GB2513908A - Method - Google Patents

Abstract

A method for determining the developmental potential of a human embryo in vitro the method comprising: a. determining at least one reference value (tref) which is a characteristic relating to the development of the embryo selected from the group consisting of: i. the time point when the pro-nuclei (PN) have disappeared; ii. the time point when the second polar body is excluding; and iii. the time point when the pro-nuclei (PN) appear; b. measuring the time point of at least one further characteristic relating to the development of the embryo (tx) wherein time zero (to) is at least one of the reference values i., ii., or iii. (tref) to establish a variable value (tvar) c. establishing a development potential for the embryo based on said at least one variable value (tvar). tvar may be calculated as tx minus tref

Description

METHOD

FIELD OF THE INVENTION

The present invention relates to a method for determining the developmental potential of a human embryo in vitro.

BACKGROUND OF THE INVENTION

Infertility affects more than 80 million people worldwide. It is estimated that 10% of all couples experience primary or secondary infertility. Assisted Reproduction Treatment (ART) is an elective medical treatment that may provide a couple who has been otherwise unable to conceive a chance to establish a pregnancy. It is a process in which eggs (oocytes) are taken from a woman's ovaries and then fertilized with sperm in the laboratory. The embryos created in this process are then placed into the uterus for potential implantation. To avoid multiple pregnancies and multiple births, only a few embryos are transferred (normally less than four and ideally only one). Selecting embryos for transfer is a critical step in any ART. Current selection procedures are almost entirely based on morphological evaluation of the embryo at different time points during development and particularly an evaluation at the time of transfer using a standard stereomicroscope. However, it is widely recognized that the evaluation procedure needs qualitative as well as quantitative improvements.

One approach is to use early cleavage' to the 2-cell stage, (i.e. before 25 -27 h post insemination/injection), as a quality indicator. In this approach the embryos are visually inspected 25 -27 hours after fertilization to determine if the first cell cleavage has been completed.

Several studies have suggested the importance of the timings of cell divisions in determining embryo quality. In 2001, Lundin et a/for example reported an early first cleavage as a strong indicator of embryo quality in human IVF (Lundin, et aL, 2001), and Meseguer et a! reported the importance of several embryo morphological parameters on subsequent implantation of the embryo (Meseguer, et aL, 2011).

Pronuclear (PN) morphology was investigated in Salumets at al 2001 to determine whether these could be used to predict embryo quality and implantation rates. However, the conclusion drawn in Salumets at a! 2001 was that there were no significant differences in embryo quality or implantation/pregnancy rates between proposed zygote classes. Scott at a/ 2003 studied non-invasive strategies for selection of human oocytes and embryos.

A time-lapse system was used in Lemmen, et a!., 2008 to study the timing and coordination of events during early development from zygote to cleavage state embryo. Early disappearance of pronuclei and onset of first cleavage after fertilization was correlated with a higher number of blastomeres on day 2 after oocyte retrieval. In addition, synchrony in appearance of nuclei after the first cleavage was signifcantly associated with pregnancy success.

Azzarello et al (Azzarello, et oil, 2012) studied PN morphology and the timing of PN breakdown (PNB) and concluded that the PN morphology changes over time and does not improve embryo selection, but that the timing of PNB should be included in embryo selection parameters.

In recent years, time-lapse equipment is used increasingly to incubate and monitor embryos during in vitro development. Time-lapse equipment is an instrument that takes photographs a time intervals (e.g. as often as every 5 minutes if necessary) during incubation which then enables the precise timing of cell events during development to be recorded, e.g. timing of cell divisions. This increased knowledge of the development of the embryo has potential for improving the selection of embryos.

However, although precise determination of the early cleavage as well as other criteria may be a quality indicator for development of an embryo there is still a need for improving the indicators for implantation success and thereby success for having a baby as a result of the ART.

SUMMARY OF THE INVENTION

The determination of embryo quality (or the developmental potential of an embryo) is based on the information obtainable from observations on the developing embryo and the fate of it.

A positive developmental potential (or good embryo quality) results in development of the embryo to blastocyst stage, implantation, pregnancy, and/or live-born babies. A negative developmental potential (or poor embryo quality) results in the embryo arresting before development to blastocyst stage, non-implantation and miscarriage. It is preferred to use non-invasive methods such as morphological characteristics in determining embryo quality.

A significant problem with determining embryo quality is the uncertainty of the fertilization time point. The normal time point used as a starting point for in v/tm fertilization (IVF) is the time point where the sperm and the oocyte are mixed. However, it can take up to 2 hours before a sperm cell has penetrated and is accepted by the oocyte, i.e. before the life of the embryo begins. It is not possible to determine the precise fertilization time point in this situation. During intracytoplasmic sperm injection (ICSI), the precise time for the penetration is easy to establish, but firstly the sperm cell is not necessarily accepted by the oocyte immediately and therefore penetration does not equate with acceptance, and secondly, the defined start time is stated as the same for a given batch of oocytes, i.e. ICSI treatment of all oocytes from one woman, and there may in reality be up to two hours between insemination of the first and the last oocyte. In addition, when frozen fertilized oocytes are used and incubated after thawing, the precise timing of the fertilization cannot be used as a parameter in determining developmental potential. The present invention can help overcome this problem.

A seminal finding of the present invention is that by using one of the following time points to normalize the timing of a subsequent time point of at least one characteristic relating to the development of an embryo, it is possible to significantly improve the ability of the method to determine the developmental potential of an embryo: the time point when the pro-nuclei (PH) have disappeared; ii. the time point when the second polar body is excluding; or iii. the time point when the pro-nuclei (PH) appear.

