Technical Field

The present disclosure generally relates to methods of determining the probability of live birth and to methods of, and compositions for, increasing the probability of live birth.

Background

Repeated IVF failure (RIF) is commonly described as the failure of two or more consecutive good-quality embryo transfer cycles and affects about 10% of couples undergoing IVF treatment. It is believed that implantation failure during assisted reproduction may be caused or associated with inappropriate immune responses in the embryo recipient.

Given the enormous financial, physical and psychological burden of IVF, further investigation is warranted and that is the reason why immune testing and therapy has such a long and persistent presence in reproductive medicine. Women themselves are particularly aware that it is their bodies that have to carry the pregnancy, and dismissing their concerns leads to IVF treatment dropouts, and engagement with alternative practitioners. Even if, as some might think, embryo quality is almost entirely responsible for IVF outcomes, unexplained RIF is a very real and distressing condition that requires sensitive and careful investigation.

Thus, there is a need for an empirical approach to immune therapy to improve treatment outcomes and increase the probability of live birth.

Summary

The present disclosure is based at least in part on the inventor’s finding that the probability of a live birth is decreased for a subject with increased NK cell count and activity. Specifically, the inventor has identified that the probability of a live birth is increased when immune therapy is administered to selected subjects having particular threshold levels of NK cell count and activity. Thus, the present disclosure relates to an improvement in assisted reproduction methods through the identification and/or selection for treatment of a specific patient sub-population that is particularly likely to benefit from immune therapy in an attempt to increase the likelihood of that subject producing a live birth. Accordingly, the present disclosure provides a method of selecting a subject as being suitable for receiving immune therapy in order to increase the probability of live birth in that subject, the method comprising determining the level and activity of NK cells in a blood sample obtained from the subject, wherein if: i) the NK cell proportion of total peripheral lymphocytes is at least 12%; and ii) the activated NK cell count is at least 12 xlO ⁶ /L, the subject is selected for immune therapy.

The present disclosure also provides a method of increasing the probability of live birth in a subject, the method comprising determining the level and activity of NK cells in a blood sample obtained from the subject, wherein if: i) the NK cell proportion of total peripheral lymphocytes is at least 12%; and ii) the activated NK cell count is at least 12 xlO ⁶ /L, the method further comprises administering immune therapy to the subject.

The present disclosure also provides a method of reducing the risk of implantation failure during assisted reproduction in a subject, the method comprising determining the level and activity of NK cells in a blood sample obtained from the subject, wherein if: i) the NK cell proportion of total peripheral lymphocytes is at least 12%; and ii) the activated NK cell count is at least 12 xlO ⁶ /L, the method further comprises administering immune therapy to the subject.

The present disclosure also provides a method of predicting pregnancy outcome in a subject, the method comprising determining the level and activity of NK cells in a blood sample obtained from the subject, wherein if: i) the NK cell proportion of total peripheral lymphocytes is at least 12%; and ii) the activated NK cell count is at least 12 xlO ⁶ /L, the subject is predicted to have lower pregnancy outcome.

The present disclosure also provides a method of increasing the probability of live birth in a subject, the method comprising administering an immune therapy to the subject, wherein the subject has previously been selected as being suitable for receiving said immune therapy by determining the level and activity of NK cells in a blood sample taken from the subject and wherein the subject has: i) an NK cell proportion of total peripheral lymphocytes of at least 12%; and ii) an activated NK cell count of at least 12 xlO ⁶ /L.

The present disclosure also provides a method of selecting a subject for treatment with immune therapy, the method comprising: a) determining whether the subject falls within one of three categories of NK cell level and activity, being: i) an NK cell proportion of total peripheral lymphocytes of 12%- 18% and/or an activated NK cell count of at least 12 x 10 ⁶ /L; ii) an NK cell proportion of total peripheral lymphocytes of at least 18% and an activated NK cell count of at least 12 xlO ⁶ /L; and iii) an NK cell proportion of total peripheral lymphocytes less than 12% and an activated NK cell count of less than 12 xlO ⁶ /L, wherein the level and activity of NK cells is or has previously been determined in a blood sample obtained from the subject; and b) selecting the subject for treatment with immune therapy if the subject falls within category i) or category ii) and not selecting the subject for treatment with immune therapy if the subject falls within category iii).

The present disclosure also provides a method of increasing the probability of live birth in a subject, the method comprising i) determining the level and activity of NK cells in a blood sample obtained from the subject; ii) selecting the subject for treatment with immune therapy if the subject has: a) an NK cell proportion of total peripheral lymphocytes of 12%- 18% and an activated NK cell count of at least 12 xlO ⁶ /L; or b) an NK cell proportion of total peripheral lymphocytes of at least 18% and an activated NK cell count of at least 12 x 10 ⁶ /L and iii) administering immune therapy to the subject.

The present disclosure also provides an immune therapy for use in increasing the probability of live birth in a subject or for use in reducing the risk of implantation failure during assisted reproduction in a subject, wherein the subject has: i) an NK cell proportion of total peripheral lymphocytes of at least 12%; and ii) an activated NK cell count of at least 12 x 10 ⁶ /L.

The NK cell proportion of total peripheral lymphocytes and the activated NK cell count may be detected or may previously have been detected in a blood sample taken from the subject.

The present disclosure also provides the use of an immune therapy in the manufacture of a medicament for increasing the probability of live birth in a subject, or in the manufacture of a medicament for reducing the risk of implantation failure during assisted reproduction in a subject, wherein the subject has: i) an NK cell proportion of total peripheral lymphocytes of at least 12%; and ii) an activated NK cell count of at least 12 xlO ⁶ /L.

