Methods for decreasing the risk of miscarriage

Abstract

The present invention relates to an immunomodulator for use in a method for decreasing the risk of miscarriage following frozen embryo transfer in a subject having an over- activated or mixed endometrial immune profile, wherein said immunomodulator is selected from glucocorticoids, intralipids, low weight heparin, and any combinations thereof. The present invention also relates to a method for determining a personalized assisted reproductive technology (ART) for decreasing the risk of miscarriage following frozen embryo transfer in a subject having a dysregulated endometrial immune profile.

Description

COMPOUNDS AND METHODS FOR DECREASING THE RISK OF MISCARRIAGEFIELD OF INVENTION

[0001] The present invention relates to the prevention of miscarriage following frozen embryo transfer in subjects undergoing an ART procedure and having a dysregulated endometrial immune profile, in particular an over-activated or mixed endometrial immune profile.BACKGROUND OF INVENTION

[0002] It is estimated that approximately one in every six people of reproductive age worldwide experience infertility in their lifetime (Infertility prevalence estimates, 1990— 2021. Geneva: World Health Organization; 2020). Assisted reproductive technologies (ART) have made considerable progress in the last few decades and have become a widely accepted treatment for infertility. However, despite the improvements in ART, the success rates remain relatively low. Indeed, the live birth rate per initiated cycle is approximately 30% for women under 35 years old and decreases drastically with age (European IVF Monitoring Consortium (EIM), for the European Society of Human Reproduction and Embryology (ESHRE); Wyns C et al., Hum Reprod Open. 2022 Jul 5;2022(3):hoac022). This leads to emotional, psychological, and financial stress with significant social and economic consequences, including loss of productivity and a decline in the quality of life for couples. Therefore, there is still a need for research and innovation to improve the success rate of ART and reduce the emotional and financial burden associated with conventional treatment.

[0003] Endometrial immune profiling aims to identify immune disturbances contributing to embryo implantation failures or pregnancy loss and guide the development of personalized treatment plans to increase embryo implantation rates. Human implantation involves the synchronized interaction of the embryo and theendometrium. The window of implantation defines the crucial time frame of uterine receptivity when the endometrium undergoes changes in response to hormonal signals from the ovary, preparing it to receive and support an embryo. Endometrial immune cells play a critical role in the process, as they contribute to the establishment of a receptive environment for the embryo to implant and develop. During this window, a crucial shift from adaptive immunity to innate immunity takes place in the endometrium. This shift creates an immunologically tolerant and fruitful environment for the developing embryo, which is a semi-allograft. The balance between Thl and Th2 cytokines plays an essential role in the success of implantation. The shift to a Th2-dominant immune environment influences the differentiation of immune cells, including macrophages, dendritic cells, uterine NK (natural killer) cells, and regulatory cells, either positively or negatively, thereby promoting or inhibiting implantation and placentation. It has been previously shown that the quantification of five biomarkers gives key information regarding the immunoregulated Th-2 / Th-l local balance, the destabilization of spiral arteries, and the mobilization and maturation of the specific uterine natural killer (uNK) cells. Endometrial immune profiling can thus be established by assessing the ratio of IL-18 / TWEAK, CD56 and the ratio of IL-15 / Fnl4 (W02014 / 013079). Using such endometrial immune profiling in extensive cohort studies focusing on subjects with a history of repeated unexplained implantation failures or unexplained recurrent miscarriages has revealed that 75-80% of these infertile subjects have a dysregulated endometrial immune profile impeding the implantation process. Personalizing ART care to address observed dysregulated endometrial immune profiles has yielded significant benefits, with a relative increase of 40-50% in live birth rates observed compared to the ones expected in these populations (Ledee N et al., Am J Reprod Immunol. 2016 Mar;75(3):388-401; Ledee N et al., Front Immunol. 2020 Jun 4; 11 : 1032; Cheloufi M et al., Front Immunol. 2021 Mar 24;12:656701).

