WO2020160612A1

WO2020160612A1 - Uterine receptivity

Info

Publication number: WO2020160612A1
Application number: PCT/AU2020/050087
Authority: WO
Inventors: Laura Lindsay; Samson Dowland; Sadaf Kalam; Christopher Murphy; Leigh Nicholson
Original assignee: University of Sydney
Current assignee: University of Sydney
Priority date: 2019-02-05
Filing date: 2020-02-05
Publication date: 2020-08-13
Anticipated expiration: 2021-08-05

Abstract

The present disclosure relates to diagnostic methods for determining uterine receptivity, comprising detecting the presence or absence of SNAP23 in a biological sample; wherein the presence of SNAP23 is indicative of uterine receptivity and the absence of SNAP23 is indicative of uterine nonreceptivity.

Description

Uterine receptivity

Field of the invention

The present disclosure relates to methods and compositions for determining uterine receptivity.

Related application

The present application claims priority from Australian provisional application AU 2019900342, the contents of which are hereby incorporated by reference in their entirety.

Background of the invention

Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of the common general knowledge in the field.

Blastocyst implantation into the uterus involves coordinated dialogue between the blastocyst and a receptive endometrium. The endometrium, and particularly the luminal uterine epithelial cells (UECs), undergo specific morphological changes which establish a receptive uterus.

The lining of the uterus is only receptive to an implanting blastocyst for a short period time during each oestrus cycle (or cycle menstrual cycle in the case of humans). In humans, this window of uterine receptivity is generally around days 19 to 23 of a typical 28-day menstrual cycle, corresponding to approximately days 5 to 9 post ovulation.

To improve the success of assisted reproductive technologies (ART) such as in vitro fertilisation (IVF), clinicians have attempted to determine when a uterus is likely to be receptive to blastocyst implantation. One approach for determining uterine receptivity involves invasive biopsy which can be uncomfortable and even painful for the female subject. Moreover, although such biopsies may provide useful insight into whether a uterus is likely to be receptive, they can damage the uterine surface and inhibit blastocyst transplantation. Thus, it is unlikely that embryo transfer or implantation could be performed within the same cycle after a biopsy has been taken. A less invasive approach for determining uterine receptivity involves measuring changes in hormone levels to determine the timing of ovulation. However, these methods are often imprecise and typically only enable determination of a broad window of time in which the uterus is receptive for blastocyst implantation.

In this context, there is a need for compositions and methods for determining uterine receptivity.

Summary of the invention

The present invention relates to the observation that the expression and distribution of soluble NSF attachment receptor (SNARE) proteins changes as a uterus becomes receptive to embryo implantation. In work leading to the present invention, the inventors surprisingly found that the SNARE protein, synaptosomal-associated protein 23 (SNAP23), is secreted by uterine epithelial cells into the uterine lumen as a uterus becomes receptive. The increased presence of SNAP23 in uterine luminal fluid (ULF) may therefore be used as a marker for determining uterine receptivity. Advantageously, the levels or amounts of SNAP23 in ULF may be detected using non-invasive techniques and without causing damage to the uterine surface.

Accordingly, in one aspect, the present invention provides a method of determining uterine receptivity in a female subject, the method comprising a step of detecting the presence or absence of SNAP23 in a biological sample obtained from the female subject, wherein the presence of SNAP23 in the sample is indicative of uterine receptivity and the absence of SNAP23 in the sample is indicative of uterine non receptivity.

More particularly, the present invention provides a method for determining the uterine receptivity profile of a female subject, the method comprising:

- providing a plurality of biological samples obtained from the female subject at different time points during the subject’s oestrous or menstrual cycle;

- determining the level or amount of SNAP23 in the samples;

- wherein the samples having the highest level or amount of SNAP23 among the plurality of samples, is indicative of a time point in the subject’s cycle at which the uterus is receptive to implantation and the samples having the lowest level or amount of SNAP23 is indicative of a time point in the subject’s cycle at which the uterus is not receptive to implantation; thereby determining the uterine receptivity profile of the subject.

Preferably, the plurality of samples comprises samples from at least 3, at least 4, at least 5, or more, different time points during the oestrus or menstrual cycle of the subject. The different time points may include time points during the pre-ovulation period of the subject. In alternative embodiments, where the subject is a human female, the different time points preferably include at least one time point in the post-ovulation period, preferably in the period 5-9 days post-ovulation. Most preferably, the different time points include at least 3 different days in the time period corresponding to prior to ovulation, at ovulation, and after ovulation (for example at the time of egg pick-up). In particularly preferred embodiments, the time period would include daily or half daily time points in the period of 5-9 days post-ovulation (corresponding to between days 19-23 of a 28 day menstrual cycle).

In preferred embodiments, the uterine receptivity profile can be used to determine a baseline level of SNAP23 in the female subject, preferably wherein the baseline level of SNAP23 is in uterine tissue and/or uterine luminal fluid.

In certain embodiments, the uterine receptivity profile of the subject can be used to predict the timing, or to determine the likelihood of successful implantation of an embryo (e.g., a blastocyst). Preferably, the subject is predicted or determined to have the greatest (or optimum) uterine receptivity at the time point in the profile having the highest level or amount of SNAP23.

Accordingly, the present invention provides a method for determining the uterine receptivity of a female subject, the method comprising:

- providing a female subject for whom uterine receptivity is to be determined;

- determining the level or amount of SNAP23 in a test biological sample obtained from the female subject;

- comparing the level or amount of SNAP23 in the test sample to the level or amount of SNAP23 in a control in the form of data representative of levels of SNAP23 in a non-receptive uterus; determining that the subject has a receptive uterus if the level or amount of SNAP23 in the test sample is higher than the levels of SNAP23 in the control; or determining that the subject does not have a receptive uterus if the level or amount of SNAP23 in the test sample is the same or lower than the level or amount of SNAP23 in the control.

Alternatively, the present invention provides a method for determining the uterine receptivity of a female subject, the method comprising:

- providing a female subject for whom uterine receptivity is to be determined;

- comparing the level or amount of SNAP23 in the test sample to the level of SNAP23 in a control in the form of data representative of the level or amount of SNAP23 in a receptive uterus; determining that the subject has a receptive uterus if the level or amount of SNAP23 in the test sample is the same or higher than the levels of SNAP23 in the control; or determining that the subject does not have a receptive uterus if the level or amount of SNAP23 in the test sample is lower than the level of SNAP23 in the control.

Preferably, the biological sample is uterine luminal fluid (ULF). Accordingly, the present invention provides a method for determining the likelihood of uterine receptivity of a female subject, the method comprising: - providing a female subject for whom uterine receptivity is to be determined;

- determining the level or amount of SNAP23 in a test sample of uterine luminal fluid obtained from the female subject;

- comparing the level or amount of SNAP23 in the test sample to the level or amount of SNAP23 in a control in the form of data representative of level or amount of SNAP23 in a non-receptive uterus; determining that the subject has a receptive uterus if the level or amount of SNAP23 in the test sample is higher than the level of SNAP23 in the control; or determining that the subject does not have a receptive uterus if the level or amount of SNAP23 in the test sample is the same or lower than the level of SNAP23 in the control.

In another aspect, the present invention provides a method of treating a female subject undergoing assisted reproduction, the method comprising:

- determining the level or amount of SNAP23 in a biological sample obtained from the female subject; and

- preparing the female subject for embryo implantation if SNAP23 is determined to be present in the biological sample.

Preferably, the female subject is prepared for embryo implantation if the level of SNAP23 in the biological sample is determined to be higher than the level or amount of SNAP23 in a control. Preferably the control is in the form of data representative of SNAP23 levels in a receptive or non-receptive uterus. Accordingly, the invention provides a method of providing assisted reproduction in a female subject, the method comprising:

- providing a female subject in need of assisted reproduction;

- determining the levels of SNAP23 in a test biological sample obtained from the female subject; and

- comparing the level or amount of SNAP23 in the test sample to the level or amount of SNAP23 in a control in the form of data representative of level or amount of SNAP23 in a non-receptive uterus;

- preparing the female subject for blastocyst implantation if the level of SNAP23 in the test sample is higher than the level of SNAP23 in the control.

In any embodiment, the control data corresponds to data on the level of SNAP23 from the female subject at an earlier time point in her oestrous or menstrual cycle, preferably from a time point before ovulation or immediately after ovulation.

In some examples, the step of preparing the female subject for embryo (blastocyst) implantation comprises inserting a catheter into the uterine cavity of the female subject. In some examples, the method further comprises implanting an embryo into the uterus of the female subject. In preferred examples, the embryo is a blastocyst.

Preferably, preparing the female subject for blastocyst implantation comprises transferring the blastocyst to the uterus of the subject.

In another aspect, the present invention provides a method of improving a step of assisted reproduction in a female subject, or for increasing the likelihood of successful implantation of a blastocyst, the method comprising:

- providing a female subject who is a candidate for implantation of a blastocyst and who is suspected of having a receptive uterus;

- comparing the level or amount of SNAP23 in the test sample to the level or amount of SNAP23 in a control sample representative of baseline levels of SNAP23 in the uterine luminal fluid of the subject; determining that the subject has a receptive uterus if the level or amount of SNAP23 in the test sample is higher than the levels of SNAP23 in the control sample and proceeding to implant the blastocyst in the subject; or determining that the subject does not have a receptive uterus if the level or amount of SNAP23 in the test sample is the same or lower than the levels of SNAP23 in the control sample and determining not to implant the blastocyst in the subject, thereby improving a step of assisted reproduction or increasing the likelihood of successful implantation of the blastocyst.

The present invention therefore also provides a method for providing an assisted reproduction technology (ART) procedure to a female subject, the method comprising:

- providing a female subject who is a candidate for implantation of an embryo and who is suspected of having a receptive uterus;

- determining the level or amount of SNAP23 in a test sample of uterine luminal fluid obtained from the female subject; - comparing the level or amount of SNAP23 in the test sample to the level or amount of SNAP23 in a control sample representative of baseline levels of SNAP23 in the uterine luminal fluid of the subject; determining that the subject has a receptive uterus if the level or amount of SNAP23 in the test sample is higher than the levels of SNAP23 in the control sample and proceeding to implant the blastocyst in the subject; or determining that the subject does not have a receptive uterus if the level or amount of SNAP23 in the test sample is the same or lower than the levels of SNAP23 in the control sample and determining not to implant the blastocyst in the subject, thereby providing an ART procedure to the female subject.

Further still, the present invention provides a method for providing an assisted reproduction technology (ART) procedure to a female subject, the method comprising:

- providing a female subject who is a candidate for implantation of an embryo;

- determining the uterine receptivity of the subject by:

o obtaining or having obtained a test sample of uterine luminal fluid from the subject;

o determining or measuring the levels of SNAP23 in the test sample; o comparing the levels of SNAP23 in the test sample to the levels of SNAP23 in a control sample representative of baseline levels of SNAP23 in the uterine luminal fluid of the subject;

- if the levels of SNAP23 in the test sample are higher than the levels of SNAP23 in the control sample, then transferring the embryo to the uterus of the subject;

- if the levels of SNAP23 in the test sample are the same or lower than the levels of SNAP23 in the control sample, then storing the embryo for transfer at a later date, thereby providing an ART procedure to the female subject.

Preferably, the embryo is a blastocyst. The subject may be suspected of having a receptive uterus based on any measure conventionally used in the art to predict uterine receptivity. In certain embodiments, the subject may be suspected of having a receptive uterus based on a measurement of hormone levels in the subject, the time since ovulation, or the particular time in the oestrous or menstrual cycle of the subject.

In any embodiment of the above described methods, the step of determining the level or amount of SNAP23 in the biological sample may comprise detecting or quantifying the level or amount of SNAP23 in the biological sample. Alternatively the step of determining the level or amount of SNAP23 may comprise observing the level or amount of SNAP23 provided in a database. In some examples, the step of quantifying the level or amount of SNAP23 in a biological sample comprises measuring the amount of SNAP23 the sample and normalising this amount to a reference standard.

In any embodiment, a“higher” or greater level or amount of SNAP23 compared to the control sample or baseline level, will be understood to include a level or amount that is at least about a 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, or 5-fold or more, increase compared to the amount in the control or baseline level.