The inventors have found that particularly normalizing subsequent timing events using the time point when the pro-nuclei (PH) disappears significantly improves the predictive capabilities of a method for determining the developmental potential of human embryo.

STATEMENTS OF THE INVENTION

According to one aspect the present invention provides a method for determining the developmental potential of a human embryo in vitro the method comprising: a. determining at least one reference value (tref) which is a characteristic relating to the development of the embryo selected from the group consisting of: i. the time point when the pro-nuclei (PH) have disappeared; ii. the time point when the second polar body is excluding; and iii. the time point when the pro-nuclei (PN) appear; b. measuring the time point of at least one further characteristic relating to the development of the embryo (t>J wherein time zero (t0) is at least one of the reference values i., ii., or iH. (tier) to establish a variable value (tvar), c. establishing a development potential for the embryo based on at least one of said variable values (tvai).

According to another aspect of the present invention there is provided a method for determining the developmental potential of a human embryo in vitro the method comprising determining the time point when the pro-nuclei (PN) have disappeared; measuring the time point of at least one further characteristic relating to the development of the embryo (tx) and subtracting the time point when the pro-nuclei (PN) have disappeared from said time point of said at least one further characteristic (tx) to establish a variable value; and establishing a developmental potential for the embryo based on said variable value or more than one said variable value.

DETAILED DISCLOSURE OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton, ot aL, DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide one of skill with a general dictionary of many of the terms used in this disclosure.

This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure.

Where a range of values is provided, it is understood that each intervening value1 to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure.

It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an embryo" includes a plurality of such candidate embryos, and so forth The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto.

All patent and non-patent references cited in the application, or in the present application, are also hereby incorporated by reference in their entirety.

The present invention relates to a method for determining the developmental potential of a human embryo in vitro the method comprising: a) determining at least one reference value (trer) which is a characteristic relating to the development of the embryo selected from the group consisting of: i. the time point when the pro-nuclei (PN) have disappeared; H. the time point when second polar body is excluding; and iii. the time point when the pro-nuclei (PN) appear; b) measuring the time point of at least one further characteristic relating to the development of the embryo (tx) wherein time zero (t0) is at least one of the reference values U., or iii. (trer) to establish a variable value (tvar), and c) establishing a development potential for the embryo based on said at least one variable value (t31).

In one embodiment of the method of the present invention the variable value (tvar) is the difference between the time point of at least one further characteristic relating to the development of the embryo (tx) and at least one of the reference values (tret). Effectively the reference value (tref) is time zero (t0).

In one embodiment of the method of the present invention, the method may comprise measuring the time point of at least one further characteristic relating to the development of the embryo (tx) and determining at least one variable value (tvar) by subtracting the time point of at least one of said reference values i., U., or iU (tf) from said time point of said at least one further characteristic (tx). In this way effectively one of the reference values i., U., or Hi becomes time zero (t0).

In one embodiment of the method of the present invention, the reference value (tret) and the time point of at the least one further characteristic relating to the development of the embryo (tx) are measured from any given time point before the reference value (tier) occurs. Any conventional time point may be used in this regard. For example, the reference value (trer) and the time point of the at least one further characteristic relating to the development of the embryo (tx) may be measured either from the time point for starting in vitro fertilization (IVF), i.e. when the sperm and the oocyte are mixed, or from the time of penetration (e.g. time of microinjection) in intracytoplasmic sperm injection (ICSI).

In one embodiment, the time point for starting IVF is when the sperm and the oocyte are mixed.

The time of penetration in ICSI as used herein means the time point when the sperm is injected (e.g. microinjected) into the oocyte.

In another embodiment of the method of the present invention, the variable value (tvar) is measured directly with at least one of the reference values i., ii., or iH (tret) commencing the measuring period as time zero (t3). In other words, the embryologist, for example, would determine the time point of the at least one reference value i., ii., or iii and would set the timer to zero minutes. The next measurement would be the time point of at least one further characteristic relating to the development of the embryo and the time from zero minutes to this measurement would be the variable value.

The term "time zero" as used herein means the time point that is considered for the purposes of the present invention as 0 minutes in the development of the embryo.

In one embodiment, time zero is the time point when the pro-nuclei (PN) have disappeared.

In another embodiment time zero is the time point when the second polar body is excluding.

In a yet further embodiment time zero is the time point when the pro-nuclei (PN) appear.

In the present method the time point of at least one further characteristic relating to the development of the embryo is also measured. This further characteristic may be any developmental time point that can be visualised, e.g. by time-lapse techniques. By way of example only the at least one further characteristic may include the time point for resolution or completion of a cell division, e.g. complete division to two cells (t2), complete division to 3 cells (t3), complete division to 4 cells (t4), complete division to 5 cells (t5), complete division to 6 cells (t3), complete division to 7 cells (t7), complete division to 8 cells (t8), complete division to nine or more cells (t9÷), compaction indicated by formation of the morula, start of blastulation, formation of blastocyst, formation of expanded blastocyst or hatching blastocyst.

In one embodiment the at least one further characteristic relating to the development of the embryo may be selected from one or more of the following: A. the time point when the embryo has divided to three cells (t3) (i.e. completes division to three cells); B. the time point when the embryo has divided to four cells (t4) (i.e. completes division to four cells); C. the time point when the embryo has divided to five cells (t5) (i.e. completes division to five cells); D. the time point when the embryo has divided to eight cells (t8) (i.e. completes division to eight cells).

