How to determine the implantation window

Abstract

The present invention relates to a method for determining the endometrial receptivity of a female subject and for selecting female subjects as candidates to receive an embryo based on dissolved gas analysis of endometrial fluid.

Description

[Technical field]

[0001] The present invention relates to the field of assisted reproductive technology, and more particularly to a method based on the analysis of dissolved gases for determining endometrial implantation competence in a female subject and a method for selecting female subjects as candidates for receiving an embryo. [Background technology]

[0002] Infertility is a global health problem affecting approximately 190 million people worldwide. Among assisted reproductive technologies (ART), in vitro fertilization (IVF) is one of the preferred treatments, where an embryo is cultured in a laboratory and then transferred into the uterus of the future mother. On average, a woman needs three complete IVF cycles to become pregnant. In Europe, more than 900,000 cycles are performed annually, but only 35% become pregnant. One of the main causes of failure is embryo implantation, with a live birth rate per embryo transfer of less than 30%.

[0003] The global IVF market is expected to reach USD 37.7 billion by 2027, expanding at a compound annual growth rate (CAGR) of 9.5%. Europe dominated the IVF procedures and equipment market in 2019. Japan, the United States, and Spain were the most active countries globally, but demand in Asia Pacific is expected to soar. As the product is targeted to be used by medical professionals, potential customers are private and public IVF clinics. There are 1,200 clinics in Europe (250 in Spain) and 480 in the United States.

[0004] Embryo quality, uterine implantation competence, and embryo-uterine synchrony are important for successful implantation. Recently, it has been possible to assess embryo maturity and quality, but estimation of endometrial development and synchrony is very imprecise, since it relies on counting the days after ovulation or the days of progesterone treatment. The period during which the endometrium is receptive is called the window of implantation (WOI) and is usually during the luteal phase, days 19–21 of the menstrual cycle. Its duration and position in the cycle depends on various factors and can vary from woman to woman and from month to month in the same woman, making it difficult to determine empirically. Accuracy of endometrial receptivity detection is essential for each woman, but not for those with displaced or shortened WOI, which account for 30% of all IVF patients. In women with repeated implantation failure, this percentage is even higher. Ultrasound / Doppler techniques to check endometrial thickness and vascularity have not been successful in measuring endometrial implantation competence and synchrony with precision, but no useful markers for endometrial implantation competence have been found using histological approaches. The advent of "omics" techniques has allowed large-scale analysis of gene expression, protein, microRNA, lipid metabolism, and secretion patterns of receptive endometrium. Commercial tests have focused on the detection of gene expression profiles that characterize the availability of receptive endometrium, e.g., ERA, ERPeak, Le Win-Test, ER Map. These methods are invasive and require several days to biopsy the endometrium and obtain the results, making it impossible to perform the test and implant during the same cycle.

[0005] There is a need to improve timely detection of endometrial implantation competence, embryo transfer coordinated with uterine development, and embryo-uterine synchrony matching to improve the success of assisted reproductive techniques. There is also a need to increase live birth rates per embryo transfer, identify women who can accept embryos, and facilitate successful assisted reproductive procedures that are shorter, less burdensome, and more affordable. Summary of the Invention

[0006] The inventors have found a novel method for determining the state of uterine receptivity, specifically the window of implantation (WOI) for embryo transfer in a female subject. WOI is manifested by the development of a receptive endometrium in said female subject and can be determined according to the present invention by measuring the dissolved gas concentration in the endometrial fluid of said female subject. It is estimated that more than 30% of women have a displaced or shortened window of implantation, usually lasting less than 48 hours, and this percentage is even higher in women with repeated implantation failure. There are no validated biomarkers or anatomical indicators for the detection of the window of implantation. Methods of gene expression analysis are widely used for the determination of endometrial implantation potential, but require the use of sophisticated equipment and tissue biopsy collection. The inventors have identified that the selection of O2 and / or CO2 as dissolved gases to measure in the endometrial fluid of a female subject allows a technically simple yet very reliable determination of endometrial implantation potential, despite the difficulties associated with cycle-to-cycle variations and physiological differences between women.

[0007] Thus, in a first aspect, the present invention relates to a method for determining endometrial receptivity in a female subject, said method comprising: i) measuring an endometrial fluid dissolved gas concentration of said female subject, thereby obtaining a dissolved gas concentration value, said dissolved gas being selected from the group consisting of O2 and CO2; ii) comparing the dissolved gas concentration values ​​with a reference value, wherein an increase in the dissolved O2 concentration value and / or a decrease in the dissolved CO2 concentration value relative to the reference value indicates that the female subject's endometrium is receptive; The present invention relates to a method comprising the steps of:

[0008] The inventors have also found that measuring the concentration of dissolved gases in the endometrial fluid of female subjects allows for the selection of female subjects undergoing assisted reproductive technology (ART) as candidates to receive embryos.

[0009] Thus, in a second aspect, the present invention provides a method for selecting a female subject for assisted reproduction procedures as a candidate to receive an embryo, said method comprising: i) determining whether the endometrium of said female subject is receptive according to the method steps of the invention; If the endometrium is receptive, ii) selecting said female subject as a candidate to receive an embryo in an embryo transfer procedure on the same day as step i); The present invention relates to a method comprising the steps of: [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 is a schematic illustration of the principle of embryo / endometrial synchrony according to the present invention showing the chronological correlation of embryo developmental stages with respect to the window of implantation and with respect to intrauterine oxygen concentration. [Diagram 2] FIG. 2 is a schematic diagram of the principles of the present invention showing oxygen diffusion from the uterus to the uterine cavity and the increase in dissolved intrauterine oxygen concentration in the uterine fluid at the opening of the implantation window. Image from Bartelmez GW. Uterine vascular shape and function in rhesus monkeys. Contrib Embryol 1957;36:154-83,courtesy of Carnegie Institution for Science,Copyright Carnegie Institution for Science. [Diagram 3] FIG. 3 is a schematic diagram of the principles of the present invention showing the correlation of pO2 variations and the occurrence of the window of implantation with planned additional testing throughout the menstrual cycle. [Figure 4-1]Figure 4 shows the intrauterine dissolved oxygen patterns observed during the luteal phase of the women in the study. Dissolved oxygen concentrations were measured using a sterilized IMP-7 optical sensor attached to the implanted catheter. Measurements were taken every 1 second for 5 minutes, with the first 120 seconds excluded due to lag time. Box and whisker plots were used. The whiskers represent 5-95% of the data; the median is represented by a line and the mean is represented by a + sign. Data outside the whiskers are represented as points. Oxygen concentrations are expressed in Torr, where 1 Torr = 133.22 Pa. Figure 4A: Data from 3 different patients combined. Oxygen levels are significantly increased from LH4 to LH5 and from LH5 to LH6 (p<0.0001, Tukey's multiple comparison test). There is no difference between LH6 and LH7, and pO2 is significantly decreased at LH8 (p<0.0001). See Tables 1-3. The window of implantation (WOI) occurs between LH6 and LH7 in natural cycles (or P5 and P6 in artificial cycles). The pO2 pattern in A is the criterion for WOI occurrence and indicates whether the WOI is open (LH5) or closed (LH8). [Figure 4-2] Figure 4B: Data represent the dissolved intrauterine pO2 patterns of volunteers with advanced and shortened WOI: pO2 maximum at LH5 and minimum at LH7 (see Tables 1-3). Figure 4C: Data represent the dissolved intrauterine pO2 profile of a healthy volunteer with no increase in oxygen levels during the luteal phase. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Under ideal conditions, the state of endometrial implantation competence, called the window of implantation (WOI), usually occurs during the luteal phase on days 19-21 of the menstrual cycle (Navot D. et al., Fertil Steril 1991, 55: 114-118; Harper MJK. Baillieres Clin Obstet Gynaecol 1992, 6: 351-371). In artificial cycles (hormone replacement therapy cycles), the window of implantation occurs 5 days after progesterone treatment (P4 + 5) (Enciso, M. et al., Sci Rep 11, 13420 (2021)). Its duration and position in the cycle depend on several factors and can vary from month to month in the same woman, making it difficult to estimate empirically. In fact, more than 30% of women have displaced or shortened WOI, and this percentage is even higher in women with repeated implantation failure. Despite the efforts of many research groups, no method for the detection of WOI by anatomical patterns or molecular biomarkers has been established. The advent of "omics" technologies in recent decades has allowed the development of novel diagnostic tests focused on the detection of gene expression profiles characteristic of WOI. However, these methods require endometrial tissue biopsy and at least one week to obtain results, which makes it impossible to perform tests during active ART cycles. In addition, "omics" technologies rely on the use of highly advanced techniques and / or the services of "omics" analysis providers. In other words, in most cases, the clinician alone cannot perform "omics" technologies. Moreover, the result is a prediction of the location of the future WOI, and to increase the accuracy of this prognosis, artificial preparation of the endometrium with exogenous hormones is recommended. It is important to emphasize that artificially prepared cycles have more obstetric and perinatal complications than natural cycles, pose risks to the health of the mother and fetus, as well as higher costs for the health system, so it is a priority for ART services to promote the use of methods that are as close as possible to the natural cycle, when the patient's physiology allows. The present invention overcomes the above problems.

