The present invention belongs to the field of reproductive medicine. The present invention relates to a kit for determining the receptivity status of an endometrium comprising means for quantifying the expression of several genes as well as to a method for determining the receptivity status of an endometrium comprising the quantification of the expression of said genes.

BACKGROUND OF THE INVENTION

One of the key processes for the establishment of a successful pregnancy is embryonic implantation into the endometrium. Implantation is a complex process that involves an intricate dialog between the embryo and the endometrial cells (Singh et al., 201 1 ). This interaction is essential for the apposition, adhesion and invasion of the blastocyst in the human endometrium (Giudice and Irwin, 1999).

The human endometrium is a highly dynamic structure, which undergoes periodical changes during the menstrual cycle in order to reach a receptive status adequate for embryonic implantation. This period of receptivity is known as the window of implantation (WOI) and occurs between Days 19 and 21 of the menstrual cycle (Navot et al., 1991 ; Harper, 1992). In any other phase of the menstrual cycle, the endometrium is not receptive to pregnancy (Garrido-Gomez et al., 2013). Successful implantation requires therefore a viable embryo and synchrony between it and the receptive endometrium (Teh et al., 2016). The correct identification and prediction of the period of uterine receptivity is essential to maximize the effectiveness of assisted reproduction treatments.

The study of endometrial receptivity is not new as histological analysis has been traditionally used for endometrial dating (Noyes et al. 1950); however, the accuracy of this method to predict endometrial receptivity has been shown to be limited (Coutifaris et al., 2004; Murray et al., 2004). Some alternative methods to evaluate endometrial receptivity have been developed in the last decade: biochemical markers (Zhang et al., 2012), soluble ligands (Thouas et al., 2015), hormone receptors (Aghajanova et al., 2009), cytokines (Paiva et al., 201 1 ), microRNAs (Sha et al., 2011 ) or HOX-class homeobox genes (Kwon and Taylor, 2004).

Other studies, focused on the understanding of the molecular mechanisms underlying the histological changes of the endometrium during the menstrual cycle, have identified specific genes responsible for the alterations observed (Talbi et al., 2006). Some other reports have addressed this molecular analysis from a wider perspective, performing a global screening of the transcriptome at different moments of the menstrual cycle (Carson, 2002; Ponnampalam et al., 2004), or under different infertility conditions (Koler et al., 2009; Altmae et al., 2010), pathologies (Kao et al., 2003) or ovarian stimulation protocols (Horcajadas et al., 2005). Valuable information about the process of endometrial proliferation can be extracted from these studies. However, even though the list of studies is long, the number of molecular diagnostic tools to identify the moment of uterine receptivity is short (Lessey et al., 1995 Dubowy et al., 2003; Diaz-Gimeno et al., 201 1 ).

Therefore, there is a need for more accurate and easy to use diagnostic tools to assess the receptivity status of an endometrium and to maximize the effectiveness of assisted reproduction treatments.

SUMMARY

In a first aspect, the present invention relates to kit for determining the receptivity status of an endometrium comprising means for quantifying the expression of the following genes: AREG, ATP5B, CAPN6, CIR1 , CMTM5, CTNNA2, CXCL6, FOXP3, HBA1 , HBG1 , HMBS, HOXB7, IL1 R1 , IL4, IL5, ITGB1 , MAPK1 , NFKBIA, PGRMC1 , PLA2G4A, RHOA, SDHA, SERPIN 1.

DESCRIPTION

The identification of the optimal time for embryo transfer is essential to maximize the effectiveness of assisted reproduction treatment. The inventors have surprisingly found a highly reliable test for endometrial receptivity, capable of determining if the status of an endometrium is receptive or non-receptive. Moreover, in the case of a non-receptive endometrium, the test developed by the inventors is capable of distinguishing between a pre-receptive and post-receptive endometrium.

The present invention provides a very useful tool for clinical practice routine to help guide embryo transfers to be performed at the best endometrial moment, guaranteeing embryo-endometrial synchrony and thus, allowing for the achievement of better assisted reproduction treatment results. Couples with repeated implantation failure or previously failed in vitro fertilization (IVF) cycles or couples with recurrent miscarriage can benefit from the detailed analysis of endometrial receptivity and embryo-endometrial synchronization. The present invention is a new step in the field of personalized medicine in human reproduction in the management of the endometrium in preparation for embryo transfer, with the final goal of achieving better assisted reproduction treatment results, increasing embryo implantation rates and the likelihood of successful pregnancies.

