US-12625145-B2 - Endometrial receptivity determination

US12625145B2US 12625145 B2US12625145 B2US 12625145B2US-12625145-B2

Abstract

Endometrial receptivity status is determined by measuring, in an uterine fluid sample taken from a woman, the amounts of at least three proteins selected among NNMT, LCN2, PGR, SLC26A2, SLC34A2, TCN1, ENPP3, GRN, STC1, DPP4, MPO, CD55, ELANE, MSLN, CTSB, RNASET2, CRISP3, MVP, MMP26, AOC1 and SDCBP2. The EM receptivity status of the woman is determined based on a comparison of the measured amounts of the at least three proteins with respective control amounts.

Inventors

Andres Salumets
Sergo KASVANDIK
Maire PETERS
Tanel KAART

Assignees

CELVIA CC AS

Dates

Publication Date: 20260512
Application Date: 20200511
Priority Date: 20190517

Claims (11)

1 . A method for treatment comprising implanting an embryo in a human female subject that has been identified as having a receptive endometrium, the method comprising: measuring, in a uterine fluid (UF) sample taken from the human female subject, a respective amount of at least three proteins selected from the group consisting of nicotinamide N-methyltransferase encoded by the gene NNMT, neutrophil gelatinase-associated lipocalin encoded by the gene LCN2, progesterone receptor encoded by the gene PGR, sulfate transporter encoded by the gene SLC26A2, sodium-dependent phosphate transport protein 2B encoded by the gene SLC34A2, transcobalamin-1 encoded by the gene TCN1, ectonucleotide pyrophosphatase/phosphodiesterase family member 3 encoded by the gene ENPP3, granulins encoded by the gene GRN, stanniocalcin-1 encoded by the gene STC1, dipeptidyl peptidase 4 encoded by the gene DPP4, myeloperoxidase encoded by the gene MPO, complement decay-accelerating factor encoded by the gene CD55, neutrophil elastase encoded by the gene ELANE, mesothelin encoded by the gene MSLN, cathepsin B encoded by the gene CTSB, ribonuclease T2 encoded by the gene RNASET2, cysteine-rich secretory protein 3 encoded by the gene CRISP3, major vault protein encoded by the gene MVP, matrix metalloproteinase-26 encoded by the gene MMP26, amiloride-sensitive amine oxidase copper-containing encoded by the gene AOC1, and syntenin-2 encoded by the gene SDCBP2; comparing the respective amount with a respective control amount of the at least three proteins; determining the human female subject has a receptive endometrium by determining that the human female subject is in a mid-secretory (MSE) phase wherein, for each protein of the at least three proteins, the respective amount of the protein is lower than the control amount if the protein is progesterone receptor encoded by the gene PGR and the respective amount of the protein is higher than the control amount if the protein is selected from the group consisting of nicotinamide N-methyltransferase encoded by the gene NNMT, neutrophil gelatinase-associated lipocalin encoded by the gene LCN2, sulfate transporter encoded by the gene SLC26A2, sodium-dependent phosphate transport protein 2B encoded by the gene SLC34A2, transcobalamin-1 encoded by the gene TCN1, ectonucleotide pyrophosphatase/phosphodiesterase family member 3 encoded by the gene ENPP3, granulins encoded by the gene GRN, stanniocalcin-1 encoded by the gene STC1, dipeptidyl peptidase 4 encoded by the gene DPP4, myeloperoxidase encoded by the gene MPO, complement decay-accelerating factor encoded by the gene CD55, neutrophil elastase encoded by the gene ELANE, mesothelin encoded by the gene MSLN, cathepsin B encoded by the gene CTSB, ribonuclease T2 encoded by the gene RNASET2, cysteine-rich secretory protein 3 encoded by the gene CRISP3, major vault protein encoded by the gene MVP, matrix metalloproteinase-26 encoded by the gene MMP26, amiloride-sensitive amine oxidase copper-containing encoded by the gene AOC1, and syntenin-2 encoded by the gene SDCBP2; and implanting an embryo in the human female subject determined to have a receptive endometrium.
2 . The method according to claim 1 , wherein measuring the respective amount comprises measuring, in the UF sample taken from the human female subject, the respective amount of nicotinamide N-methyltransferase encoded by the gene NNMT and the respective amount of at least two proteins selected from the group consisting of, neutrophil gelatinase-associated lipocalin encoded by the gene LCN2, progesterone receptor encoded by the gene PGR, sulfate transporter encoded by the gene SLC26A2, sodium-dependent phosphate transport protein 2B encoded by the gene SLC34A2, transcobalamin-1 encoded by the gene TCN1, ectonucleotide pyrophosphatase/phosphodiesterase family member 3 encoded by the gene ENPP3, granulins encoded by the gene GRN, stanniocalcin-1 encoded by the gene STC1, dipeptidyl peptidase 4 encoded by the gene DPP4, myeloperoxidase encoded by the gene MPO, complement decay-accelerating factor encoded by the gene CD55, neutrophil elastase encoded by the gene ELANE, mesothelin encoded by the gene MSLN, cathepsin B encoded by the gene CTSB, ribonuclease T2 encoded by the gene RNASET2, cysteine-rich secretory protein 3 encoded by the gene CRISP3, major vault protein encoded by the gene MVP, matrix metalloproteinase-26 encoded by the gene MMP26, amiloride-sensitive amine oxidase copper-containing encoded by the gene AOC1, and syntenin-2 encoded by the gene SDCBP2.
