation d'embryon - Google Patents

Procédé d'évaluation d'embryon Download PDF

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WO2015001458A1
WO2015001458A1 PCT/IB2014/062677 IB2014062677W WO2015001458A1 WO 2015001458 A1 WO2015001458 A1 WO 2015001458A1 IB 2014062677 W IB2014062677 W IB 2014062677W WO 2015001458 A1 WO2015001458 A1 WO 2015001458A1
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pregnant
embryos
embryo
minutes
deselection
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PCT/IB2014/062677
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English (en)
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Csaba Pribenszky
Eszter LOSONCZI
Hubert JORIS
Anne-Li SIGVARDSSON
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Vitrolife Kft
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Publication of WO2015001458A1 publication Critical patent/WO2015001458A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0604—Whole embryos; Culture medium therefor

Definitions

  • the present invention relates generally to mammalian assisted reproductive technology (ART) and in particular of human IVF and to methods utilizing time-lapse to de-select embryos with low or no probability of development and to complete a successful pregnancy.
  • the method provides a method to de-select embryos at the 8-cell stage or at the blastocyst stage to increase the likelihood of successful pregnancy in IVF.
  • In vitro fertilization is a technique used to overcome various forms of male and female infertility.
  • the process involves fertilizing an oocyte with a sperm in vitro and subsequently transferring one or more of the developing embryos into the female body.
  • IVF Steptoe, P.C. and Edwards, R.G., Lancet 2(8085): 366 (1978)
  • the process faces continuing challenges of low implantation and pregnancy rates.
  • the success rate has only increased to 28% per started cycle for women under 35, based on national IVF registers in Sweden 2010.
  • Simple media are those, such as Earle's and human tubal fluid (HTF), which are balanced salt solutions with added carbohydrate energy sources such as pyruvate, lactate, glucose, antibiotics and proteins.
  • Complex media such as Ham's F-10, further include non-essential and essential amino acids as well as other additives, such as vitamins, hormones and other components present in oviductal and uterine fluid.
  • the medium system used as well as other environmental factors as temperature and gas pressures in the incubator, are well known to affect the embryo viability. In addition those factors also affect the morphokinetics of the embryo.
  • the morphological selection principles of embryos are based on static scoring system upon viewing the embryos on a microscope before transfer selection and/or during a daily embryo monitoring routine.
  • a morphological scoring system can be found in the "Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting 2011".
  • Cleavage stage embryos can be graded as A, B, C or D. Where A is recognized as a top quality embryo, B is a good quality embryos, C is an impaired quality embryo and D is an embryo not recommended for transfer.
  • parameters relied upon in the scoring is percentage of fragmentation, blastomere symmetry, presence of multinucleation, presence of vacuoles and the appearance of the zona pellucida.
  • Blastocysts are scored based on stage of blastocyst development eg, early blastocyst, blastocyst, expanded blastocyst and hatched or hatching blastocyst. The inner cell mass and throphectoderm are then scored independently. Other scoring systems have also been clinically used and are published elsewhere. Proteomics and metabolomics are other potential methods for embryo evaluation. Those methods are still under development and not yet in clinical use. For example measuring of amino acid turn over, oxygen utilization and glucose metabolism have been shown to have predictive value for embryo viability.
  • Ramsing et al (WO2012/163363) has proposed to use the timing from fertilization to the five-cell stage in combination with a ratio calculated between two time intervals related to morphological events in the embryo between the five and eight cell stages.
  • Rubio et al (2012) also investigates the time between the 1 st and second mitosis, and proposes that this morphokinetic parameter has a valid clinical use.
  • the present invention provides improved embryo de-select methods for mammalian embryos and in particular for human embryos.
  • it provides de-select parameters of early embryonic events that prevents deselection of embryos perfectly capable of leading to pregnancy and birth of healthy babies.
  • the embryo de-select principles are based on timings of the development between the 2- to 4-cell stages and/or timings of the development between the 5- to 7-cell stages or between the 5- to 8- cell stages. In a further embodiment it is based on the timing from fertilization to blastocyst stage.
  • the inventors realized that a narrow select principle is not suitable in a clinical setting. Although there might be an optimal timing for developmental events, the variation among the embryos that are able to implant and become a healthy baby born, is larger than what would be expected from the prior art. The inventors realized that only the worst embryos, in term of not complying with their developmental plan, should be de-selected, when there are alternative embryos to select.