In one preferred embodiment the time point of the further characteristic relating to the development of the embryo is the time point when the embryo has divided to five cells (tS). In other words the time point of the further characteristic relating to the development of the embryo may be the time point when the embryo completes division to 5 cells. Division to 5 cells demonstrate the beginning of the 4th cell cycle Alternatively, the time point of the further characteristic relating to the development of the embryo is the time point when the embryo has divided to three cells (t3). In other words the time point of the further characteristic relating to the development of the embryo may be the time point when the embryo completes division to 3 cells. Division to 3 cells demonstrate the beginning of the 3 cell cycle.

The term cell division period" as used herein means the time from the first observation of indentations in the cell membrane (indicating onset of cytoplasmic division) to the cytoplasniic cell division is complete so that the cytoplasm of the ensuing daughter cells is segregated into two separate daughter cells.

The time point when the embryo has divided is the point when the cell division is complete so that the cytoplasm of the ensuing daughter cells is segregated into two separate daughter cells.

The term "cell cycle period" as defined herein means the time period between consecutive cell divisions e.g. from 1-*2 cells, from 2-4 cells, from 4--.8 cells, and from 8-*16 cells.

The "first cell cycle period' is time period between fertilisation until completion of the cell division to two cells (or two blastomeres). n

The "second cell cycle period' is the time period between completion of division to two cells (two blastomeres) until completion of the cell division to four cells (or four blastomeres).

The third cell cycle period" is the time period between completion of division to four cells (four blastomeres) until completion of the cell division to eight cells (or eight blastomeres).

The "fourth cell cycle period" is the time period between completion of division to eight cells (eight blastomeres) until completion of the cell division to sixteen cells (or sixteen blastomeres) Cell division represents the end of one cell cycle and the commencement of the next cell cycle for the two new cells.

Within the second cell cycle period there are two separate cell cycles; one for the fastest blastomere (e.g. fastest blastomere to divide) and the second for the slower blastomere (e.g. the slowest blastomere to divide). These two cell cycles are referred to herein as 2a and 2b cell cycle periods, respectively. In other words the 2a cell cycle period is the time period between the completion of division to two cells (two blastomeres) until the completion of the cell division to three cells by the fastest of the two blastomeres. This time point (i.e. to completion of the cell division to three cells is referred to herein as t3). This t3 cell division also demonstrates the beginning of the third cell cycle period for the fastest blastomere.

The 2b cell cycle period is the time period between completion of division to two cells (two blastomeres) until the completion of the cell division to four cells (four blastomeres) (e.g. for the slower blastomere).

Within the third cell cycle period there are four separate cell cycles; one for the fastest blastomere (e.g. fastest blastomere to divide) and one for each of the other slower blastomeres. These four cell cycles are referred to herein as 3a, 3b, 3c and 3d cell cycle periods, respectively. In other words the 3a cell cycle period is the time period between the completion of division to three cells (three blastomeres) until the completion of the cell division to five cells by the fastest of the four blastomeres. This time point (i.e. to completion of the cell division to five cells is referred to herein as t5). This t5 cell division also demonstrates the beginning of the fourth cell cycle period for the fastest of the four blastomeres. The 3b cell cycle period is the time period between completion of division to three cells (three blastorneres) until the completion of the cell division to six cells (six blastomeres) (e.g. for the next fastest blastomere) and so forth.

Notably t3 (completion of the cell division to three cells) and tS (completion of the cell division to five cells) represent very important characteristics in the development of the embryo. In particular, if one studies embryo development, typically an embryo will remain in as a single cell for a period of time then will divide to two cells. The embryo may then remain as two cells for a period of time before the first of the two blastomeres divides (into 3 cells -t3). Typically (e.g. as often seen in good quality embryos) the second of the two blastomeres will divide rapidly after the first, preferably at more or less the same time, e.g. these cell divisions are preferably synchronised. The embryo may then remain as four cells for a period of time before the first of the four blastorrieres (cells) divides (into 5 cells -t5). Again the remaining 3 blastomeres will often divide rapidly after the first.

t3 (completion of the cell division to three cells) and t5 (completion of the cell division to five cells) also represent the beginning of the third cell cycle period or fourth cell cycle period, respectively.

In some embodiments t5 (completion of the cell division to five cells) is the preferred (or one of the preferred) further characteristic to measure. t5 can be a particularly useful characteristic because it is a characteristic which occurs relatively late in the development of the embryo and thus by this time point many embryos with a negative developmental potential will have already arrested or otherwise ceased developing.

However in some clinics embryos are transferred at day 2 (when the embryo is only at the 3 or 4 cell stage). Clearly in such circumstances the t5 measurement cannot be used in the assessment of the embryo to be transferred. In such circumstances t3 (completion of the cell division to three cells) is the preferred (or one of the preferred) further characteristics to measure.

Although the present invention may be used for any embryo, it is preferred that the present invention is for use with human embryos.