3 . The method according to claim 2 , wherein measuring the respective amount comprises measuring, in the UF sample taken from the human female subject, the respective amount of nicotinamide N-methyltransferase encoded by the gene NNMT, neutrophil gelatinase-associated lipocalin encoded by the gene LCN2, progesterone receptor encoded by the gene PGR; and comparing the respective amount comprises comparing the respective amount with the respective control amount of nicotinamide N-methyltransferase encoded by the gene NNMT, neutrophil gelatinase-associated lipocalin encoded by the gene LCN2, progesterone receptor encoded by the gene PGR.
4 . The method according to claim 3 , wherein measuring the respective amount comprises measuring, in the UF sample taken from the human female subject, the respective amount of nicotinamide N-methyltransferase encoded by the gene NNMT, neutrophil gelatinase-associated lipocalin encoded by the gene LCN2, progesterone receptor encoded by the gene PGR and an amount of sulfate transporter encoded by the gene SLC26A2 and/or of sodium-dependent phosphate transport protein 2B encoded by the gene SLC34A2; and comparing the respective amount comprises comparing the respective amount with the respective control amount of nicotinamide N-methyltransferase encoded by the gene NNMT, neutrophil gelatinase-associated lipocalin encoded by the gene LCN2, progesterone receptor encoded by the gene PGR and control amount of sulfate transporter encoded by the gene SLC26A2 and/or of sodium-dependent phosphate transport protein 2B encoded by the gene SLC34A2.
5 . The method according to claim 4 , wherein measuring the respective amount comprises measuring, in the UF sample taken from the human female subject, the respective amount of nicotinamide N-methyltransferase encoded by the gene NNMT, neutrophil gelatinase-associated lipocalin encoded by the gene LCN2, progesterone receptor encoded by the gene PGR and the amount of sulfate transporter encoded by the gene SLC26A2; and comparing the respective amount comprises comparing the respective amount with the respective control amount of nicotinamide N-methyltransferase encoded by the gene NNMT, neutrophil gelatinase-associated lipocalin encoded by the gene LCN2, progesterone receptor encoded by the gene PGR and control amount of sulfate transporter encoded by the gene SLC26A2.
6 . The method according to claim 2 , wherein measuring the respective amount comprises measuring, in the UF sample taken from the human female subject, the respective amount of nicotinamide N-methyltransferase encoded by the gene NNMT, neutrophil gelatinase-associated lipocalin encoded by the gene LCN2, progesterone receptor encoded by the gene PGR and an amount of sulfate transporter encoded by the gene SLC26A2 and/or of sodium-dependent phosphate transport protein 2B encoded by the gene SLC34A2; and comparing the respective amount comprises comparing the respective amount with the respective control amount of nicotinamide N-methyltransferase encoded by the gene NNMT, neutrophil gelatinase-associated lipocalin encoded by the gene LCN2, progesterone receptor encoded by the gene PGR and control amount of sulfate transporter encoded by the gene SLC26A2 and/or of sodium-dependent phosphate transport protein 2B encoded by the gene SLC34A2.
7 . The method according to claim 6 , wherein measuring the respective amount comprises measuring, in the UF sample taken from the human female subject, the respective amount of nicotinamide N-methyltransferase encoded by the gene NNMT, neutrophil gelatinase-associated lipocalin encoded by the gene LCN2, progesterone receptor encoded by the gene PGR and the amount of sulfate transporter encoded by the gene SLC26A2; and comparing the respective amount comprises comparing the respective amount with the respective control amount of nicotinamide N-methyltransferase encoded by the gene NNMT, neutrophil gelatinase-associated lipocalin encoded by the gene LCN2, progesterone receptor encoded by the gene PGR and control amount of sulfate transporter encoded by the gene SLC26A2.
8 . The method according to claim 1 , wherein measuring the respective amount comprises measuring, in the UF sample taken from the human female subject, the respective amount of at least three but no more than six proteins selected from the group; and comparing the respective amount comprises comparing the respective amount with the respective control amount of the at least three but no more than six proteins.
9 . The method according to claim 1 , wherein measuring the respective amount comprises measuring, in the UF sample taken from the human female subject, the respective amount of the at least three proteins selected from the group using a respective antibody that specifically binds to the respective protein of the at least three proteins selected from the group.
10 . The method according to according to claim 1 , wherein measuring the respective amount comprises: separating proteins from the UF sample taken from the human female subject on a two-dimensional gel electrophoresis gel; identifying the at least three proteins selected from the group on the two-dimensional gel electrophoresis gel; and measuring a respective amount of the identified at least three proteins selected from the group on the two-dimensional gel electrophoresis gel.
11 . The method according to claim 1 , further comprising: measuring, in the UF sample taken from the human female subject, an amount of elongation factor 1-alpha 1 (EEF1A1); and normalizing the respective amount of the at least three proteins based on the amount of EEF1A1, wherein comparing the respective amount comprises comparing the respective normalized amount with a respective control amount of the at least three proteins.