  • the present invention provides methods and algorithms for monitoring and evaluation of gametes, zygotes and/or embryos in vitro prior to implantation.
  • culture medium is used throughout the present specification to refer to aqueous media containing salts and carbohydrates prepared to a defined osmolality and pH which is used for in vitro culture of mammalian embryos such as, gametes, zygotes, cleavage-stage embryos, morulas and blastocysts.
  • embryo is used to refer to cells in all stages of development from a fertilized egg up to the first 5 or 6 days or to the expanded or hatched blastocyst stages.
  • pregnancy defined herein as an endpoint for assessing successful implantation, and detected by diagnosing fetal heart beat at week 18 of pregnancy.
  • evaluation or “monitoring” or “assessment” is used to refer to a way to establish quality criteria or viability criteria for an embryo or to rank embryos in relation to other embryos among the set of embryos, that belong to a couple, having IVF treatment. It can also relate to a method to select the best or de-select the worst embryos among a set of embryos from a couple having IVF treatment, with the aim to improve chances of a healthy baby being born.
  • the 1 st cell cycle (ccl) is usually defined as the time from fertilization to the completion of the first cytokinesis, resulting in two daughter cells, blastomeres.
  • each blastomere is regarded as a distinct cell.
  • the cell cycle or cell-division cycle, is the series of events that take place in that cell leading to its division and duplication (replication) that produces two daughter cells.
  • the cell cycle can be divided in three periods: interphase— during which the cell grows, accumulating nutrients needed for mitosis preparing it for cell division and duplicating its DNA— and the mitotic (M) phase, during which the cell splits itself into two distinct cells (daughter cells) and the final phase, cytokinesis, where the new cell is completely divided.
  • each cell (blstomere) is followed up individually, as it is seen in Fig. 1.
  • Interphase or interdivision cycle is observed in between the cleavage cycles.
  • An interphase lasts in between cleavage (mitotic) cycles.
  • a mitotic cycle lasts from when the first blastomer in the given cycle cleaves until the last one does (e.g. from t3 to t4, from t5 to t8, from t9 to tl6)
  • interphase I lasts from fertilization till the onset of the 1 st cleavage (12);
  • interphase II lasts from t2 till t3, interphase III lasts from t4 to t5. This may not be correct biologically, but assuming that individual blastomeres in an intact early human embryo are usually synchronized in developmental programming, this might be an easy and practical approach.
  • Approach I and III observes the embryo as a set of cells
  • approach II observes the individual blastomeres.
  • the 3 rd cleavage/ mitotic cycle or s3 lasts from t5 till t8. Describing this cycle means the annotation of the 5 cell stage, and the annotation of the 8 cell stage. The time difference of t8 and t5 would describe correctly the length of this cycle.
  • correct recognition of 8 blastomeres is difficult using current technical solutions.
  • Blastomeres might overlap or completely cover each other in an eight cell embryo. To exemplify this issue, three independent embryologists were observing 8 cell stage embryos and their judgment was 40 % consistent. This consistency was 97% at judging the 7 cell stage.
  • t2 is the time from fertilization until the embryo has two individual cells.
  • t3 is the time from fertilization until the embryo has three individual cells.
  • t4 is the time from fertilization until the embryo has four individual cells.
  • t5 is the time from fertilization until the embryo has five individual cells.
  • t7 is the time from fertilization until the embryo has seven individual cells.
  • de-select means that an embryo within the de-select criteria should not be transferred back to the uterus, unless there are no alternative embryos without any positive de-select criteria.
  • An embryo with a positive de-select criteria should not be transferred back to the uterus, unless there are no alternative embryos without any positive de-select criteria.
  • the present invention provides a ranking method based on the de-select criteria to identify the embryos with the least chance for implantation.
  • a true positive de-select embryo is defined as an embryo that falls within at least one of the de-select criteria and did not result in pregnancy.
  • a true negative de-select embryo is an embryo that does not fall within any of the de-select criteria and does implant and result in pregnancy.
  • An embryo with false negative de-select criteria is an embryo that does not fall within any of the de-select criteria, but does not implant or results in pregnancy.
  • An embryo with a false positive de-select criteria is an embryo that falls within any of the de-select criteria and still implant and result in pregnancy.