One advantage of the present invention is that the variable values are normalised using the reference value i., ii., or Hi, making it possible for the method to accurately predict good developmental potential of an embryo. A further advantage of the present invention is that the variable values are normalised using the reference value i., ii., or Hi, making it possible for the method to predict good developmental potential more accurately than conventional methods which use either the time point starting in vitro fertilization (IVE), i.e. when the sperm and the oocyte are mixed, the time of penetration (e.g. time of microinjection) in intracytoplasrnic sperm injection (ICSI) or the time point of thawing. As noted above, the conventional methods are problematical when trying to determine the developmental potential of an embryo because there is a large degree of uncertainty associated wEth the fertilization time point. The normal time point used as a starting point for in vitro fertilization (IVF) embryo monitoring is the time point where the sperm and the oocyte is mixed.

However, it can take up to 2 hours before a sperm cell has penetrated and is accepted by the oocyte, i.e. before the life of the embryo begins. It is not possible to determine the precise fertilization time point in this situation. During intracytoplasmic sperm injection (lCSl), the precise time for the penetration (e.g. by microinjection) is easy to establish, but firstly, the sperm cell is not necessarily accepted by the oocyte immediately and therefore penetration does not equate with acceptance, and secondly, the defined start time is stated as the same for a given batch of oocytes, i.e. lCSl treatment of all oocytes from one woman, and there may in reality be up to two hours between insemination of the first and the last oocyte. In addition, depending on the country and the clinic, some clinics can freeze fertilized oocytes immediately after fertilization. When these frozen fertilized oocytes are used and incubated after thawing, the precise timing of the fertilization cannot be used as a parameter in determining developmental potential. The present invention overcomes all of these problems, by determining a reference value for each individual embryo (tf) and using this reference value as time zero (t0) for subsequent measured timings.

This leads to an improved method which increases the predictive nature of the method to identify embryos with good developmental potential (e.g. to identify good (or high) quality embryos).

The term "developmental potential" as defined herein means the likelihood of an embryo to develop to blastocyst stage, to implant1 to result in pregnancy, andlor to result in a live-born baby. In one embodiment the development potential may be a determination of embryo quality. Developmental potential may be equated with embryo quality. Embryo quality (or the developmental potential of an embryo) is based on the information obtainable from observations on the developing embryo and the fate of it. A positive developmental potential (or good (or high) embryo quality) results in development of the embryo to blastocyst stage, results in successful implantation, development of the embryo in the uterus after transfer, results in pregnancy, and/or results in live-born babies (preferably at least results in successful implantation). A negative developmental potential (or poor embryo quality) results in the embryo arresting before development to blastocyst stage, non-implantation and miscarriage. It is preferred to use non-invasive methods such as morphological characteristics in determining embryo quality.

Once the variable value(s) has been determined the values are employed to determine the developmental potential of the embryo.

A positive developmental potential (or good (or high) embryo quality) means that the embryo is statistically likely to develop to blastocyst stage, to result in successful implantation, to develop in the uterus after transfer, to result in pregnancy, and/or to result in a live-born baby (preferably at least to result in successful implantation).

Embryos of good (or high) quality have a higher probability of successfully implanting and/or of developing in the uterus after transfer compared with 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.

Developmental potential or embryo quality (or viability) is a parameter intended to reflect the quality (or viability) of an embryo such that embryos with high values of developmental potential 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 developmental potential only have a low probability of having a high quality (or viability) and a high probability of being low quality (or viability).

Cell division (e.g. complete division) and/or the time point when the embryo has divided may be referred to herein as the cleavage time and is defined as the first observed time point when the newly formed blastomeres are completely separated by confluent cell membranes, the cleavage time is therefore the time of completion or resolution of a blastomere cleavage.

The terms "faded" and disappeared" in relation to the pro-nuclei (PN) are used herein interchangeably.

The embryo in the present method is preferably monitored regularly to obtain the relevant information, preferably at least once per hour, such as at least twice per hour, such as at least three times per hour. In some embodiments, the embryo in the present method may be monitored every ten minutes. In some embodiments, the embryo in the present method may be monitored every minute. The monitoring is preferably conducted while the embryo is situated in the incubator used for culturing the embryo. This is preferably carried out through image acquisition of the embryo, such as time-lapse methods.

Determination of the reference value and/or the at least one further characteristic relating to the development of the embryo can be done for example by visual inspection of the images of the embryo and/or by (semi)-automated methods such as described in detail in the W02007/042044. Furthermore, other methods to determine the reference value and/or the at least one further characteristic relating to the development of the embryo can be done by determining the position of the cytoplasm membrane by envisioned e.g. by using FertiMorph software (lmageHouse Medicall Copenhagen, Denmark). The described methods can be used alone or in combination with visual inspection of the images of the embryo and/or with automated methods as described above.

The method of the present invention is preferably carried out and/or the values are measured by time-lapse microscopy. A suitable system for measuring the values by time-lapse microscopy is described in W020071042044 (which is incorporated herein by reference).

The resulting different images can be used to quantify the amount of change occurring between consecutive frames in an image series.

The invention may be applied to analysis of difference image data, where the changing positions of the cell boundaries (i.e. cell membranes) as a consequence of cellular movement causes a range parameters derived from the difference image to rise temporarily (see WO 2007/042044). These parameters include (but are not restricted to) a rise in the mean absolute intensity or variance. Cell cleavages and their duration and related cellular re-arrangement can thus be detected by temporary change, an increase or a decrease, in standard deviation for all pixels in the difference image or any other of the derived parameters for "blastomere activity listed in WO 20071042044. However the selection criteria may also be applied to visual observations and analysis of time-lapse images and other temporally resolved data (e.g. excretion or uptake of metabolites, changes in physical or chemical appearance, diffraction, scatter, absorption etc.) related to the 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 become a feotus. According to this definition the fertilized oocyte is often called a pre-embryo until implantation occurs.