[0012] Oxygen homeostasis is essential for the survival of eukaryotic organisms. Increased cellular oxygen levels result in the production of toxic oxygen species, and oxygen deficiency causes inhibition of mitochondrial respiration. In particular, the embryo must be exposed to oxygen dissolved in the uterus or endometrial fluid, which is regulated by the uterine system through endometrial thickening and vascularization, and is in close coordination with the embryonic metabolic needs. In fact, embryonic metabolism changes from anaerobic to aerobic in just 5 days.

[0013] The inventors have developed a method to identify the implantation window, which is manifested by the development of receptive endometrium, in relation to the dissolved gas concentration in endometrial fluid. The inventors provide evidence that the change in the trend of dissolved gas concentration in endometrial fluid throughout the menstrual cycle allows the elucidation of the appropriate timing for embryo implantation (embryo / endometrial synchrony) in a given female subject. The inventors have also found that the uterine environment is hypoxic during the embryo's anaerobic stage, and when the endometrium becomes receptive, the intrauterine oxygen pressure increases, coinciding with blastocyst development and implantation, where the embryo's oxygen consumption increases dramatically.

[0014] Therefore, the present invention provides a method for determining endometrial implantation competence in a female subject, and therefore a method for identifying the implantation window and evaluating embryo implantation outcome and pregnancy. The method of the present invention provides a reliable determination of endometrial implantation competence with the advantage that it is no longer necessary to obtain an endometrial biopsy. In particular, the method allows clinicians to identify the implantation window and perform embryo transfer within a few hours, i.e., the same day. The method of the present invention does not require expensive or bulky equipment, but standard medical gas measurement equipment that can be operated by hand, so that it can be applied in a wider range of clinics, even with lower costs. Furthermore, the simple and rapid identification of the implantation window makes the prediction of WOI in the next cycle more accurate, thus reducing the need to administer unnecessary hormone therapy, which is related to more frequent pregnancy complications with natural cycles. Therefore, the method is applied in assisted reproduction centers and in vitro fertilization units to improve embryo implantation rate and pregnancy rate, accelerating the clinical decision process, as well as reducing the costs and complications induced from the administration of hormone therapy in women who can benefit from natural cycles.

[0015] 1. Method for determining endometrial receptivity in a female subject The inventors have found that determining the concentration of dissolved gases in endometrial fluid in a female subject allows for a reliable identification of the implantation window in said female subject. Thus, in a first aspect, the present invention provides a method for determining endometrial receptivity in a female subject (first method of the present invention), said method comprising:

[0016] i) measuring an endometrial fluid dissolved gas concentration of said female subject, thereby obtaining a dissolved gas concentration value, said dissolved gas being selected from the group consisting of O2 and CO2; ii) comparing the dissolved gas concentration values ​​with a reference value, wherein an increase in the dissolved O2 concentration value and / or a decrease in the dissolved CO2 concentration value relative to the reference value indicates that the female subject's endometrium is receptive; The present invention relates to a method comprising the steps of:

[0017] The first step involves measuring the endometrial fluid dissolved gas concentration of the female subject, thereby obtaining a dissolved gas concentration value.

[0018] As used herein, the expression "determining endometrial implantation competence" relates to the assessment or prediction of the physiological or clinical state of the endometrium, in particular with regard to pregnancy outcome (e.g. the likelihood of pregnancy or of implantation-competent embryo survival).

[0019] As used herein, the term "endometrial implantation competence" refers to the state in which the endometrium of a female subject is prepared for embryo implantation. The term "endometrial implantation competence" can refer to the state during which the endometrium acquires a functional state that allows blastocyst implantation and leads to pregnancy. Implantation consists of three stages: (1) appoposition, when the blastocyst contacts the implantation site; (2) adhesion, when the blastocyst binds to the receptive endometrial epithelium; and (3) invasion, when the invasive trophoblast cells of the blastocyst cross the endometrial epithelium and invade the endometrial stroma. The term "implantation window" can refer to the period during which the uterus of said female subject is receptive for embryo implantation. The term "receptive endometrium" can also refer to the state of the endometrium that is characterized by the period known as "implantation window" in female subjects. The term "endometrial receptivity" may also refer to the state of the endometrium during which the window of implantation occurs such that embryo implantation has a high probability of resulting in pregnancy.

[0020] The term "subject" or "individual" or "animal" or "patient" or "mammal" is understood as any subject for which a determination or diagnosis or prognosis is desired, particularly a mammal. Mammalian subjects include humans, livestock, farm animals, and zoo, sports, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, etc. In a preferred embodiment of the invention, the subject is a mammal. In a more preferred embodiment of the invention, the subject is a human.

[0021] As used herein, the term "female subject" refers to a female mammal whose endometrial implantation potential is to be determined. Typically, said mammal is a human (i.e., female), but may relate to other mammals, such as primates, dogs, cats, pigs, sheep, cows, etc.

[0022] The endometrium is the inner epithelial lining of the mammalian uterus, which, together with its mucosa, forms a cavity. As used herein, the term "endometrial fluid" (EF) or "endometrial cavity fluid" (ECF) or "uterine fluid" or "uterine cavity fluid" refers to the collection of fluid within the endometrial cavity.

[0023] As used herein, the term "dissolved gas" may refer to the pressure of a gas in a body fluid. The term "gas pressure" may refer to the partial pressure of a gas in said body fluid. In a gas mixture, each constituent gas has a "partial pressure" which is the conceptual pressure of that constituent gas if it were to occupy the entire volume of the original mixture alone at the same temperature. In the context of the present invention, the amount of a given gas dissolved in a given liquid, particularly endometrial fluid, is directly proportional to the partial pressure of the gas in contact with said liquid. As used herein, the partial pressure of a gas in a solvent is also proportional to the concentration of the gas dissolved in a given liquid, particularly endometrial fluid. In the context of the present invention, the terms "dissolved gas concentration", "gas pressure", "partial pressure" or "partial pressure" are used interchangeably. Methods for measuring "dissolved gas concentration" are known in the art.

[0024] According to the invention, the method includes measuring oxygen (O2) pressure (PxO2) and / or carbon dioxide (CO2) pressure (PxCO2). By convention, the subscript "x" in each symbol represents the tissue or organ containing the bodily fluid for which the dissolved gas is to be measured: for example, "a" stands for arterial (blood), "v" stands for venous (blood), and "c" stands for capillary (blood). In some embodiments, PuO2 with "u" meaning uterine fluid or PeO2 with "e" meaning endometrial fluid may be used interchangeably to represent the oxygen pressure in the bodily fluid contained in the endometrial cavity. In some embodiments, PuCO2 with "u" meaning uterine fluid or PeCO2 with "e" meaning endometrial fluid may be used interchangeably to represent the carbon dioxide pressure in the bodily fluid contained in the endometrial cavity.

[0025] According to the present invention, the partial pressure of oxygen in endometrial fluid can be expressed in units of mmHg. In some embodiments, the partial pressure of oxygen is interchangeably expressed as a concentration value (percentage (%)) of dissolved oxygen. In some embodiments, the partial pressure of oxygen in endometrial fluid can be expressed in units of kPa or Torr. Methods of converting units are known in the art. For example, it is well known that 1 Torr is 1001325 / 760 Pascal, and therefore, approximately 133.22 Pa.

[0026] According to the present invention, the partial pressure of carbon dioxide in endometrial fluid may be expressed in units of mmHg. In some embodiments, the partial pressure of carbon dioxide is interchangeably expressed as a concentration value (percentage (%)) of dissolved carbon dioxide. In some embodiments, the partial pressure of carbon dioxide in endometrial fluid may be expressed in units of kPa.