  • the de-select principle is when t3-t2 (timing of three cells minus timing of two cells) is ⁇ 180 minutes, or within ⁇ 120 minutes. In one embodiment the de-select principle is when t3-t2 (timing of three cells minus timing of two cells) is >900 minutes. In another embodiment the 2xt3-t2-t4 equation (two times the timing of three cells minus the timing of two cells minus the timing of four cells) is ⁇ 0 minutes or >900 minutes. In another embodiment t4-t2 (timing of the four cells minus timing of the two cells) is ⁇ 600 minutes or t4-t2 >900 minutes.
  • the de-select principle is when t7-t5 (timing of seven cells minus timing of five cells) is >500 minutes.
  • a further de-select principle is when an expanded blastocyst has not been formed within 7000 minutes or within 8000 minutes from fertilization. Any combination of those parameters such as a logical algorithm comprising (t3-t2 ⁇ 120 minutes or t3-t2>900 or t7-t5>500 minutes or 2xt3-t2-t4 ⁇ 0 minutes or 2xt3-t2-t4 >900 minutes or t4-t2 ⁇ 600 minutes or t4-t2 >900 minutes) could be used.
  • An embryo falling within any one or more of the de-select criteria are to be classified as de-select embryos.
  • An alternative algorithm includes the blastocyst development and then comprises (t3-t2 ⁇ 120 minutes or t3-t2>900 or t7-t5>500 minutes or 2xt3-t2-t4 ⁇ 0 minutes or 2xt3-t2-t4 >900 minutes or t4-t2 ⁇ 600 minutes or t4-t2 >900 minutes or T-blastocyst >7000 minutes).
  • An embryo falling within any one or more of the de-select criteria are to be classified as a de-select embryo.
  • the t3-t2 ⁇ 120 minutes and the t3-t2>900 could be removed from the algorithm and instead embryonic arrest at the three cell stage could be included as a parameter. Embryonic arrest at t3, could be visualised as no t4, no t5 and no t7 values.
  • Such an alternative algorithm could be (embryo arrest at t3 or t7-t5>500 minutes or 2xt3-t2-t4 ⁇ 0 minutes or 2xt3-t2-t4 >900 minutes or t4-t2 ⁇ 600 minutes or t4-t2 >900 minutes).
  • an embryo falling within any one or more of the de-select criteria are to be classified as de-select embryos.
  • the algorithm could be (embryo arrest at t3 or t7-t5>500 minutes or 2xt3-t2-t4 ⁇ 0 minutes or 2xt3-t2-t4 >900 minutes or t4-t2 ⁇ 600 minutes or t4-t2 >900 minutes or T-blastocyst >7000 minutes).
  • An embryo falling within any one or more of the de-select criteria are to be classified as de-select embryos.
  • An alternative limit for the t4-t2 ⁇ 550 minutes could be used, which would reduce sensitivity, but could also decrease risk of lower specificity in a larger embryo population.
  • An algorithm could then for example be (embryo arrest at t3 or t7-t5>500 minutes or 2xt3-t2-t4 ⁇ 0 minutes or 2xt3-t2-t4 >900 minutes or t4-t2 ⁇ 550 minutes or t4-t2 >900 minutes). Any other combination of two or more de-select parameters could be used. For example (t3-t2 ⁇ 120 minutes or t3-t2>900 or t7-t5>500 minutes) could be used. Combinations between the mentioned morphokinetic parameters and morphology scoring and/or metabolomics are also suggested by the invention.
  • the best practice to allow proper de-selection in a medical practice is to establish specific criteria for the given situation.
  • criteria may be determined to be mostly environment independent (i.e. the effect of the clinic, culture medium, patients' age, diagnosis, etc. are eliminated)
  • the person skilled in the art will understand that to allow better deselection, the cut-off values may be specified at lower values where the embryos assessed on a retrospective basis e.g. at a given clinic. After the appropriate number of cases, the practitioner may establish his or her own de-select criteria to provide the optimal results.
  • the process may involve graphing all embryos first resulting in non-pregnancy, followed with embryos resulting in pregnancy, by showing the time value corresponding to each embryo.