In any embodiment, a“lower” or lesser level or amount of SNAP23 compared to a reference level, control or baseline level, will be understood to include a level or amount that is at least about a 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, or 5-fold, or more, decrease compared to the amount in the control or baseline level.

In any embodiment, a“higher” or greater level or amount of SNAP23 compared to a reference level, control or baseline level, will be understood to include a level or amount that is at least about 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more, greater than the amount in the control or baseline level..

In any embodiment, a“lower” or lesser level or amount of SNAP23 compared to a reference level, control or baseline level, will be understood to include a level or amount that is at least about 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more, less than the amount in the control or baseline level.

In any embodiment, an amount or level that is the“same as” the level or amount of SNAP23 compared to a reference, control or baseline level will be understood to be a level or amount that is no more than about 1 %, 2%, 3%, 4%, 5% or 10% more or less than the control or baseline level.

In certain examples, the levels of SNAP23 may not be detectable in the biological sample, indicating that the subject does not have a receptive uterus at that time. In further examples, the control sample indicative of the levels of SNAP23 in a non- receptive uterus or indicative of a pre- or post- receptivity of the subject, may not have detectable levels of SNAP23.

In any embodiment, the control, in the form of data representative of levels of SNAP23 in a receptive or non-receptive uterus may be data from one or more individuals for whom uterine receptivity was previously determined. Alternatively, the data may be from the subject for whom uterine receptivity is being determined. In certain embodiments, the control may be data from the subject obtained at an earlier time point in her oestrous or menstrual cycle.

In any embodiment, measuring the level or amount of SNAP23 may be by any method conventional in the art for quantifying a specific protein in a sample. In certain embodiments, the method for determining the level or amount of SNAP23 is selected from the group consisting of: enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, immunofluorescence, latex agglutination, hemagglutination, lateral flow immunoassay, immunoprecipitation, mass spectrometry or Western blot analysis.

In certain embodiments, measuring the levels of SNAP23 may comprise contacting the biological sample with an anti-SNAP23 antibody or an antigen-binding fragment thereof and detecting the formation of a complex between the SNAP23 and anti-SNAP23 antibody. In some examples, the antibody is a monoclonal antibody. The antibody or antigen-binding fragment thereof may be immobilised to a solid support. The antibody or antigen-binding fragment thereof may be conjugated to a detectable label.

In particularly preferred embodiments, the step of detecting the levels of SNAP23 in the biological sample comprises: contacting a first anti-SNAP23 antibody or an antigen-binding fragment thereof with the biological sample such that the first anti-SNAP23 antibody or antigen-binding fragment thereof forms a complex with SNAP23 when SNAP23 is present in the sample; and detecting the presence of the complex.

Preferably, detecting the presence of the complex includes detecting the first anti-SNAP23 antibody or antigen-binding fragment thereof, when the antibody is conjugated to a detectable label. Alternatively, detecting the presence of the complex includes contacting the complex with a second antibody or an antigen-binding fragment thereof, wherein the second antibody or antigen-binding fragment thereof specifically binds to SNAP23 or to the first anti-SNAP23 antibody or antigen-binding fragment thereof and wherein the second antibody or antigen-binding fragment thereof may be conjugated to a detectable label.

The detectable label may be an enzyme (eg, horseradish peroxidase or alkaline phosphatase), a gold nanoparticle, a fluorophore, a radioisotope, a latex bead, a carbon nanoparticle, biotin or a quantum dot

In some examples, the first anti-SNAP23 antibody or antigen-binding fragment thereof is immobilized to a solid support. In certain examples, contacting the first anti- SNAP23 antibody or antigen-binding fragment thereof with the biological sample mobilizes the first anti-SNAP23 antibody or antigen-binding fragment thereof.

In some examples, the second antibody or antigen-binding fragment thereof is immobilized to a solid support.

In another aspect, the present invention provides an assay comprising a step of detecting the presence or absence of SNAP23 in a biological sample obtained from a female subject, preferably for use in accordance with the methods of the invention. In particularly preferred embodiments, the assay comprises determining the levels of SNAP23 in a biological sample obtained from the subject.

In preferred embodiments, the steps of detecting the presence or absence of SNAP23 (or determining the levels of SNAP23) in the biological sample comprise contacting the biological sample with an anti-SNAP23 antibody or an antigen-binding fragment thereof. The antibody may be a monoclonal antibody. The antibody or antigen- binding fragment thereof may be immobilised to a solid support. The antibody or antigen-binding fragment thereof may be conjugated to a detectable label.

In some examples, the step of detecting the presence or absence of SNAP23 in the biological sample comprises: contacting a first anti-SNAP23 antibody or an antigen-binding fragment thereof with the biological sample such that the first anti-SNAP23 antibody or antigen-binding fragment thereof forms a complex with SNAP23 when SNAP23 is present in the sample; and contacting the complex with a second antibody or an antigen-binding fragment thereof, wherein the second antibody or antigen-binding fragment thereof specifically binds to SNAP23 or to the first anti-SNAP23 antibody or antigen-binding fragment thereof, and detecting the presence of the complex; wherein, optionally, the first anti-SNAP23 antibody or antigen-binding fragment thereof is conjugated to a detectable label or the second antibody or antigen-binding fragment thereof is conjugated to a detectable label.

In some examples, the step of detecting the presence or absence of SNAP23 comprises quantifying the level of SNAP23 in the biological sample. The assay may comprise comparing the level of SNAP23 in the biological sample to a reference level of SNAP23. In another aspect, the present invention provides a device for detecting the levels of SNAP23 in a biological sample, preferably for use in accordance with the methods of the invention.

The biological sample may be uterine fluid (i.e., endometrial fluid), vaginal fluid, or peritoneal fluid. Preferably, the biological sample is uterine fluid or vaginal fluid.

In some examples, the device comprises an anti-SNAP23 antibody or an antigen binding fragment thereof.

In some examples, the device is a lateral flow device. The lateral flow device may comprise:

- a pad region for receiving the biological sample, wherein the pad region comprises a first anti-SNAP23 antibody or an antigen-binding fragment thereof; and

- a test region comprising a second anti-SNAP23 antibody or an antigen binding fragment thereof, wherein the first anti-SNAP23 antibody or antigen-binding fragment thereof is mobilizable along the device and the second anti-SNAP23 antibody or antigen-binding fragment thereof is immobilised to the device.

The first anti-SNAP23 antibody or antigen-binding fragment thereof may be conjugated to a detectable label.

The first anti-SNAP23 antibody may be a monoclonal antibody. The second anti- SNAP23 antibody may be a monoclonal antibody.

The second anti-SNAP23 antibody or antigen-binding fragment thereof may be conjugated to a detectable label.

The present invention also provides a kit for detecting the presence or absence, or levels of SNAP23 in a biological sample such as uterine fluid or vaginal fluid, for use according to a method as described herein. The kit may include an anti-SNAP23 antibody or antigen-binding fragment thereof. The anti-SNAP23 antibody or antigen binding fragment thereof may be conjugated to a detectable label, such as an enzyme (eg, horseradish peroxidase or alkaline phosphatase), a gold nanoparticle, a fluorophore, a radioisotope, a latex bead, a carbon nanoparticle, biotin or a quantum dot, or the kit may comprise a second antibody or fragment which is conjugated to a detectable label and which binds to the anti-SNAP23 antibody or antigen-binding fragment thereof. Preferably the kit also comprises written instructions for the use thereof in a method of the invention.

In further embodiments, the methods of the present invention comprise detecting the presence or absence of a SNARE protein in a biological sample obtained from the female subject, preferably detecting the levels of a SNARE protein or SNARE complex in the biological sample, and comparing the levels to a control in order to determine a relative level of the SNARE protein or SNARE complex.

In some examples, the step of detecting the levels of the SNARE protein or SNARE complex comprises quantifying expression of a gene encoding the SNARE in a biological sample. The biological sample may comprise cells obtained from the uterus of the subject, including cells obtained directly from tissue, or indirectly (e.g., cells present in a fluid sample obtained from the subject, such as vaginal or uterine fluid). Expression of the gene may be quantified using polymerase chain reaction, nucleic acid sequencing or a microarray.

In some examples, the step of detecting the levels of the SNARE protein or SNARE complex in the biological sample comprises contacting the biological sample with an anti-SNARE antibody or an antigen-binding fragment thereof. The antibody may be a monoclonal antibody. The antibody or antigen-binding fragment thereof may be immobilised to a solid support. The antibody or antigen-binding fragment thereof may be conjugated to a detectable label, optionally wherein the detectable label is selected from an enzyme (eg, horseradish peroxidase or alkaline phosphatase), a gold nanoparticle, a fluorophore, a radioisotope, a latex bead, a carbon nanoparticle, biotin or a quantum dot.

In some examples, the step of detecting the levels of the SNARE protein or SNARE complex in the biological sample comprises: contacting a first antibody or an antigen-binding fragment thereof specific to the SNARE protein or a protein in the SNARE complex with the biological sample such that the first antibody or antigen-binding fragment thereof forms a complex with the SNARE protein/complex when the SNARE protein/complex is present in the sample; and contacting the complex with a second antibody or an antigen-binding fragment thereof, wherein the second antibody or antigen-binding fragment thereof specifically binds to the SNARE protein/complex or to the first antibody or antigen-binding fragment thereof.

In some examples, the first antibody or antigen-binding fragment thereof is conjugated to a detectable label.

In some examples, the second antibody or antigen-binding fragment thereof is conjugated to a detectable label.

In some examples, the first antibody or antigen-binding fragment thereof is immobilized to a solid support. Contacting the first antibody or antigen-binding fragment thereof with the biological sample may mobilize the first antibody or antigen binding fragment thereof.

In some examples, the method comprises comparing the level of the SNARE in the biological sample to a reference level of the SNARE.

Preferably, the SNARE protein is a component of a t-SNARE complex. Preferably, the t-SNARE component is SNAP23 or a syntaxin (such as syntaxin-2, syntaxin-3 or syntaxin-4).

In any embodiment of the invention, the female subject is preferably a human subject. In other embodiments, the female subject is a non-human subject in need of assisted reproductive technology. The non-human subject may be selected from the group consisting of: a non-human primate, a canine (dog), feline (cat), murine (mouse or rat), guinea pig, hamster, bovine (cow), equine (horse), ovine (sheep), caprine (goat) or other.

In some examples, the method further comprises the step of obtaining the biological sample from the female subject.

It will be understood that any method described herein can be performed ex vivo methods or in vitro for the determination of SNAP23.

In certain embodiments, the method comprises the use of device for insertion into the uterus and for measurement of the levels of SNAP23 in the subject (such as a fibre- optic device adapted to detect SNAP23 or to obtain samples of luminal fluid in which SNAP23 levels can be determined).

As used herein, the terms“level” or“amount” of SNAP23 will be understood to include levels, concentrations or amounts of SNAP23 that are normalised against a reference standard. Preferably the reference standard comprises the total amount of protein in the sample, or the concentration, level or amount of a reference protein in the sample. The reference protein may be a house-keeping protein, such as but not limited to actin, albumin or the like.

In any method of the invention described herein, and unless specifically stated otherwise, the biological sample is selected from the group consisting of: a biopsy, uterine fluid (i.e., uterine luminal fluid which may also be referred to as endometrial fluid or endometrial luminal fluid), vaginal fluid, endometrial fluid or peritoneal fluid. Preferably, the biological sample is uterine fluid or vaginal fluid.

Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.

Brief description of the drawings

Figure 1. Syntaxin-2 localisation in rat uterus. (A, B) Day 1 and 3.5 of pregnancy shows diffuse cytoplasmic localisation of syntaxin-2 in UECs. (C, D) On day 5.5 and 6 of pregnancy, syntaxin-2 is concentrated in the apical region of UECs. (E) On day 7 of pregnancy, syntaxin-2 is apically cytoplasmic. (F) Non-immune control shows no staining in UECs. (G) Unpaired two-tailed Student’s t-test found syntaxin-2 has a significantly greater intensity on day 5.5 compared to day 1 of pregnancy in UECs. (^* P<0.05) n=5; error bar is the mean ± S.E.M. All scale bars are 20pm. (L= lumen, E= epithelium and S= stroma).