Again, the NK cell proportion of total peripheral lymphocytes and the activated NK cell count may be detected or may previously have been detected in a blood sample taken from the subject.

In any of the methods disclosed herein, the NK cell proportion of total peripheral lymphocytes may be at least 12%.

In any of the methods disclosed herein, the NK cell proportion of total peripheral lymphocytes may be between 12-18%.

In any of the methods disclosed herein, the NK cell proportion of total peripheral lymphocytes may be at least 18%.

In any of the methods disclosed herein, the NK cell may be a CD56 ⁺ CD3‘ NK cell. Alternatively or in addition, in any of the methods disclosed herein, the activated NK cell may be a CD69 ⁺ CD56 ^Dim NK cell.

In any of the methods disclosed herein, the immune therapy may comprise administering any one or more of an immunosuppressive agent, a corticosteroid, an anticoagulant, a disease-modifying anti-rheumatic drug or an intralipid infusion composition. In one example, the immune therapy comprises a corticosteroid and an anticoagulant. The corticosteroid may be prednisolone. The anticoagulant may be enoxaparin sodium.

Thus, the immune therapy may comprise administering prednisolone and enoxaparin sodium (Clexane).

The prednisolone may be administered or provided for administration at a dose of 10 mg daily prior to ovulation and 20 mg daily after ovulation, and/or the enoxaparin sodium may be administered or provided for administration at a dose of 20 mg daily after ovulation.

In any of the methods disclosed herein, the subject may be a subject undergoing assisted reproduction. The assisted reproduction may be IVF, ICSI or IUI. In one example, the assisted reproduction is IVF. In another example, the assisted reproduction is ICSI. Alternatively, the subject may not be undergoing assisted reproduction. Thus, the subject may wish to increase their chances of live birth without having previously had any history of attempted or failed reproduction.

In any of the methods disclosed herein, the subject may be a subject who has suffered from miscarriage or from recurrent miscarriages. Alternatively or in addition, the subject may have had recurrent implantation failure.

In any of the methods disclosed herein, the subject may be a mammalian female subject. For example, the subject may be a human female. The human female subject may be of any reproductive age. In one particular example in which the methods and compositions disclosed herein have been shown to be particularly advantageous, the female human subject may be between 38 to 40 years old.

In any of the methods disclosed herein, adjunct therapy to improve live birth rate may be administered separately, simultaneously or sequentially with the immune therapy.

Brief description of the drawings

The following figures form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these figures in combination with the detailed description of specific embodiments presented herein. Figure 1. Cycle outcomes following administration or no administration of the “Bondi protocol” treatment in three age groups: <38 years, 38-39.9 years, and >40 years, a. Pregnancy and live births within each age group are shown for all cycles, b. Pregnancy and live births within each age group are shown PGD single transfer cycles only.

Figure 2. Cycle outcomes according to age and patient treatment groups a. Pregnancy and live birth rates for the three patient groups (Bondi, normal and mixed) are shown per patients, with patients being categorised by patient age. b. Cycle outcomes are shown according to patient age and treatment groups.

Figure 3. Live birth rate per cycle trend

The impact of the Bondi protocol in women with increasing NK activity is shown, with over 4-fold difference in live birth outcomes for the high NK group.

Detailed description

General Techniques and Definitions

Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g. in immunology, reproductive immunology, immunohistochemistry, biochemistry, embryology and pharmacology).

Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features. Thus, each feature of any particular aspect or embodiment of the present disclosure may be applied mutatis mutandis to any other aspect or embodiment of the present disclosure.

The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally equivalent products, compositions and methods are clearly within the scope of the disclosure, as described herein. Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

As used herein, the singular forms of “a”, “and” and “the” include plural forms of these words, unless the context clearly dictates otherwise.

The term “and/or”, e.g., “X and/or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

Throughout this specification, the word “comprise” or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Selected definitions

As used herein, the term "assisted reproduction" refers to clinical and laboratory techniques used to enhance fertility in humans and animals, including, but not limited to, in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), gamete intrafallopian transfer (GIFT), artificial insemination and the like.

The term "in vitro fertilization (IVF)" refers to the procedure involving ovarian hyperstimulation, oocyte retrieval from the mother-to-be or a donor, fertilization outside the subject's body, embryo culture and embryo transfer. The fertilization may be through intracytoplasmic sperm injection (ICSI) or traditional IVF. As used herein, embryo transfer refers to the procedure involving transfer to a subject's uterus, of the developing or cleaving embryos or pre-embryos, also termed preimplantation embryos. In one example, the assisted reproduction is intrauterine insemination (IUI). In another example, the assisted reproduction is IVF. In another example, the assisted reproduction is ICSI.

As used herein, the term "embryo" refers to the fertilized egg which is transferred into the uterus. The embryo may be any stage in development. For example, the embryo may be a cleavage stage embryo, a morula or a blastocyst. In one example, the embryo is a blastocyst. The blastocyst may be a fresh blastocyst (fresh cycle) or blastocyst that has been cryopreserved (frozen cycle). The blastocyst may have undergone pre-implantation genetic testing.

The term "miscarriage" refers to delivery or loss of the product of conception before the 20th week of pregnancy. The term miscarriage includes but is not limited to spontaneous miscarriage, threatened miscarriage, inevitable spontaneous miscarriage, incomplete spontaneous miscarriage, habitual or recurrent spontaneous miscarriage or missed miscarriage. Miscarriage is intended to be synonymous with the term “spontaneous abortion”. The term “recurrent miscarriage” is defined as three or more consecutive pregnancy losses prior to 20 weeks’ gestation.