  • the vertical demarcation line separates the two groups. To the right from the line, all embryos resulted in pregnancy, therefore the highest time value of those can be set as the upper cut-off value. Similarly, the lowest time value can be set as the lower cut-off value. Based on visual evaluation, this process results in cut-off values of about 900 minutes and 500 minutes, respectively. Where a low cut-off value is established, analogously the lowest value is selected.
  • the person skilled in the art understands that these types of determinations should be based on appropriate statistical analysis. As we discussed above, the outcome of the assessment may result in any of the possible variations of true positives, true negatives, false negatives and false positives.
  • the de-select criteria may be determined to allow for a pre-determined statistical sensitivity and specificity.
  • the present invention provides said de-select cut-off values to match these specific sensitivity and specificity values.
  • Experiment I was designed as a pilot experiment to confirm previously published results that state, that timing of certain embryonic events are relevant for pregnancy. We have unexpectedly found that none of the previously published parameters were found to be relevant for predicting pregnancy. Experiment I involved four clinics and 95 transferred embryos altogether. All transferred embryos were considered, irrespectively whether the implantation resulted 100% success rate in terms of pregnancy or less (i.e. 2 transferred embryos resulting in a singleton pregnancy or 3 transferred resulting in a twin or singleton pregnancy).
  • Experiment II was designed to confirm the finding of Experiment I and explore useful alternatives of the state - of-the-art techniques.
  • Experiment II was conducted on 109 transferred embryos with known implantation (100% or 0% implantation, i.e. after one transferred embryo there is no or a singleton pregnancy, in case of 2 transferred embryos there is a twin pregnancy or no pregnancy, in case of 3 transferred embryos there is a triplet or no pregnancy).
  • Experiment III aimed to extend the case number of Experiment II, and further 308 embryos with known implantation were assessed. Altogether Experiment II and III involved the analysis of 417 embryos from 257 women, from 8 clinics from different countries from Europe (Poland, Check republic, Germany, Russia, Greece, Hungary).
  • Tables I and II show all relevant data for all embryos examined in our years-long study.
  • the study according to Experiment II therefore involved 109 embryos, and resulted in the findings presented in the invention as filed in the priority application.
  • the cutoff values were determined as established in Experiment II. During the time since elapsed, further embryos' data were collected, and the analysis was carried out (Experiment III). It was evident from the results (see Example 3) that the cutoff values previously established for 100% specificity resulted in several false determinations, leading to decreased specificities, but still usually well over 90%.
  • the practicing the de-selection method can result in at least two approaches: (i) either the cut-off values are adjusted as more data are collected during daily practice to the pre-determined statistical level (i.e. 100% if complete certainty sought, or any preferred value, such as 99.5%, 99%, 98%, 97%, 95%, 92%, 905) or (ii) the cut-off values are fixed, and outlier values result in increased number of false results.
  • the pre-determined statistical level i.e. 100% if complete certainty sought, or any preferred value, such as 99.5%, 99%, 98%, 97%, 95%, 92%, 905
  • the cut-off values are fixed, and outlier values result in increased number of false results.
  • the present invention provides a method which involves the determination of the specific de-select criteria to be used for the evaluation of the embryos, comprising determining the timing values of the cellular events as defined above for a large set of embryos monitored in vitro, calculating the deselection parameters as defined above for each of the embryos, monitoring the success of implantation of the transferred embryos, and carrying out a retrospective analysis to determine an upper and a lower (if necessary) cut-off limit for a deselection parameter; and identifying said deselection criteria as the time values that define a range where a predetermined percentage of the embryos resulting in successful pregnancy falls.
  • the improvement provided by the present inventions is, that embryos that have hardly no chance to implant and become a healthy baby born are removed based on the morphokinetic analysis, without de-selecting embryos that, although not having perfect morphokinetics, are viable and can result in a successful pregnancy.
  • the disclosed de-select method about 20 - 50%, preferably 30% to 40% of the embryos otherwise being transferred based on morphological data alone, could have been categorized as de-select embryos.
  • a better choice of embryo for transfer could have been made, based on this new viability assessment. Not every couple with embryos that would be de-selected with the disclosed de-select criteria have alternative embryos for transfer.
  • an alternative aspect of the invention provides further guidance to maximizing the chances of implantation and successful pregnancy.
  • This alternative approach is based on the ranking of the embryos based on the de-select criteria according to the invention.