Figure 2. SNAP23 localisation in rat uterus. (A, B) On day 1 and 3.5, SNAP23 is diffusely cytoplasmic in UECs. (C, D) On day 5.5 and 6, SNAP23 is concentrated in the apical region of UECs. (E) On day 7, SNAP23 is cytoplasmic. (F) Non-immune control shows no staining in UECs. (G) Unpaired two-tailed Student’s t-test found SNAP23 had a significantly greater intensity on day 5.5 compared to day 1 in UECs. (^*P<0.05) n=5; error bar is the mean ± S.E.M. All scale bars are 20 pm. (L= lumen, E= epithelium and S= stroma).

Figure 3. Western blot analysis of syntaxin-2 and SNAP23 in isolated UECs. (A) Syntaxin-2 is present at 33 kDa in isolated UECs during day 1 , 3.5, 5.5, 6 and 7. (B) SNAP23 is present at 57 kDa in isolated UECs during day 1 , 3.5, 5.5, 6 and 7. b-actin was used as a loading control. (C) Densitometric and statistical analysis (one-way ANOVA) found a significant increase in syntaxin-2 on day 5.5 compared to day 1 (^*P<0.05) n=5. (D) Densitometric and statistical analysis (one-way ANOVA) found a significant increase in SNAP23 on day 5.5 compared to day 1. SNAP23 also showed a significantly higher level of SNAP23 on day 3.5 and 5.5 compared to day 7 (^*P<0.05, ^***P<0.0001 ) n=5. Error bar is the mean ± S.E.M.

Figure 4. Triple labelling of SNAP23 and Phalloidin in UECs at day 5.5. (A) Merged channels showing the localisation of Phalloidin, SNAP23 and nuclei in UECs on day 5.5. (B) Green channel shows SNAP23 (arrow) is present in secretions within the luminal space, outside of the UECs. (C) Red channel shows that Phalloidin (arrow head) localises apically in UECs. (D) Blue channel shows the nuclei of UECs. All scale bars are 20 pm. (L= lumen, E= epithelium and S= stroma). (E) Western blot showing presence of SNAP23 in luminal fluid (lane 1 ) and in UECs (lane 2) from a pregnant rat at day 5.5.

Figure 5. SNAP23 and Phalloidin co-localisation coefficient and controls. (A) Pearson co-localisation coefficient (PCC) scatter plot showing that SNAP23 does not co- localise with Phalloidin, with an average PCC of 0.1 17, n=5. (B) SNAP23 staining showing that there is no cross talk (bleed through) from the red channel. (C) Phalloidin staining showing that there is no cross talk (bleed through) from the green channel. (D) Non- immune control shows no staining in UECs. All scale bars are 20 miti. (L= lumen, E= epithelium and S= stroma).

Figure 6. SNAP23 in UECs in vitro. (A) Maximum intensity projection micrograph of HEC1 A cells shows that SNAP23 is cytoplasmic with prominent punctate staining. (B) Maximum intensity projection micrograph of RL95-2 cells shows that SNAP23 is cytoplasmic. (C, D) Z-slices of HEC1 A and RL95-2 cells show that SNAP23 staining is cytoplasmic and punctate. (E) Non-immune control showed no staining in HEC1 A in vitro. (F) SNAP23 is present at 23 kDa and 57 kDa. b-actin was used as a loading control. (G) Densitometry and statistical analysis (Student’s t-test) showed that SNAP23 significantly decreased in RL95-2 compared to HEC1 A cells, (^** P<0.01 ) n=3.

Figure 7. Transmission electron microscopy of extracellular vesicles in UECs on days 1 , 5.5 and 6 of early pregnancy. Extracellular vesicles were present on days 1 , 5.5 and 6 of pregnancy. (A, B) On day 1 of pregnancy, exosomes (arrows) and microvesicles (MV) were observed within the luminal space of the uterus. (C, D, E, F) On day 5.5 and 6 of pregnancy, microvesicles were observed budding off from the UECs .

Figure 8. Transmission electron microscopy of extracellular vesicles in UECs on day 5.5 of early pregnancy. (A, B) A large number of microvesicles (MV) of various sizes and membrane compositions are secreted into the luminal space on day 5.5 of pregnancy. These microvesicles are found near pinopods (P) and appear to aggregate and accumulate together.

Detailed description of the embodiments

The lining of the uterus is only receptive to an implanting blastocyst for a short period of time during the oestrous or menstrual cycle. In humans, the window of uterine receptivity is approximately days 19 to 23 of a typical 28-day menstrual cycle or around days 5 to 9 post-ovulation. Flowever, these time periods only provide an approximation of the likely time during which the uterine lining (endometrium) is at its most receptive and there is therefore a need for methods which more accurately predict the optimum time for blastocyst implantation. The present inventors have surprisingly found that levels of the protein SNAP23 in the endometrium and endometrial fluid are correlated with increased uterine receptivity. More importantly, the inventors have found that SNAP23 is secreted into the uterine lumen, with peak levels of SNAP23 observed in uterine luminal fluid (ULF) at a time when the uterus is primed for blastocyst implantation. Thus, the present invention is thought to enable a more accurate prediction of the optimum time for blastocyst transfer to the subject and may therefore be useful for increasing the prospects of successful implantation.

A particular advantage of the present invention is the finding by the inventors that SNAP23 is present in the ULF and can therefore be quantified using non-invasive procedures. This provides a significant advantage over existing methods for determining the profile of uterine receptivity in an individual, which typically rely on collection of uterine tissue via biopsy, which renders the uterine lining non-viable for implantation until the next oestrous or menstrual cycle. Thus, the present invention enables real-time determination of uterine receptivity such that clinicians can potentially obtain information on uterine receptivity on the day that the subject presents to the clinic for embryo transfer, or whether to wait until a later time in the cycle or indeed until another cycle.

Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that the intention is not to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the present invention as defined by the claims.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described. Definitions

The articles“a” and“an” are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example,“a cell” means one cell or more than one cell.

The term "about" is understood to refer to a range of +/- 10%, preferably +/- 5% or +/- 1 % or, more preferably, +/- 0.1 %.

The term“assisted reproduction” refers to clinical and laboratory techniques used to enhance fertility in human or animals, including, but not limited to, in vitro fertilization (IVF), frozen embryo transfer (FET), intracytoplasmic sperm injection (ICSI), intra cytoplasmic morphologically selected sperm injection (IMSI), gamete intrafallopian tube transfer (GIFT), intrauterine insemination (IUI) and zygote intrafallopian tube transfer (ZIFT).

As used herein, the term “uterine” will be understood to mean relating to the uterus.“Uterine receptivity” will therefore be understood to refer to the receptivity of the uterus to implantation of an embryo.

The skilled person will understand that uterine receptivity may also be referred to as“endometrial receptivity”, wherein the endometrium is the mucous membrane lining the uterus, which thickens during the oestrous or menstrual cycle in preparation for the possible implantation of an embryo. Accordingly, the terms“uterine receptivity” and “endometrial receptivity” will be understood to be used interchangeably.

As used herein, the term“uterine receptivity” refers to whether the uterus of the female subject is likely to be receptive to implantation of an embryo, or likely to not be receptive (i.e., non-receptive) to implantation of an embryo.

The term“uterine implantation window” refers to the very short period beginning about 4 to 5 days after ovulation and lasting about 4 days. This window defined as the implantation window defines the period in the oestrous or menstrual cycle of uterine receptivity to the embryo. During the mild luteal phase, uterine remodelling events required for a successful pregnancy begin before implantation with the decidualisation of the endometrium, which occur even in the absence of a fertilized conceptus in human.

As used herein“blastocyst” refers to a structure formed in the early development of mammals. A blastocyst comprises an inner cell mass which subsequently forms the embryo. The outer layer of the blastocyst consists of cells collectively called the trophoblast. This layer surrounds the inner cell mass and a fluid-filled cavity called the blastocoel and wherein the trophoblast gives rise to the placenta. In humans, blastocyst formation begins at about 5 days after fertilisation. About 7 days ager fertilisation, the blastocyst undergoes implantation, embedding into the endometrium of the uterine wall.

The use of blastocysts in in vitro fertilisation (IVF), involves culturing a fertilised egg for five days before transferring it into the uterus for implantation. Accordingly, determination of whether the uterus is receptive to implantation is a critical step in successful IVF.

As used herein, the term "antibody" refers to any form of antibody that exhibits the desired biological activity. Thus, it is used in a broad sense and includes, but is not limited to, monoclonal antibodies (including full length monoclonal antibodies comprising two light chains and two heavy chains), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies and camelized single domain antibodies. Single domain antibodies are composed of single VFI or VL domains.

Naturally occurring antibody structural units typically comprise a tetramer. Each such tetramer typically comprises two pairs of polypeptide chains, each pair having one full- length "light" and one full-length "heavy" chain. The amino-terminal portion of each chain typically includes a variable region of about 100 to 1 10 or more amino acids that typically is responsible for antigen recognition. The carboxy-terminal portion of each chain typically defines a constant region that may be responsible for effector function. Fluman light chains are typically classified as kappa and lambda light chains. Heavy chains are typically classified as mu, delta, gamma, alpha or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA and IgE, respectively. IgG has several subclasses, including, but not limited to, lgG1 , lgG2, lgG3 and lgG4. IgM has subclasses including, but not limited to, lgM1 and lgM2. IgA is similarly subdivided into subclasses including, but not limited to, lgA1 and lgA2. Within full-length light and heavy chains, often, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids. See, eg, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). The variable regions of each light/heavy chain pair typically comprise the antigen binding site.

As used herein, the term "antigen binding protein" refers to a protein that specifically binds to one or more target antigens. An antigen binding protein can include an antibody and binding fragments thereof. An "antigen binding fragment" or "antigen binding portion" used interchangeably in certain contexts herein with "binding fragment" or "fragment" is a portion of an antibody that lacks at least some of the amino acids present in a full-length heavy chain and/or light chain, but which is still capable of specifically binding to an antigen. An antigen binding fragment includes, but is not limited to, a single-chain variable fragment (scFv), a nanobody (eg, VFI domain of camelid heavy chain antibodies; VHH fragment), a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment and a Fd fragment, and may be derived, for example, from a mammalian source, such as human, mouse, rat, rabbit or camelid. Antigen binding fragments may compete for binding to a target antigen with an intact antibody and the fragments may be produced by the modification of intact antibodies (eg, enzymatic or chemical cleavage) or synthesized de novo using recombinant DNA technologies or peptide synthesis.

An antigen binding protein may also include a protein comprising one or more antigen binding fragments incorporated into a single polypeptide chain or into multiple polypeptide chains. For example, antigen binding proteins may include, but are not limited to, a diabody (see, e.g., EP 404,097; WO 93/1 1 161 ; and Hollinger et al, Proc. Natl. Acad. Sci. USA, Vol. 90:6444- 6448, 1993), an intrabody, a domain antibody (single VL or VFI domain or two or more VFI domains joined by a peptide linker; see, eg, Ward et al, Nature, Vol. 341 :544-546, 1989), a maxibody (two scFvs fused to Fc region, see, e.g., Fredericks et al, Protein Engineering, Design & Selection, Vol. 17:95-106, 2004 and Powers et al, J. Immunol. Meth. 2001. 251 : 123-135), a triabody, a tetrabody, a minibody (scFv fused to CH3 domain; see, eg, Olafsen et al., Prot. Eng. Des. Sel. 2004. 17: 315-23), a peptibody (one or more peptides attached to an Fc region, see, eg, WO 00/24782), a linear antibody (a pair of tandem Fd segments (VH-CH1 -VH-CH1 ) which, together with complementary light chain polypeptides, form a pair of antigen binding regions (see, eg, Zapata et al. Protein Eng. 1995. 8: 1057-1062), a small modular immunopharmaceutical (see, e.g., U.S. Patent Publication No. 20030133939), and immunoglobulin fusion proteins (e.g., IgG-scFv, IgG- Fab, 2scFv-lgG, 4scFv-lgG, VH-lgG, IgG-VH, and Fab-scFv-Fc; see, e.g., Spiess et al, Mol. Immunol., Vol. 67(2 Pt A):95-106, 2015).

