WO2018175885A1

WO2018175885A1 - Method for embryo selection

Info

Publication number: WO2018175885A1
Application number: PCT/US2018/024023
Authority: WO
Inventors: David Bar-Or; Raphael Bar-Or; Charles DORSEY
Original assignee: Aytu Bioscience, Inc.
Priority date: 2017-03-23
Filing date: 2018-03-23
Publication date: 2018-09-27

Abstract

Methods and systems for measuring and using the oxidation-reduction potential (ORP) of a biological sample are provided. The ORP measurements are used to determine the quality of an oocyte or a fertilized egg. Knowledge of the quality of a fertilized egg allows the selection of eggs most likely to result in a successful pregnancy. Such knowledge can also be used to determine the optimal time for transferring a fertilized egg into the uterus, increasing the likelihood of a successful pregnancy.

Description

METHOD FOR EMBRYO SELECTION

FIELD

The present invention relates to methods using the oxidation-reduction potential (ORP) of a fluid sample containing a fertilized egg to improve the success rate of in vitro fertilization procedures.

BACKGROUND

Scientific advancements in assisted reproduction have increased the ability of couples having trouble conceiving a child through the normal method, to realize their dream of having a child. One such advance is in vitro fertilization (IVF). In the process of in vitro fertilization, the ovary is stimulated with hormones such that more than one egg matures, and is released. The released eggs (oocytes) are collected, maintained in culture medium and the most promising (i.e., most competent) are fertilized by contacting them with sperm obtained from a collected sample. The fertilized eggs (zygotes) are then typically cultured for another 2 (cleavage stage) to 5 (blastocyst stage) days at which time the most promising zygotes (or blastocysts) are transferred to the recipient's uterus, where they implant and develop.

While such techniques are often successful, the selection of fertilized eggs

(zygotes) with the best potential for implantation remains a challenge. Improving the chances that a particular fertilized egg will successfully implant and develop normally would maximize the take-home baby rate, while minimizing the incidence of multiple pregnancies and its associated complications. The current gold standard for embryo selection is morphological selection, which involves visual examination of the

morphology of a fertilized egg under a microscope (Gardner and Balaban, 2016). While various other methods for embryo selection, using both invasive and non-invasive procedures, have been described, none of these methods has been conclusively

demonstrated to be better than morphological selection.

One alternative method of embryo selection is measurement of the oxidation- reduction potential (ORP) of an egg and/or the medium in which the egg is cultured. An oxidation-reduction system, or redox system, involves the transfer of electrons from a reductant to an oxidant according to the following equation:

oxidant + n€ → reductant (1) where ne equals the number of electrons transferred. At equilibrium, the redox potential (E), or oxidation-reduction potential (ORP), is calculated according to the Nernst-Peters equation:

E(ORP) = Eo - ΚΎ/nF In [reductant]/[oxidant] (2) where R (gas constant), T (temperature in degrees Kelvin) and F (Faraday constant) are constants. E₀ is the standard potential of a redox system measured with respect to a hydrogen electrode, which is arbitrarily assigned an E₀ of 0 volts, and n is the number of electrons transferred. Therefore, ORP is dependent on the total concentrations of reductants and oxidants, and ORP is an integrated measure of the balance between total oxidants and reductants in a particular system.

Many biological fluids, such as whole blood, plasma, serum, uterine, seminal fluid, and vaginal fluids, have oxidation-reduction potentials (ORP). Clinically, the ORP of such fluids provides the oxidative status of an animal. Moreover, the ORP of such fluids has been shown to be related to health, disease, and the status of biological tissues and processes of the animal.