  • the present finding is as described in detail above that durations and/or timings for a given event or cleavage period/interval that were outside of the cut-off values had zero chance for pregnancy in our retrospective dataset. Timings for the relevant events representing pregnancy were also representing embryos that did not implant. These values can be referred as normal values.
  • de-selection may be carried out with high specificity, while selection is much less certain.
  • the ultimate goal is to determine the most viable embryo, or a few embryos for implantation giving the highest probability of pregnancy.
  • the present invention facilitates this decision by eliminating the embryos with limited chance for resulting in pregnancy.
  • all embryos of a couple are ranked based on their distance from the cut-off value.
  • the cut-off value is established to eliminate embryos that has no or limited chance for implantation and successful pregnancy, but according to our finding, the further away we go from the normal values, crossing the cut-off point, more and more embryos fall into the non-pregnant group. It is clear, therefore, that the term 'distance' has a direction, always directed from the cut-off point to the normal range. Accordingly the distance may also be a negative number, and means an actual value falling outside the cutoff value and signifies an embryo to be eliminated, i.e. having zero chance for implantation.
  • the ranking of the embryos has usually no added value for either selection or deselection purposes, and other assessment techniques dominate the selection process (such as morphological scoring, metabolomics assessment, glucose utilization, amino acid turn over, and others).
  • other assessment techniques dominate the selection process (such as morphological scoring, metabolomics assessment, glucose utilization, amino acid turn over, and others).
  • several embryos may fall outside of the normal range, but all may fall within the cut-off point. In these situations, a ranking method is preferred to eliminate those embryos that have the least development potential and chance for successful implantation/pregnancy.
  • the present invention provides a method which involves the ranking of the embryos, comprising calculating the distances of the timing values determined from the cutoff value, ordering the distances calculated from the smallest to the largest value; and identifying the one or more embryos as having increasing chance for resulting in successful pregnancy according to the order determined.
  • the measurement of the distance from the cut-off criterion may be based on any appropriate mathematical approach.
  • said distances are simple differences between the cutoff value and the actual value.
  • the actual values may be represented as the percentage of the cut-off value.
  • the actual timing value is compared not to the cutoff value itself, but its position is assessed in relation to both the 'normal range' as well established in the art and the cutoff value.
  • the reference point for the normal range may be replaced by its midpoint, its limit closer to the cut-off value, or alternatively, rather than using literature defined values, the data set used for the calculation of the cutoff values may be used to define a normal range/value which is appropriate to the actual clinic.
  • this pre-determined normal value may be the average timing value of all embryos that resulted in successful pregnancy, or alternatively, the median value thereof, or the endpoint of the normal range closer to the cut-off value.
  • the calculation may be based on percentage, ratio or more advanced statistical methods.
  • a sliding scale of specificities may be applied to determine the ranking of the embryos.
  • the embryos are de-selected with reference to a cutoff value established for decreasing level of specificity. For example, if an embryo is deselected based on a cutoff value determined for 100% specificity, said embryo ranked as having the least chance for implantation and resulting in successful pregnancy.
  • the embryos ar again de-selected using a cutoff value determined for, e.g., 99.5% specificity. If another embryo may be deselected in this step, said second embryo ranked second as having the least chance for implantation and resulting in successful pregnancy.
  • steps may be iterated or adjusted to allow for establishing a specific order of the embryos with respect to their de-selection ranking.
  • the present invention is concerned with the elimination of unviable embryos, and this ranking method only gives guidance in this respect, and a set of embryos from a given couple may only contain a few embryos which may be ranked this way. Nevertheless, the ranking system may still be useful to eliminate the least probable embryo candidates from consideration for transfer.
  • these difference-values may be summed, and this sum is used for the ranking of the embryos.
  • Alternative calculations may also be used for determining a cumulative de- select/viability score for each embryo.
  • the de-select criteria are combined with an embryo or blastocyst scoring system.
  • the embryo scoring system could be the above mentioned scoring systems or an alternative morphological scoring system.
  • the de-select criteria are combined with an embryo or blastocyst metabolism analysis.
  • the metabolomics analysis could be glucose consumption, oxygen consumption, amino acid consumption or turn-over rate of one or more individual amino acids.