In a preferred embodiment of the present invention, the kit of the first aspect further comprises means for quantifying the expression of at least one of the following genes: ANXA4, AQP3, ARG2, CXCL1 , GPX3, ITGA9, MAOA, MMP9, MT1 F, MT1 H, MT1 L, MT1 X, PGR, PTGS1 , RPL13A, SCGB2A2, TFF3.

In a preferred embodiment of the present invention, the kit of the first aspect comprises means for quantifying the expression of the following genes: AREG, ATP5B, CAPN6, CIR1 , CMTM5, CTNNA2, CXCL6, FOXP3, HBA1 , HBG1 , HMBS, HOXB7, IL1 R1 , IL4, IL5, ITGB1 , MAPK1 , NFKBIA, PGRMC1 , PLA2G4A, RHOA, SDHA, SERPIN1 , ANXA4, AQP3, ARG2, CXCL1 , GPX3, ITGA9, MAOA, MMP9, MT1 F, MT1 H, MT1 L, MT1X, PGR, PTGS1 , RPL13A, SCGB2A2, TFF3.

In a preferred embodiment of the present invention, the kit of the first aspect comprises means for quantifying the expression of substantially only the following genes: AREG, ATP5B, CAPN6, CIR1 , CMTM5, CTNNA2, CXCL6, FOXP3, HBA1 , HBG1 , HMBS, HOXB7, IL1 R1 , IL4, IL5, ITGB1 , MAPK1 , NFKBIA, PGRMC1 , PLA2G4A, RHOA, SDHA, SERPIN1 , ANXA4, AQP3, ARG2, CXCL1 , GPX3, ITGA9, MAOA, MMP9, MT1 F, MT1 H, MT1 L, MT1 X, PGR, PTGS1 , RPL13A, SCGB2A2, TFF3. As used herein, the expression "substantially only" means that if any other gene is quantified, then said other gene or genes is or are reference genes, which are only quantified for serving as a reference for the quantification of the expression, as a reference for the sample load.

In a preferred embodiment of the present invention the means for quantifying the genes comprise a pair of primers for each one of the genes.

In a preferred embodiment of the present invention, the kit of the first aspect further comprises a pair of primers for at least one reference gene. Preferably, the reference gene or genes is or are selected from actin, beta-2 microglobulin, cytochrome C1 , EMG1 N1 -specific pseudouridine methyltransferase, glyceraldehyde-3-phosphate dehydrogenase, TATA-box binding protein, topoisomerase (DNA) I, tyrosine 3- monooxygenase/tryptophan 5-monooxygenase activation protein zeta and combinations thereof. In a preferred embodiment, the kit of the first aspect comprises means for quantifying the expression of all the eight reference genes mentioned above. In another preferred embodiment, the reference genes are cytochrome C1 , EMG1 N1 -specific pseudouridine methyltransferase and glyceraldehyde-3-phosphate dehydrogenase (CYC1 , EMG1 , GAPDH).

Therefore, in a preferred embodiment, the kit of the invention consists essentially of means for quantifying the expression of the following genes: AREG, ATP5B, CAPN6, CIR1 , CMTM5, CTNNA2, CXCL6, FOXP3, HBA1 , HBG1 , HMBS, HOXB7, IL1 R1 , IL4, IL5, ITGB1 , MAPK1 , NFKBIA, PGRMC1 , PLA2G4A, RHOA, SDHA, SERPIN1 , ANXA4, AQP3, ARG2, CXCL1 , GPX3, ITGA9, MAOA, MMP9, MT1 F, MT1 H, MT1 L, MT1X, PGR, PTGS1 , RPL13A, SCGB2A2, TFF3, actin, beta-2 microglobulin, cytochrome C1 , EMG1 N1 -specific pseudouridine methyltransferase, glyceraldehyde-3-phosphate dehydrogenase, TATA-box binding protein, topoisomerase (DNA) I and tyrosine 3- monooxygenase/tryptophan 5-monooxygenase activation protein zeta. As used herein, the expression“consists essentially of” means that no other genes are quantified and that the kit may contain other reagents such as buffers, polymerase enzymes, nucleotides mixes, etc, which are necessary for the analysis of the expression of the above mentioned genes.

In a preferred embodiment of the present invention, the kit of the first aspect further comprises a master mix comprising a DNA-binding dye.

In another preferred embodiment of the present invention, the kit of the first aspect further comprises a specific probe for each one of the genes. Preferably the probe is labeled, preferably dual-labeled. As used herein, the term “labeled” means that the probe is marked by means of a fluorescent reporter dye on the 5' base and a quencher located on the 3' base.