[0027] According to the present invention, the measurement of endometrial fluid dissolved gas concentration represents the absolute or relative determination of the concentration of dissolved gas in endometrial fluid by any conventional measuring device suitable for detecting and quantifying the presence of dissolved gas in body fluid.Non-limiting examples of techniques suitable for measuring dissolved gas concentration are conventional devices used in medicine in the analysis of arterial / venous blood and breathed air, polarographic microelectrodes, electrochemical electrodes, optical sensors, fiber optic sensors, fluorophore quenching sensors.Further non-limiting examples of techniques suitable for measuring dissolved gas concentration include dynamic contrast magnetic resonance imaging, blood oxygen level-dependent imaging and electron paramagnetic resonance imaging.Such sensors are well known in the art and can be easily applied by those skilled in the art to perform the measurement of functions related to the present invention.

[0028] In some embodiments, the dissolved gas concentration is measured optically, preferably by phase fluorometry. As used herein, the term "optical method" refers to any method in which light is used to detect, characterize and / or quantify gas. Optical methods can be based on absorption, fluorescence intensity or fluorescence lifetime measurement. Illustrative non-limiting examples of optical methods that can be used for measuring O2 and CO2 dissolved concentrations are usually based on fluorescence quenching in the presence of oxygen. These include intensity quenching detection, or lifetime quenching detection, both in the time or frequency domain. Measurement and analysis methods for time-resolved fluorescence tracing include phase-modulated or pulsed fluorometry (direct emission decay measurement, single-photon time resolution, streak camera measurement, fluorescence upconversion, and optical Kerr gating). Optical methods and fluorophores for measuring dissolved gas concentrations are known in the art.

[0029] As used herein, the term "phase fluorometry" refers to a method in which a sample is excited with sinusoidally modulated light at a high frequency. The fluorescence response of the dye is also sinusoidally modulated at the same frequency, but with a time delay or phase shift relative to the excitation signal, which in turn depends on the pO2 in contact with the dye on the surface of the sensor probe.

[0030] As used herein, the measurement of endometrial fluid dissolved gas concentration can refer to the determination of oxygen tension in the endometrial fluid.As used herein, the measurement of endometrial fluid dissolved gas concentration can refer to the determination of carbon dioxide tension in the endometrial fluid.As used herein, the measurement of endometrial fluid dissolved gas concentration can refer to the determination of oxygen tension and carbon dioxide tension in the endometrial fluid.

[0031] In a second step of the first method of the invention for determining endometrial receptivity in a female subject, the endometrial fluid dissolved gas concentration value is compared to a reference value.

[0032] The term "reference value" or "reference level" as used herein in the context of the method of the present invention refers to a value used as a reference for the value obtained from the measurement of dissolved gas concentration to classify within a particular physiological or clinical state, or to classify as high or low the probability of entering a physiological or clinical state, or to classify as good or bad the prognosis of entering a physiological or clinical state. The reference value or reference level according to any of the methods of the present invention can be an absolute value; a relative value; a value with upper and / or lower limits; a range of values; an average value; a median value; a mean value, or a value compared to a specific control or baseline value. The reference value will be compared to said reference value based on individual sample values, for example values ​​obtained from female subjects that are being tested but at an earlier time point than the measurement under consideration. The reference value can be based, for example, on multiple samples from a population of subjects in a chronological age-matched group, or on a pool of samples that includes or excludes the sample to be tested. When determining the reference value of a marker, various considerations are taken into account. Among such considerations are menstrual cycle, age, weight, sex, the general physical condition of the patient, and the like. For example, preferably the equivalent of the above considerations, eg a group of at least 2, at least 10, at least 100, preferably up to more than 1000 subjects classified according to various age categories, serves as a reference group.

[0033] As used herein, the term "reference value" may refer to a concentration value of a dissolved gas in the endometrial fluid under consideration in a given subject at a given time point. In a preferred embodiment, the reference value corresponds to a concentration value of O2 in the endometrial fluid at a given time point. In another embodiment, the reference value corresponds to a concentration value of CO2 in the endometrial fluid at a given time point. In another embodiment, the reference value corresponds to the individual concentration values ​​of O2 and CO2 in the endometrial fluid at a given time point.

[0034] In a preferred embodiment, the reference value is the concentration value of the dissolved gas in endometrial fluid for a given female subject at a given time point prior to the start of the window of implantation in said female subject.

[0035] In some embodiments, the reference value may represent a value obtained from a pool of concentration values ​​of dissolved gases in endometrial fluid obtained from normal or healthy female subjects prior to the start of the implantation window. In some embodiments, the reference value may represent a value obtained from a pool of concentration values ​​of dissolved gases in endometrial fluid in female subjects undergoing assisted reproduction procedures and / or IVF prior to the start of the implantation window.

[0036] The term "reference value" can refer to a series of concentration values ​​of dissolved gas in endometrial fluid obtained at different successive time points.As used herein, "reference value" can refer to the concentration value of dissolved gas in endometrial fluid obtained from a subject that is undergoing endometrial implantation competence determination or a group of subjects known to have a known physiological or clinical condition, preferably two or more subjects different.Appropriate reference concentration value of dissolved gas in endometrial fluid can be determined by measuring the concentration of dissolved gas in several suitable subjects at a given time point, and such reference level can be adjusted to a specific subject population or clinical setting.

[0037] Usually, the reference value can be a threshold value. Usually, the "threshold value" can be experimentally, empirically, or theoretically determined. The threshold value can also be arbitrarily selected based on current experimental and / or clinical conditions, as recognized by those skilled in the art. The threshold value must be determined to obtain optimal selectivity and specificity according to the function of the test and the benefit-risk balance (false positive and false negative clinical outcomes). Usually, the optimal selectivity and specificity (and therefore the threshold value) can be determined using a receiver operating characteristic (ROC) curve based on experimental data. In one embodiment of the present invention, the threshold value is derived from the concentration value (or ratio, or score) of dissolved gas in endometrial fluid determined in one or more female subjects at a predetermined time point with high probability of pregnancy outcome. In a particular embodiment, the reference value or threshold value is selected from the group consisting of 30 Torr (4.00 KPa), 35 Torr (4.67 KPa), and 40 Torr (5.33 KPa).

[0038] In a particular embodiment, the reference value is a concentration value of dissolved O2 and / or CO2 in endometrial fluid in the female subject measured at least once during a period comprised between 2 days before the onset of ovulation and the 2nd or 3rd day of the luteal phase in a natural cycle, or between 2 days before the onset of ovulation and the 1st or 2nd day after administration of progesterone in an artificial cycle.

[0039] The term "at least once" includes 1 time, 2 times, 3 times, 4 times, 5 times or more.

[0040] The term "ovulation" refers to the stage of the menstrual cycle in which an egg is released from the ovary, which may or may not be fertilized by a sperm. If fertilized, the egg travels to the uterus and can implant resulting in pregnancy. If it remains unfertilized, the egg breaks down and the uterine lining is shed during the period. Ovulation usually occurs around day 14 of a normal 28-day menstrual cycle. However, the present invention contemplates menstrual or non-menstrual conditions and female subjects in which the exact timing of the ovulatory phase can be highly variable. The ovulation process usually begins with the body's release of follicle-stimulating hormone (FSH) between days 6 and 14 of the menstrual cycle, which contributes to the maturation of the egg inside the ovary, causing the production of estrogen in the follicle. Once the egg has matured, the body releases a surge of luteinizing hormone (LH), causing the release of the egg. Under normal conditions, ovulation can occur 28 to 36 hours after the LH surge. The luteinizing hormone (LH) surge can be determined by uterine and / or blood tests using methods known in the art. In certain embodiments, the LH surge is defined as a serum LH concentration of at least 15 mIU / mL (milli-International Units per milliliter), and in more particular embodiments, at least 20 mIU / mL.

[0041] As used herein, the term "natural cycle" refers to a cycle in the absence of hormone replacement therapy. The luteal phase is the later part of the menstrual cycle. The luteal phase begins with the formation of the corpus luteum and ends with either pregnancy or luteal involution (also luteolysis), which is the structural and functional breakdown of the corpus luteum (CL) and occurs at the end of the luteal phase of the menstrual cycle in the absence of pregnancy. The main hormone associated with this stage is progesterone, which is significantly higher in the luteal phase than in other phases of the cycle. The luteal phase begins with ovulation. Thus, as used herein, day 1 of the luteal phase (LH+1) corresponds to the day after ovulation, day 2 of the luteal phase (LH+2) corresponds to the second day after ovulation, day 3 of the luteal phase (LH+3) corresponds to the third day after ovulation, etc. Typically, the window of implantation in the natural cycle occurs from LH+6 to LH+9.