The term "implantation failure" refers to the failure of an embryo produced by assisted reproduction to implant in the uterus of a recipient subject. As used herein, recurrent implantation failure refers to cases in which women have had three failed in vitro fertilization (IVF) attempts with good quality embryos.

As used herein, the term "subject" refers to animals such as mammals, including, but not limited to, primates (such as humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice and the like. Typically, the terms "subject" and "patient" are used interchangeably, particularly in reference to a human subject. The subject is typically a female. For example, the subject may be a human female. The subject may be (but is not necessarily) a subject suffering from, suspected of suffering from, or predisposed to, recurrent miscarriage or recurrent implantation failure. The subject may be any mammalian subject at risk of a spontaneous abortion. The subject may have previously had one or more miscarriages. The subject may have previously had two or more miscarriages. The subject may have had recurrent miscarriages, i.e., two or more, or three or more miscarriages.

The subject can be any subject in a population at risk for miscarriage or recurrent implantation failure. For instance, the subject can be a human female in an age group at risk for miscarriage or recurrent implantation failure. The subject can be a human female greater than 30 years of age, greater than 35 years of age, greater than 40 years of age or greater than 45 years of age. Alternatively, the subject can be a human female less than 35 years of age or less than 30 years of age or less than 25 years of age. In one example, the subject is under 40 years old. In another example, the subject is over 40 years old. In one example, the subject is a human female aged between 30 and 45 years of age, such as between 35 and 45 years of age, or between 35 and 40 years of age, or between 38 and 40 years of age.

Alternatively or in addition, the subject can be of any age and the egg age can be any of the ages or age ranges described herein. Further, the egg age may be the age of the woman from whom the egg was collected, at egg collection and freezing.

The subject can also be in any other population at risk for miscarriage as determined by a practitioner of skill in the art. For example, the subject may have had an implantation failure during a previous assisted reproduction procedure. In one example, the subject is suffering from primary infertility. In another example, the subject is suffering from secondary infertility.

As used herein, the terms "therapy", "treating", "treat" or "treatment" and variations thereof, refer to clinical intervention designed to alter the natural course of the individual being treated during the course of clinical pathology including infertility or reduced fertility. Desirable effects of treatment include reducing the risk of implantation failure, reducing the probability of miscarriage, increasing the probability of live birth or improving prognosis. Any one or more of these effects may be measured to provide an indication of a subject’s response to immune therapy in the methods disclosed herein.

Immune therapy

Without wishing to be bound by theory, it is believed that embryo implantation failure may be caused or associated with inappropriate immune responses in the embryo recipient. As such, modulating the immune system by immune therapy may increase the probability of live birth and/or reduce the risk of implantation failure in a subject with an increased NK cell level and/or activity.

In one example, immune therapy comprises the administration of any one or more of an immunosuppressive agent, a corticosteroid, an anticoagulant, an intralipid composition or a disease-modifying anti-rheumatic drug.

Immunosuppresive agents are known in the art. Examples of immunosuppressive agents include, but are not limited to, calcineurin inhibitors such as cyclosporine and tacrolimus and purine analogues such as azathioprine. Without wishing to be bound by theory, corticosteroids may have anti-inflammatory, immunosuppressive, anti -proliferative, and vasoconstrictive effects. Suitable corticosteroids are known in the art. Examples of suitable corticosteroids include, but are not limited to, cortisone, prednisone (CAS ID no. 53-03-2), prednisolone (CAS ID no. 50-24-8), methylprednisolone (CAS ID no. 83-43-2), dexamethasone (CAS ID no. 50-02-2), betamethasone (CAS ID no. 378-44-9) and hydrocortisone (CAS ID no. 50-23-7).

In one example, the corticosteroid is prednisone, whose structure is illustrated in the following Formula I:

In another example, the corticosteroid is prednisolone, whose structure is illustrated in the following Formula II:

In another example, the corticosteroid is dexamethasone, whose structure is illustrated in the following Formula III: These and other corticosteroids are readily available in the art, for example from commercial sources, and can be made by methods known in the art.

Anticoagulants decrease the clotting ability of blood. Suitable anticoagulants are known in the art, and are readily available in the art, for example from commercial sources, and/or can be made by methods known in the art. Examples of anticoagulants include, but are not limited to, heparin (CAS ID no. 9005-49-6, low-molecular weight heparin (LMWH) and warfarin (CAS ID no. 81-81-2). In one example, the anticoagulant is a LMWH. In one example, the anticoagulant is enoxaparin sodium (CAS ID no. 9041-08-1, referred to interchangeably as “enoxaparin”).

In another example, the anticoagulant is enoxaparin sodium, whose structure is illustrated in the following Formula IV:

In another example, the anticoagulant is dalteparin (CAS ID no. 9041-08-1). Preferably, the anticoagulant is enoxaparin sodium (CAS ID no. 9041-08-1).

Disease-modifying anti -rheumatic drugs (DMARDs) modulate the immune system’s response. Suitable DMARDs include, but are not limited to, hydroxychloroquine (CAS ID no. 118-42-3), sulfasalazine (CAS ID no. 599-79-1) and adalimumab (Humira, CAS ID no. 331731-18-1). In one example, the DMARD is hydroxchloroquine. In another example, the DMARD is adalimumab. Intralipids (referred to herein interchangeably as the singular composition, ‘intralipid’) are typically a sterile fat emulsion comprising egg yolk phospholipids, soybean oil, glycerin and water, often given as an intravenous infusion. Without wishing to be bound by theory, it is believed that intralipids can help to deactivate the natural killer cells, allowing the embryo to implant on the uterine wall and grow normally and keep the natural killer cells deactivated until the pregnancy can override the signals being sent by the immune system. Intralipid may be administered as an intravenous infusion of 100 mg in a 500vml bag of normal saline over 2 hours. It may be administered once before embryo transfer, and once after a positive pregnancy test. Thus, in one example, an intralipid infusion is administered prior to embryo transfer. In another example, an intralipid infusion is administered after embryo transfer. In another example, an intralipid infusion is administered prior to embryo transfer and after embryo transfer.