In a preferred embodiment of the present invention, the kit of the first aspect further comprises at least one of the following: a DNA polymerase, a nucleotide mix, a buffer, DNase-free water.

In a second aspect, the present invention relates to a method for determining the receptivity status of an endometrium comprising the quantification of the expression of the following genes: AREG, ATP5B, CAPN6, CIR1 , CMTM5, CTNNA2, CXCL6, FOXP3, HBA1 , HBG1 , HMBS, HOXB7, IL1 R1 , IL4, IL5, ITGB1 , MAPK1 , NFKBIA, PGRMC1 , PLA2G4A, RHOA, SDHA, SERPIN 1 , in a sample.

In a preferred embodiment of the second aspect, the method further comprises the quantification of the expression of at least one of the following genes: ANXA4, AQP3, ARG2, CXCL1 , GPX3, ITGA9, MAOA, MMP9, MT1 F, MT1 H, MT1 L, MT1X, PGR, PTGS1 , RPL13A, SCGB2A2, TFF3.

In a more preferred embodiment of the second aspect of the invention, the expression of the following genes is quantified: AREG, ATP5B, CAPN6, CIR1 , CMTM5, CTNNA2, CXCL6, FOXP3, HBA1 , HBG1 , HMBS, HOXB7, IL1 R1 , IL4, IL5, ITGB1 , MAPK1 , NFKBIA, PGRMC1 , PLA2G4A, RHOA, SDHA, SERPIN1 , ANXA4, AQP3, ARG2, CXCL1 , GPX3, ITGA9, MAOA, MMP9, MT1 F, MT1 H, MT1 L, MT1 X, PGR, PTGS1 , RPL13A, SCGB2A2, TFF3.

In a preferred embodiment of the present invention, the method of the second aspect further comprises quantifying the expression of at least one reference gene in the sample. Preferably, the reference gene or genes is or are selected from actin, beta-2 microglobulin, cytochrome C1 , EMG1 N1 -specific pseudouridine methyltransferase, glyceraldehyde-3-phosphate dehydrogenase, TATA-box binding protein, topoisomerase (DNA) I, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta and combinations thereof. In another preferred embodiment, the reference genes are cytochrome C1 , EMG1 N1 -specific pseudouridine methyltransferase and glyceraldehyde-3-phosphate dehydrogenase (CYC1 , EMG1 , GAPDH).

In a preferred embodiment of the second aspect of the invention, the quantification is made by qPCR, preferably by RT-qPCR.

In a more preferred embodiment of the second aspect of the invention, the sample is a cDNA sample, preferably a cDNA obtained from an RNA sample from a female subject, preferably a female human. More preferably, the RNA has been obtained from an endometrial biopsy.

In a preferred embodiment of the method of the invention, the results of the gene expression levels can classify an endometrium into:“receptive” or“non receptive”. When an endometrium is classified as“receptive”, it means that the window of implantation matches the day on which the biopsy was taken. The embryo transfer should be scheduled to be performed on the same day and type of cycle on which the biopsy was taken. If an endometrium is classified as“non-receptive” (either pre-receptive or post- receptive), it means that the WOI is displaced. A new endometrial biopsy on the day estimated needs to be obtained so that the WOI is identified and a personalized embryo transfer is scheduled. Another aspect of the present invention is a method for improving the pregnancy success of an IVF treatment comprising:

(i) determining the receptivity status of an endometrium following the method of the second aspect of the present invention,

(ii) determining the day in which the endometrium is receptive following the method of the present invention, and

(iii) transferring at least one embryo, preferably at blastocyst stage, at the day determined in step (ii).

The present invention relates to a method for determining if the status of an endometrium is receptive or non-receptive, and in case it is non-receptive, if it is pre-receptive or post- receptive. The method comprises analyzing in a sample obtained from an endometrium, preferably a human endometrium, the gene expression levels of the following genes: AREG, ATP5B, CAPN6, CIR1 , CMTM5, CTNNA2, CXCL6, FOXP3, HBA1 , HBG1 , HMBS, HOXB7, IL1 R1 , IL4, IL5, ITGB1 , MAPK1 , NFKBIA, PGRMC1 , PLA2G4A, RHOA, SDHA, SERPIN1 , ANXA4, AQP3, ARG2, CXCL1 , GPX3, ITGA9, MAOA, MMP9, MT1 F, MT1 H, MT1 L, MT1 X, PGR, PTGS1 , RPL13A, SCGB2A2, TFF3.