The terms "comprise", "comprises", "comprised" or "comprising", "including" or "having" and the like in the present specification and claims are used in an inclusive sense, i.e., to specify the presence of the stated features but not preclude the presence of additional or further features.

The term “isolated” as used herein refers to material that is substantially or essentially free from components that normally accompany it in its native state. For example, an“isolated polynucleotide” as used herein refers to a polynucleotide which has been purified from the sequences which flank it in a naturally-occurring state, e.g., a DNA fragment which has been removed from the sequences that are normally adjacent to the fragment. Alternatively, an“isolated peptide” or an“isolated polypeptide” and the like, as used herein, refer to in vitro isolation and/or purification of a peptide or polypeptide molecule from its natural cellular environment, and from association with other components of the cell, i.e., it is not associated with in vivo substances.

The terms “treatment” and “treat” include both prophylactic or preventive treatment (that prevent and/or slow the development of a targeted pathologic condition or disorder) and curative, therapeutic or disease-modifying treatment, including therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a pathologic condition or disorder; and treatment of patients at risk of contracting a disease or suspected to have contracted a disease, as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition. The terms“treatment” and“treat” do not necessarily imply that a subject is treated until total recovery. The terms“treatment” and“treat” also refer to the maintenance and/or promotion of health in an individual not suffering from a disease but who may be susceptible to the development of an unhealthy condition. The terms“treatment” and “treat” are also intended to include the potentiation or otherwise enhancement of one or more primary prophylactic or therapeutic measures. As non-limiting examples, a treatment can be performed by a patient, a caregiver, a doctor, a nurse, or another healthcare professional.

Soluble NSF Atachment Protein Receptor

Soluble NSF attachment proteins (SNAPs) and SNAP receptor (SNARE) proteins are a large superfamily of proteins that mediate vesicle fusion. SNARE proteins, along with Ca2+ signalling, also regulate exocytosis.

Secretion involves the release of vesicular content following fusion between a vesicle membrane and a target membrane. Fusion generally requires a vesicle SNARE (v-SNARE), such as vesicle associated membrane protein (VAMP), located on the vesicle membrane, and one or more target SNAREs (t-SNARE), such as a syntaxin and synaptosomal-associated protein 23 (SNAP23) or SNAP25, located on the target membrane, to form a SNARE core complex.

SNAP23 is a homolgue of SNAP25 and plays important roles in neurotransmitter release from neurons and insulin secretion from pancreatic b cells. SNAP23 also regulates exocytosis in a range of nonneuronal cells, such as surfactant release from alveolar epithelial cells, glucose transporter GLUT4 translocation in adipocytes, and Ig release from plasma cells.

Two isoforms of SNAP23 have been identified in humans, including a 211 amino acid isoform (SEQ ID NO. 1 ) and a 158 amino acid isoform (SEQ ID NO. 2) as set forth below.

MDNLSSEEIQQRAHQITDESLESTRRILGLAIESQDAGIKTITMLDEQKEQLNRIE EG LDQINKDMRETEKTLTELNKCCGLCVCPCNRTKNFESGKAYKTTWGDGGENSPC NVVSKQPGPVTNGQLQQPTTGAASGGYIKRITNDAREDEMEENLTQVGSILGNLK DMALNIGNEIDAQNPQIKRITDKADTNRDRIDIANARAKKLIDS (SEQ ID NO. 1 )

MDNLSSEEIQQRAHQITDESLESTRRILGLAIESQDAGIKTITMLDEQKEQLNRIE EG LDQINKDMRETEKTLTELNKCCGLCVCPCNSITNDAREDEMEENLTQVGSILGNLK DMALNIGNEIDAQNPQIKRITDKADTNRDRIDIANARAKKLIDS (SEQ ID NO. 2) As used herein, the term SNAP23 will be understood to encompass any isoform or sequence variant of SNAP23 and the present disclosure is not limited to any particular isoform nor to SEQ ID NO. 1 or SEQ ID NO. 2. Those skilled in the art will understand that the sequence of SNAP23 may vary between individuals, and that the methods and compositions of the present disclosure are applicable to such variants.

Determination of uterine receptivity

The present inventors have identified SNAP23 and in particular, secreted SNAP23, as a biomarker that can be used to determine uterine receptivity. As will be clear from the present disclosure, the presence of SNAP23 in a biological sample obtained from a female subject may be indicative of a receptive uterus. Preferably, an increased level of SNAP23 relative to a baseline control is indicative of a receptive uterus, Concentrations or levels of SNAP23 that are indicative of uterine receptivity may vary depending on a number of factors such as the biological sample used (e.g., uterine fluid, vaginal fluid or peritoneal fluid), the stage of development of the embryo to be implanted, the manner in which the biological sample is prepared and the patient (e.g., age, weight, medical history), as well as the professional judgement of the practitioner preforming the diagnostic method. In some examples, the presence of SNAP23 at a concentration of at least about 1 ,000 pg/mL in the biological sample (e.g., uterine fluid) is diagnostic of a receptive uterus. In some examples, the presence of SNAP23 at a concentration of at least about 10 pg/mL, such as at least about 25 pg/mL, at least about 50 pg/mL, at least about 100 pg/mL, at least about 500 pg/mL, at least about 1 ,000 pg/mL, at least about 1 ,500 pg/mL, at least about 2,500 pg/mL, at least about 5,000 pg/mL or at least about 10,000 pg/mL in the biological sample (e.g., uterine fluid) is diagnostic of a receptive uterus. In such examples, a clinician may decide to transfer a blastocyst to the uterus of a subject as the uterus is deemed to be receptive for implantation. Alternatively, in examples where SNAP23 is absent or present at low concentrations, a clinician may decide to freeze the blastocyst for use in a later oestrous or menstrual cycle.

It will be understood by those skilled in the art that the presence of SNAP23 at a high concentration in the biological sample may be indicative that the uterus of the subject is suitable for blastocyst stage (e.g., 5-6 days post-fertilisation) transfer, but that it may not be receptive to an earlier embryo (e.g., 2 days post-fertilisation) as the uterus is too advanced for transfer of an immature embryo. Preferably, the time point at which the highest amount of SNAP23 is observed or a rapid increase in SNAP23 is observed, is considered indicative of optimum uterine receptivity.

In some examples, the level of SNAP23 detected in a biological sample is compared to a reference level of SNAP23. The reference level of SNAP23 may be based on data obtained from female subjects for whom embryo transfer led to a successful pregnancy. Alternatively, or in addition, the reference level of SNAP23 may be based on data obtained from female subjects for whom embryo transfer did not lead to a successful pregnancy. In certain examples, the level of SNAP23 is compared to a knowledge base which comprises data in the form of correlations between levels of SNAP23 and pregnancy outcomes.

It will be understood that the diagnostic methods described herein may be performed more than once over a period of time such as daily, once every two, three, four, five or six days, or weekly, in order to detect dynamic changes in SNAP23 levels. For example, by performing the methods of the present disclosure on more than one occasion it may be determined that SNAP23 levels are rising and that the uterus is becoming more receptive. Alternatively, a subject may find that SNAP23 levels are decreasing and that the uterus is becoming less receptive. In the latter situation, the subject may decide to delay blastocyst implantation until a subsequent cycle.

The compositions and methods described herein may also be useful to couples seeking to conceive naturally. For example, the compositions and methods of the present disclosure may be used to determine whether the uterus of a female subject is passing through the window of receptivity and may indicate whether the female is capable of conceiving naturally.

Methods for determining levels of SNAP23

Those skilled in the art will appreciate that levels of SNAP23 may be determined in many different ways and the present invention contemplates the use of any method for determining SNAP23 levels in a relevant biological sample. In certain preferred examples, levels of SNAP23 may be quantified using enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, immunofluorescence, latex agglutination, hemagglutination, lateral flow immunoassay, mass spectrometry or Western blot analysis. Alternatively, qRT-PCR or related methodologies known in the art may be used to determine levels of expression of the SNAP23 gene, for example by amplifying cell-free nucleic acids present in uterine or vaginal fluid, or by amplifying nucleic acids in cells present in tissue or fluid samples.

ELISAs are known in the art (see, eg, Crowther, John R. 2009. "The ELISA Guidebook." 2nd ed. Humana Press and Lequin R. 2005. "Enzyme immunoassay EIA)/enzyme-linked immunosorbent assay (ELISA)". Clin. Chem. 51 (12): 2415-8). Suitable ELISA formats that may be used to detect the presence or absence of SNAP23 include direct ELISA, indirect ELISA, sandwich ELISA and competitive ELISA.

Matrix-assisted laser-desorption (MALDI) time-of-flight (TOF) mass spectrometry (MS) with delayed extraction and a reflectron in the TOF chamber is one form of MS that may be used to detect the presence or absence of SNAP23 in a biological sample. Preferably, MALDI assays are performed on silicon arrays.

Other suitable immunoassays may include electrochemiluminescence, fluorescence polarization, Luminex LabMAP and Bioplex immunoassays. The LabMAP system incorporates polystyrene microspheres that are dyed internally with two spectrally distinct fluorochromes. Using precise ratios of these fluorochromes, an array is created comprising 100 different microsphere sets with specific spectral addresses. Each microsphere set can possess a different reactant on its surface. Because microsphere sets can be distinguished by their spectral addresses, they can be combined, allowing 100 different analytes to be measured simultaneously in a single reaction vessel. A third fluorochrome coupled to a reporter molecule quantifies the biomolecular interaction that has occurred at the microsphere surface. Microspheres are interrogated individually in a rapidly flowing fluid stream as they pass by two separate lasers in the Luminex analyzer. High-speed digital signal processing classifies the microsphere based on its spectral address and quantifies the reaction on the surface in a few seconds per sample.

In examples where an antibody is used to detect SNAP23, the antibody may be a commercial antibody (eg, Abeam ab166808; Creative Diagnostics ABPR-0874), or the antibody may be developed using techniques known in the art. The antibody or fragment thereof may be conjugated to a detectable label such as an enzyme (eg, horseradish peroxidase), a gold nanoparticle, a fluorophore, a radioisotope, a latex bead, a carbon nanoparticle, biotin or a quantum dot.

Those skilled in the art will be aware of various methods that may be used to generate antibodies and antigen binding proteins. For example, antibodies may be produced by immunising a non-human animal, eg, a rodent, with SNAP23 or a fragment thereof. In certain embodiments, the anti-SNAP23 antibody is a monoclonal antibody. Monoclonal antibodies can be produced using a variety of techniques known in the art including by using hybridoma technologies, recombinant DNA technologies, and phage display technologies, or a combination thereof. Other techniques for producing human monoclonal antibodies include the trioma technique, the human B-cell hybridoma technique and the EBV-hybridoma technique. Phage display is described in U.S. Patent No. 5,223,409; Clackson et al. Nature. 1991. 352: 624-628; Marks et al. J. Mol. Biol. 1991. 222:581 -597.

Those skilled in the art will be aware of several techniques for producing monoclonal antibodies using hybridoma technology. By way of non-limiting example, splenocytes and/or lymph node cells from immunized mice may be isolated and fused to an appropriate immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas can be screened for the production of antigen-specific antibodies. For example, single cell suspensions of splenic lymphocytes from immunized mice can be fused to Sp2/0 nonsecreting mouse myeloma cells (ATCC CRL 1581 ) with 50% PEG. Cells may be plated at approximately 2 x 10⁵ in a flat bottom microtiter plate, followed by a two week incubation in selective medium containing 10% fetal Clone Serum, 18% "653" conditioned media, 5% origen (IGEN), 4 mM L-glutamine, 1 mM sodium pyruvate, 5mM FIEPES, 0.055 mM 2- mercaptoethanol, 50 units/ml penicillin, 50 mg/ml streptomycin, 50 mg/ml gentamycin and 1 X FIAT (Sigma). After approximately two weeks, cells can be cultured in medium in which the FIAT is replaced with FIT. Individual wells can then be screened by ELISA for human monoclonal IgM and IgG antibodies. Once extensive hybridoma growth occurs, the medium may be observed after about I Q- 14 days. The antibody secreting hybridomas can be re-plated, screened again, and if still positive for human IgG, the monoclonal antibodies can be subcloned at least twice by limiting dilution. The stable subclones can then be cultured in vitro to generate small amounts of antibody in tissue culture medium for characterization. To purify monoclonal antibodies, selected hybridomas can be grown in two litre spinner-flasks for monoclonal antibody purification. Supernatants can be filtered and concentrated before affinity chromatography with protein A-Sepharose. The buffer solution can be exchanged into PBS, and the concentration can be determined by OD280 using 1.43 extinction coefficient.