[0042] As used herein, the term "artificial cycle" refers to a cycle under the stimulation of hormone replacement therapy. In an artificial cycle, progesterone is administered during the luteal phase, and as used herein, P+1 corresponds to the first day after progesterone administration, P+2 corresponds to the second day after progesterone administration, etc. The implantation window in an artificial cycle usually occurs from P+4 to P+6. There are also natural cycles in which progesterone is administered as a supplement. In each cycle in which progesterone is administered to develop or support the luteal phase, the implantation window usually occurs from P+4 to P+6.

[0043] In some embodiments, the reference value is a concentration value of dissolved O2 and / or CO2 in endometrial fluid of another female subject or group of female subjects measured at least once during a period comprised between 2 days before the onset of ovulation and the 2nd or 3rd day of the luteal phase in a natural cycle, or between 2 days before the onset of ovulation and the 4th to 6th day after administration of progesterone in an artificial cycle. The another female subject or group of female subjects may be healthy or normal female subjects or female subjects undergoing assisted reproductive procedures and / or IVF.

[0044] In accordance with the present invention, the measurement of endometrial fluid dissolved gas concentration does not involve direct contact or physical interaction with the endometrium of said female subject. The method contemplates measuring dissolved gas concentration solely by interaction with endometrial fluid, but by avoiding any direct contact with endometrial tissue in the measuring step.

[0045] As used herein, a deviation of a dissolved gas concentration value relative to a reference value may represent an "increase" in the concentration value relative to the reference value.

[0046] The term "increased concentration value" or "increased dissolved gas concentration value" as used herein in relation to dissolved gas concentration values ​​in endometrial fluid relates to a condition in which the concentration value of said dissolved gas is increased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% when compared to a corresponding reference value obtained in said female subject at a previous time point.

[0047] In a preferred embodiment, an increased concentration value can be obtained in a given female subject with respect to a reference value obtained in said female subject at a previous time point.As used herein, an increased concentration value can indicate an implantation-synchronous endometrium for an implantation-competent embryo that enters aerobic metabolism and thus requires higher oxygen concentration.The term "implantation-synchronous endometrium" refers to the state of the endometrium that is suitable for allowing the implantation of an embryo that has developed physiologically and temporarily so that it can interact with the receptive endometrium to proceed with pregnancy.

[0048] In one embodiment, the dissolved gas concentration value may be unchanged relative to the reference value. As used herein, a dissolved gas concentration value that is "unchanged" relative to the reference value may indicate that the endometrium is not receptive.

[0049] As used herein, a deviation of a dissolved gas concentration value relative to a reference value may represent a "decrease" in the concentration value relative to the reference value.

[0050] As used herein in relation to dissolved gas concentration values ​​in endometrial fluid, the term "concentration value decrease" or "dissolved gas concentration value phenomenon" relates to a condition in which the concentration value of said dissolved gas is decreased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% when compared to a corresponding reference value obtained in said female subject at a previous time point.

[0051] In some embodiments, a decrease in concentration value may be obtained in a given female subject relative to a reference value obtained in said female subject at a previous time point. As used herein, a decrease in concentration value may indicate an endometrium that is not implantation synchronous with respect to an implantation-competent embryo. The term "endometrium that is not implantation synchronous" refers to an endometrium that is not suitable for allowing the implantation of an embryo, so that pregnancy does not occur.

[0052] In a preferred embodiment, the terms "receptive endometrium" or "implantation-synchronous endometrium", as used interchangeably herein with respect to concentration values ​​of dissolved O2 in endometrial fluid, refer to a condition in which the concentration value of dissolved O2 is increased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% when compared to a corresponding reference value obtained in said female subject at a previous time point.

[0053] In some embodiments, the term "endometrium is receptive" as used herein with respect to dissolved CO2 concentration values ​​relates to a condition in which the dissolved CO2 concentration value is decreased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% when compared to a corresponding reference value obtained in said female subject at a previous time point.

[0054] In some embodiments, the term "endometrium is receptive" as used herein with respect to concentration values ​​of dissolved O and CO in endometrial fluid means that the endometrium is receptive to at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% increase and at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% decrease when the concentration value of dissolved CO2 is compared to a corresponding reference value obtained in said female subject at a previous time point.

[0055] In some embodiments, the term "non-receptive endometrium" or "non-implantation-synchronous endometrium" as used herein with respect to concentration values ​​of dissolved O2 in endometrial fluid refers to a condition in which the concentration value of dissolved O2 is decreased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% when compared to a corresponding reference value obtained in said female subject at a previous time point.

[0056] In some embodiments, the term "non-receptive endometrium" or "non-implantation-synchronous endometrium" as used herein with reference to concentration values ​​of dissolved CO in endometrial fluid relates to a condition in which the concentration value of dissolved CO is increased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% when compared to a corresponding reference value obtained in said female subject at a previous time point.

[0057] In some embodiments, the term "endometrium is not receptive" as used herein with respect to concentration values ​​of dissolved O and CO in endometrial fluid means that the level of dissolved CO is at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 10 ... at least 80%, at least 90% or at least 100% increase and at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% decrease when the level of dissolved O2 is compared to a corresponding reference value obtained in said female subject at a previous time point.

[0058] As used herein, the term "endometrium is receptive" relates to a condition in which the endometrium of a female subject exhibits at least a 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% probability of acquiring a functional state that will allow blastocyst attachment and result in pregnancy upon implantation of a viable embryo.

[0059] As used herein, the term "endometrium is not receptive" refers to a state in which the endometrium of a female subject exhibits at least a 10%, at least a 20%, at least a 30%, at least a 40%, at least a 50%, at least a 60%, at least a 70%, at least a 80%, at least a 90%, at least a 100% probability of not attaining a functional state that would allow blastocyst attachment and lead to pregnancy upon implantation of a viable embryo. The term "pregnancy" is understood as the visualization of a gestational sac 4 weeks after embryo transfer.

[0060] In some embodiments, once the reference value is established, the concentration value of the dissolved gas in the endometrial fluid of the female subject whose endometrial receptive capacity is to be determined can be compared to the reference value and thus assigned a fold change of "increase", "decrease" or "equivalent" to the reference value obtained in said female subject at a previous time point. For example, a level increase of at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.6 times, at least 1.6 times, at least 1.7 times, at least 1.8 times, at least 1.9 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times or even more above the reference value compared to the reference value obtained in said female subject at a previous time point is considered to be an "increased" concentration value. On the other hand, a level decrease below the reference value of at least 0.9 times, 0.75 times, 0.2 times, 0.1 times, 0.05 times, 0.025 times, 0.02 times, 0.01 times, 0.005 times or even less compared to the reference value is considered to be a "decreased" concentration value obtained in said female subject at a previous time point. If the difference between the concentration value and the reference value obtained in said female subject at a previous time point is less than 1%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.1%, less than 0.05% or less, the concentration value can be considered as "equivalent" to the reference value.

[0061] As the skilled artisan will understand, such a determination does not usually require that it be correct for all (i.e., 100%) of the subjects that will be identified. However, the term requires that a statistically significant portion of the subjects can be identified (e.g., a cohort in a cohort study). The skilled artisan can easily determine if a portion is statistically significant using several well-known statistical evaluation tools, such as determining confidence intervals, determining p-values, Student's t-test, Mann-Whitney test, and others. Details can be found in Dowdy and Wearden, Statistics for Research, John Wiley and Sons, New York 1983. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99%. The p-value is preferably 0.1, 0.05, 0.01, 0.005 or 0.0001. More preferably, at least 60%, at least 70%, at least 80% or at least 90% of the subjects of the population can be suitably identified by the method of the present invention.

[0062] In some embodiments, endometrial fluid dissolved gas concentrations are measured continuously for a period of 1 to 10 minutes, preferably 2 to 7 minutes, and more preferably 3 to 5 minutes.