  • the de-select criteria is suitable for human embryo use. More specifically, the invention provides a method for carrying out in vitro fertilization on a patient, wherein the method comprises the embryo assessment method according to the invention, and the additional step of implanting at least one embryo into the uterus of a female patient.
  • the invention provides a computer program product for embryo assessment, the computer program product comprising a computer-readable medium having a plurality of computer program instructions stored therein, which are operable to cause a computer to perform the steps of any one of the method disclosed herein.
  • the invention provides a non-transitory computer program product for embryo assessment, the computer program product comprising a computer-readable medium having a plurality of computer program instructions stored therein, which are operable to cause a computer to perform the steps of the methods according to the invention.
  • Fig. 1 shows a schematic representation of cell divisions during preimplantation embryo development.
  • Fig. 2 shows that the timing of cleavages, developmental stages (2 cell stage, 5 cell stage, blastocyst stage) is falling into the same range regardless of where the treatment was performed (clinic) or regardless of the fertility outcome. Most importantly, embryos resulting in pregnancy vs. resulting in no pregnancy have no visible difference in the timing of the cellular events. The statistical analysis confirmed this observation, see Example l. The vertical line separates the groups of embryos resulting in no pregnancy (to the left) and the embryos resulting in pregnancy (to the right). The three panels show the time values until the two-cell, five-cell and blastocyst stages.
  • Fig. 3 shows that the timing of cleavages, developmental stages (2 cell stage, 5 cell stage, blastocyst stage) is falling into the same range regardless of what type of media was used for embryo culture or regardless of the fertility outcome.
  • the panels are to be interpreted as on Fig. 2.
  • Fig. 4 shows that the timing differences as calculated are able to differentiate between certain embryos within the group of embryos resulting in no pregnancy, irrespectively the clinic performing the IVF procedure.
  • the vertical line separates the groups of embryos resulting in no pregnancy (to the left) and the embryos resulting in pregnancy (to the right).
  • the three panels show the time difference values between the two- and three cell stages, three- and four-cell stages, and five- and seven-cell stages.
  • Fig. 5 is the analysis according to Fig.4, except it shows the data sorted by the type of embryo culture medium used.
  • This study (also referred to as Experiment I) was designed as a pilot experiment to confirm previously published results that timing of certain embryonic events are relevant for pregnancy.
  • the study involved four clinics and 95 transferred embryos altogether. All transferred embryos were considered, irrespectively whether the implantation resulted 100% success rate in terms of pregnancy or less (i.e. 2 transferred embryos resulting in a singleton pregnancy or 3 transferred resulting in a twin or singleton pregnancy).
  • the WOW dish (Primo Vision Microwell Group Culture Dish, Vitrolife Kft, Szeged, Hungary) consists of a 35-mm diameter polystyrene Petri dish containing nine or sixteen microwells aligned in three (four) rows and three (four) columns in the centre of the bottom of the dish.
  • the approximate dimensions and function of the microwells follows the principle described in earlier publications (Vajta et al., 2000, 2008); however, the dishes used in this study were industrially produced.
  • the wells are 470 ⁇ in diameter, 280 ⁇ deep, flat U-shaped to achieve the appropriate optical clarity. The distance between the wells meets optimal embryo density requirements according to earlier publications (Gopichandran and Leese, 2006; Stokes et al., 2005).
  • This design of the culture system may provide the benefits of communal culture while permitting individual continuous morphological monitoring of embryos during development.
  • a purpose-made, compact, digital inverted microscopes (Primo Vision; Vitrolife Kft. Szeged, Hungary) was placed inside traditional C02 incubators.
  • the outer dimensions of the microscopes are 220 ⁇ 80 ⁇ 110 mm for length, width and height, respectively.
  • the microscopes (consisting of a custom designed and produced optics, a 5 megapixel charge- coupled device and a 560 nm green light emitting diode light source) are covered completely by corrugated aluminum except for a glass window for the inverted objective and a shielded USB cable for intermittent power source and communication.
  • the USB cable of the microscope was led through the factory -made side port of the incubator and was connected to an electric controlling unit that was able to control up to six of these microscopes.
  • the images were transferred through this controlling unit to a PC via a USB cable.
  • the custom-made software running on the PC made it possible to adjust the interval of the image acquisitions and the duration of the recording.
  • the WOW dishes were placed on the top of the glass window, into the sample holder of the microscope and the objective was focused.