Oxidative stress in animals results from an alteration in the relative balance between reductants and oxidants in the animal. Such an alteration in the balance can result from higher production of reactive oxygen and reactive nitrogen species, or a decrease in endogenous protective antioxidative capacity. Several investigations have shown a close association between the oxidative status of a patient suffering from an illness, and the patient's outcome. See Roth et al., Current Opinion in Clinical Nutrition and Metabolic Care, 7: 161-168 (2004). Further, oxidative stress has been shown to be associated with various diseases and aging, has been found to occur in all types of illnesses, and has been shown to affect numerous biological processes including conception and pregnancy. See, e.g., Veglia et al., Biomarkers, 11(6): 562-573 (2006); Roth et al., Current Opinion in Clinical Nutrition and Metabolic Care, 7: 161-168 (2004); U.S. Patent No. 5,290,519 and U.S. Patent Publication No. 2005/0142613; Agarwal et al., Reproductive Biology and Endocrinology, 3 :28: 1-21 (2005); Rad et al., J. Caring Sciences, 2(4): 287-294 (2013).

Oxidative stress in patients has been evaluated by measuring various individual markers. See, e.g., Veglia et al., Biomarkers, 11(6): 562-573 (2006); Roth et al., Current Opinion in Clinical Nutrition and Metabolic Care, 7: 161-168 (2004); U.S. Patent No. 5,290,519 and U.S. Patent Publication No. 2005/0142613. However, such measurements are often unreliable and provide conflicting and variable measurements of the oxidative status of a patient. See Veglia et al., Biomarkers, 11(6): 562-573 (2006); Roth et al., Current Opinion in Clinical Nutrition and Metabolic Care, 7: 161-168 (2004). Consequently, measurement of multiple markers, which are then used to provide a score or other assessment of the overall oxidative status of a patient, has been developed to overcome the problems of using measurements of single markers. See Veglia et al., Biomarkers, 11(6): 562-573 (2006); Roth et al., Current Opinion in Clinical Nutrition and Metabolic Care, 7: 161-168 (2004). Although such composite approaches are more reliable and sensitive than measurements of a single marker, they are complex and time consuming.

The use of measuring static ORP (sORP) and/or capacity ORP (cORP) for determination of sperm and egg fertility potential has been described in PCT publication WO2016/085997, which is herein incorporated by reference. While PCT publication WO2016/085997 discusses the use of ORP to evaluate the quality of a fertilized egg, the timing of when to measure ORP (sORP and/or cORP) was not discussed. In particular, the optimal time for measuring the ORP of fertilized eggs (i.e. embryos) to determine which are most suitable for implantation, has not been directly addressed. Thus, there is a need for optimization of ORP measurement, and the use of such measurements to determine which embryo(s) to transfer, and when it is/they are to be transferred. The present invention addresses this need and provides other, related benefits as well.

SUMMARY

One embodiment of the invention is a method of selecting a fertilized egg for implantation in an in vitro fertilization procedure, comprising measuring the oxidation- reduction potential (ORP) of medium in which the fertilized egg is being cultured, at a optimal time after fertilization; comparing the measured ORP to a reference value; and selecting and preparing the egg for implantation in an IVF procedure based on the comparison. In certain aspects, if the measured ORP value is less than, or equal to, a predetermined reference value, the egg is selected for transfer. In certain aspects, the step of measuring is conducted on day 3, 4, 5, or 6, after fertilization. In one aspect, the measured ORP is static ORP (sORP). In one aspect, the measured ORP is capacity ORP (cORP).

In yet another aspect, the method comprises selecting a fertilized egg from among two or more fertilized eggs under consideration, wherein the reference value comprises the measured ORP values of the two or more fertilized eggs under consideration, and wherein the step of selecting comprises selecting the fertilized egg with the lowest ORP value. One embodiment of the invention is a method of determining the quality of a fertilized egg, comprising measuring the oxidation-reduction potential (ORP) of medium in which the fertilized egg is being cultured, at a specific time after fertilization;

comparing the measured ORP to a reference value; and identifying the quality of the fertilized egg based on the comparison. In certain aspects, if the measured ORP value is less than, or equal to, a pre-determined reference value, the egg is identified as being of high quality. In certain aspects, if the measured ORP value is greater than a predetermined reference value, the egg is identified as being of low quality.