[0063] In some embodiments, endometrial fluid dissolved gas concentration is measured at least once, including at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or more times during the period from 2 days before ovulation to day 12 of the luteal phase, preferably at least once during the period from the day of ovulation to day 10 of the luteal phase, more preferably at least once during the period from day 2 to day 8 of the luteal phase, and even more preferably at least once during the period from day 4 to day 7 of the luteal phase.

[0064] In some embodiments, endometrial fluid dissolved gas concentrations are measured at least once two days before the onset of ovulation, at least once at the time of ovulation, and / or at least once on the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 10th, or 12th day of the luteal phase.

[0065] In a particular embodiment, measurements taken 2 days before the onset of ovulation to 2 days after ovulation may be used as reference values. In this particular embodiment, detection of a deviation, in particular an increase in the concentration value of dissolved O2 and / or a decrease in the concentration value of CO2, indicates that the endometrium is receptive in one or more of the measurements taken after ovulation compared to the measurements taken before ovulation.

[0066] In some embodiments, measurements taken on the second day of ovulation and on the day of ovulation may be used as reference values.

[0067] In some embodiments, measurements taken on the day of ovulation and the day after ovulation may be used as reference values.

[0068] In some embodiments, measurements taken on the day of ovulation and two days after ovulation may be used as reference values.

[0069] Without wishing to be bound by any particular theory, it is believed that during the proliferative phase of the menstrual cycle, oxygen diffusion from the spiral arteries to the uterine cavity is reduced as a result of endometrial thickening. This physiological intrauterine hypoxia protects the embryo from toxic oxygen species. However, when the morula reaches the uterus and the blastocyst begins its development, the oxygen concentration in the endometrial fluid should increase to meet the metabolic needs of the embryo. Thus, in some embodiments, the reference value is the value of dissolved O2 at a concentration of physiological hypoxia in the endometrial fluid, and an increase in the dissolved O2 concentration value relative to physiological hypoxia indicates that the endometrium of the female subject is receptive. As used herein, "physiological hypoxia" refers to a state in which the oxygen concentration in the endometrial fluid is significantly lower than the oxygen concentration in the atmosphere. Alternatively, as used herein, "physiological hypoxia" refers to a state in which the oxygen concentration in the tissue is less than 40 mmHg (pO2 5%), more particularly less than pO2 2%, which is physiologically achieved.

[0070] In some embodiments, the female subject undergoes assisted reproductive procedures, including the step of embryo transfer. As used herein, the phrase "assisted reproductive technology" or "assisted reproductive technology" or "assisted reproductive procedures" (ART) refers to a number of treatments aimed at improving fertility. Non-limiting examples of ART include intrauterine sperm injection (IUI), intracytoplasmic sperm injection (ICSI), in vitro fertilization (IVF), embryo transfer into a female subject, preimplantation genetic testing, hormone replacement therapy (HRT) cycles (e.g., exogenous administration of progesterone and estrogen), in vitro oocyte maturation, gamete intrafallopian tube transfer (GIFT), zygote intrafallopian tube transfer (ZIFT), and surrogate pregnancy. In vitro fertilization (IVF) is the in vitro combination of a female egg and a male sperm. An IVF cycle includes several steps; step 1: stimulation, also called superovulation; step 2: egg retrieval; step 3: fertilization (such as IVF or ICSI); step 4: embryo culture; and step 5: embryo transfer. As used herein, the term "embryo transfer" refers to the process of implanting an embryo produced by any fertilization method into the uterus of a female recipient.

[0071] In some embodiments, the method of the present invention further comprises the step of detecting ovulation in said female subject by any suitable method, including luteinizing hormone levels in blood or urine, ultrasound, basal body temperature (BBT) method and methods based on blood quantification of other hormones such as progesterone and estrogen, prior to measuring endometrial fluid dissolved gas concentration in said female subject. In certain embodiments, ovulation is detected by measuring luteinizing hormone (LH) levels and / or ultrasound monitoring. Methods of measuring luteinizing hormone (LH) levels and / or ultrasound monitoring are known in the art. Luteinizing hormone levels can represent blood levels of LH or urinary levels of LH. In both cases, ovulation occurs within 12-36 hours after the LH surge occurs. Blood and urinary LH levels can be determined by routine testing of protein quantification, preferably by using an antibody capable of specifically binding to the protein to be assayed, in this case LH, such as Western blot or immunoblot, ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), competitive EIA (enzyme-linked immunosorbent assay), DAS-ELISA (double antibody sandwich ELISA), two-dimensional gel electrophoresis, capillary electrophoresis, immunocytochemistry and immunohistochemistry techniques, immunoturbidimetry, immunofluorescence, techniques based on the use of biochips or protein macroarrays containing specific antibodies or colloidal precipitation-based assays in formats such as reagent paper and antibody-bound quantum dot-based assays.Other forms of protein detection and quantification include, for example, affinity chromatography techniques or ligand binding assays.In certain embodiments, urinary LH levels can be determined by lateral flow devices.

[0072] Ovulation can be detected by ultrasound, which can be abdominal or transvaginal. This method is based on monitoring follicular growth by measuring follicular dimensions. Ovulation generally occurs when the follicle reaches a size of approximately 1.8-2.5 cm in diameter.

[0073] In some embodiments, the method of the present invention further comprises measuring parameters selected from the group consisting of urinary luteinizing hormone (LH) concentration, blood progesterone, estrogen and / or vascular endothelial growth factor (VEGF) concentration, the presence of follicles and / or corpora lutea in the ovaries, the thickness and appearance of the endometrium, and the resistive index and pulsatility index of the uterine spiral arteries. Methods for measuring the above parameters are known in the art.

[0074] In particular, the concentrations of progesterone, estrogen and VEGF in the blood may be determined by any suitable method for the quantification of proteins in a blood sample, as explained above for LH quantification.

[0075] The presence of follicles and / or corpus luteum in ovary can be determined by transvaginal or abdominal ultrasound.As used herein, the term "follicle" refers to the structure in ovary that contains and nurtures oocytes or eggs.As used herein, the term "corpus luteum" refers to the mass of cells that form in ovary after follicle releases egg and contributes to the production of progesterone in early pregnancy.Ovulation can be detected by monitoring the growth of follicles and the formation of corpus luteum.

[0076] The thickness and appearance of the endometrium can be analyzed by ultrasound.

[0077] The resistive index (RI) and pulsatility index (PI) of the uterine and spiral arteries can be determined by transvaginal Doppler ultrasound. The term "pulsatility index", also known as the Gosling index, is a flow parameter calculation in ultrasound derived from the maximum, minimum, and average Doppler frequency shifts during a defined cardiac cycle. The pulsatility index is calculated by one of the following formulas: PI=(V max -V min ) / (V mean ) PI = (peak systolic velocity - end diastolic velocity) / (mean velocity).

[0078] The term "resistive index", also known as the Pourcelot index, refers to a flow parameter calculation in ultrasound derived from the maximum, minimum, and mean Doppler frequency shifts during a defined cardiac cycle. The resistive index was calculated using the formula: RI=(PSV-EDV) / PSV, where PSV means peak systolic blood velocity and EDV means end diastolic blood velocity.

[0079] In some embodiments, endometrial fluid dissolved gas concentrations are measured in the fundus and / or cervix of said female subject, preferably in the cervical canal of the uterus.

[0080] As used herein, the term "fundus" refers to the broad, curved, upper region of the uterus where the fallopian tubes connect to the uterus. In some embodiments, dissolved gas concentration measurements are taken at least 1 cm away from the fundus.

[0081] As used herein, the term "cervix" refers to the lower portion of the uterus that extends downward from the isthmus until it opens into the vagina. In some embodiments, the step of measuring endometrial fluid dissolved gas concentration is performed by a sensor capable of measuring endometrial fluid dissolved gas concentration, and the sensor is not in contact with the endometrium of the female subject. Non-limiting examples of sensors capable of measuring endometrial fluid dissolved gas concentration are commercially available sensors such as PreSens Oxygen Microsensors, Neotrend, Paratrend, Neurotrend, NEUROVENT-PTO and Licox.

[0082] In some embodiments, the step of measuring endometrial fluid dissolved gas concentration is carried out in vitro in a sample of endometrial fluid. As used herein, the term "in vitro" refers to a method that is not carried out in the body of a human or animal subject, but rather in cells or body fluids or test tubes isolated from said subject. Methods for obtaining endometrial fluid samples are well known to those skilled in the art, for example, by aspiration of the body fluid contained in the uterine cavity. To carry out this particular embodiment of the method of the present invention, contact between the endometrial fluid sample and the atmosphere should be avoided so that the dissolved gas content in the endometrial fluid sample is not altered.