Thus, in one example, immune therapy comprises administering a corticosteroid and an anticoagulant to the subject. In one particular example, immune therapy comprises administering prednisolone and enoxaparin sodium to the subject. Prednisolone may be administered prior to ovulation at a dose of at least 10 mg daily or at least 15 mg daily or at least 20 mg daily or at least 25 mg daily. Prednisolone may be administered prior to egg collection at a dose of at least 10 mg daily or at least 15 mg daily or at least 20 mg daily or at least 25 mg daily. In one example, prednisolone may be administered prior to ovulation at a dose of 10 mg daily. In another example, prednisolone may be administered prior to egg collection at a dose of 10 mg daily. After ovulation, the dose of prednisolone may be increased to at least 20 mg daily or at least 25 mg daily or at least 30 mg daily. After ovulation, enoxaparin sodium may be administered at a dose of at least 20 mg daily or at least 40 mg daily or at least 60 mg daily. Thus, in one example, immune therapy comprises administering prednisolone at a dose of 10 mg daily prior to ovulation and 20 mg daily after ovulation, and administering enoxaparin sodium at a dose of 20 mg daily after ovulation.

Any combinations of these alternative doses may suitably be employed in the methods of the present disclosure.

In another example, immune therapy further comprises an intralipid infusion. The present disclosure also provides an immune therapy for use in increasing the probability of live birth in a subject or for use in reducing the risk of implantation failure during assisted reproduction in a subject, wherein the subject has: i) an NK cell proportion of total peripheral lymphocytes of at least 12%; and ii) an activated NK cell count of at least 12 xlO ⁶ /L.

The subject may have previously been selected as being suitable for receiving said immune therapy by determining the level and/or activity of NK cells in a blood sample taken from the subject. The immune therapy may be any immune therapy disclosed herein.

The present disclosure further provides the use of an immune therapy in the manufacture of a medicament for increasing the probability of live birth in a subject, or in the manufacture of a medicament for reducing the risk of implantation failure during assisted reproduction in a subject, wherein the subject has: i) an NK cell proportion of total peripheral lymphocytes of at least 12%; and ii) an activated NK cell count of at least 12 xlO ⁶ /L.

Again, the subject may have previously been selected as being suitable for receiving said immune therapy by determining the level and/or activity of NK cells in a blood sample taken from the subject.

The immune therapy compositions or medicaments disclosed herein may be prepared by any suitable means known in the art of pharmaceutical preparations. Thus, the compositions or medicaments may comprise one or more pharmaceutically acceptable carriers or excipients.

The immune therapy compositions or medicaments may be prepared as combinations of active agents, for example, a corticosteroid and an anticoagulant as disclosed herein. Thus, in one example, the immune therapy compositions or medicaments may be prepared as a combination of prednisolone and enoxaparin sodium at any of the dosages disclosed herein. It will be appreciated that the combination of such active agents may be achieved in part through the provision of literature describing the intent and benefit of the combined administration of the multiple active agents in order to effect the treatment disclosed herein.

Natural Killer (NK) cells

Natural killer (NK) cells are innate immune cells that show strong cytolytic function against physiologically stressed cells such as tumor cells and virus-infected cells. NK cells are strikingly suppressed in normal early pregnancy (Szereres-Barthos and Wegmann, 1996; Sacks et al., 2003). Without wishing to be bound by theory, it is hypothesized that NK cells that are not suppressed (or are indeed activated) could cause a type 1 (cell-mediated) shift and miscarriage in some women (Chaouat, 2008).

NK cells are innate lymphocytes with a CD3 CD56 ⁺ phenotype. They can be generally divided into two subsets based on the relative surface density of the CD56 antigen. The CD56 ^+Bnght NK subset is CD 16", has a high IL-2 affinity and produces cytokines. The CD56 ^+Dim subset is CD16 ⁺ , has moderate IL-2 affinity and orchestrates NK cytotoxicity (Cooper et al., 2001). These subsets also express different monocyte-derived cytokine and MHC-recognizing receptors including the activating receptor CD69 (Cooper et al., 2001; Coulam and Roussev, 2003). NK cells are present in the peripheral blood and in the uterine tissue, where they appear to regulate trophoblast invasion (Moffett-King, 2002; Moffett et al., 2004).

Without wishing to be bound by theory, women with recurrent miscarriage or implantation failure have been identified as having a higher blood NK cell levels as a proportion of lymphocytes and high levels of activation as expressed by the CD69 surface marker relative to women who do not have recurrent miscarriage or implantation failure. The present disclosure demonstrates that live birth rate is improved when immune therapy is administered to patients who have: i) an NK cell proportion of total peripheral lymphocytes of at least 12%; and ii) an activated NK cell count of at least 12 xlO ⁶ /L.

In one example, the NK cells is a CD3 CD56 ⁺ NK cell. Suitable methods of detecting a CD3 CD56 ⁺ NK cell and the CD56 ^+Dim and CD56 ^+Bnght subsets and thus measuring their levels in peripheral blood are known in the art. For example, immunohistochemistry and/or flow cytometry may be used.