One embodiment of the invention is a method of determining when to transfer a fertilized egg in an in vitro fertilization procedure, comprising measuring the oxidation- reduction potential (ORP) of medium in which the fertilized egg is being cultured, at a specific time after fertilization; comparing the measured ORP to a reference value; and deciding to transfer the egg, or not, based on the comparison. In certain aspects, if the measured ORP value is greater than, or equal to, a pre-determined reference value, the egg is transferred. In certain aspects, if the measured ORP value is less than a pre-determined reference value, the egg is not transferred, and may be incubated for an additional amount of time.

One embodiment of the invention is a method of improving the likelihood that a fertilized egg will establish and maintain a successful pregnancy in an IVF procedure, comprising measuring the oxidation-reduction potential (ORP) of medium in which the fertilized egg is being cultured, at a specific time after fertilization; comparing the measured ORP to a reference value; and, based on the comparison, treating the fertilized egg, or the medium in which it is incubated, to improve the ORF characteristics of the egg. In certain aspects, if the measured ORP value is less than, or equal to, a pre-determined reference value, no treatment is administered. In certain aspects, if the measured ORP value is greater than a pre-determined reference value, the egg or the medium in which it is incubated, is treated to improve the ORF characteristics of the egg.

In certain aspects of the afore-mentioned inventions, the medium being tested can come from a fertilized egg that was cryogenically stored, thawed and cultured. Such samples are well known in the field of reproductive biology. Moreover, it is appreciated that cryogenic storage involves the use of cryoprotectant agents, such as glycerol and dimethyl sulfoxide. Recent evidence suggests that certain cryoprotectants may affect stored cells. Thus, methods of the present invention can also be used to determine the effect of storage on fertilized egg competence and to identify useful methods and compositions for such storage. In one embodiment, the ORP characteristics are used to measure the effect of various storage conditions on fertilized egg competence. In certain embodiments, the storage conditions are varied by the addition of antioxidants, such as for example, ascorbic acid and a-tocopherol.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1. Shows the correlation between sORP and embryo score (ES) for all embryos in group.

Fig. 2 shows the correlation between sORP and ES at 48 hours.

Fig. 3 shows the correlation between sORP and ES at 72 hours.

Fig. 4 shows the correlation between sORP and ES at 96 hours.

DETAILED DESCRIPTION

The present invention relates to a method of selecting one or more fertilized eggs for transfer in an IVF procedure. It also relates to the timing of embryo transfer, and a method of improving the likelihood that a fertilized egg will implant and develop normally in an in vitro fertilization procedure. Specifically, the present invention is based on using the ORP value of medium in which a fertilized egg has been cultured, to determine which fertilized egg(s) is/are most likely to successfully implant in the uterus, and to proceed through normal, in utero, development. The inventor has unexpectedly discovered that, not only can the ORP measurement be used to select fertilized eggs for transfer, but that the timing of such measurement is important for selecting those fertilized eggs having the highest potential for successful implantation and normal development. Accordingly, the present invention can generally be practice by measuring the oxidation-reduction potential (ORP) of medium in which a fertilized egg has been cultured, at an optimal time after fertilization, comparing the measured ORP value to a reference value, and selecting fertilized eggs to be transferred based on the comparison. The reference value can be an ORP value that is known to be associated with eggs that implant successfully, develop normally, and result in successful delivery of a baby. In certain aspects of the invention, the ORP is measured at 3, 4, 5 or 6 days post-fertilization. In certain aspects of the invention, the measured ORP is static ORP (sORP). In certain embodiments, the measured ORP is capacity ORP (cORP).

Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the claims.

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. For example, a nucleic acid molecule refers to one or more nucleic acid molecules. As such, the terms "a", "an", "one or more" and "at least one" can be used interchangeably. Similarly the terms "comprising", "including" and "having" can be used interchangeably. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

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 invention belongs. In addition to the above, unless specifically defined otherwise, the following terms and phrases, which are common to the various embodiments disclosed herein, are defined as follows:

As used herein, the terms, egg, oocyte, ovum, and the like, can be used interchangeably to refer to a female reproductive cell that has not yet been fertilized. Likewise, the terms sperm and spermatozoa can be used interchangeably to refer to a male reproductive cell.

As used herein, the term fertilized egg and zygote can be used interchangeably, and refer to a cell that has been produced by the union of an egg and a sperm cell.