[0083] In another specific embodiment, a sensor for measuring dissolved O2 or CO2 is placed inside the catheter and an endometrial fluid sample is aspirated into the catheter such that measurements are taken inside the catheter while in the uterus, preferably in the cervical canal.

[0084] 2. Method for selecting female subjects undergoing ART procedures as candidates for receiving embryos The inventors have found that the measurement of dissolved gas concentration in the endometrial fluid of a female subject allows the selection of the female subject undergoing the ART procedure as a candidate for receiving an embryo, which is performed on the same day as the measurement of dissolved gas. The identification of the female subject whose endometrium is receptive or not receptive is the basis for clinical decisions in certain steps of the assisted reproduction procedure. Such clinical decisions may include the selection of an embryo capable of implantation and the transfer of said embryo to the uterus of the female subject, if the endometrium is receptive. Conversely, if the endometrium is not (yet) receptive, the subject is asked to return the next day for another measurement. And after receptivity, the selection of being capable of implantation is omitted and the embryo transfer is no longer planned. This method provides the basis for reliable clinical decisions within a short time, even within a few minutes. The planning of the subsequent steps in the assisted reproduction procedure can be evaluated much more quickly than conventional methods. In addition, the measurement of dissolved gas concentration in the endometrial fluid of a female subject to evaluate endometrial implantation potential at a given time does not require the collection of an endometrial biopsy. The measurement may be performed using standard equipment used for in vitro fertilization procedures, more particularly for the embryo transfer step into the uterus of the female subject. The measurement technique according to the present invention is a very mild procedure that is generally not associated with pain or tissue damage in the female subject. Thus, the step of measuring the dissolved gas concentration can be compatible with the subsequent embryo transfer in the same female subject, if the endometrium of said female subject is determined to be receptive using the first method of the present invention within a minimum of a few hours of the measurement step.

[0085] Thus, in a further aspect, the present invention relates to a method for selecting a female subject undergoing assisted reproduction procedures as a candidate to receive an embryo (second method of the invention), said method comprising: i) determining whether the endometrium of said female subject is receptive according to the method steps of one method of the present invention; If the endometrium is receptive, ii) selecting said female subject as a candidate to receive an embryo in an immediate embryo transfer procedure on the same day as step i); The present invention relates to a method comprising the steps of:

[0086] As used herein, the term "immediate treatment" refers to the earliest possible time point at which a given procedure may be performed, without being limited to a later time point relative to said earliest time point. The term "immediately on the same day as step i)" is understood to mean that according to the present invention, the earliest time point for embryo transfer may be the day of making the determination of endometrial receptivity, but is not limited to a later time point, for example, 1 day after the determination of endometrial receptivity, 2 days after the determination of endometrial receptivity, etc.

[0087] In a first step of the second method of the invention, the concentration of dissolved gas in endometrial fluid is measured and the receptive capacity of the endometrium of the female subject is determined as previously described in connection with the first method of the invention. The method for measuring the concentration of dissolved gas in endometrial fluid previously described in connection with the first method of the invention is applicable to the second method of the invention.

[0088] Embodiments disclosed in the context of the first method of the invention are also contemplated for the second method of the invention.

[0089] 3. Methods of Achieving Pregnancy in a Female Subject The inventors have found that measuring the dissolved gas concentration in the endometrial fluid of a female subject provides the possibility of pregnancy in the female subject, particularly in the female subject undergoing assisted reproduction treatment.As used herein, the expression "determining endometrial implantation potential" refers to the evaluation of the physiological or clinical state of the endometrium with respect to pregnancy outcome (e.g., the possibility of pregnancy or of survival of a viable embryo capable of implantation).As used herein, the term "pregnancy" is understood as the visualization of a gestational sac 4 weeks after embryo transfer.The term "pregnancy" may also be understood as the result of the final outcome of a fertilization event.

[0090] As used herein, the term "method of achieving pregnancy" refers to any type of preventative or therapeutic measure, including any class of measures, such as hygienic, pharmaceutical, surgical or physical measures, that have the purpose of promoting pregnancy in a female subject.

[0091] Thus, in a further aspect, the present invention relates to a method of achieving pregnancy in a female subject (third method of the invention), said method comprising: i) measuring an endometrial fluid dissolved gas concentration of said female subject, thereby obtaining a dissolved gas concentration value, said dissolved gas being selected from the group consisting of O2 and CO2; ii) comparing the dissolved gas concentration values ​​with a reference value, wherein an increase in the dissolved O2 concentration value and / or a decrease in the dissolved CO2 concentration value relative to the reference value indicates that the female subject's endometrium is receptive; If the dissolved gas concentration value deviates from the reference value, iii) implanting the embryo into the receptive endometrium of said female subject; iv) allowing the pregnancy to progress; and The present invention relates to a method comprising the steps of:

[0092] Alternatively, the present invention provides a method for treating infertility in a female subject, the method comprising: i) measuring an endometrial fluid dissolved gas concentration of said female subject, thereby obtaining a dissolved gas concentration value, said dissolved gas being selected from the group consisting of O2 and CO2; ii) comparing the dissolved gas concentration value with a reference value; If there is an increase in the dissolved O2 concentration value and / or a decrease in the CO2 concentration value compared to the reference value, iii) treating infertility by transferring an embryo to the receptive endometrium of said female subject to allow implantation of the embryo; The present invention relates to a method comprising the steps of:

[0093] In a first step of the third method of the invention, the concentration of dissolved gas in endometrial fluid is measured and the receptive capacity of the endometrium of the female subject is determined as previously described in connection with the first method of the invention. The method for measuring the concentration of dissolved gas in endometrial fluid previously described in connection with the first method of the invention is applicable to the third method of the invention.

[0094] Embodiments disclosed in the context of the first method of the invention are also contemplated for the third method of the invention.

[0095] The present invention will now be described in detail by the following examples, which are merely illustrative and not in any way limiting the scope of the invention. EXAMPLES

[0096] Due to the absence of reliable methods for WOI detection, the multifactorial complexity of endometrial implantation potential, and the lack of known biomarkers, biophysical parameters such as oxygen and carbon dioxide tensions have been considered as indicators of a healthy and properly functioning endometrium. These parameters have a dynamic behavior inside the uterus, and importantly, they must be synchronized with the requirements of the embryo at different stages of its development. In this sense, when the embryo is present in the fallopian tube, up to the morula stage, the oxygen requirement is low, but when the embryo becomes a blastocyst, the oxygen requirement increases dramatically and the use of glucose as an energy source to ensure its biosynthetic activity increases. Nutrient availability, oxygen concentration and the redox state of the blastocyst regulate its metabolism and implantation potential. Considering that the oxygen supply of the embryo depends on the oxygen dissolved in the fallopian tube and in the endometrial fluid, the oxygen concentration changes in the uterus must be synchronized with the metabolic development of the embryo. Thus, during the proliferative phase of the menstrual cycle, the endometrium thickens and oxygen diffusion from the spiral arteries into the uterine cavity becomes more difficult. This physiological intrauterine hypoxia protects the embryo from toxic oxygen species, and if hypoxic conditions are not established, such as when the endometrium is thin, pregnancy is prevented. However, when the morula reaches the inside of the uterus and the blastocyst begins to develop, the oxygen concentration in the endometrial fluid must increase, since low oxygen concentrations inhibit mitochondrial activity and glucose metabolism, which have a negative effect on blastocyst development and implantation. In other words, the intrauterine oxygen pressure must be synchronized with the metabolic demands of the embryo depending on its developmental stage. That is, the cell stage must be in a hypoxic state, and the blastocyst stage to implantation must be in a higher oxygen concentration to synchronize with the metabolic demands of the embryo.