In one example, the NK cell proportion of total lymphoctyes is determined by one or more immunohistochemical methods. For example, the level NK cells may be determined by contacting a sample with an antibody capable of binding specifically to the surface markers on the NK cell, which antibody is conjugated to a detectable label (e.g., a fluorescent label). The label may be detected by any means known in the art. In one example, the label is detected by flow cytometry. The activity of NK cells may be determined by measuring the level of expression of the CD69 surface marker in the CD56 ^+Dim subset. Without wishing to be bound by theory, it is believed that activated NK cells express CD69. As such, measuring the number of CD56 ^+Dim cells that express CD69 may give a measure of NK cell activity in a biological sample from a subject.

The activity of the NK cells may be determined by measuring the number of cells expressing the activation marker, CD69. The level of expression of CD69 may be determined by one or more protein quantitation methods. For example, the level of expression of CD69 may be determined by mass spectrometry, flow cytometry or immunohistochemistry.

Any suitable number of NK cells and/or lymphocytes may be sampled in order to provide a statistically meaningful measurement of level of NK cell and/or activity. For example, at least about 1000, or at least about 2000, or at least about 3000, or at least about 4000, or at least about 5000, or at least about 6000, or at least about 7000, or at least about 8000, or at least about 9000, or at least about 10 000 NK cells may be sampled. In one example, 2000 NK cells are sampled for NK subset determination. In another example, 10 000 NK cells may be sampled to measure activation.

It will be understood by a person skilled in the art that any suitable method may be used to determine the level of NK cell and/or activity in a subject (including in a blood sample taken from a subject) as long as it is equivalent to an NK cell proportion of total peripheral lymphocytes and an activated NK cell count as determined by the methods disclosed herein, such as the methods disclosed in the examples herein. Thus, the methods disclosed herein may suitably be performed by determining the level and activity of NK cells by following the method of King et al., 2010. It will also be appreciated by the person skilled in the art, that the level of NK cell and/or activity may be determined by the same party or by separate parties.

The level and activity of NK cells may be measured in a biological sample taken from the subject. The biological sample may comprise of one or more cells derived from the subject. In one example, the biological sample is blood. Any of the methods disclosed herein may comprise a step of taking a biological sample from a subject and determining the level and/or activity of NK cells in the sample. Any suitable pre-treatment steps may be applied to the blood sample before the NK cell level and activity is determined.

In any of the methods disclosed herein, the NK cell proportion of total peripheral lymphocytes may be at least 12%, or at least 13%, or at least 14%, or at least 15%, or at least 16%, or at least 17%, or at least 18%, or at least 19%, or at least 20%. In one example, the NK cell proportion of total peripheral lymphocytes may be at least 12%. In one example, the NK cell proportion of total peripheral lymphocytes may be between 12-18%. In another example, the NK cell proportion of total peripheral lymphocytes may be at least 18%. These threshold levels have been shown herein, for the first time, to be particularly informative indicators of an increased likelihood of improved reproductive outcomes following administration of a suitable immune therapy.

In any of the methods disclosed herein, an informative threshold level of the activated NK cell count may be below 12 xlO ⁶ /L, or at least 12 xlO ⁶ /L. In one example, the activated NK cell count is at least 12 xlO ⁶ /L.

In any of the methods disclosed herein, the NK cell proportion of total peripheral lymphocytes may be at least 12% and the activated NK cell count may be at least 12 xlO ⁶ /L. Alternatively, the NK cell proportion of total peripheral lymphocytes may be between 12- 18% and the activated NK cell count may be at least 12 xlO ⁶ /L. In a further alternative, the NK cell proportion of total peripheral lymphocytes may be at least 18% and the activated NK cell count may be at least 12 xlO ⁶ /L.

The present disclosure also provides a method of calibrating a new threshold level for a particular patient population (e.g., of a particular geographical region) that is equivalent to the threshold levels determined herein, which are based on a heterogeneous Australian population. Thus, for example, a representative sample of a particular patient population can be subjected to the same assays disclosed herein for determining NK cell level and activity, and the determined measurements can be used to prepare alternative threshold levels for the performance of the methods disclosed herein on subjects from that particular patient population. For example, if a patient population from a particular geographical region has a baseline NK cell level and/or activity that is higher than the Australian population, the methods disclosed herein can be calibrated using a representative sample of subjects from that new, geographically defined population to provide new threshold levels appropriate for that population that are equivalent to the threshold levels disclosed herein.

Methods of subject selection

The inventor has surprisingly shown for the first time that the administration of immune therapy to women with: i) an NK cell proportion of total peripheral lymphocytes of at least 12%; and ii) an activated NK cell count of at least 12 xlO ⁶ /L, in their blood increases the probability of live birth.

Based on this finding, the inventor has developed and provides herein at least: (i) methods of selecting a subject as suitable for immune therapy; (ii) methods of increasing the probability of live birth in a subject; (iii) methods of reducing the risk of implantation failure during assisted reproduction in a subject and (iv) methods of predicting pregnancy outcome in a subject.

The probability of live birth may be increased any measurable amount. For example, the probability of live birth may be increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or at least 55% following administration of immune therapy.

The risk of implantation failure may be reduced by any measurable amount. For example, the risk of implantation failure may be reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or at least 55% following administration of immune therapy.

Adjunct therapy

Adjunct therapy is often given to subjects undergoing assisted reproduction. In one example, the subject has been treated with adjunct therapy to improve live birth rate. In another example, the subject is undergoing adjunct therapy to improve live birth. Adjunct therapies to improve live birth rate are known in the art. Suitable adjunct therapies include, but are not limited to, low dose aspirin, acupuncture, folate, melatonin, metformin, EmbryoGlue, granulocyte colony stimulating factor (G-CSF) and high dose progesterone. Thus, in any of the methods disclosed herein, the adjunct therapy may comprise any one or more of low dose aspirin, acupuncture, folate, melatonin, metformin, EmbryoGlue, granulocyte colony stimulating factor (G-CSF) or high dose progesterone. In one example, the adjunct therapy comprises acupuncture.