[0004] The Inventors have now obtained data indicating that endometrial immune profiling and the subsequent personalized ART care it allows are particularly beneficial to decrease the risk of miscarriage following frozen embryo transfer. A decrease in the risk of miscarriage following frozen embryo transfer was notably observed in subjects identified as having an over-activated or mixed endometrial immune profile and receivinga personalized ART care comprising an immunomodulator selected from glucocorticoids, intralipids, low-molecular-weight heparin, and any combination thereof.

[0005] The present invention thus relates to a method for determining a personalized assisted reproductive technology (ART) for decreasing the risk of miscarriage following frozen embryo transfer in a subject having a dysregulated endometrial immune profile, wherein said method comprises:a) determining the dysregulated endometrial immune profile of the subject prior to frozen embryo transfer by:measuring the expression levels of IL-18, TWEAK, CD56, IL-15 and Fnl4 in an endometrial sample of the subject,evaluating first the IL-18 / TWEAK ratio, then CD56, and finally the IL-15 / Fnl4 ratio; andb) determining a personalized ART adapted to the dysregulated endometrial immune profile of the subject.In particular, the present invention relates to an immunomodulator for use in a method for decreasing the risk of miscarriage following frozen embryo transfer in a subject having an over-activated or mixed endometrial immune profile, wherein said immunomodulator is selected from glucocorticoids, intralipids, low-molecular-weight heparin, and any combinations thereof, and wherein said over-activated or mixed endometrial immune profile is determined by:measuring in an endometrial sample of the subject the expression levels of IL- 18, TWEAK, CD56, IL-15 and Fnl4,evaluating first the IL- 18 / T WEAK ratio, then CD56, and finally the IL-15 / Fnl4 ratio.SUMMARY

[0006] The present invention relates to an immunomodulator for use in a method for decreasing the risk of miscarriage following frozen embryo transfer (FET) in a subject having an over-activated or mixed endometrial immune profile, wherein said immunomodulator is selected from glucocorticoids, intralipids, low-molecular-weightheparin, and any combinations thereof, and wherein said over-activated or mixed endometrial immune profile is determined by:measuring the expression levels of IL-18, TWEAK, CD56, IL-15 and Fnl4 in an endometrial sample of the subject, andevaluating first IL-18 / TWEAK ratio, then CD56, and finally IL-15 / Fnl4 ratio.

[0007] In some embodiments, evaluating the IL-18 / TWEAK ratio comprises calculating the IL- 18 / TWEAK ratio and comparing said ratio to a reference IL- 18 / TWEAK ratio obtained from a fertile female subject. In some embodiments, evaluating the CD56 expression level comprises comparing said expression level to a reference CD56 expression level obtained from a fertile female subject. In some embodiments, evaluating the IL-15 / Fnl4 ratio comprises calculating the IL-15 / Fnl4 ratio and comparing said ratio to a reference IL-15 / Fnl4 ratio obtained from a fertile female subject.

[0008] In some embodiments, the over-activated endometrial immune profile or the mixed endometrial immune profile is characterized by a high IL- 18 / TWEAK ratio.

[0009] In some embodiments, the immunomodulator is for administration in combination with an increased supplementation of progesterone during the luteal phase of the FET cycle.

[0010] The present invention also relates to a method for determining a personalized assisted reproductive technology (ART) for decreasing the risk of miscarriage following frozen embryo transfer (FET) in a subject having a dysregulated endometrial immune profile, wherein said method comprises:a) determining the dysregulated endometrial immune profile of the subject prior to frozen embryo transfer by:measuring the expression levels of IL 18, TWEAK, CD56, IL-15 and Fnl4 in an endometrial sample of the subject,evaluating first IL-18 / TWEAK ratio, then CD56, and finally IL-15 / Fnl4 ratio; andb) determining a personalized ART adapted to the dysregulated endometrial immune profile of the subject.

[0011] In some embodiments, evaluating the IL- 18 / TWEAK ratio comprises calculating the IL- 18 / TWEAK ratio and comparing said ratio to a reference IL- 18 / TWEAK ratio obtained from a fertile female subject. In some embodiments, evaluating the CD56 expression level comprises comparing said expression level to a reference CD56 expression level obtained from a fertile female subject. In some embodiments, evaluating the IL-15 / Fnl4 ratio comprises calculating the IL-15 / Fnl4 ratio and comparing said ratio to a reference IL-15 / Fnl4 ratio obtained from a fertile female subject.