[0097] Previous studies have shown that between ovulation and the luteal phase, there is a significant growth and coiling of the spiral arteries, maturation of the subepithelial capillary network and an increased density of vascular branching points, which coincide with the production of progesterone and a significant increase in the angiogenic factor VEGF and its receptors. This process can promote the diffusion of oxygen from the blood vessels into the endometrial fluid, resulting in an increase in intrauterine oxygen concentration. Studies in humans suggest an increase in oxygen and a decrease in carbon dioxide in the luteal phase versus the proliferative phase, but no differences have been observed between measurements made in the fundus or cervix. Furthermore, the balance of these gases appears to be altered in some pathological conditions of the uterus, such as fibroids, cervical cancer or endometrial cancer. Oxygen homeostasis is a complex process that reflects the proper functioning of the ecosystem as a whole. Oxygen homeostasis can be altered by infection, inflammation, cancer, or by abnormalities in angiogenesis or hormonal regulation. It is also known that i) the embryo consumes oxygen dissolved in the endometrial fluid in vivo, ii) the oxygen concentration in this fluid depends on the diffusion of gas from the blood vessels into the uterine cavity, and iii) this diffusion depends on the distance between the blood vessels and the cavity, and therefore on the thickness of the endometrium and the development of these blood vessels.

[0098] However, there is no research on the trend change of dissolved oxygen concentration in endometrial fluid as an indicator of the beginning of endometrial implantation competence.Therefore, the present invention provides a method for detecting the implantation window in a female subject based on the trend change of dissolved oxygen concentration in endometrial fluid, and the reference value obtained before in the same subject is related to the occurrence of endometrial implantation competence.In other words, the inventors have found that oxygen measurement in endometrial fluid is a reliable method for evaluating whether endometrium has developed properly and is synchronous and receptive to blastocyst at the timing of embryo transfer in ART procedure, with the aim of achieving improved implantation rate.

[0099] Importantly, the method of the present invention is advantageous over conventional techniques because it does not rely on the analysis of biopsy samples, and therefore does not affect the integrity of the endometrial tissue and preserves a suitable environment for subsequent embryo implantation. The method of the present invention is advantageous because it is a method of detection of WOI, can be performed in the same cycle in which the embryo is transferred, is compatible with the natural cycle, and then allows for the personalization of ART protocols focused on improving the success of treatment, promotes the natural cycle, and reduces the number of transfers required to achieve pregnancy, thereby reducing the impact on the physical and mental health of the family and the economic costs to the health system.

[0100] The method of the present invention aims to detect endometrial implantation potential and synchrony by non-invasive measurement of intrauterine oxygen pressure in endometrial fluid, for example by optical means.Oxygen optical sensors are safe, accurate, and are used in different clinical applications, such as continuous blood gas monitoring in sedated patients and critically ill newborns, and cerebral oxygen monitoring in patients with acute brain injury.Measurement of dissolved oxygen concentration by means of oxygen optical sensors can be successfully carried out on the day of embryo transfer using a transfer catheter (such as the catheter used for embryo transfer in IVF) to place an optical microsensor in the fundus, which is in contact with endometrial fluid but not with endometrium.

[0101] material and method Target population Five young (18-35 years) healthy female volunteers with BMI<30, regular cycles, non-smokers and normal uterine morphology. Exclusion criteria: -Medical conditions: insulin-dependent diabetes mellitus, Cushing's syndrome, thyroid dysfunction, hepatic and / or renal insufficiency, medical conditions contraindicating ovarian stimulation and / or pregnancy and antiphospholipid syndrome. -Uterine conditions (endometriosis, cancer, congenital anomalies, polyps, fibroids). - Smoker or drug user. - Treatment with oral contraceptives or an IUD in the past 3 months.

[0102] Experimental design A sterile optical oxygen sensor is used that has been used previously in the detection of intrauterine oxygen tension (Ottosen et al. “Observations on intrauterine oxygen tension measured by fiber-optic microsensors” Reproductive biomedicine online vol.13,3(2006):380-5). The method in Ottosen et al. for the detection of intrauterine oxygen in women uses a fiber-optic microsensor and an implanted catheter for its introduction into the uterus. The type of oxygen sensor used is also part of several approved medical devices for the detection of oxygen in the blood and brain: Neurotrend, Neotrend, Paratrend, NEUROVENT-PTO, Raumedic neuro-icu (https: / / www.raumedic.com / neuromonitoring / neuro-icu).

[0103] The measuring properties are determined by the manufacturer. The measuring range of the sensor is 0-500% air saturation. Due to the measuring principle of the sensor, the resolution varies with partial pressure. The resolution at 1, 30, 100 and 250% air saturation was 0.05, 0.1, 0.5 and 1.7% air saturation, respectively. Thus, the resolution in the uterine cavity is better than 0.1% air saturation. The accuracy is within ±1% air saturation. The 0-90% response time is <5-10 seconds. The sensor shows no cross-sensitivity to carbon dioxide or any ionic species (www.presense.de).

[0104] Intrauterine oxygen tension is measured using a specially designed type of fiber-optic microsensor fabricated from a thin, flexible, insulated optical fiber with an outer diameter of 0.9 mm. These miniaturized implantable probes are suitable for various applications, e.g., for insertion into blood circuits or tissues, and provide online real-time oxygen measurements. The detection principle is based on the quenching by oxygen of a fluorescent dye (fluorophore) fixed to the fiber tip and then coated with an optical insulator to avoid interference from intrinsic fluorescence (PreSens, Regensburg, Germany, www.presens.de). The decrease in the fluorescence quantum yield of the fluorophore [ruthenium(II)-tris-(4,7-diphenyl-1,10-phenanthroline], as well as the fluorescence decay time, are proportional to the concentration of oxygen bound to the matrix in which the dye is immobilized. The fluorescence decay time is recorded using an oxygen meter OXY-1-ST (www.presens.de) connected to a laptop computer. In detail, a catheter coupling device containing a rotating male luer lock end is fixed to the sensor in a position that allows exposure of less than 1 mm of the sensor tip to the endometrial fluid when the luer lock rotates. An Embryo Transfer Catheter from Labotect containing a luer connection at the proximal end is attached to the sensor. The guide cannula of the Set is used to enter the uterine cavity under ultrasound assistance. When the cannula end is 1 cm away from the fundus, the microsensor is introduced with it and the luer lock is rotated, allowing the microsensor tip to "swim" in the endometrial fluid but never come into contact with the endometrial tissue. The rounded distal end of the labotect guide cannula has been found to be particularly useful in this sense. After immediate extension of less than 1 mm of the sensor tip, the measurement is started and after retraction the sensor is removed from the uterine cavity.

[0105] This precise extension of the sensor tip, obtained by fixing the catheter coupling device to the sensor, avoids manually exposing the sensor to the uterine cavity. In particular, the controlled extension of the sensor to less than 1 mm into the endometrial fluid is advantageous, since it substantially improves the measurement accuracy with respect to known methods. This procedure guarantees safety and reproducibility of results: it ensures that the sensor tip is not broken or damaged, that it does not come into contact with endometrial tissue that is only exposed to uterine fluid, and it ensures that the measurement is always performed in the same position at the fundus.

[0106] When measurements are made, the catheter is withdrawn from the uterus into the cervical canal to measure oxygen levels at the cervix.

[0107] These determinations are made once during the early follicular phase (days 3-5 of the menstrual cycle) and once every 48 hours on days 14-26 of the cycle. The dissolved oxygen concentration in the endometrial fluid is measured in the fundus and in the cervix. The values ​​obtained are compared with the following parameters: presence of follicles and corpora lutea, endometrial thickness and appearance, resistive and pulsatility indices, uterine LH concentration and blood progesterone and estrogen.

[0108] Experimental design At home: -Uterine LH detection daily on days 10-17 of the cycle using lateral flow strips (LH lateral flow strips).

[0109] At the IVF clinic: -Ultrasound monitoring of follicular and luteal appearance, as well as endometrial thickness and appearance. In addition, resistive and pulsatility indices are determined by Doppler ultrasound. This monitoring is performed on days 3-5 of the cycle and every other day from days 14-26 of the menstrual cycle in the IVF clinic. -Oxygen measurements in the endometrial fluid are carried out according to the method of the invention using a micrometer optical fiber inserted into the uterine cavity by means of an implanted catheter. The insertion is monitored by abdominal ultrasound. Measurements are carried out for 5-10 minutes at the fundus and for another 5-10 minutes at the cervix. These measurements are carried out on days 3-5 of the cycle and every other day from days 14-26 of the menstrual cycle with IVF consultations.

[0110] In the treatment room: -Blood samples will be collected after an 8-hour fast on days 3-5 of the cycle and on alternate days 14-26 of menstrual cycle A. -Determination of blood oxygen saturation using a pulse oximeter on days 3-5 of the cycle and on alternate days between days 14-26 of the menstrual cycle.