The mRNA of an endometrial biopsy is extracted and purified by methods known in the art. The expression levels of the above mentioned genes are determined and said expression levels are analyzed by a computer software comprising a specific prediction model which classifies and determines the status of the endometrium depending on the gene expression levels. The prediction model is a mathematical system using different algorithms, formulas, to distinguish between a receptive profile, a pre-receptive profile and a post-receptive profile.

The prediction model is designed from the data obtained from the analysis of the expression of the above mentioned genes in different samples, as explained in the examples“Patient selection and sample collection” and“Principal component analysis and discriminant functional analysis” sections.

EXAMPLES

RNA extraction and cDNA preparation

Total RNA was extracted using RNeasy mini kit (Qiagen, London, UK); RNA purity and concentration was confirmed by NanoDrop 2000 Spectophotometer (Thermo Scientific, Waltham, MA, USA) and RNA integrity was assessed using Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) following the manufacturer’s instructions. RNAs were reverse-transcribed into cDNA using Fluidigm Reverse Transcription Master Mix (Fluidigm, San Francisco, CA, USA) and either immediately used or stored at -20°C until further downstream processing.

Gene expression analysis

The pairs of primers targeting the selected and reference genes were designed and are indicated in the sequence listing. Table 2 includes the primer sequences.

Specific target amplification (STA) was carried out on cDNA samples using Fluidigm PreAmp Master Mix and DELTAgene assays following the manufacturer’s instructions (Fluidigm, San Francisco, CA). RT-qPCR reactions were performed following the Fast Gene Expression Analysis Using Evagreen on the Biomark HD Systemand 96.96 Dynamic Array™ IFC (Fluidigm, San Francisco, CA).

Data was collected with Fluidigm® Real-Time PCR analysis software using linear baseline correction method and global auto Cq threshold method. Data were then exported to Excel as.csv files and Cq values were normalized using the mean of the reference genes selected.

Study design

Gene expression data from endometrial biopsies obtained at different moments of the menstrual cycle from healthy fertile donors (Group A) and subfertile women (Group B) were analyzed.

Patient selection and sample collection

Group A consisted of 96 healthy fertile donors (18-34 years), with regular menstrual cycles and normal BMI (25-30). Endometrial biopsies from this group were obtained on two different days of the same natural menstrual cycle: LH + 2, i.e. 2 days after the luteinizing hormone surge and LH + 7, i.e. 7 days after the LH surge. Group B consisted of 120 subfertile patients (30-42 years) undergoing hormone-replacement therapy (HRT) cycles. Endometrial biopsies from this group of patients were obtained after five full days of progesterone administration (P ₄ +5). Endometrial biopsies of approximately 30 mg were obtained from the uterine fundus using a Pipelle catheter (Gynetics, Namont- Achel, Belgium) under sterile conditions. Tissue was then placed in a CryoTube® (Nunc, Roskilde, Denmark) containing 1 ml RNAIater® (Sigma-Aldrich, St Louis, MO, USA) and stored at -20 °C until further processing. Principal component analysis and discriminant functional analysis

Differential expression of genes from LH + 2 and LH + 7 groups was assessed by comparing ACq values (paired t-test (P < 0.05)). Fold change (-AACq) was calculated to determine upregulated and downregulated genes in the WOI. In order to assess if receptivity status could be established with a reduced number of genes, a principal component analysis (PCA) of the genes showing significant fold change between LH + 2 and LH + 7 was performed. Discriminant functional analysis (DA) was then used to evaluate the ability of the genes with the highest absolute coefficient value from each of the leading principal components to accurately discriminate samples into the following states: proliferative, receptive, pre-receptive and post-receptive. A Split- Sample validation of the DA was performed to assess the reliability and robustness of discriminant findings. Both fertile and infertile patient samples were split into two subsets. One data set (70% of the samples) was used as a training set and the other one as testing set (remaining 30% of the samples). The percentage of correct classifications was calculated to determine the reliability of the DA model. Data analyses were performed by using IBM SPSS Statistics software version 19.0.

Clinical experience: reproductive outcomes

Analysis of the outcomes after the first embryo transfer (blastocyst) when using the method of the invention indicated a significantly higher positive pregnancy rate (gestational sac at 8 weeks and heartbeat positive at 12 weeks) in the group of patients that followed the progesterone pretreatment recommendation using the method of the invention, compared to those that deviated more than 12 h from this recommendation. In total, 741 cases were analysed, with an average of 1.16 embryos transferred and a mean age of the women of 41.41 years for the group that followed the progesterone pretreatment recommendation using the method of the invention, and an average of 1.28 embryos transferred and a mean age of the women of 41.06 years for the group that deviated more than 12 h from this recommendation.