Hybridomas may be screened using standard methods, such as ELISA and surface plasmon resonance (BIACORE), to identify hybridomas that produce an antibody that specifically binds to a particular target such as SNAP23. Surface plasmon resonance may also be used to increase the efficiency of phage antibodies which bind to an epitope of SNAP23.

For antibodies expressed by hybridomas, DNA encoding the light and heavy chains of the antibody may be obtained by standard PCR amplification or DNA cloning techniques. For antibodies obtained from an immunoglobulin gene library (eg, using phage display techniques), nucleic acid encoding the antibody may be recovered from the library. Once DNA fragments encoding VH and VL segments are obtained, they may be manipulated by standard recombinant DNA techniques, for example, to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes or to a scFv gene. In these manipulations, a VL- or VH-encoding DNA fragment may be linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker.

The isolated DNA encoding the VH region may be converted to a full-length heavy chain gene by linking the VH-encoding DNA to another DNA molecule encoding heavy chain constant regions (hinge, CH1 , CH2 and/or CH3), the sequences of which for humans are known (see, eg, Kabat, E. A., el al. (1991 ) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91 -3242). The heavy chain constant region may be, for example, an lgG1 , lgG2, lgG3, lgG4, IgA, IgE, IgM or IgD constant region. For a Fab fragment heavy chain gene, the VH- encoding DNA may be linked to another DNA molecule encoding only the heavy chain CH1 constant region. The isolated DNA encoding the VL region may also be used to express a full-length light chain gene (as well as a Fab light chain gene) by linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL.

The antibody or sample may be immobilized on a carrier or solid support such as nitrocellulose, glass, polyacrylamides, gabbros or magnetite. The support material may be, for example, a microtiter well, magnetic bead, non-magnetic bead, column, matrix, membrane, glass, polystyrene, dextran, nylon, amylase, natural or modified cellulose, polyacrylamide, agarose, magletite, fibrous mat composed of synthetic or natural fibers (e.g., glass or cellulose-based materials or thermoplastic polymers, such as, polyethylene, polypropylene, or polyester), sintered structure composed of particulate materials (e.g., glass or various thermoplastic polymers), or cast membrane film composed of nitrocellulose, nylon, polysulfone or the like (generally synthetic in nature). The substrate materials can be used in suitable shapes, such as films, sheets, or plates, or they may be coated onto or bonded or laminated to appropriate inert carriers, such as paper, glass, plastic films, or fabrics. Suitable methods for immobilizing antibodies on solid phases include ionic, hydrophobic, covalent interactions and the like. In some examples, the immobilization involves binding the agent (eg, the anti-SNAP23 antibody) to the support member. In other examples, the immobilization involves adsorbing the antibody to the support member. Such adsorption methods may be performed, for example, by incubating the antibody in a buffer in the wells of a multi-well plate. In some examples, the agent such as the antibody is provided in a coating buffer and incubated in the wells of a multi-well plate. Coating buffers will be evident to one of skill in the art and may be prepared or obtained from a commercial source. Non-limiting examples of coating buffers include 50 mM sodium bicarbonate, pH 9.6; 0.2 M sodium bicarbonate, pH 9.4; phosphate buffered solution (50 mM phosphate, pH 8.0, 0.15 M NaCI); carbonate-bicarbonate solution; and TBS (50 mM TRIS, pH 8.0, 0.15 M NaCI).

In some examples, the support is dissolvable such that upon addition of a biological sample, the antibody is released from the solid support. Such examples may be useful for use in at home or point-of-care devices such as a lateral flow device.

In some examples the present disclosure provides a method of quantifying the amount of SNAP23 in a sample the method comprising: contacting a first SNAP23- binding protein (eg, an antibody) with the sample to form a complex, wherein the first SNAP23- binding protein is attached to a solid support; contacting the complex with a second SNAP23-binding protein (eg, antibody), wherein the second SNAP23-binding protein is conjugated to a detectable label; and detecting a signal released by the detectable label, directly or indirectly.

In certain examples, the SNAP23 is detected using an immunoassay adapted for home testing, point of care testing or laboratory testing. The device may be an immunochromatographic device comprising a membrane (cellulosic or non-cellulosic) at least partially enclosed in a plastic holder. Preferably, the immunochromatographic assay device includes a control to indicate that the assay has proceeded correctly. The control can be a specific binding reactant at a region more distal from the sample pad region on the solid phase support than the test region that will bind to labelled reagent in the presence or absence of analyte, thus indicating that the mobilizable receptor has migrated a sufficient distance with the liquid sample to give a meaningful result.

Valkirs et al. (U.S. Pat. No. 4,632,901 ) discloses a device comprising an antibody, specific to an antigen analyte, bound to a porous membrane or filter to which is added a liquid sample. As the liquid flows through the membrane, target analytes bind to the antibody. The addition of the sample is followed by the addition of a labelled antibody. The visual detection of the labelled antibody provides an indication of the presence of the target analyte in the sample. Another example of a flow-through device is disclosed by Kromer et al. (EP-A 0 229 359), which describes a reagent delivery system comprising a matrix saturated with a reagent or components thereof dispersed in a water soluble polymer for controlling the dissolution rate of the reagent for delivery to a reaction matrix positioned below the matrix. In migration type assays, the solid phase support, e.g., membrane may be impregnated with the reagents needed to perform the assay. A test region may be provided in which labelled analyte is bound and the results of the assay are read. For example, see Tom et al. (U.S. Pat. No. 4,366,241 ), and Zuk (EP-A 0 143 574). Migration assay devices may incorporate within them reagents that have been attached to colored labels such as colloidal gold or carbon, thereby permitting visible detection of the assay results without addition of further substances. In certain examples, the present disclosure provides a lateral flow device for detecting the presence or absence of SNAP23 in a biological sample. The device preferably includes a pad region for receiving the biological sample, wherein the pad region comprises a first anti-SNAP23 antibody or antigen-binding fragment thereof; and a test region comprising a second anti-SNAP23 antibody or antigen-binding fragment thereof, wherein the first anti-SNAP23 antibody or fragment thereof is mobilizable along the device and the second anti-SNAP23 is immobilised to the device. It will be appreciated that application of the biological sample to such devices mobilises the first anti-SNAP23 antibody, which then migrates along the device, for example, by capillary flow, towards the second anti-SNAP23 antibody. As used herein, the term "anti-SNAP23 antibody" refers to an antibody that specifically binds to SNAP23. An antibody is said to "specifically bind" if it reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular target antigen (eg, SNAP23) than it does with alternative targets in a sample.

Kits

The present disclosure also provides a kit for detecting the presence or absence of SNAP23 in a biological sample such as uterine fluid or vaginal fluid. The kit may include an anti-SNAP23 antibody or antigen-binding fragment thereof. The anti-SNAP23 antibody or antigen-binding fragment thereof may be conjugated to a detectable label, such as an enzyme (eg, horseradish peroxidase or alkaline phosphatase), a gold nanoparticle, a fluorophore, a radioisotope, a latex bead, a carbon nanoparticle, biotin or a quantum dot, or the kit may comprise a second antibody or fragment which is conjugated to a detectable label and which binds to the anti-SNAP23 antibody or antigen-binding fragment thereof.

In certain examples, the present disclosure provides a method of diagnosing uterine receptivity in a female subject the method comprising the step of detecting the presence or absence of SNAP23 in uterine fluid or vaginal fluid obtained from the subject. In some examples, the present disclosure provides a method of diagnosing uterine receptivity in a female subject the method comprising: contacting an anti- SNAP23 antibody with a sample of uterine fluid or vaginal fluid obtained from the subject, wherein the anti- SNAP23 antibody is conjugated to a detectable label; and detecting a signal released by the detectable label, directly or indirectly. In some examples, the present disclosure provides a method of diagnosing uterine receptivity in a female subject the method comprising the step of contacting an anti-SNAP23 antibody with a sample of uterine fluid or vaginal fluid obtained from the subject, wherein the anti- SNAP23 antibody is immobilised to a solid support. In some examples, the present disclosure provides a method of diagnosing uterine receptivity in a female subject the method comprising the step of contacting a monoclonal anti-SNAP23 antibody with a sample of uterine fluid or vaginal fluid obtained from the subject. In some examples, the present disclosure provides a method of detecting SNAP23 in vaginal fluid or uterine fluid obtained from a female subject the method comprising: contacting a first anti- SNAP23 antibody with a sample of the vaginal fluid or uterine fluid to thereby form an antibody-SNAP23 complex, wherein the first anti-SNAP23 antibody is attached to a solid support; contacting the antibody-SNAP23 complex with a second anti-SNAP23 antibody, wherein the second anti-SNAP23 antibody is conjugated to a detectable label; and detecting a signal released by the detectable label, directly or indirectly. In some examples, the present disclosure provides a method of detecting SNAP23 in vaginal fluid or uterine fluid obtained from a female subject the method comprising: contacting a first anti-SNAP23 antibody with a sample of the vaginal fluid or uterine fluid to thereby form an antibody-SNAP23 complex, wherein the first anti- SNAP23 antibody is conjugated to a detectable label; contacting the antibody-SNAP23 complex with a second anti-SNAP23 antibody, wherein the second anti-SNAP23 antibody is attached to a solid support; and detecting a signal released by the detectable label, directly or indirectly.

As will be appreciated from the present disclosure, the expression of SNARE proteins changes as a uterus becomes receptive to embryo implantation. Accordingly, the present disclosure also provides methods of diagnosing uterine receptivity in a subject the method comprising the step of detecting the presence or absence of a SNARE in a biological sample obtained from the subject. As described herein, increased levels of SNAP23 or syntaxin-2 may be indicative of uterine receptivity. Accordingly, the present disclosure provides methods of diagnosing uterine receptivity in a subject the method comprising the step of quantifying the expression of a gene encoding a SNARE (eg, SNAP23 or syntaxin-2). Gene expression may be quantified at the protein level, for example, using ELISA, immunohistochemistry, lateral flow immunoassay, mass spectrometry or Western analysis. In other examples, expression of a gene encoding the SNARE may be quantified at the nucleic acid (eg, RNA) level, for example, using polymerase chain reaction (eg, quantitative or semi-quantitative PCR), northern blotting, nucleic acid sequencing or a microarray. Those skilled in the art will be aware that other methods may be used to quantify expression of a gene encoding a SNARE.

In some examples, the present disclosure provides a method of diagnosing uterine receptivity in a female subject the method comprising the step of detecting the presence or absence of syntaxin-2 or SNAP23 in a biological sample obtained from the subject. In some examples, the present disclosure provides a method of diagnosing uterine receptivity in a female subject the method comprising the step of quantifying the level of syntaxin-2 or SNAP23 in a biological sample obtained from the subject. In some examples, the present disclosure provides a method of diagnosing uterine receptivity in a female subject the method comprising the step of quantifying expression of a gene encoding syntaxin-2 or SNAP23 in a biological sample obtained from the subject.

Biological samples

It will be appreciated that the methods described herein do not require invasive biopsy or cause damage to the uterine surface. Preferred biological samples that may be used in accordance with the methods of the invention include, for example, uterine fluid (which may also be referred to as uterine luminal fluid, ULF or endometrial fluid), vaginal fluid and peritoneal fluid. Other biological samples may include urine, ascites, tissue exudate, blood, plasma, saliva, serum and lymph fluid. The biological sample may be untreated, precipitated, fractionated, separated, diluted, concentrated or purified.