[0111] result One-way ANOVA followed by Tukey's multiple comparison test was performed using GraphPad Prism version 9.3.1 for macOS (GraphPad Software, San Diego, CA, USA, www.graphpad.com).

[0112] The study was performed in the Assisted Reproductive Unit of the Vall d'Hebron Hospital in Barcelona. As a result, we obtained the first human data showing intrauterine oxygen patterns throughout the luteal phase of the menstrual cycle in fertile women. pO2 patterns showed WOI occurrence in 80% of participants (Figure 4: A, B), indicated by peaks in dissolved oxygen concentration. Descriptive analysis of the data presented in Figure 4 is shown in Tables 1, 2 and 3.

[0113] [Table 1]

[0114] [Table 2]

[0115] [Table 3]

[0116] Near the day of ovulation or shortly after ovulation occurs, physiological hypoxia is achieved, with dissolved oxygen tension reaching values ​​below 10 Torr (1.4% or 1333 Pa) at the fundus of the uterine cavity. Women following the standard pattern (Figure 4A) have pO2 values ​​of approximately 20 Torr by day LH+4, increasing to 30 Torr on day LH+5. On day LH+6, pO2 reaches a maximum value (40-45 Torr) and remains stable on day LH+7. Oxygen levels then begin to decrease on day LH+8, again reaching values ​​of approximately 30 Torr. This pattern correlates with medical history data collected by anatomical pathology and gene expression, indicating that the window of implantation occurs from LH+6 to LH+9 in natural cycles or P+4 to P+7 in hormone replacement therapy (HRT) cycles. The uterine environment was prepared for the embryo on LH+6 and LH+8 days, and the WOI was found to begin to close (Figure 4A).

[0117] Crosstalk between the receptive endometrium and the embryo is important for successful embryo implantation and ongoing pregnancy. In one aspect, the endometrium secretes multiple factors, proteins, or vesicles into the uterine fluid that are required for blastocyst development and trophoblast invasion. In another aspect, the embryo regulates the endometrium for successful implantation. Several cytokines (such as placental growth factor) are upregulated in the human trophectoderm at day 5 of the blastocyst. The receptive epithelium mounts a transcriptional response to trophoblast challenge, inducing early and transient transcriptional upregulation in the receptive epithelium when combined with the endometrial epithelium.

[0118] Thus, the decreasing pO2 levels at LH+8 are a response to the absence of an embryo in the uterine cavity and are necessary to trigger the signaling of WOI closure. Invasive placentation and pregnancy are known to occur in the presence of a viable embryo. However, in its absence, the endometrium breaks down, remodels, and regenerates.

[0119] Figure 4B shows the pO2 profile of a woman with shortened and advanced WOI. As shown, physiological hypoxia occurred at the beginning of the luteal phase. However, on day LH+5, the dissolved oxygen level was already in the range of 40-45 Torr, which corresponds to the pO2 values ​​of days LH+6 and LH+7 in the standard profile (Figure 4A). In addition, the oxygen concentration quickly decreases, reaching pO2 values ​​below 10 Torr on day LH+7. Thus, the WOI would have already closed if an embryo transfer was performed on LH+7 for this woman. In addition, 20% of participants did not show an increase in pO2, but WOI disruption is suspected. Women who show this pO2 profile should undergo a detailed evaluation of endometrial function.

[0120] In addition, at least two other parameters can be combined with pO2-WOI detection to treat infertility and recommend embryo transfer: blood progesterone levels and endometrial thickness.

[0121] Low serum progesterone levels on the day of embryo transfer were associated with poorer outcomes after fresh or frozen embryo transfer, with progesterone levels of 10-20 ng / mL on the day of embryo transfer being optimal. Thus, individualized progesterone support after embryo transfer may benefit these patients. Furthermore, a thin endometrium (≦7 mm) in the late follicular phase may be associated with implantation failure and lower live birth rates.

[0122] Intrauterine oxygen measurements, used alone or in combination with progesterone levels and / or endometrial thickness before embryo transfer, would allow a recommendation of infertility treatment. The options could be to proceed with embryo transfer, to supplement with progesterone in an individualized way, to treat the thin endometrium, to obtain a detailed analysis of endometrial function, or to interrupt transfer for the next natural or artificial IVF cycle.

Claims

1. 1. A method for determining endometrial receptivity in a female subject, the method comprising: i) measuring a dissolved gas concentration in endometrial fluid of said female subject, thereby obtaining a dissolved gas concentration value, said dissolved gas being O ２ and CO ２ and ii) comparing the dissolved gas concentration value with a reference value, determining the dissolved O concentration relative to the reference value. ２ and / or dissolved CO ２ a decrease in the concentration value of indicates that the endometrium of said female subject is receptive; and A method comprising:

2. 10. The method of claim 1, wherein the step of measuring endometrial fluid dissolved gas concentration does not involve contact with the endometrium of the female subject.

3. 3. The method of claim 1 or 2, wherein deviation of the dissolved gas concentration value relative to the reference value is indicative of an implantation-synchronous endometrium for a viable embryo.

4. The reference value is the dissolved O2 in the endometrial fluid of the female subject measured at least once during the period from 2 days before the start of ovulation to the second or third day of the luteal phase. ２ or CO ２ The method according to any one of claims 1 to 3, wherein the concentration value is

5. The reference value is the dissolved O2 in the endometrial fluid of another female subject or group of female subjects measured at least once during the period from 2 days before the start of ovulation to the second or third day of the luteal phase. ２ or CO ２ The method according to any one of claims 1 to 4, wherein the concentration value is

6. 6. A method according to any one of claims 1 to 5, wherein the endometrial fluid dissolved gas concentration is measured continuously for a period of from 1 to 10 minutes, preferably from 2 to 7 minutes, more preferably from 3 to 5 minutes.

7. The method according to any one of claims 1 to 6, wherein the endometrial fluid dissolved gas concentration is measured at least once during the period from 2 days before ovulation to the 12th day of the luteal phase, preferably at least once during the period from the day of ovulation to the 10th day of the luteal phase, more preferably at least once during the period from the 2nd day to the 8th day of the luteal phase, and even more preferably at least once during the period from the 3rd day to the 6th day of the luteal phase.

8. 8. The method of claim 1, wherein the endometrial fluid dissolved gas concentration is measured at least once two days before the onset of ovulation, at least once at the time of ovulation, and / or at least once on the 2nd, 4th, 6th, 8th, 10th or 12th day of the luteal phase.

9. The reference value is the dissolved O2 concentration at physiological hypoxia in the endometrial fluid. ２ and the dissolved O ２ The method of any one of claims 1 to 9, wherein an increased concentration value is indicative of receptive endometrium of said female subject.

10. The method according to any one of claims 1 to 9, wherein the female subject is undergoing an assisted reproductive procedure comprising a step of embryo transfer.

11. 11. The method according to any one of claims 1 to 10, further comprising the step of detecting ovulation in said female subject prior to step i) by measuring the level of luteinizing hormone (LH) and / or by ultrasound monitoring.

12. The method according to any one of claims 1 to 11, further comprising measuring parameters selected from the group consisting of urinary LH concentration, blood concentrations of progesterone, estrogen and / or VEGF (vascular endothelial growth factor), the presence of follicles and / or corpora lutea in the ovaries, the thickness and appearance of the endometrium, and the resistive index and pulsatility index of the uterine spiral arteries.

13. 13. The method of any one of claims 1 to 12, wherein the endometrial fluid dissolved gas concentration is measured in the fundus and / or cervix of the female subject.

14. 14. The method of any one of claims 1 to 13, wherein the step of measuring the endometrial fluid dissolved gas concentration is performed by a sensor capable of measuring the endometrial fluid dissolved gas concentration, the sensor not in contact with the endometrium of the female subject.

15. The method according to any one of claims 1 to 14, wherein the dissolved gas concentration is measured optically, preferably by phase fluorometry.

16. A method according to any one of claims 1 to 15, wherein the step of measuring the endometrial fluid dissolved gas concentration is performed in vitro in a sample of endometrial fluid.

17. 1. A method for selecting a female subject undergoing assisted reproductive technology (ART) as a candidate to receive an embryo, said method comprising: i) determining whether the endometrium of said female subject is receptive according to the method steps of any one of claims 1 to 14; If the endometrium is receptive, ii) selecting said female subject as a candidate to receive an embryo in an immediate embryo transfer procedure on the same day as step i); A method comprising:

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