However, the term "embryo" as used herein will have a broader definition, which includes the pre-embryo phase. The term "embryo" as used herein encompasses all developmental stages from the fertilization of the oocyte through morula, blastocyst stages, hatching and implantation.

An embryo is approximately spherical and is composed of one or more cells (blastomeres) surrounded by a gelatine-like shell, the acellular matrix known as the zona pellucida. The zona pellucida performs a variety of functions until the embryo hatches, and is a good landmark for embryo evaluation. The zona pellucida is spherical and translucent, and should be clearly distinguishable from cellular debris.

An embryo is formed when an oocyte is fertilized by fusion or injection of a sperm cell (spermatozoa). The term is traditionally used also after hatching (i.e. rupture of zona pelucida) and the ensuing implantation. For humans the fertilized oocyte is traditionally called a zygote or 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 fetus. However the distinction between zygote, embryo and fetus is not generally well defined. The terms embryo and zygote are used herein interchangeably.

The embryo evaluated in the present method may be previously frozen, e.g. embryos cryopreserved immediately after fertilization (e.g. at the 1-cell stage) and then thawed.

Alternatively, they may be freshly prepared, e.g. embryos that are freshly prepared from oocytes by IVF or lCSl techniques for example.

Fertilization is the time point where the sperm cell is recognized and accepted by the oocyte.

The sperm cell triggers egg activation after the meiotic cycle of the oocyte has been suspended in metaphase of the second meiotic division. This results in the production and extrusion of the second polar body. This is the time point referred to herein as the second polar body excluding (see reference value ii.). Some hours after fusion of sperm and ovum, DNA synthesis begins. Male and female pronuclei (RN) appear. This is the time point referred to as when the pronuclei (PN) appear (see reference value iii.). The RN move to the centre of the egg and the membranes breakdown and the PN disappear. This is the time point referred to herein as the time point when the pro-nuclei have disappeared (or faded) (see reference value i.). This combination of the two genomes is called syngamy. Hereafter, the cell divisions begin.

During embryonic development, blastomere numbers increase geometrically (1-2-4-8-16-etc.). Synchronous cell cleavage is generally maintained to the 8-cell stage in human embryos. After that, cell cleavage becomes asynchronous and finally individual cells possess their own cell cycle. Human embryos produced during infertility treatment are usually transferred to the recipient before 8-blastomere stage. In some cases human embryos are also cultivated to the blastocyst stage before transfer. This is preferably done when many good quality embryos are available or prolonged incubation is necessary to await the result of a pre-implantation genetic diagnosis (PGD). However, there is a tendency towards prolonged incubation as the incubation technology improves.

Accordingly, the term embryo is used in the following to denote each of the stages fertilized oocyte, zygote, 2-cell, 4-cell, 8-cell, 16-cell, compaction, morula, blastocyst, expanded blastocyst and hatched blastocyst, as well as all stages in between (e.g. 3-cell or 5-cell).

A final analysis step may include a comparison of the observations, e.g. the variable values, obtained by the method with similar observations of embryos of different quality and development competence. It has been found useful if observations (e.g. the variable value) for a given embryo are compared with the data sets for that variable value obtained from the same clinic (or from clinics which use the same parameters, e.g. egg collection techniques, insemination techniques, culture conditions for the embryo, culture media for embryo culture and/or transfer techniques for instance) together with information on whether the embryo was a good quality embryo. Parameters, such as egg collection techniques, insemination techniques, culture conditions for the embryo, culture media for embryo culture and/or transfer techniques for instance, can influence the specific timings of embryo development. In other words time points of characteristic(s) relating to the development of the embryo can change depending on these parameters or factors. Thus, when making comparisons to determine the time range for a variable value to use for a given characteristic which is indicative of a good quality embryo, it is advantageous for the skilled person to establish this time range for the variable value based on data from a specific clinic (e.g. typical developmental timings for embryos in that clinic together with information on whether each embryo developed to blastocyst stage, implanted, resulted in pregnancy, and/or resulted in a live-born baby). These specific timings for that clinic for instance can then be used to determine a variable value range which is indicative of positive developmental potential for each characteristic for that clinic (or for clinics that use comparable parameters, e.g. comparable egg collection techniques, insemination techniques, culture conditions for the embryo, culture media for embryo culture and/or transfer techniques). This preferred time range for the variable values are then used during the statistical analysis of the variable value(s) obtained using the method of the present invention to determine the developmental potential of an embryo (e.g. from the specific clinic(s)).

The variable values obtained by the method of the present invention may be used to evaluate the developmental potential of an embryo in the same way as timings have been conventionally used by one skilled in the art. However, the "normalized" timings (e.g. variable value) from the reference value as taught herein may provide a more accurate guide to the developmental potential of an embryo compared with the timings previously used. For example W02012/163363 teaches conventional methods and is incorporated herein by reference.

The developmental potential (quality) can be based on a single variable value, or multiple variable value (more than one, preferably more than two variable values).

As well as comparing variable values obtained by the method with similar observations of embryos of different quality and development competence, one may also compare the variable values for a given embryo with other quantitative measurements made on the same embryo. This may include a comparison with online measurements such as blastomere motility, respiration rate, amino acid uptake etc. A combined dataset of blastomere motility analysis, respiration rates and other quantitative parameters are likely to improve embryo selection and reliably enable embryologist to choose the best embryos for transfer.