  • the WOW culture dish enabled all the embryos to be positioned in the field of view. Illumination was provided by a reflected 560 nm green light.
  • the system was set to take a single picture every 10 min Exposition time was 100ms. Apart from these expositions, no electricity passed through the USB cable to the microscope, because both the power source of the light and the camera were shut down in the electric controlling unit.
  • the computer screen displayed the actual developmental stages of the embryos, while all the images recorded earlier were saved to be analyzed later with software. The embryos were not moved or disturbed in any way for the whole period of development, completely eliminating sheer stress. Lubricants were not used; moreover, neither additional heat nor continuous low voltage electric currents were affecting embryo development.
  • Timing of early events during in vitro embryo development has been shown to have correlation with embryo viability and implantation potential.
  • studies incorporated fixed conditions for culture, or mostly followed a routine of a single clinic.
  • the aim of this study was to collect data from different clinics using different procedures including different culture media, incubator and examine the relevance of the timing of morphokinetic events during in vitro embryo development to pregnancy.
  • Time-lapse recordings of 95 transferred embryos were analyzed. Following art-recognized, embryos were cultured in microwell dishes, and monitored by automated time-lapse microscopes (Primo Vision, Hungary) inside of conventional incubators as described above. Duration between fertilization and 2-cell, 3-cell, and 5-cell stages, and between 3 to 4-cell, and 5 to 8-cell stages were recorded. The effects of patients' age, infertility diagnosis and treatment protocol, method of fertilization and culture media and the occurrence of ongoing pregnancy were investigated on the timing of these events. Mixed effects model was used for statistical analysis, P ⁇ 0.05 was regarded as significant.
  • T2C relevance of of the timing of the 2 cell stage (1st cytokinesis) to pregnancy
  • treatment referencia: agonist long
  • Example 1 The negative experience in Example 1 in relation to the published literature regarding the relevance of the timings - durations of early cleavages to pregnancy lead us to perform a new clinical study to investigate what criteria to use instead to obtain a better discrimination between an embryo that will implant and result in pregnancy and one that will not.
  • the aim was especially to develop de-select criteria that are capable of deselecting non-viable embryos without de-selecting viable embryos.
  • the time-lapse equipment used was according to Example 1.
  • Table I Retrospective morphokinetic analysis of the transferred embryos of experiment II.
  • non-pregnant 1434 2460 not vis. not vis. not vis. n.a.
  • non-pregnant 1531 2211 2251 3051 3281 non-pregnant 1631 2331 2351 3041 3311 non-pregnant 1761 2431 2491 2551 3281 non-pregnant 1651 2411 2421 2431 3331 non-pregnant 1589 2279 2289 3039 3209 non-pregnant 1449 2109 2189 2769 2969 non-pregnant 1350 2120 2150 2890 3270 non-pregnant 1200 1900 1910 2630 2810 non-pregnant 1401 2011 2021 2861 3021 6261 non-pregnant 1451 2151 2171 2961 3301 6621 non-pregnant 1882 2752 2882 4402 n.a.
  • the combined de-select criteria for blastocyst culture (t3-t2 ⁇ 120 minutes or t3-t2>900 or t7-t5>500 minutes or 2xt3-t2-t4 ⁇ 0 minutes or 2xt3-t2-t4 >900 minutes or t4-t2 ⁇ 600 minutes or t4-t2 >900 minutes or T-blastocyst
  • Experiment II To verify the results of Experiment II, we collected a further set of data. The study design was basically the same, but the scope increased: an additional 308 embryos with known implantation were assessed. All together, Experiments II and III involved the analysis of 417 embryos from 257 women, from 8 clinics from different countries (Poland, Check republic, Germany, Russia, Greece, Hungary, Brazil).
  • Table II Retrospective morphokinetic analysis of the transferred embryos of experiment III.
  • non-pregnant 1522 2262 2282 208 non-pregnant 1707 2367 2387
  • Example 4 Impact of the determination of criteria on the specificity and sensitivity of de-selection
  • Example 5 Estimation of females with transferred de-select embryos with alternatives Based on the data obtained Experiment II, 31 embryos could have been deselected out of the transferred ones, based on our new deselect criteria. These embryos were transferred to 23 patients, essentially resulting in no pregnancy. Out of that group 12 patients had embryos that would have had a better (non-deselect) choice. Altogether 45 better embryos were available in those 12 patients.