Within the group of cases that followed the recommendation using the method of the present invention, clinical pregnancies were almost two times higher than the ones from the group whose transfers deviated more than 12 h from the recommendation (that is, that deviated more than 12 h from the WOI). These differences were statistically significant (44.5% vs 26%, p=0.002, n=741 ). Pregnancy rates of the group of transfers performed with a deviation of more than 12 h were studied in further detail. This group was divided into:

(i) transfers performed between 12 and 24 h from the recommendation using the method of the invention (WOI), and

(ii) transfers performed with more than 24 h deviation from this window.

For the group that deviated from 12 to 24 h from the recommendation, an average of 1.29 embryos were transferred and the mean age of the women was 41.40 years. For the group that deviated from more than 24 h from the recommendation, an average of 1.27 embryos were transferred and the mean age of the women was 40.62 years. The group were the transfer was performed with more than 24 h of deviation from the WOI showed a significant decline in pregnancy rates. The pregnancy rates of the three groups were the following: following the method of the invention: 44.5 %

from 12 to 24 h deviation: 33.3 %

- more than 24 h deviation: 15.6 %

These differences in the pregnancy rates of the three groups were significant (p=0.001 ).

Within the group of cases that followed the recommendation using the method of the present invention, clinical pregnancies were almost four times higher than the ones from the group whose transfers deviated more than 24 h from the recommendation. Successful pregnancies after following the embryonic transfer recommendations of the test of the invention

Endometrial biopsies from 11 women were obtained after four to six full days of progesterone administration (P ₄ + 6). All the women were in a hormone replacement therapy cycle (HRT). The receptivity of the endometrium was analysed using the method of the invention and the results of the following table were obtained.

When the endometrium was found to be Receptive, it was recommended to transfer the embryo (at blastocyst stage) after the same number of days of progesterone administration as when the biopsy was obtained. Table 1 . Examples of successful pregnancies after using the method of the invention.

HRT; hormone-replacement therapy (HRT) cycle

P _fi +5: obtained after five full days of progesterone impregnation b-hCG positive result >25 IU/1; Sac and heartbeat positive determined by ultrasound at 8 and 12 weeks respectively.

When the endometrium was found to be Pre-Receptive, it was recommended to transfer the embryo (at blastocyst stage) after one day or half a day more in respect to the number of days of progesterone administration when the biopsy was obtained.

When the endometrium was found to be Post-Receptive, it was recommended to transfer the embryo (at blastocyst stage) after one day or half a day less in respect to the number of days of progesterone administration when the biopsy was obtained.

Following this strategy, pregnancy and healthy births were achieved in all 1 1 cases (table 1 )·

Clinical case reports Case l :

A woman with a history of 2 gestations, 2 miscarriages and no deliveries had received 5 IVF treatments without any pregnancy outcome and 5 IVF after preimplantation genetic testing for aneuploidy (PGT-A), without any pregnancy either.

The method of the invention was performed on an endometrial biopsy from this woman at day P ₄ +7 (7 full days of progesterone administration) and was determined to be receptive. It was recommended to transfer the embryos at day P ₄ +7. Following this recommendation, pregnancy was achieved (positive b-hGC (beta human chorionic gonadotropin) and positive heartbeat).

Case 2: A woman with a history of no gestations, no miscarriages and no deliveries had received 4 artificial insemination treatments, 9 IVF+PGT-A treatments and 1 oocyte donation without any pregnancy outcome.

The method of the invention was performed on an endometrial biopsy from this woman at day P ₄ +5.5 (5.5 days of progesterone administration) and was determined to be Pre- receptive. It was recommended to transfer the embryos at day P ₄ +6.5. Following this recommendation, pregnancy was achieved (positive b-hGC (beta human chorionic gonadotropin), heartbeat and birth). Case 3:

A woman with a history of 5 gestations, 5 miscarriages and no deliveries had received 3 artificial insemination treatments followed by 2 miscarriages, 1 IVF+PGT-A treatment followed by miscarriage and 1 oocyte donation + PGT-A followed by 2 cryotransfers and 2 biochemical miscarriages.

The method of the invention was performed on an endometrial biopsy from this woman at day P ₄ +5.5 (5.5 days of progesterone administration) and was determined to be Pre- receptive. It was recommended to transfer the embryos at day P ₄ +7. Following this recommendation, pregnancy was achieved (positive b-hGC (beta human chorionic gonadotropin) and positive heartbeat).

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