Thus, in one embodiment the method according to the invention may be combined with other measurements in order to evaluate the embryo in question, and may be used for selection of competent embryos for transfer to the recipient.

Such other measurements may be by way of example only selected from the group of respiration rate, amino acid uptake, motility analysis, blastomere motility, morphology, blastomere size, blastomere granulation, fragmentation, multinucleation, blastomere colour, polar body orientation, nucleation, spindle formation and integrity, and numerous other qualitative measurements. The respiration measurement may be conducted as described in In a preferred embodiment the observations are conducted during cultivation of the cell population, such as wherein the cell population is positioned in a culture medium. Means for culturing cell population are known in the art. An example of culturing an embryo is described in PCI publication no. WO 2004/056265.

The embryos may be cultured under any conventional conditions known in the art to promote survival, growth and/or development of the embryo. Such conditions may be any known in the art, for instance may include the method and device and conditions taught in W02004/056265, which is incorporated herein by reference.

The invention further relates to a data carrier comprising a computer program directly Ioadable in the memory of a digital processing device and comprising computer code portions constituting means for executing the method of the invention as described above.

The data carrier may be a magnetic or optical disk or in the shape of an electronic card as for example the type EEPROM or Flash, and designed to be loaded into existing digital processing means.

The present invention further provides a method for selecting an embryo for transplantation.

The method implies that the embryo has been monitored as discussed above to determine its developmental potential.

The selection or identifying method may be combined with other measurements for example as described above in order to evaluate the quality of the embryo The important criteria in a morphological evaluation of embryos are: (1) shape of the embryo including number of blastomeres and degree of fragmentation; (2) presence and quality of a zona pellucida; (3) size; (4) colour and texture; (5) knowledge of the age of the embryo in relation to its developmental stage, (6) blastomere membrane integrity, and (7) multinucleation.

The transplantation may then be conducted by any suitable method known to the skilled person.

The time point of at least one further characteristic relating to the development of the embryo may be the formation of the morula, start of blastulation, formation of blastocyst, formation of expanded blastocyst or hatching blastocyst.

Typically formation of the morula or compaction is defined as the first time where no plasma-membrane between any blastomeres is visible. Ideally 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 (this time point is S considered as the formation of the 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 (33) but most often compaction/Morula is seen after S3 close to or right in the beginning of the fourth synchrony period (34). Rarely do the embryos cleave to 16 cell or more before compaction occurs Onset of cavitation/early blastocyst/blastocyst (BI) (also referred to herein as start of blastulation) 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.

Onset of expansion of the blastocyst (EB) or formation of expanded 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 (HB) is defined as the first time a trophectoderm cell has escaped I penetrated the zona pellucida.

Fully hatched blastocyst (FH) is defined as when hatching is completed with shedding zona pellucida.

In humans embryonic gene activation (EGA) typically occurs on day 3, around the 8-cell stage, Before EGA embryos are observed to translate only maternally inherited mRNA, i.e. that mRNA which is present in the oocyte when it is fertilized. The mRNA is localized in different parts of the oocyte, so that as the oozytelzygote divides it is segregated into different blastomeres. This segregation is thought to underlie much of the differentiation of cells that occurs before EGA. After EGA the embryo begins to transcribe its own DNA, cells become motile and cell division becomes asynchronous. Since the cells are now transcribing their own DNA, this stage is where differential expression of paternal genes is first observed. The transition around EGA is also referred to as midblastula or midblastula transition.

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 implanting and developing 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.

The invention will now be described, by way of example only, with reference to the following Figures and Examples.

EXAMPLES

Materials and methods Data were collected from the clinic Maigaard (Aarhus, DK).

The present study included 945 embryos which were generated in 578 ART treatment cycles.

The embryos were obtained after fertilization by Intra Cytoplasmic Sperm Injection (ICSI) or In Vitro Fertilisation (IVF) and were part of the clinic's normal treatments. Time-lapse images were acquired of all embryos, but only transferred embryos with known implantation (i.e. either 0% implantation or 100% implantation) were investigated by detailed time-lapse analysis measuring the exact timing of the developmental events in hours-postferUlization.

Ovum pick-up, ICSI and IVF were done according to the standard procedure used in the clinic. These standard procedures are known to the skilled person.

After IVF or ICSI, the oocytes were placed in pre-equilibrated slides (EmbryoSlide® Unisense FertiliTech, Aarhus Denmark).

Incubation The slides are constructed with a central depression containing 12 straight-sided cylindrical wells each containing a culture media droplet of 20 pL medium. The depression containing the 12 wells was filled with an overlay of 1.4 mL mineral oil to prevent evaporation. The slides were prepared at least 4 hrs in advance and left in an incubator to pre-equilibrate at 37 °C in the 5,0 % C02 atmosphere. After pre-equilibration all air bubbles are meticulously removed before the oocytes are placed individually in droplets and incubated in the time-lapse monitoring system until embryo transfer 72 hour later approximately.