When other therapeutic agents are employed in combination with those disclosed herein, they may be used for example in amounts as noted in the Physicians’ Desk Reference (PDR) or as otherwise determined by one of ordinary skill in the art.

It will be understood, however, that the specific dose level and frequency of dosage for any particular subject may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, gender, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the subject undergoing therapy.

Examples

Example 1.

This study is a first retrospective review of the successes and failures of a treatment protocol known as “the Bondi protocol” previously administered to women undergoing embryo transfers by a single clinician (GS) at IVF Australia between February 2016 and April 2020. This included 1806 transfer cycles across 853 patients: 467 cycles with Bondi protocol treatment and 1339 ‘normal’ cycles without Bondi protocol treatment.

In this clinical study, repeated IVF failure was defined as two or more blastocyst transfer failures. Couples were investigated with a comprehensive endocrine, thrombophilia and autoantibody screen for the women (full blood count, cardiolipin antibodies, lupus anticoagulant, activated protein C resistance, protein C, protein S, antithrombin III, prothrombin gene mutation, Factor V Leiden mutation, MTHFR mutation, thyroid antibodies and hormone, fasting glucose, insulin and homocysteine), karyotype screening for both partners, hysteroscopy and endometrial sampling, and sperm DNA fragmentation testing. After three or more transfer failures, women were offered a further blood test for natural killer activity as described below.

As described elsewhere, fresh IVF/ICSI cycles were routine antagonist (80%), long protocol (6%) or flare protocol (14%). Frozen embryo transfer cycles were routine natural (30%), stimulated (64%) or hormone replacement (6%). There were eleven cycles associated with egg thaws.

Ages reported in this study refer to age of the egg at fertilisation. For fresh cycles, this was the age of the woman at the start of her IVF cycle. For frozen cycles, this was the age of the woman at egg collection and freezing.

For embryo quality analysis, using the Gardner criteria (Gardner et al., 2015), two categories of ‘good embryos’ and ‘poor embryos’ were formed, combining stage and grade. ‘Good embryos’ were defined as stage 3 and above with AA, AB, BA, or BB combinations, or frozen collapsed embryos with higher than 60% cell survival rate. Everything else was defined as ‘poor’ .

Clinical notes were used to determine previous transfer history prior to the study timeframe and often at previous clinics. This information was used as background only. Cycle analysis was performed on all cycles done at IVF Australia in the study period without exception. Many patients continued with therapy after the endpoint of this study.

As previously described in King et al., 2010, blood NK (bNK) cell test results consisted of a combination of the total NK cell count as a percentage of lymphocytes and the concentration of activated NK cells. Briefly, blood samples were taken in the mid-luteal phase. According to one protocol, prior to staining for surface marker analysis, a white cell count and lymphocyte percentage was obtained from the Sysmex XE2100 Haematology analyser (Roche Diagnostics). Blood samples were collected in Streck Cyto-Chex BCT cell stabilisation tubes (Streck, La Vista, New England, USA), gently mixed, then transported to the lab at room temperature. Surface marker analysis on peripheral blood samples was performed using direct immunofluorescence and a lyse/wash protocol and was performed within 4 h or 36 h of collection. Four tubes were set up per patient using the mouse IgG PE- Cy 7, mouse IgGi FITC monoclonal antibodies to CD45-PerCP or CD45-APC-H7, CD3- APC, CD19-APC, CD56-PE, CD69-FITC, CD14-PerCP and CD16-Pe-Cy 7 (Becton Dickinson, San Jose, CA). According to one protocol, CD14-PE and CD16-FITC (Immunotech, Marseilles, France) were also used. Isotype and matched controls (Becton Dickinson) were used as negative controls. Antibodies were used at the volumes recommended by the manufacturer and incubated for 10 min with a volume of peripheral blood to give a concentration of 1X10' ⁶ white cells per tube. Cells were then lysed and fixed with FACS lysing solution (Becton Dickinson) for 15 min before being washed three times prior to analysis. Flow cytometric analysis was performed on the Becton Dickinson FACSCalibur or FACSVerse dual laser flow cytometer (488/633 nm) using CellQuest software (Becton Dickinson). According to one protocol, white cell counts were performed on the FACSVerse flow cytometer. Correlation has been performed between the FACSVerse and the FACSLyric systems (Becton Dickinson). Either can suitably be used. For NK subset determination, 2000 NK cells (CD3 negative, CD56 positive) were collected, and 10 000 NK cells for measurement of activation using CD69. Total NK cells and the CD56 ^+Dim subset were expressed as a percentage of lymphocytes as well as absolute number. The activated CD69 ⁺ CD56 ⁺ Dim subset was expressed as a percentage of CD56 ^+Dim cells as well as an absolute number.

Considering both numbers, three clinical categories were created for NK cell activity: normal, borderline, and high. A ‘normal’ NK result was where the total percentage was less than 12% and the activated count was less than 12 xlO ⁶ /L. A ‘high’ result was where the total percentage was over 18% and the activated count was over 12 xlO ⁶ /L. And a ‘borderline’ result was where one or other marker was not ‘normal’ but the combination did not reach criteria for ‘high’.