As used herein, the phrase "egg quality", and the like, refers to the ability of a fertilized egg to establish and maintain a viable pregnancy. As such, a fertilized egg having a high quality is more likely than not to divide appropriately, maintain a diploid state, successfully implant in the endometrium of the uterus, and proceed to at least the fetal stage of development. A fertilized egg that is determined to have a high quality rating is also more likely than not to develop normally. In contrast, a fertilized egg that is determined to be of low quality, is more likely than not to be unable to complete at least one of the above-listed steps. Transfer of low quality eggs often results in spontaneous termination of a pregnancy prior to the fetal stage of development.

As used herein, the phrase "ORP characteristics" generally refers to the ORP value of a sample, such as the egg or medium in which the egg is cultured. In certain aspects, ORP characteristics can include the change in an ORP value over time. The ORP can be cORP, sORP, or a combination of both.

As used herein, static ORP (sORP) refers to a measure of the balance between total oxidants and reductants in a biological system. As used herein, capacity ORP (cORP) refers to the amount of antioxidant reserves present in a system. sORP and cORP are described in U.S. patent NO. 9,360,446, which is incorporated herein by reference.

As used herein, the term "significantly", with regard to ORP refers an ORP value that differs from another ORP value by at least greater than 5%, and preferably at least 10%. Likewise, the term "approximately", worth regard to ORP values, refers to a difference in ORP values of 5% or less.

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 the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.

One embodiment of the present invention is a method for assessing the quality of a fertilized egg, the method comprising measuring the ORP characteristics of a sample of the medium in which the fertilized egg is cultured, at a particular time, and evaluating if the ORP characteristics are significantly different from the ORP characteristics of a reference ORP value. The quality of the fertilized egg is then determined from this evaluation. In such a method, a measured ORP value greater than a pre-determined reference ORP (threshold ORP) value, or cut-off value, indicates the egg is of low quality. A measured ORP value less than a pre-determined reference ORP (threshold ORP) value, or cut-off value, indicates the egg is of high quality. A measured ORP value less than, but approximately equal to a pre-determined reference ORP value (i.e., within 5% of the reference value), indicates the egg is of optimal quality. The threshold ORP value represents an ORP value above which, the chance of establishing and maintaining a successful pregnancy, is significantly reduced compared to the chance of establishing and maintaining a successful pregnancy observed at the threshold, or lower, ORP values. Correspondingly, the threshold ORP value represents an ORP value below which, the egg being measured is likely to establish and maintain a successful pregnancy. The threshold ORP can be determined, for example, from historical ORP data obtained from previous successes or failures in transferring fertilized eggs. Thus, in certain aspects, if the measured ORP is greater than the reference ORP, the egg is identified being of low quality. In certain aspects, if the measured ORP is less than, or equal to, the reference ORP, the egg is identified being of high quality.

In a further embodiment, if the egg is deemed high quality, the egg is selected, prepared and transferred into a uterus.

The inventors have unexpected found that the timing of measuring the medium

ORP value can be important with regard to identifying fertilized eggs having the highest quality. Thus, in certain aspects of the invention, the fertilized egg being tested is a zygote. In certain aspects, the fertilized egg being tested has reached the cleavage stage. In certain aspects, the fertilized egg is a blastocyst. In certain aspects, the fertilized egg is a morula. In certain aspects, the fertilized egg is a blastula. In certain aspects, the fertilized egg is a blastocyst.

In certain aspects, of the invention the ORP value of the medium is determined at least two days post-fertilization. In certain aspects, the ORP value of the medium is determined at least three days post-fertilization. In certain aspects, the ORP value of the medium is determined at least four days post-fertilization. In certain aspects, the ORP value of the medium is determined at least five days post-fertilization. In certain aspects, the ORP value of the medium is determined at least six days post-fertilization.

In order to determine the trend of ORP characteristics in a cultured egg over time, without limitation, the ORP characteristics of the culture medium may be checked at suitable intervals. In certain aspects, the ORP characteristics of the medium in which the egg has been cultured can be checked every 30 minutes, hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours or 18 hours. In certain aspects, the change and/or the rate of change in ORP over time is determined.