The, EmbryoScope® time-lapse instrument (ES), (Unisense Fertililech, Aarhus, Denmark) is a tn gas oocyte/embryo incubator with a built in microscope to automatically acquire images of up to 72 individual embryos during development.

imaging system The imaging system in the ES uses low intensity red light (635 nm) from a single LED with short illumination times of 3D ms per image to minimize embryo exposure to light and to avoid damaging short wavelength light. The optics comprise of a modified Hoffmann contrast with a 20x speciality objective (Leica Place) to provide optimal light sensitivity and resolution for the red wavelength. The digital images are collected by a highly sensitive CGD camera (1280 x 1024 pixels) with a resolution of 3 pixels per pm. Image stacks were acquired at 5 equidistant focal planes every 15 minutes during embryo development inside the ES (i.e. from about 1 hr after fertilization to transfer on day 3 about 72 hrs after fertilization). Embryo exposure to light during incubation was measured with a scalar irradiance microsensor with a tip diameter of 100 pm placed within the EmbryoScope at the position of the embryo in the EmbryoSlide. Similar measurements were made on standard microscopes used in fertility clinics. The total exposure time in the time-lapse system during 3 day culture and acquisition of 1420 images were 57 seconds, which compares favourably with the 167s microscope light exposure time reported for a standard ART treatment, Ottosen et al 2007. As the light intensity measured within the ES with the scalar irradiance microsensor was much lower than the light intensity in microscopes used in IVF clinics, the total light dose during 3 day incubation in the time-lapse system was found to be 20 J/m2 (i.e. 0.24 pJ/embryo) as opposed to an exposure of 394 J/m2 during microscopy in normal IVF treatments (i.e. 4.8 pJ/embryo) based on average illumination times from Ottosen et al 2007 and measured average intensities with the scalar irradiance microsensor. Furthermore, the spectral composition of the light in the ES was confined to a narrow range centered around 635 nm, and thus devoid of low wavelength light below 550 nm, and comprise about 15% of the light encountered in a normal IVF microscope.

Time-lapse evaluation of morphokinetic parameters Retrospective analysis of the acquired images of each embryo was made with an external computer, EmbryoViewer ® workstation (EV), (Unisense FertiliTech, Aarhus, Denmark) using image analysis software in which all the considered embryo developmental events were annotated together with the corresponding timing of the events in hrs after ICSI microinjection or IVF treatment. Subsequently the EV was used to identify the precise timing of disappearance of 2PN, and the cell divisions t3 (completion of the cell division to three S cells) and t5 (completion of the cell division to five cells). Time of the events were defined as the first observed timepoint I image frame where the 2PN had disappeared, and newly formed blastomeres were completely separated by confluent cell membranes. All events are expressed as hours post ICSI microinjection or IVE treatment.

The detailed analysis was performed on transferred embryos with 100% implantation (i.e. where the number of gestational sacs confirmed by ultrasound matched the number of transferred embryos); and on embryos with 0% implantation, where no biochemical pregnancy was achieved.

Embryo transfer The number of embryos transferred was normally one, but in some cases 2 or 3 embryos were transferred because of embryo quality or patient wishes. The 13-hOG (13-Human Chorionic Gonadotropin) value was determined 13 days after embryo transfer and the clinical pregnancy was confirmed when a gestational sac with fetal heartbeat was visible after 7 weeks of pregnancy.

Statistical Analysis To describe the distribution of the probabilities of implantation, timings were converted from continuous variables into a categorical variable using quartiles for all observations of each of the measured parameters. A system based on ordinations giving four categories (timing quartiles) with equal number of observations in each of them was used to obtain these categories. By this procedure, bias due to differences in the total number of embryos in each category was avoided. Hereafter the percentage of embryos that implanted for each timing quartile was calculated to assess the distribution of implantation in the different categories.

For each of the categories, the odds ratio (OR) was calculated. OR is calculated as the implantation rate in the quartile containing the highest rate of implanting embryos divided with the quartile containing the lowest rate of implanting embryos. Thus the OR is a measure of the ability to distinguish between the embryos that implanted from the ones that did not.

Statistical analysis was performed using the statistical package R, version 2.13.2, The R Foundation for Statistical Computing.

Results A single gestational sac was frequently observed after dual embryo transfer and similar one to two after transferring three embryos. As it was not possible to ascertain with certainty, which of the two or three transferred embryos implanted, these embryos were excluded from further analysis. All remaining embryos with known implantation were selected for further retrospective analysis. From the remaining embryos, 156 gave successful implantation out of the total 945 transferred, giving rise to a 16.5% implantation rate on this subset of data with known outcome, based on combined gestational sac and fetal heart beat observations.

The relatively low implantation rate was obtained in the calculations, as this clinic in the examined period had many transfers with three embryos. None of these gave three embryos implantation but all situations with no implanted embryos were part of the data. This reduced the calculated implantation rate as only embryos with an ascertain fate were used in the calculations.

Table 1. The implantation rate based on Fetal Head Beat (FHB) for the four timing quartiles of each events, based either on time from IGSI or IVF (t3 and t5) or on time from PN disappearance (t3pN and tSpN). The odds ratio of each event is shown in the last row, where the amount of ÷ demonstrate how high the odds ratio is. In this table, all patient groups are included.

Quartiles t3pN t3 t5pN t5 Qi 98% 14,1% 8,5% 7,5% 02 15,2% 13,6% 15,1% 14,7% 03 11,8% 9,5% 15,2% 15,7% 04 7,8% 7,3% 6,6% 7,6% Odds ratio +++++ ÷i-÷+ ++++++++ ++++++ Table 2. The implantation rate based on Fetal Heart Beat (FHB) for the four timing quartiles of each events, based either on time from ICSI or IVF (t3 and t5) or on time from PN disappearance (t3pN and tSpN). The odds ratio of each event is shown in the last row, where the amount of + demonstrate how high the odds ratio is. In this table, only patients with age below 38 years are included.