In certain examples, the methods of the invention are performed at the time of egg collection from a female subject. In such examples, uterine lavage obtained during egg collection may be used as a biological sample for detecting the presence or absence of SNAP23. In some examples, ovulation has been induced in the female subject, for example, by administration of hCG or GnRHa. Typically, the levels of SNAP23 observed at the time of egg collection will be understood to be indicative of the baseline levels of SNAP23. In other words, the“baseline” level of SNAP23 observed in the biological sample (e.g., ULF) will be understood to be the level of SNAP23 representative of a uterus that is not ready for implantation. In preferred embodiments, the levels of SNAP23 can be measured in a biological sample (preferably uterine tissue or most preferably uterine luminal or vaginal fluid) in order to obtain a uterine receptivity profile of the female subject. It will be understood that the profile can be used to ascertain the baseline levels of SNAP23 in the subject or the range of variation of SNAP23 levels in the subject over the course of the oestrous cycle or menstrual cycle.

It will be understood that in order to determine the uterine receptivity profile of the subject, a plurality of samples obtained at different point during the oestrous or menstrual cycle of the subject will be obtained, and in which levels of SNAP23 will be determine. Preferably the plurality of samples comprises samples from at least 3, at least 4, at least 5, or more, different time points during the oestrus or menstrual cycle of the subject. The different time points may include time points during the pre-ovulation period of the subject, which preferably can also be used to identify the baseline level of SNAP23 in the subject.

In alternative embodiments, where the subject is a human female, the different time points preferably include at least one time point in the post-ovulation period, preferably in the period 5-9 days post-ovulation. Most preferably, the different time points include at least 3 different days in the time period corresponding to prior to ovulation, at ovulation, and after ovulation (for example at the time of egg pick-up).

It will be appreciated that given the non-invasive nature of the methods of the present invention, it is possible to obtain samples of uterine luminal or vaginal fluid from the subject at each day (including at multiple time points each day) during the period that is predicted to correspond to the period of uterine receptivity (ie, the period that is typically understood to be 5-9 days post-ovulation). Thus, in particularly preferred embodiments, the time period includes daily or at least half daily time points in the period of 5-9 days post-ovulation (corresponding to between days 19-23 of a 28 day menstrual cycle). Still further, the time periods for which samples are obtained could include every day in the oestrous or menstrual cycle of the subject.

In other embodiments, a baseline level of SNAP23 is an amount that is determined in a uterus after the optimum period for implantation has passed (i.e., the uterus is in a“post-receptive” phase). It will generally be understood that the SNAP23 levels observed in pre- and post-receptive samples will be very low or may not be detectable compared to the level of SNAP23 observed at the period of optimum receptivity, where a spike or rapid increase in SNAP23 is observed in uterine fluid for the subject will be understood to be indicative of uterine receptivity.

In any embodiment, a“higher” or greater level or amount of SNAP23 compared to a reference level, control or baseline level, will be understood to include a level or amount that is at least about a 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, or 5-fold or more, increase compared to the amount in the control or reference sample.

In any embodiment, a“lower” or lesser level or amount of SNAP23 compared to a reference level, control or baseline level, will be understood to include a level or amount that is at least about a 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, or 5-fold, or more, decrease compared to the amount in the control or reference sample.

In any embodiment, a“higher” or greater level or amount of SNAP23 compared to a reference level, control or baseline level, will be understood to include a level or amount that is at least about 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more, greater than the amount in the control or reference sample.

In any embodiment, a“lower” or lesser level or amount of SNAP23 compared to a reference level, control or baseline level, will be understood to include a level or amount that is at least about 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more, less than the amount in the control or reference sample.

In any embodiment, an amount that is the“same as” the level or amount of SNAP23 compared to a reference, control or baseline level will be understood to be a level or amount that is no more than about 1 %, 2%, 3%, 4%, 5% or 10% more or less than the reference or control sample.

Uterine (i.e., endometrial fluid) may be collected using methods known in the art, for example, by aspiration or using a syringe. Vaginal fluid may be collected, for example, by a swab (e.g., flocked vaginal swab), a douche method, a vaginal wash, a syringe etc. The biological sample is preferably obtained from a human female subject. However, it will be appreciated that the present invention applies equally to veterinary applications. In that regard, the female subject may be a canine (dog), feline (cat), rodent (mouse, rat, guinea pig, hamster or other), bovine (cow), equine (horse), ovine (sheep), caprine (goat), porcine (pig), non-human primate animal, or other non-human animal and the levels of SNAP23 in the uterine fluid from that subject can be used to determine the optimum time of uterine receptivity in a veterinary application of assisted reproductive technology.

Examples Animals and mating

The present study used female virgin Wistar rats aged 10-12 weeks. All procedures were approved by The University of Sydney Animal Ethics Committee. Rats were housed in plastic cages at 21 °C under a 12-hour light-dark cycle and were provided with free access to food and water. Pro-oestrus female rats were mated overnight with males of proven fertility to induce uterine receptivity. The presence of sperm in a vaginal smear the following morning indicated successful mating and this was designated day 1 of pregnancy. Uterine tissues were collected from 5 rats each on days 1 , 3.5, 5.5, 6 and 7 of pregnancy for immunofluorescence and western blot analysis. Further uterine tissue was collected from 4 rats each on days 1 , 5.5 and 6 for transmission electron microscope (TEM) analysis.

Tissue and fluid collection

To collect uterine tissue, rats were administered 20 mg/kg of sodium pentobarbitone (Vibac Animal Health, NSW, Australia) intraperitoneally and the uterine horns were collected under deep anaesthesia, before euthanasia. The uterine horns were used for immunofluorescence, western blotting and TEM analysis. Uterine luminal fluid was obtained from pregnant rats by injection of saline into the uterine lumen and collection of that fluid into an Eppendorf tube. This luminal fluid was immediately aliquoted, frozen in liquid nitrogen and stored in a -80°C freezer.

Cell culture HEC1 A cells (a non-receptive human endometrial adenocarcinoma cell line; ATCC HTB-1 12TM) were grown at 37°C and 5% CO2 in McCoy’s 5A Medium (1X)/ L- glutamine (GIBCO, Grand Island, NY) supplemented with 10% fetal bovine serum (Bovogen Biologicals Pty, Essendon, VIC, Australia) and 1 % streptomycin and penicillin (Invitrogen, Carlsbad, CA) until confluent.

RL95-2 cells (a receptive human endometrial adenocarcinoma cell line; ATCC CRL-1671TM) were grown at 37°C and 5% CO2 in DMEM/F-12 (Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12) containing FIEPES and L-glutamine (GIBCO) supplemented with 10% fetal bovine serum (Bovogen Biologicals Pty), 0.005 mg/ml insulin (I0516-5ML, Sigma Aldrich) and 1 % streptomycin and penicillin (Invitrogen) until confluent.

Immunofluorescence of rat uterus

Uterine horns (5 mm pieces) were coated with OCT (Tissue Tek, CA, USA), snap frozen in supercooled isopentane, and stored under liquid nitrogen until use. Frozen sections were cut (7 pm) using a Leica CM 3050 cryostat (Leica, Fleerbrugg, Switzerland) and air-dried on gelatine-chrome alum-coated slides.

Syntaxin-2 sections were fixed in 4% formaldehyde in 0.1 M phosphate buffer and SNAP23 sections were fixed in 75% methanol in phosphate buffer saline (PBS) for 10 minutes at room temperature (RT). All sections were washed with PBS and blocked with 1 % bovine serum albumin (BSA; Sigma Aldrich, MO, USA) in PBS for 30 minutes at RT. Sections were incubated overnight at 4°C with rabbit monoclonal anti -syntaxin-2 antibody (7.8 pg/ml; Abeam, Cambridge, England, UK: ab170852) or goat anti-SNAP23 antibody (1.25 pg/ml; Abeam, ab166808), diluted in 1 % PBS/BSA. Concurrently, control sections were incubated with non-immune IgG (Sigma Aldrich) at the same concentration as the primary antibody. Sections were washed in PBS and incubated with FITC-conjugated goat anti-mouse IgG (3 pg/mL; Jackson ImmunoResearch, PA, USA) or Alexa Fluor 488 conjugated donkey anti-goat IgG (0.02 pg/ mL; Life Technologies Australia Pty Ltd, Mulgrave, VIC, Australia) for 30 minutes at RT. Sections were washed in PBS and mounted with Vectashield with DAPI (Vector Laboratories, CA, USA) and coverslipped (No. 1 thickness). Zeiss Axio Imager M2 Microscope (Carl Zeiss, Australasia) was used to image the sections. Images were acquired using a Zeiss AxioCam HR digital monochrome CCD camera (Carl Zeiss) and ZEN 2013 (Blue edition) software (Carl Zeiss). The camera exposure time was set on the brightest sample per protein and the same parameters were used to image all other samples. Those images were then used to quantify the fluorescence intensity.

Image analysis

Intensity measurements were made using FIJI. All non-uterine epithelial tissue was cropped out before any intensity measurement. Cell count was obtained from the DAPI channel by thresholding the nuclei, and a particle size limit was set to eliminate peripherally cut cells in the section. The channel with the protein staining was thresholded at the same range for all images, and a grey scale value was obtained for each image. These were then standardised to the number of nuclei to obtain a measurement of fluorescence intensity per cell. Three randomly selected high magnification images per rat were analysed and averaged to provide an average intensity per set. P<0.05 was determined to be significant. All graphs were generated using Graph Pad Prism Software (Version 7.02, Graph Pad Software, Inc., CA, USA) and error bars represent mean ± SEM.

Co-localisation analysis

For co-localisation of SNAP23 and Phalloidin, sections were fixed in 50/50 methanol/PFA for 10 minutes at RT. Sections were then washed with PBS and blocked with 1 % BSA (Sigma Aldrich) in PBS for 30 minutes at RT. Sections were incubated overnight at 4°C with goat anti-SNAP23 (1 .25 gg/ml; Abeam, ab166808), diluted in 1 % PBS/BSA. Sections were washed in PBS and incubated with Alexa Fluor 488 donkey anti-goat IgG (0.02 gg/mL; Life Technologies) for 30 minutes at RT. Concurrently, control sections were incubated with non-immune IgG (Sigma Aldrich) at the same concentration as the primary antibody. Sections were washed in PBS, then incubated for 1 hour with tetramethylrhodamine isothiocyanate (TRITC)-conjugated Phalloidin (Sigma-Aldrich) diluted to 0.5 pg/mL in 1 % BSA/PBS to stain filamentous actin (F-actin). Sections were then washed with PBS and mounted using Vectashield with DAPI (Vector Laboratories) and coverslipped (No. 1 thickness). SNAP23-only and Phalloidin-only sections were prepared alongside as controls. Confocal images were taken using a Zeiss LSM 800 confocal microscope (Carl Zeiss). Pearson co-localisation coefficient (PCC) was calculated using the co-localisation module in the ZEN 2 software. PCC close to one indicated protein co- localisation.

Immunofluorescence of cell culture

Confluent monolayer cells grown on no. 1 coverslips were fixed with 2% paraformaldehyde for 20 minutes at RT. Cells were washed in PBS and incubated for 30 minutes with 100 mM glycine at RT. The cells were then permeablised at RT with 0.1 % triton for 5 minutes and incubated with a 2%BSA/PBS/0.1 % triton blocking solution for 1 hour at RT. Cells were incubated with goat anti-SNAP23 antibody (1.25 pg/ml; Abeam, ab166808), diluted in 2%BSA/PBS/0.1 % triton at 4°C overnight. Randomly selected coverslips were incubated with non-immune IgG (Sigma Aldrich) at the same concentration as the primary antibody. Cells were washed with PBS and incubated with the secondary antibody, Alexa Fluor 488-conjugated donkey anti-goat IgG (0.02 pg/mL; Life Technologies) for 30 minutes at RT. Coverslips were washed in PBS and mounted using Vectashield with DAPI (Vector Laboratories) onto glass slides. Z-series optical sections of the cells were taken with a Zeiss LSM 510 Meta Confocal microscope (Carl Zeiss) and images were acquired using the Zeiss LSM software (Carl Zeiss).