One embodiment of the invention is a method of selecting a fertilized egg for implantation in an in vitro fertilization procedure, the method comprising measuring the oxidation-reduction potential (ORP) characteristics of medium in which a fertilized egg is cultured, at a particular time; comparing the measured ORP to a reference value; and selecting and preparing the egg for transfer in an IVF procedure based on the comparison. In such a method, a measured ORP value greater than a pre-determined reference ORP (threshold ORP) value, or cut-off value, indicates the egg is of low quality. The threshold ORP value represents an ORP value above which, the chance of establishing and maintaining a successful pregnancy, is significantly reduced compared to the chance of establishing and maintaining a successful pregnancy observed at the threshold, or lower, ORP values. Correspondingly, the threshold ORP value represents an ORP value below which, the egg being measured is likely to establish and maintain a successful pregnancy. The threshold ORP can be determined, for example, from historical ORP data obtained from previous successes or failures in transferring fertilized eggs. Thus in certain aspects, if the measured ORP is significantly lower than, or equal to, the reference ORP, the egg being measured is selected for transfer. In certain aspects, if the measured ORP is significantly higher than the reference ORP, the egg is not selected for transfer. In a further embodiment, the egg is transferred into a uterus.

In vitro fertilization usually involves obtaining and culturing more than one egg at the same time. Thus, in one aspect of the invention, the method comprises selecting a fertilized egg from among two or more fertilized eggs under consideration, wherein the reference value comprise the measured ORPs of the two or more fertilized eggs under consideration, and wherein the step of selecting comprises selecting the fertilized egg with the lowest ORP value.

In certain aspects, the fertilized egg being tested is a zygote. In certain aspects, the fertilized egg being tested has reached the cleavage stage. In certain aspects, the fertilized egg is a blastocyst. In certain aspects, the fertilized egg is a morula. In certain aspects, the fertilized egg is a blastula. In certain aspects, the fertilized egg is a blastocyst.

In certain aspects, the ORP value of the medium is determined at least two days post-fertilization. In certain aspects, the ORP value of the medium is determined at least three days post-fertilization. In certain aspects, the ORP value of the medium is determined at least four days post-fertilization. In certain aspects, the ORP value of the medium is determined at least five days post-fertilization. In certain aspects, the ORP value of the medium is determined at least six days post-fertilization.

In order to determine the trend of ORP characteristics in a cultured egg over time, without limitation, the ORP characteristics of the culture medium may be checked at suitable intervals. In certain aspects, the ORP characteristics of the medium in which the egg has been cultured can be checked every 30 minutes, hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours or 18 hours. In certain aspects, ORP characteristics can include the change in an ORP value over time. The ORP can be cORP, sORP, or a combination of both.

Because a decrease in egg quality can reduce the chances that a fertilized egg will establish and maintain a successful pregnancy, methods of the present invention can be used to improve the likelihood that a fertilized egg will establish and maintain a successful pregnancy. Thus, one embodiment of the present invention is a method of improving the likelihood that a fertilized egg will establish and maintain a successful pregnancy, the method comprising measuring the ORP characteristics of a sample of the medium in which the fertilized egg is cultured, evaluating if the ORP characteristics are significantly different from the ORP characteristics of the reference ORP value, and if the ORP characteristics are significantly different from the ORP characteristics of a reference ORP value, treating the culture medium, or the fertilized egg, to improve the ORP characteristics of the fertilized egg. In certain aspects of the invention, if the measured ORP is significantly greater than a reference ORP, the egg, or the medium containing the egg, is treated to improve the ORP characteristics of the egg. In certain aspects, if the measured ORP is not significantly greater than a reference ORP, then no treatment is administered. In such embodiments, treatment of the cultured fertilized egg can comprise treating the culture medium (e.g., with antioxidants, or other suitable compounds) to improve the ORP characteristics of the medium, or treating the egg with a compound (e.g., a pharmaceutical compound, or a natural compound) to improve the quality of the egg. Treatments can include supplementing or refreshing the medium in which the egg is incubated (i.e., removing the medium and replacing it with fresh medium).