Description

The sequence listing submitted herewith, entitled “Nov-15-2021-Sequence-Listing_ST25.txt”, created Nov. 15, 2021 and having a size of 13,687 bytes, is incorporated herein by reference. TECHNICAL FIELD The present invention generally relates to determination of endometrial receptivity, and in particular to a uterine fluid protein panel that can be used in such a determination. BACKGROUND The uterine micromilieu and its main medium, the uterine fluid (UF), play an important role in reproductive success, influencing sperm movement through the uterus to the fallopian tubes, embryo development and implantation processes. The UF is a complex mixture of molecules secreted primarily by the endometrial (EM) glandular epithelial cells, but also by the immune cells and exosomes derived from EM cells. Additionally, detached or non-adherent cells and passively diffused molecules may likely contribute to the final repertoire of molecules found in the UF. The lower molecular weight fraction has been described to consist of compounds, such as amino acids, lactate, pyruvate, oxygen, glucose, antioxidants, ions, growth factors, hormones and lipids. To date, high-throughput proteomics studies have established that the UF contains at least 600-1,500 different proteins, depending on the sampling procedures and analysis methods used. The proteomic component of the UF reflects not only protein expression patterns in the EM tissue but may also contain components from other reproductive tract fluids, such as cervicovaginal fluid and fallopian tubes or even of peritoneal origin. Regardless of the origin of the compounds identified in the UF, all of them provide a suitable buffer for the developing embryo in transit and facilitate its arrival to the correct intrauterine location for subsequent implantation. During the menstrual cycle, successful implantation is considered to be possible in a short period of time, known as the window of implantation (WOI), starting on cycle days 19 or 20 and lasting for about 4-5 days. Determination of the WOI has an utmost importance in the in vitro fertilization (IVF) procedure to increase chances of successful commencement of pregnancy. Recurrent implantation failure (RIF) patients form one of the most complex groups of patients whose conception are cumbersome and can require large amount of resources and time, while causing emotional stress to both infertile couples and clinicians. RIF is defined when at least three implantation failures with good quality embryo transfers have occurred or when conception was not achieved after transfer of at least ten good-quality embryos. In some patients with RIF, WOI may be temporally displaced, leading to asynchrony between the developing embryo and the EM tissue that may result in implantation failure. Alternatively, the endometrial RIF may also arise from a molecularly disrupted WOI without a temporal shift. Currently, there are few approaches in clinical use that enable determination of EM receptivity by gene expression profiling of endometrial tissue. U.S. Pat. No. 10,081,840 discloses an endometrial receptivity array (ERA) that allows evaluation of the receptive state of a human endometrium. ERA requires taking an endometrial sample by biopsy from the fundus of the uterus of a woman 7 days after her endogenous luteinizing hormone (LH) surge (LH+7) and then measuring the expression of 238 genes from the tissue sample. The endometrium is determined to be receptive based on a fold change greater than or equal to about three for the 238 genes when compared to a non-receptive endometrial sample. The gene expression profiling methods exemplified by ERA, however, are invasive methods requiring taking an EM tissue sample. This means that such biopsy-based methods exclude embryo transfer during the same menstrual cycle as biopsy sampling. WO 2018/198054 discloses a method and a diagnostic kit for analyzing the inflammatory state and endometrial receptivity in women suffering from unexplained spontaneous recurrent abortion and/or infertility. The ratio between the levels of expression of the NALP-3 and thrombomodulin proteins in the endometrium is used in the analysis. A ratio of greater than 1 is indicative of an increased condition of endometrial inflammation compared to a fertile subject and is associated with reduced endometrial receptivity. SUMMARY It is a general objective to determine EM receptivity status without the need for taking biopsies. This and other objectives are met by the embodiments. The present invention is defined in the independent claim. Further embodiments of the invention are defined in the dependent claims. An aspect of the embodiments relates to a method for determining endometrial (EM) receptivity status of a human female subject. The method comprises measuring, in a uterine fluid (UF) sample taken from the human female subject, a respective amount of at least three proteins selected from the group consisting of nicotinamide N-methyltra