Isolation of rat uterine luminal epithelial cells

UECs were isolated as previously described (Kaneko et al. 2008. Reprod. Fertil. Dev. 20: 892-899) and immediately placed into lysis buffer (50 mM Tris-HCI, pH 7.5, 1 mM EDTA, 150 mM NaCI, 0.1 % SDS, 0.5% Deoxycholic acid, 1 % Igepal and 1 % protease inhibitor cocktail; Sigma Mammalian Cell lysis kit, Sigma Aldrich) with 10% PhosSTOP phosphatase inhibitor (Roche, NSW, Australia). The isolated cells were homogenised using a 23-gauge needle and a 1 ml_ syringe (Livingstone International, Rosebery, NSW, Australia) and centrifuged at 8,000 g at 4°C for 3 minutes. The supernatant was collected and frozen immediately in liquid nitrogen and stored at -80°C until use for western blotting. Cell culture lysate

Confluent cells were processed with lysis buffer (50 mmol/l Tris-HCI, pH 7.5, 1 mmol/l EDTA, 150 mmol/l NaCI, 0.1 % SDS, 0.5% deoxycholic acid, 1 % igepal, and 1 % protease inhibitor cocktail; Mammalian Cell lysis kit; Sigma Aldrich) and homogenised using a 23-gauge needle and a 1 ml_ syringe (Livingstone International) and centrifuged at 8,000 g at 4°C for 3 minutes. The supernatant was collected and frozen immediately in liquid nitrogen and stored at -80°C until use for western blotting.

Western blotting

Protein concentration was determined using the BCA protein assay (Micro BCATM protein assay kit; Thermo Fisher Scientific, MA, USA) and CLARIOstar microplate reader (BMG labtech Durham, NC, USA) according to the manufacturer’s instructions. To detect syntaxin-2, protein samples (20 pg) and sample buffer for syntaxin-2 (8% glycerol, 50 mM Tris-HCI, pH 6.8, 1.6% SDS, 0.024% bromophenol blue, 4% dithiotheitol (DTT)) were heated at 95°C for 10 minutes prior to loading. To detect SNAP23, protein samples (20 pg) and sample buffer (8% glycerol, 50 mM Tris- HCI, pH 6.8, 1.6% SDS, 0.024% bromophenol blue, 4% b-mercaptoethanol) were heated at 95°C for 5 minutes prior to loading. 12% FastCast SDS-polyacrylamide gels (Bio-Rad Laboratories, Hercules, CA, USA) were used, and protein was separated by electrophoresis at 200 V for 40 minutes. Proteins were transferred to a polyvinylidene dilfluoride (PVDF) membrane (Immunobilon™ transfer membrane; Millipore, Bedford, MA, USA) at 100 V for 1.5 hours. Syntaxin-2 membranes were blocked with 1 % skim milk in TBS-t (10 mM Tris-HCI, pH 7.4, 150 mM NaCI, 0.05% Tween 20) and SNAP23 membranes were blocked in 5% skim milk for 1 hour at RT with constant agitation. Membranes were then incubated with anti-syntaxin-2 (1.95 pg/ml, Abeam, ab170852) or anti-SNAP23 (0.25 pg/ml, Abeam, ab166808) primary antibodies diluted in 1 % skim milk in TBST overnight at 4°C on a rocking platform. The membranes were washed in TBS-t and subsequently incubated with goat anti-rabbit IgG horseradish peroxidase- conjugated secondary antibody (0.5 pg/ml; Dako, VIC, Australia) or rabbit anti- Goat IgG horseradish peroxidase-conjugated secondary antibody (0.125 pg/ml; Dako) for 2 hours at RT with constant agitation. Protein bands were detected with Immobilon Western HRP Substrate (Merck Millipore) and images were captured with a CCD camera and the Bio-Rad ChemiDoc MP System (Bio-Rad). Membranes were then incubated in stripping buffer [62.5 mM Tris-HCI (pH 6.7), 2% SDS and 100 mM b- mercaptoethanol] at 60°C for 45 minutes and re-probed with mouse monoclonal anti-b- actin antibody (0.4 pg/ml; Sigma Aldrich) overnight at 4°C and HRP-conjugated goat anti-mouse IgG (0.2 pg/mL; GE Healthcare) for 2 hours at RT to ensure equal loading.

Densitometry analysis

Protein band intensities were quantified using the Volume Analysis Tool with local background subtraction from the Bio-Rad Image Lab 4.0 software (Bio-Rad) and were normalised to b-actin band intensities from the same lane. Statistical analysis was performed on normalised intensities using GraphPad Prism Software (Version 7.02, GraphPad Software). Changes in quantity from day 1 , 3.5, 5.5, 6 and 7 were analysed using one-way ANOVA. For multiple comparisons, Turkey’s post hoc test was applied (reporting multiplicity-adjusted P-values) to determine which pairs of means were significantly different. Changes in abundance between HEC1 A and RL95-2 were analysed using unpaired two-tailed Student’s t-test. P<0.05 was determined to be significant. All graphs were generated using GraphPad Prism Software and error bars represent mean ± SEM.

Transmission electron microscopy

Uteri were cut into 5 mm pieces and were directly fixed in Karnovsky’s fixative (2.5% glutaraldehyde (ProSciTech, Australia), 2% paraformaldehyde (ProSciTech) in 0.1 M Sorenson’s phosphate buffer (PB, pH 7.4)) for 45 minutes at RT. The tissue was further cut into 0.5-1 mm slices under a droplet of fixative and returned to fresh fixative for another 45 minutes. The tissue was washed in 0.1 M PB, then washed again with 0.1 M maleate buffer (MB). The tissue was then post-fixed with 1 % tannic acid in 0.1 M MB. Tissue was rinsed in 0.1 M MB, then dH20 and further fixed with 1 % uranyl acetate in dH20. Tissue was rinsed again in dH20 and dehydrated with a graded series of ethanol, then infiltrated with Spurr’s resin (SPI supplies, Leicestershire, England, UK). Uterine slices were embedded in fresh Spurr’s resin in BEEM® capsules (ProSciTech), and polymerised at 60°C for 24 hours. Two blocks per animal were cut at 60-70 nm using a Leica Ultracut S ultramicrotome (Leica) and mounted onto 400-mesh copper grids. Sections were post- stained with 2% uranyl acetate in dH20 for 10 minutes and then with Reynold's lead citrate for 10 minutes. Sections were examined in a Jeol 1400 TEM (Jeol Ltd., Japan) at 100 kV.

Example 1 : Svntaxin-2 is present in UECs

Indirect immunofluorescence revealed that syntaxin-2 is present in UECs during early pregnancy in the rat uterus (Figure 1 ). On day 1 and day 3.5 of pregnancy, syntaxin- 2 is found cytoplasmically throughout the cell (Figure 1 A and 1 B). On day 5.5 and day 6, syntaxin-2 is localised in the cytoplasm and apically in UECs (Figure 1 C and 1 D). On day 7 of pregnancy, syntaxin-2 is localised cytoplasmically (Figure 1 E). Non- immune controls were performed with all immunofluorescence protocols and showed no staining in UECs. A representative image of day 5.5 non-immune controls is shown in Figure 1 F. Measurements of fluorescence intensity between day 1 and day 5.5 revealed that syntaxin- 2 levels were significantly greater on day 5.5 compared to day 1 of pregnancy; ^*p <0.05; n=5 (Figure 1 G).

Example 2: SNAP23 is present in UECs SNAP23 is present in UECs during early pregnancy when examined by indirect immunofluorescence and western blot analysis (Figure 2). On day 1 and day 3.5 of pregnancy, SNAP23 is cytoplasmic throughout the UECs (Figure 2A and 2B). On day 5.5 and 6, SNAP23 is localised apically (Figure 2C and 2D). On day 7, SNAP23 returns to its cytoplasmic localisation (Figure 2E). Non-immune controls were performed with all immunofluorescence protocols and showed no staining in UECs. A representative image of day 5.5 non-immune controls is shown in Figure 2F. Fluorescence intensity measurements revealed that SNAP23 is significantly more intense on day 5.5 compared to day 1 of pregnancy in UECs; ^*p <0.05; n=5 (Figure 2G).

Example 3: Western blotting Western blot analyses showed that syntaxin-2 (33 kDa) is present in UECs on all days of early pregnancy and is significantly more abundant on day 5.5 compared to day 1 (Figure 3A and 3C).

Western blot analyses showed that SNAP23 (57 kDa) is present in UECs on all days of early pregnancy (Figure 3B) and is significantly higher on day 5.5 compared to day 1 and day 7. A significant decrease in SNAP23 was also observed from day 3.5 to day 7 (Figure 3D).

These results indicate that the levels of SNAP23 in uterine tissue is typically low before pregnancy, in the very early stages of pregnancy (i.e., at day 1 , data not shown) and then after pregnancy has been established (i.e., after day 7). However, there is a short-lived, but rapid increase or spike in SNAP23 levels at day 5.5 of rat pregnancy which correlates with the time of optimum uterine receptivity for blastocyst implantation.

Example 4: SNAP23 is present in luminal secretions

Co-localisation experiments between SNAP23 and Phalloidin in UECs on day 5.5 showed no co-localisation of SNAP23 and actin (Figure 4). SNAP23 staining was localised in the entire cavity of the luminal space on day 5.5 (Figure 4A and 4B). Phalloidin stained apically and at the apico-lateral junctional areas on day 5.5 of pregnancy in UECs (Figure 4A and 4C). The observation that SNAP23 and actin do not co-localise in UECs, and the observation of SNAP23 staining in the entire cavity of the luminal space on day 5.5, suggests that SNAP23 is secreted from the UECs into the luminal fluid.

Figure 4E shows the presence of SNAP23 in luminal fluid taken from a day 5.5 pregnant rat using Western blot techniques. In uterine luminal fluid, SNAP23 was observed as bands at 70 kDa and 57 kDa.

An average PCC of 0.1 17 was calculated; n=5 (Figure 5A). SNAP23 only (Figure 5B) and Phalloidin only (Figure 5C) control were also imaged with all three filters and the same parameters, and there was no cross talk or bleed through observed between the channels. Non-immune controls were conducted and no staining was observed (Figure 5D).

These results demonstrate that secreted SNAP23 may be detected using non- invasive techniques, and that its presence, for example, in uterine fluid, may be indicative of uterine receptivity. Example 5: SNAP23 is present in human UECs

SNAP23 is present in beth HEC1 A and RL95-2 human immertalised endemetrial cell lines (Figure 6). Immuncflucrescence staining fcund that SNAP23 is present in discrete punctate dcts thrcughcut the cytcplasm in bcth HEC1A and RL95-2 cells (Figure 6A tc 6D). Nc staining was cbserved in ncn-immune ccntrcls. A representative image cf FIEC1A ncn-immune ccntrcl is shewn in Figure 6E. Western belt analysis revealed that SNAP23 is present at 23 kDa and 57 kDa in FIEC1 A and RL95-2 cells (Figure 6F). The 23 kDa band remained unchanged and the 57 kDa decreased in abundance in RL95-2 cells ccmpared tc FIEC1 A cells (Figure 6G).

Example 6: Extracellular vesicles

Generally speaking, there are three main categcries cf extracellular vesicles (EVs); exesemes (apprex. 50-100 nm), micrcvesicles (MVs) (apprex. 100 nm-1 pm) and apcptctic vesicles (apprex. 50 nm-5 pm) (Crescitelli et al. 2013. J. Extracell. Vesicles. 2: 20677; Denzer et al. 2000. J. Cell. Sci. 1 13 Pt 19: 3365-3374; EL Andalcussi et al. 2013. Nat. Rev. Drug Disccv. 12: 347-357). Other EVs that fall in between cr cutside cf these categories are often named according to their tissue of origin (Di Vizio et al. 2012. Am. J. Pathol. 181 : 1573-1574; Morello et al. 2013. Cell Cycle. 12: 3526-3536; Ronquist and Brody. 1985. Biochim. Biophys. Acta. 822: 203-218). In secretory cells, exosomes are formed in endosomal compartments called multivesicular bodies (MVBs). The fusion of MVBs to the plasma membrane releases smaller vesicles which are the exosomes (Bobrie et al. 201 1. Traffic. 12: 1659-1668). MVs are regions of plasma membrane that bud out and are pinched off as EVs (Antonyak and Cerione. 2014. Meth. Mol. Biol. 147- 173; Cocucci et al. 2009. Trends Cell Biol. 19: 43-51 ). Both of these EVs carry and transfer regulatory molecules such as microRNAs, proteins and lipids that may mediate intercellular communication (Bebelman et al. 2018. Pharmacol. Ther. 188: 1 -1 1 ; Marca and Fierabracci. 2017. Int. J. Mol. Sci. 18: 1974; Raposo and Stoorvogel. 2013. J. Cell. Biol. 200: 373-383).