One embodiment of the present invention is a method determining when to transfer a fertilized egg in culture, at a particular time, the method comprising measuring the ORP characteristics of a sample from the individual, evaluating if the ORP characteristics are significantly different from the ORP characteristics of a reference ORP value, and using the evaluation to determine the optimal time for transferring the fertilized egg. In such a method, an increase in the ORP characteristics to a pre-determined ORP threshold value, or cut-off value, indicates it is time to transfer the fertilized egg into the uterus. The threshold ORP value represents an ORP value at which the fertilized egg is more likely than not to establish and maintain a successful pregnancy. The threshold ORP can be determined, for example, from historical ORP data obtained from previous successes or failures in transferring fertilized eggs. Thus, in certain aspects of the invention, if the measured ORP is less than the reference ORP, the egg is can, but may not be transferred. Instead, the egg may be incubated for an additional amount of time. In certain aspects of the invention, if the measured ORP is approximately equal to (i.e., within 5%), the reference ORP, the egg is transferred. In certain aspects, if the measured ORP is greater than the reference ORP, the egg may be transferred, but such an egg has a lower likelihood of successfully implanting and developing than does an egg having a measured ORP approximately equal to the reference ORP. In certain aspects, if the measured ORP is greater than the reference ORP, the egg is not transferred.

In order to determine the trend of ORP characteristics in a cultured egg over time, without limitation, the ORP characteristics of the culture medium may be checked at suitable intervals. In certain aspects, the ORP characteristics of the medium in which the egg has been cultured can be checked every 30 minutes, hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours or 18 hours.

In certain aspects of the aforementioned inventions, the medium being tested can come from a fertilized egg that was cryogenically stored, thawed and cultured. Such samples are well known in the field of reproductive biology. Moreover, it is appreciated that cryogenic storage involves the use of cryoprotectant agents, such as glycerol and dimethyl sulfoxide. Recent evidence suggests that certain cryoprotectants may affect stored cells. Thus, methods of the present invention can also be used to determine the effect of storage on fertilized egg competence and to identify useful methods and compositions for such storage. In one embodiment, the ORP characteristics are used to measure the effect of various storage conditions on fertilized egg competence. In certain embodiments, the storage conditions are varied by the addition of antioxidants, such as for example, ascorbic acid and a-tocopherol. It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

EXAMPLES

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the embodiments, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for.

This example demonstrates the use of measuring sORP levels for embryo selection in patients undergoing intracytoplasmic sperm injection (ICS!) by comparing it to the morphological assessment of the embryos.

Materials and Methods

A total of 43 embryos cultured individually from 10 patients undergoing ICSI treatment were studied. The sORP of the spent culture medium was determined at various stages of embryo culture and compared to the morphological assessment of the embryo using the Veeck's modified scoring system at 48, 72 and 96 hours after insemination (Veeck, 1991). Res jilts

There was a fair correlation between the sORP of the spent culture medium and the embryo score when all embryos were included (r= 0.448). The correlation coefficient was 0.315 at 48 hours, 0.415 at 72 hours and 0.706 at 96 hours after insemination. See Figs. 1- 4 and Table 1.

In Table 1 , the morphology scoring column refers to the cleavage state. Higher numbers are indicative of higher cell cleavage. Combined with morphology grading, this guides which and how many embryos will be implanted. In the Veeck grading criteria the morphology (grade) is as follows: Grade I: blastomeres of equal size and no cytoplasmic fragments

Grade II: blastomeres of equal size with minor cytoplasmic fragments or blebs

Grade III: blastomeres of distinctly unequal size with few to no cytoplasmic fragments Grade IV: blastomeres of equal or unequal size with significant cytoplasmic fragmentation Grade V: few blastomeres of any size and severe or complete fragmentation

The three-letter codes in the first column are sample identifiers.