  • Example 7 correlation between morphological scoring and pregnancy outcome
  • De-select note that this particular embryo would have been de-selected using the disclosed de-select criteria.
  • Fragmentation was retrospectively analyzed for 23 embryos that resulted in pregnancy. Blastocyst scoring according to Gardner et al 1999 grading was also done on the 15 of those embryos that were cultured to blastocyst stage.
  • the morphological data shows that none of the embryos that resulted in pregnancy had more than 20% fragmentation, whereas 3 of the embryos in the non-pregnant group had 30% or more fragmentation.
  • HOLM P ET AL "Development kinetics of the first cell cycles of bovine in vitro produced embryos in relation to their in vitro viability and sex" THERIOGENOLOGY, vol. 50, no. 8, December 1998 (1998-12), pages 1285-1299 LEQUARRE A S ET AL: "Cell cycle duration at the time of maternal zygotic transition for in vitro produced bovine embryos: Effect of oxygen tension and transcription inhibition.” BIOLOGY OF REPRODUCTION, vol. 69, no. 5, November 2003 (2003-11), pages 1707-1713
  • Rubio I et al "Limited implantation success of direct-cleaved human zygotes: a time-lapse study.” Fertil Steril.
  • SCHATTEN ET AL "The significance of mitochondria for embryo development in cloned farm animals" MITOCHONDRION, ELSEVIER, AMSTERDAM, NL, vol. 5, no. 5, October 2005 (2005-10), pages 303- 321
  • VAN BLERKOM J ET AL "A microscopic and biochemical study of fragmentation phenotypes in stage- appropriate human embryos.”
  • HUMAN REPRODUCTION OXFORD, ENGLAND

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Abstract

La présente invention concerne généralement la technologie de reproduction assistée chez les mammifères et en particulier la fécondation in vitro (FIV) chez l'homme, ainsi que des procédés utilisant un contrôle à intervalles pour évaluer et désélectionner des embryons présentant peu ou pas de probabilités de développement, et pour mener à terme une grossesse réussie.
PCT/IB2014/062677 2013-07-04 2014-06-27 Procédé d'évaluation d'embryon WO2015001458A1 (fr)

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CN116561627A (zh) * 2023-05-11 2023-08-08 中南大学 用于确定胚胎移植类型的方法、装置、处理器及存储介质

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WO2011025736A1 (fr) * 2009-08-22 2011-03-03 The Board Of Trustees Of The Leland Stanford Junior University Imagerie et évaluation d'embryons, d'ovocytes et de cellules souches
WO2012163363A1 (fr) * 2011-05-31 2012-12-06 Unisense Fertilitech A/S Evaluation de la qualité des embryons basée sur le clivage des blastomères et morphologie

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Publication number Priority date Publication date Assignee Title
WO2011025736A1 (fr) * 2009-08-22 2011-03-03 The Board Of Trustees Of The Leland Stanford Junior University Imagerie et évaluation d'embryons, d'ovocytes et de cellules souches
WO2012163363A1 (fr) * 2011-05-31 2012-12-06 Unisense Fertilitech A/S Evaluation de la qualité des embryons basée sur le clivage des blastomères et morphologie

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Title
HERRERO JAVIER ET AL: "A time to look back: analysis of morphokinetic characteristics of human embryo development", FERTILITY AND STERILITY, vol. 100, no. 6, 29 September 2013 (2013-09-29), pages 1602, XP028795497, ISSN: 0015-0282, DOI: 10.1016/J.FERTNSTERT.2013.08.033 *
SANDRINE CHAMAYOU ET AL: "The use of morphokinetic parameters to select all embryos with full capacity to implant", JOURNAL OF ASSISTED REPRODUCTION AND GENETICS, vol. 30, no. 5, 13 April 2013 (2013-04-13), pages 703 - 710, XP055142511, ISSN: 1058-0468, DOI: 10.1007/s10815-013-9992-2 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116561627A (zh) * 2023-05-11 2023-08-08 中南大学 用于确定胚胎移植类型的方法、装置、处理器及存储介质
CN116561627B (zh) * 2023-05-11 2024-04-16 中南大学 用于确定胚胎移植类型的方法、装置、处理器及存储介质

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