Women with a clinical history of repeated transfer failure (at least two as described above) and ‘high’ or ‘borderline’ blood NK cell activity were offered empirical therapy with the ‘Bondi protocol’ for their next treatment cycles. In the Bondi protocol, women take 10 mg of prednisolone daily from the start of the cycle. This is increased to 20mg the day after egg collection or ovulation, and 20 mg clexane injections are started. Prednisolone is continued until 12 weeks of pregnancy or a negative pregnancy test, and clexane injections are continued until 16 weeks of pregnancy or a negative pregnancy test.

Statistical analyses

Statistical analysis was performed on GraphPad Prism software, using Fisher’s exact and Mann-Whitney u-tests.

Ethical approval

The study was approved by the IVF Australia R&D Committee. Results

Cycles

13 patients involving 20 cycles were excluded from analysis due to incomplete information, leaving 1786 cycles over 840 patients as the basis for the study. An overall summary is shown in Table 1.

Table 1. Overview of the database

¹ Bondi/normal age p=0.0336

² Bondi/intralipid age p=0.0008 ³ normal/intralipid age p=<0.0001 ⁴ Bondi/normal transfers p=<0.0001 ⁵ normal/intralipid transfers p=0.0003

* ¹ Age refers to the age of the egg at fertilisation * ² IVF/ICSI are for fresh transfers only

* ³ is calculated based on different between pregnancy and live birth, including both biochemical and clinical pregnancy losses

Amongst all women in the study, the mean age was 37.0, 40% of transfers were fresh, 76% were single embryo transfers and the remainder were all double, 20% of cycles were with euploid embryos, and live birth rate was 27% per cycle.

There were no significant differences in pregnancy rates or live birth rates between ‘Bondi’ (32%, 26%) and ‘normal’ (34%, 28%) cycles. However, women (or the ages of the eggs at fertilisation) undergoing ‘Bondi’ cycles were significantly older than those undergoing ‘normal’ cycles. ‘Bondi’ cycles also had significantly more double embryo transfers, although the multiple pregnancy rate was low (1.7%) and the same across both groups. In a sub-analysis of single euploid embryo transfer cycles, the pregnancy and livebirth rates for ‘Bondi’ (41%, 36%, n=90) and ‘normal’ cycles (43%, 39%, n=241) were also not significantly different.

An alternative immune therapy - intralipid - was trialled on its own in 52 cycles in this study, and these were excluded from the main analysis of the study. Intralipid cycles were associated with significantly higher mean age and more double embryo transfers. The live birth rate for those intralipid cycles was relatively low (15%) but not significantly different to Bondi cycles (26%). A further 72 cycles had joint therapy of Bondi protocol and intralipid and were regarded as simply Bondi cycles.

Table 2 shows the live birth rates in fresh IVF, fresh ICSI and frozen transfer cycles. The highest success rates were in the frozen groups for both ‘Bondi’ and ‘normal’, and those included all euploid embryo transfers. In a sub-analysis of women under age 40, there was no difference in IVF, ICSI and frozen success rates in ‘normal’ cycles. But in ‘Bondi’ cycles the ICSI group had a significantly lower live birth rate (19%) than both IVF (50%) and frozen (34%). 40 yrs IVF/ICSI p=0.0048 40 yrs ICSI/frozen p=0.0215

³ normal all IVF/frozen p=0.0199

⁴ normal all ICSI/frozen p=0.0077

Egg age was further examined across three groups: <38 years, 38-39.9 years, and >40 years. Pregnancy and livebirth rates within each age group are shown in Figure la. In both ‘Bondi’ and ‘normal’ groups, livebirth rates were significantly higher in the <38 group, at 33% and 36%, dropping to 10% and 13% in the >40 group. Livebirth rates were higher in ‘Bondi’ cycles (32%) for women aged 38-39.9 compared to normal cycles (20%). In euploid transfer cycles, the pregnancy and livebirth rates remained steady across all age groups (Figure lb). Miscarriages occurred in both ‘Bondi’ cycles and euploid transfer cycles at a rate of 5-9%, with no significant differences.

Patient groups

Excluding nine patients who had intralipid cycles, there were 831 patients (1734 cycles) who were divided into three groups: the ‘Bondi’ group (n=160) consisting of patients who only ever had ‘Bondi protocol’ cycles, the ‘normal’ group (n=573) who only ever had ‘normal’ cycles, and the mixed group (n=98) who had both kinds of cycles. Note this only accounted for cycles completed within the timeframe of this study. Their characteristics are displayed in Table 3.

Table 3 , Outcomes and number of cycles for the three patient groups

'normal/mixcd age p=<0.0001

² bondi/normal previous cycles p=<0.0001

³ bondi/mixed previous cycles p=<0.0001

⁴ normal/mixed live birth p=0.0289

⁵ bondi/mixed mean number of cycles p=<0.0001

⁶ normal/mixed mean number of cycles p=<0.0001

During the study timeframe, livebirth rates were 52% for Bondi patients, 54% for normal patients and 42% for mixed patients. Bondi patients had significantly more previous cycles (3.5) than the normal group (1.6) and the mixed group (2.3). During the study timeframe, the mixed group had significantly more cycles (3.85) than both the Bondi group (1.76) and the normal group (1.88).

Combining previous and current therapy, women in the Bondi group had significantly more cycles (5.3) than the normal group (3.5, p=<0.0001). The mixed group had the highest overall number of cycles (6.1).

The pregnancy and livebirth rates were assessed for the three patient groups in Figure 2a, categorised by age. During the study timeframe, 62% of women on the Bondi protocol in the <38 group had a live birth, dropping to 55% for ages 38-39.9 and 20% for >40. This was achieved with cycle success rates of 40% for <38, 36% for 38-39.9, and 12% for >40 (Figure 2b). This was not significantly different to women on normal protocols.