Quartiles t3FN t3 t5pt4 t5 QI 15,1% 21,5% 14,8% 12,3% 02 22,4% 16,7% 21,3% 20,5% 03 19,2% 16,3% 20,5% 22,1% 04 12,0% 14,1% 10,7% 12,4% Odds ratio ++++ Discussion From this data it was clear that t3pN and tSpN e.g. time points when the embryo has divided to 3 cells or 5 cells respectively normalised with the time point of when the pro-nuclei (PN) had S disappeared gave a much better prediction of good embryo development potential compared with the conventional measurements t3 or t5 (e.g. time points when the embryo has divided to 3 cells or 5 cells respectively based either on time from ICSI (e.g. time of injection or penetration), IVF (e.g. time of mixing the sperm and oocyte) or thawing.

Thus surprisingly using the time point of when the pro-nuclei (RN) have disappeared to normalise the timings of subsequent further characteristics relating to the development of an embryo, can substantially improve the predictive nature of a method when determining the developmental potential of the embryo (e.g. the potential for the embryo to implant).

All publications mentioned in the above specification are herein incorporated by reference.

Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in embryology, biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.

REFERENCES

Azzarello A, Hoest T & Mikkelsen AL (2012) The impact of pronuclei morphology and dynamicity on live birth outcome after time-lapse culture. Hum Reprod 27: 2649-2657.

S Lemmeri JG, Agerholm I & Ziebe S (2008) Kinetic markers of human embryo quality using time-lapse recordings of IVF/ICSI-fertilized oocytes Reprod Biomed Online 17: 385-391.

Lundin K, Bergh C & Hardarson T (2001) Early embryo cleavage is a strong indicator of embryo quality in human IVF. Hum Reprod 16: 2652-2657.

Meseguer M, Herrero J, Tejera A, Hilligsoe KM, Ramsing NB & Remohi J (2011) The use of morphokinetics as a predictor of embryo implantation. Hum Reprod 26: 2658-2671.

Ottosen LD, Hindkjaer J & lngerslev J (2007) Light exposure of the ovum arid preimplantation embryo during ART procedures. J Assist Reprod Genet 24, 99-103.

Salumets A, I-Iyden-Granskog C, Suikkari AM, Tiitinen A & Tuuri T (2001) The predictive value of pronuclear morphology of zygotes in the assessment of human embryo quality. Hum Reprod 16: 2177-21 81.

Scott L (2003) Pronuclear scoring as a predictor of embryo development. Reprod Biomed Online 6: 201 -21 4.

Claims (13)

1. CLAIMS1. A method for determining the developmental potential of a human embryo in v/tro the method comprising: a. determining at least one reference value (tier) which is a characteristic relating to the development of the embryo selected from the group consisting of: i. the time point when the pro-nuclei (RN) have disappeared; IL the time point when the second polar body is excluding; and iU. the time point when the pro-nuclei (RN) appear; b. measuring the time point of at least one further characteristic relating to the development of the embryo (t>) wherein time zero (t0) is at least one of the reference values i., li.1 or Di. (tier) to establish a variable value (t1), c. establishing a development potential for the embryo based on at least one of said variable values (tvar).
2. 2. A method according to claim 1 wherein the variable value is the difference between the time point of at least one further characteristic relating to the development of the embryo (tx) and at least one of the reference values (tier).
3. 3. A method according to claim 1 or claim 2 wherein the reference value (tier) and the time point of at least one further characteristic relating to the development of the embryo (tx) are measured from any given time point before the reference value (tier) occurs
4. 4. A method according to claim 3 wherein the reference value (tier) and the time point of at least one further characteristic relating to the development of the embryo (tx) are measured either from the time point for starting in vitro fertilization (IVF) when the sperm and the oocyte are mixed or the time of penetration in intracytoplasmic sperm injection (ICSI).
5. 5. A method according to any one of claims ito 4 wherein step b. of the method further comprises measuring the time point of at least one further characteristic relating to the development of the embryo (tx) and determining at least one variable value (t) by subtracting the time point of at least one of said reference values L, ii., or UI (t from said time point of said at least one further characteristic (tx).
6. 6. A method according to claim 1 wherein the variable value (tvar) is measured in step b.directly with at least one of the reference values i., ii., or iii. (trer) commencing the measuring period as time zero (t0).
7. 7. A method according to any one of claims ito 6 wherein said time point of the further S characteristic relating to the development of the embryo is selected from one or more of the following: A. the time point when the embryo has divided to three cells (t3); B. the time point when the embryo has divided to four cells (t4); C. the time point when the embryo has divided to five cells (t5); D. the time point when the embryo has divided to eight cells (t8).
8. 8. A method according to any one of claims ito 7 wherein the time point of the further characteristic relating to the development of the embryo is the time point when the embryo has divided to five cells (t5).
9. 9. A method according to any one of claims ito 7 wherein the time point of the further characteristic relating to the development of the embryo is the time point when the embryo has divided to three cells (t3).
10. 10. A method according to any one of the preceding claims wherein the reference value (trer) is the time point when the pro-nuclei (RN) have disappeared.
11. 11. A method according to any one of claims Ito 9 wherein the reference value (tref) is the time point when the second polar body is excluding.
12. 12. A method according to any one of claims ito 9 wherein the reference value (tref) is the time point when the pro-nuclei (RN) appears.
13. 13. A method according to any one of the preceding claims wherein the values are measured by time-lapse microscopy.