EVs were found to be present on day 1 , day 5.5 and day 6 of pregnancy in the luminal cavity of the uterus when examined using the TEM (Figure 7 and Figure 8; Table 1 ). EVs observed to be 100 nm and under fell within the classification of exosomes. Exosomes were observed in the lumen on day 1 of pregnancy (Figure 7A). These exosomes exhibited two different types of membranes and were thus classified as exosome type 1 and type 2. Exosome type 1 ranged in size from 20 nm to 100 nm with a membrane thickness up to 25 nm. Exosome type 2 ranged in size from 10 nm to 60 nm with a membrane thickness up to 10 nm (Table 1 ). EVs larger than 100 nm were classified as MVs. MVs are seen in the lumen on day 1 , day 5.5 and day 6 of pregnancy with three different types of membranes (Figure 7; Table 1 ). They were classified as MVs type 1 , 2 and 3. MVs generally range from 100 nm to 1000 nm. However the MVs observed on day 1 , day 5.5 and day 6 ranged from 100 nm to 4100 nm. The range in diameter size for MV type 1 is 100 nm to 2500 nm with a membrane thickness up to 25 nm. MV type 2 ranged in size from 100 nm to 4100 nm with a membrane thickness up to 300 nm. MV type 3 ranged in size from 100 nm to 1000 nm with a membrane thickness up to 150 nm (Table 1 ).

Table 1 : Extracellular vesicle classification found on day 1 , 5.5 and 6 of pregnancy.

Conclusion

The results of the work described herein, indicate that SNAP23 levels expressed by uterine epithelial cells increases dramatically at day 5.5 of rat pregnancy. The time at which the highest levels of SNAP23 (day 5.5 in a pregnant rat) corresponds to a time in early pregnancy when the uterus is most receptive to embryo implantation and when the uterus is primed for blastocyst implantation. Accordingly, the inventors believe that observation of a similar spike in SNAP23 levels in other non-human animals and human would be indicative of the optimum time for implantation of a blastocyst.

Uterine luminal fluid is secreted by the uterine glands and the uterine epithelial cells. The inventors have found that in addition to being expressed by uterine epithelial cells, SNAP23 is secreted into uterine luminal fluid. Their studies demonstrate that SNAP23 plays a role in uterine luminal secretions and subsequently the microenvironment for blastocyst implantation. Further, these findings suggest that levels of SNAP23 representative of uterine expression of SNAP23 can be quantified using non-invasive techniques and that SNAP23 levels in the uterine fluid can be used to ascertain the receptivity of the uterine/endometrial tissue.

The present invention thus finds application in assisted reproductive technologies where clinicians must determine the optimum time for transfer of an embryo (e.g., blastocyst) into the uterus. The measurement of SNAP23 levels in uterine fluid is expected to assist clinicians/veterinarians in determining whether the uterus is likely to be receptive to embryo attachment (and therefore whether to transfer an embryo into the subject or whether to defer transfer until a later time point in the oestrous or menstrual cycle, or indeed defer transfer until an earlier time point in a later cycle). More specifically, by obtaining an understanding of the basal levels of SNAP23 in ULF of candidates for ART, and subsequently observing for a spike in SNAP23 levels in ULF, clinicians/veterinarians can determine with greater accuracy the time of optimum uterine receptivity for blastocyst implantation.

It will be appreciated by those skilled in the art that the methods and compositions described herein may be embodied in many other forms.

It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

Claims

1. A method of determining uterine receptivity in a female subject, the method comprising a step of detecting the presence or absence of SNAP23 in a biological sample obtained from the female subject, wherein the presence of SNAP23 in the sample is indicative of uterine receptivity and the absence of SNAP23 in the sample is indicative of uterine non-receptivity.

2. A method for determining the uterine receptivity profile of a female subject, the method comprising:

- determining the level or amount of SNAP23 in the samples;

- wherein the samples having the highest level or amount of SNAP23 among the plurality of samples, are indicative of a time point in the subject’s cycle at which the uterus is receptive to implantation and the samples having the lowest level or amount of SNAP23 are indicative of a time point in the subject’s cycle at which the uterus is not receptive to implantation; thereby determining the uterine receptivity profile of the subject.

3. A method for determining the uterine receptivity of a female subject, the method comprising:

- providing a female subject for whom uterine receptivity is to be determined;

- comparing the level or amount of SNAP23 in the test sample to the level or amount of SNAP23 in a control in the form of data representative of levels of SNAP23 in a non-receptive uterus; determining that the subject has a receptive uterus if the level or amount of SNAP23 in the test sample is higher than the levels of SNAP23 in the control; or determining that the subject does not have a receptive uterus if the level or amount of SNAP23 in the test sample is the same or lower than the level or amount of SNAP23 in the control, thereby determining the uterine receptivity of the subject.

4. A method for determining the uterine receptivity of a female subject, the method comprising:

- providing a female subject for whom uterine receptivity is to be determined;

- comparing the level or amount of SNAP23 in the test sample to the levels of SNAP23 in a control in the form of data representative of levels of SNAP23 in a receptive uterus; determining that the subject has a receptive uterus if the level or amount of SNAP23 in the test sample is the same or higher than the levels of SNAP23 in the control; or determining that the subject does not have a receptive uterus if the level or amount of SNAP23 in the test sample is lower than the levels of SNAP23 in the control, thereby determining the uterine receptivity of the subject.

5. The method of any one the preceding claims, wherein the step of determining the level or amount of SNAP23 in the biological sample comprises measuring or detecting the level or amount of SNAP23 in the biological sample or observing the level or amount of SNAP23 provided in a database.

6. The method of any one of the preceding claims wherein the level or amount of SNAP23 in the control in the form of data representative of SNAP23 in a non- receptive uterus, is a non-detectable level of SNAP23.

7. The method of any one of claims 2 to 6, wherein the control, in the form of data representative of levels of SNAP23 in a receptive or non-receptive uterus is data from one or more individuals for whom uterine receptivity was previously determined.

8. The method of any one of claims 2 to 6, wherein the control is in the form of data from the subject for whom uterine receptivity is being determined, optionally wherein the control is data from the subject obtained at an earlier time point in their oestrous or menstrual cycle.

9. A method of treating a female subject undergoing assisted reproduction, the method comprising:

10. A method of providing assisted reproduction in a female subject, the method comprising:

- providing a female subject in need of assisted reproduction;

- determining the level or amount of SNAP23 in a test biological sample obtained from the female subject; and

- comparing the level or amount of SNAP23 in the test sample to the level or amount of SNAP23 in a control in the form of data representative of level or amount of SNAP23 in a non-receptive uterus; preparing the female subject for embryo implantation if the level of SNAP23 in the test sample is higher than the level of SNAP23 in the control.

1 1. The method of claim 10, wherein the control data corresponds to data on the level of SNAP23 from the female subject at an earlier time point in her oestrous or menstrual cycle, preferably from a time point before ovulation or immediately after ovulation.

12. The method of claim 10 or 1 1 wherein the step of preparing the female subject for embryo implantation comprises inserting a catheter into the uterine cavity of the female subject.

13. The method of any one of claims 10 to 12 wherein the method further comprises transferring an embryo into the uterus of the female subject.

14. The method of any one of the preceding claims wherein the biological test sample is selected from the group consisting of: uterine luminal fluid (ULF), vaginal fluid or peritoneal fluid.

15. The method of any one of the preceding claims wherein the biological test sample is ULF.

16. A method of improving a step of assisted reproduction in a female subject, or for increasing the likelihood of successful implantation of an embryo, the method comprising:

- comparing the level or amount of SNAP23 in the test sample to the level or amount of SNAP23 in a control sample representative of baseline levels of SNAP23 in the uterine luminal fluid of the subject; determining that the subject has a receptive uterus if the level or amount of SNAP23 in the test sample is higher than the levels of SNAP23 in the control sample and proceeding to implant the embryo in the subject; or determining that the subject does not have a receptive uterus if the level or amount of SNAP23 in the test sample is the same or lower than the levels of SNAP23 in the control sample and determining not to implant the embryo in the subject, thereby improving a step of assisted reproduction or increasing the likelihood of successful implantation of the embryo.

17. A method for providing an assisted reproduction technology (ART) procedure to a female subject, the method comprising:

- comparing the level or amount of SNAP23 in the test sample to the level or amount of SNAP23 in a control sample representative of baseline levels of SNAP23 in the uterine luminal fluid of the subject; determining that the subject has a receptive uterus if the level or amount of SNAP23 in the test sample is higher than the levels of SNAP23 in the control sample and proceeding to implant the embryo in the subject; or determining that the subject does not have a receptive uterus if the level or amount of SNAP23 in the test sample is the same or lower than the levels of SNAP23 in the control sample and determining not to implant the embryo in the subject, thereby providing an ART procedure to the female subject.

18. A method for providing an assisted reproduction technology (ART) procedure to a female subject, the method comprising:

- providing a female subject who is a candidate for implantation of an embryo;

- determining the uterine receptivity of the subject by:

- if the levels of SNAP23 in the test sample are higher than the levels of SNAP23 in the control sample, then transferring the embryo to the uterus of the subject; or - if the levels of SNAP23 in the test sample are the same or lower than the levels of SNAP23 in the control sample, then storing the embryo for transfer at a later date, thereby providing an ART procedure to the female subject.

19. The method of any one of claims 10 to 18 wherein the embryo is a blastocyst.

20. The method of any one of claims 1 to 19, wherein the method comprises obtaining the biological test sample from the subject.

21. The method of any one of claims 9 to 20, wherein the subject is a candidate for implantation of an embryo or is suspected of having a receptive uterus based on a measurement of hormone levels in the subject, the time since ovulation, or the particular time in the oestrous or menstrual cycle of the subject.

22. The method of any one of the preceding claims wherein determining the presence or absence of, or the level or amount of SNAP23 comprises measuring the amount of SNAP23 in the biological test sample.

23. The method of any one of claims 1 to 21 wherein determining the presence or absence of, or the level or amount of SNAP23 comprises determining the amount of SNAP23 for the biological test sample provided in a database.

24. The method of claim 22 wherein the measuring comprises performing one or more of: enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, immunofluorescence, latex agglutination, hemagglutination, lateral flow immunoassay, immunoprecipitation, mass spectrometry or Western blot analysis.

25. The method of any one of claims 1 to 22 wherein detecting the presence or absence of SNAP23, or determining the level or amount of SNAP23 in the biological sample, comprises contacting the biological sample with an anti-SNAP23 antibody or an antigen-binding fragment thereof to form a SNAP23-antibody complex, and detecting the presence of the complex.

26. The method of claim 25 wherein the antibody is a monoclonal antibody.

27. The method of claim 25 or 26 wherein the antibody or antigen-binding fragment thereof is immobilised to a solid support.

28. The method of any one of claims 25 to 27 wherein the anti-SNAP23 antibody or antigen- binding fragment thereof is conjugated to a detectable label.

29. The method of any one of claims 25 to 28 wherein the method comprises contacting the complex with a second antibody or an antigen-binding fragment thereof, wherein the second antibody or antigen-binding fragment thereof specifically binds to SNAP23 or to the first anti-SNAP23 antibody or antigen-binding fragment thereof, and detecting the presence of the complex.

30. The method of claim 29 wherein the second antibody or antigen-binding fragment thereof is conjugated to a detectable label.

31. A kit for determining the uterine receptivity of a female subject, wherein the kit comprises a means for determining the level or amount of SNAP23 in a test biological sample obtained from a female patients, optionally including written instructions for determining the level or amount of SNAP23 in the sample according to the method of any one of claims 1 to 30.

32. The method of any one of claims 1 to 30 or the kit of claim 31 , wherein the female subject is a human subject.