Live birth outcomes

Overall 54% (n=428) of patients achieved a live birth within the timeframe of the study and 46% (n=369) did not (Table 4). Across all three patient groups (Bondi, normal and mixed), patients who failed had a much higher mean age of 38.7, compared to 35.4 in the live birth group. It was noteworthy that in the Bondi group, patients who failed also had significantly fewer cycles (1.6 vs 1.9).

Table 4. Comparison of successful and unsuccessful patients

' Bondi age p=<0.0001

² normal age p=<0.0001

³ mixed age p=<0.0001

⁴ Bondi cycles p=0.0246

Mixed group

Mixed group patients had tried both ‘Bondi’ and ‘normal’ therapies during the timeframe of the study. For this subgroup, embryo quality was assessed as an additional variable. There was no significant difference in the percentage of ‘good’ embryos in Bondi cycles (77.5%) versus normal cycles (78.3%). For all embryos that succeeded, 41/43 (95%) were ‘good embryos’; whereas, for embryos that failed, only 206/274 (75%) were ‘good embryos’ (p=0.0015). For Bondi cycles that succeeded, 26/28 embryos (93%) were categorised as ‘good embryos’, compared with 15/15 (100%) ‘good embryos’ in ‘normal’ cycles that succeeded.

In this mixed group, 23/38 (82%) patients had between one and four failed normal cycles, followed by success with the ‘Bondi protocol’. The other fifteen women experienced a mixed approach of ‘Bondi’ and ‘normal’ cycles, with three more patients succeeding on a Bondi cycle, and the remaining twelve succeeding on normal cycles.

NK cell test results Overall 554 women in the database had NK cell testing (66%). Clinical interpretation was 46% normal, 39% borderline and 15% high.

All 98 patients in the mixed group received an NK cell test, 158 patients in the Bondi group received an NK cell test, and 298/573 (52%) patients in the normal group had an NK cell test. Tables 5-8 represent patients with NK cell tests only.

Most patients in the normal group had normal NK cell tests with only 3% having high NK levels (Table 5). In contrast, in the Bondi group 35% had high bNK cell activity and only 13% had normal bNK cell activity. The mixed group had a more even spread.

Pregnancy outcomes according to NK result and treatment group are shown in Table 5.

Table 5, Clinical interpretation of the NK cell test results Table 6 shows the outcomes associated with NK cell results.

Table 6, Outcomes associated with NK cell results per patient group

In the mixed group, livebirth outcomes were even across all three NK groups (38%-44%). In the ‘Bondi’ group, the livebirth rates increased from normal NK (40%) to borderline NK (58%) and high NK (49%). In the ‘normal’ group, the livebirth rates decreased from normal NK (50%) to borderline NK (42%) to high NK (11%). The livebirth rate of 11% in the high NK group was significantly lower than the 50% in the normal NK group (p=0.036).

In women with high NK cell levels, livebirth rates in the ‘Bondi’ group were higher (49%) than the normal group (11%), but this did not account for the higher number of cycles in the Bondi group (1.8 vs 1.3).

Cycle data and NK test results were further explored in Table 7.

Table 7, Outcomes associated with NK cell results per cycle

'mixed group Bondi/normal high NK live birth rate p=0.0412

Within the mixed group, there was a trend favouring the ‘Bondi’ protocol in the borderline and high NK groups. Livebirth rates were 12% and 8% for ‘Bondi’ and ‘normal’ cycles respectively for women with normal bNK activity, 17% and 8% for those with borderline bNK activity, and 23% and 5% forthose with high bNK activity. This was statistically significant.

A similar trend was observed comparing the ‘Bondi’ and ‘normal’ groups. In the normal NK group, livebirth rates were 22% for the Bondi group and 26% for the normal group. This was reversed in the borderline NK group, with livebirth rates of 36% and 25% for Bondi and normal cycles, and even greater divergence in the high bNK group with rates of 30% and 8%.

In a sub-analysis of women under 40 years old, this trend of increasing success with the Bondi protocol with increasing NK cell activity was even stronger. The difference in success rates between ‘Bondi’ and ‘normal’ cycles increased from -2% (29% vs 31%) for normal NK, to 13% (44% vs 31%) for borderline NK, to 26% (37% vs 11%) for high NK. This was in favour of Bondi protocol therapy and amounted to over a 3 -fold difference.

Combining cycles from all three patient groups, larger numbers were obtained. Outcomes associated with all ‘Bondi’ and ‘normal’ cycles are shown in Table 8.

Table 8, Outcomes associated with Bondi cycles and normal cycles borderline NK pregnancy rate p=0.0117 borderline NK live birth rate p=0.0098 ³ high NK pregnancy rate p=0.0072 ⁴ high NK live birth rate p=0.0007

In the normal NK group, livebirth outcomes were better in ‘normal’ cycles (23%) than ‘Bondi’ cycles (16%), though not significant. However, there was a striking and highly significant switch in women with abnormal bNK activity. In those with borderline bNK results, the livebirth rate was significantly higher with the Bondi protocol (28%) than normal protocols (18%, p<0.01). And in those with high bNK results, the livebirth rate with the Bondi protocol was also 28%, but in normal protocols it was only 6% (p<0.001). In this analysis, 672 cycles were analysed in women with borderline and high bNK results, 184 of them in the high group. The impact of the Bondi protocol in women with increasing bNK activity, culminating in an over 4-fold difference in livebirth outcomes for the high NK group is illustrated in Figure 3, demonstrating that immune therapy can improve the probability of live birth.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

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