Conclusions

The measurement of oxidative stress, specifically the sORP, in the spent culture medium using this simplified methodology is a useful method for embryo selection in patients treated with ICSI. Correlation between the ORP and the embryo score is good at 72 hours and much better at 96 hours after insemination.

While various embodiments of the present invention have been described in detail, it is apparent that modifications and adaptations of those embodiments will occur to those skilled in the art. It is to be expressly understood, however, that such modifications and adaptations are within the scope of the present invention, as set forth in the following exemplary claims.

References:

1. Gardner DK, Balaban B . Assessment of human embryo development using

morphological criteria in an era of time-lapse, algorithms and OMICS': is looking good still important? Mol Hum Reprod. 2016 Oct;22(10):704-718.

2. Wiener-Megnazi Z, Shiloh H, Avraham L, Lahav-Baratz S, Koifman M, Reznick AZ, Auslender R, Dirnfeld M. Oxidative parameters of embryo culture media may predict treatment outcome in in vitro fertilization: a. novel applicable tool for

improving embryo selection. Fertil Steril. 2011 Mar l;95(3):979-84.

3. Agarwal A, Sharma R, Roychoudhury S, Du Plessis S, Sabanegh E. MiQXSYS: a novel method of measuring oxidation reduction potential in semen and seminal plasma. Fertil Steril. 2016 Sep l;106(3):566-573.

4. Veeck LL. Atlas of the human oocyte and early conceptus. Vol. 2. Baltimore:

Williams and Wilkins, 1991 :427-44.

Claims

What is claimed:

1. A method of selecting a fertilized egg for transfer in an in vitro fertilization (IVF) procedure, comprising

a. measuring the oxidation-reduction potential (ORP) of medium in which the fertilized egg is being cultured on day 3, or later, after fertilization; b. comparing the measured ORP to a reference ORP value; and,

c. selecting the egg for implantation based on the comparison.

2. The method of claim 1, wherein if the measured ORP is significantly lower than, or equal to, the reference ORP, selecting the egg for transfer.

3. The method of claim 1, wherein if the measured ORP is significantly higher than the reference ORP, identifying the egg as not suitable for transfer.

4. The method of claim 1, wherein the method comprises selecting a fertilized egg from among two or more fertilized eggs under consideration, wherein the reference value comprises the measured ORP values of the two or more fertilized eggs under consideration and wherein the step of selecting comprises selecting the fertilized egg with the lowest ORP value.

5. A method of determining the quality of a fertilized egg, comprising

a. measuring the oxidation-reduction potential (ORP) of medium in which the fertilized egg is being cultured on day 3, or later, after fertilization; b. comparing the measured ORP to a reference ORP value; and

c. determining the quality of the egg based on the comparison.

6. The method of claim 1, wherein if the measured ORP is significantly higher than the reference ORP, identifying the egg as having low quality.

7. The method of claim 1, wherein if the measured ORP is significantly lower than, or equal to, the reference ORP, identifying the egg as having high quality.

8. A method of determining when to transfer a fertilized egg in an INF procedure, comprising:

a. measuring the oxidation-reduction potential (ORP) of medium in which the fertilized egg is being cultured on day 3, or later, after fertilization; and, b. comparing the measured ORP to a reference ORP value;

wherein if the measured ORP value is significantly lower than, or

approximately equal to a reference ORP value, transferring the egg.

9. A method of improving the likelihood that a fertilized egg will establish and maintain a successful pregnancy in an IV procedure, comprising

wherein if the measured ORP value is significantly higher than a reference ORP value, treating the fertilized egg, or the medium containing the fertilized egg, to improve the ORP characteristics of the medium.

10. The method of any one of claims 1-9, wherein the ORP being measured is static ORP (sORP)

11. The method of any one of claims 1-9, wherein the ORP being measured is capacity ORP (cORP)

12. The method of any one of claims 1-11, wherein the ORP is measured on day 4 following fertilization.

13. The method of any one of claims 1-11, wherein the ORP is measured on day 5 following fertilization.

14. The method of any one of claims 1-11, wherein the ORP is measured on day 6 following fertilization.

15. The method of any one of claims 1-14, wherein the fertilized egg is a human egg.