[0012] In some embodiments, the dysregulated endometrial immune profile is determined at most 12 months prior to frozen embryo transfer.

[0013] In some embodiments, the dysregulated endometrial immune profile determined at step a) is an over-activated endometrial immune profile characterized by a high or normal IL-18 / TWEAK ratio and a high or normal IL-15 / Fnl4 ratio, with at least one of IL 18 / TWEAK ratio, CD56 expression level and IL-15 / Fnl4 ratio being high.

[0014] In some embodiments, the dysregulated endometrial immune profile determined at step a) is a mixed endometrial immune profile characterized by a high IL- 18 / TWEAK ratio and a low IL-15 / Fnl4 ratio.

[0015] In some embodiments, the personalized ART determined at step b) is adapted to the over-activated and mixed endometrial immune profile and comprises recommendations for downregulating the local activity of local immune cells. In some embodiments, recommendations for downregulating the local activity of local immune cells comprise recommending an immunomodulating therapy, preferably glucocorticoids, intralipids and / or low-molecular-weight heparin.DEFINITIONS

[0016] In the present disclosure, the following terms have the following meanings:

[0017] “ART” stands for assisted reproductive technology.

[0018] “CD56” stands for cluster of differentiation 56.

[0019] “CI” stands for confidence interval.

[0020] “ET” stands for embryo transfer and “FET” stands for frozen embryo transfer.

[0021] “Fnl4” stands for fibroblast growth factor-inducible 14.

[0022] “hCG” stands for human chorionic gonadotropin and “BhCG” stands for beta human chorionic gonadotropin.

[0023] “ICSI” stands for intracytoplasmic sperm injection.

[0024] “IL-15” stands for interleukin- 15 and “IL-18” stands for interleukin- 18.

[0025] “IQR” stands for interquartile range.

[0026] “ITT” stands for intention-to-treat and “mITT” stands for modified intention-to-treat.

[0027] “IUI” stands for intrauterine insemination.

[0028] “IVF” stands for in vitro fertilization.

[0029] “LBR” stands for live birth rate.

[0030] “LH” stands for luteinizing hormone.

[0031] “LMWH” stands for low-molecular-weight heparin.

[0032] “OR” stands for odds ratio.

[0033] “PCR” stands for polymerase chain reaction and “RT-PCR” stands for reverse transcription polymerase chain reaction.

[0034] “SD” stands for standard deviation.

[0035] “TWEAK” stands for TNF weakly inducer of apoptosis (sometimes also referred to as TNF-like weak inducer of apoptosis).

[0036] “uNK cells” stands for uterine natural killer cells

[0037] The terms “a” and “an” refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0038] “About”, when preceding a figure, encompasses plus or minus 10%, or less, of the value of said figure. It is to be understood that the value to which the term “about” refers is itself also specifically, and preferably, disclosed.

[0039] “Comprising” or “comprise” is to be construed in an open, inclusive sense, not limited to the features following this term.

[0040] “Consisting of’ or “consist” is to be construed in a close, non-inclusive sense, limited to the features following this term.

[0041] “Cycle of the frozen embryo transfer (FET)” or “FET cycle” means the menstrual cycle during which the transfer of a frozen embryo takes place.

[0042] “Marker”, in particular “biomarker” or “biological marker” refers to a variable that can be measured in a biological sample from a subject.

[0043] “Measuring” or “measurement”, or alternatively “detecting” or “detection”, mean assessing the presence, absence, quantity, or amount (which can be an effective amount) of a given substance, e.g., IL-18, TWEAK, CD56, IL-15 and Fnl4, within a biological sample from a subject. “Measuring” or “measurement”, or alternatively “detecting” or “detection” as used herein include the derivation of the qualitative or quantitative concentration of said substance, e.g., IL-18, TWEAK, CD56, IL-15 and Fnl4, within the biological sample.

[0044] “Luteal phase” of the menstrual cycle corresponds to the extent of time between ovulation and the first day of the next period. The luteal phase generally has a duration of about 14 days.

[0045] “Pharmaceutically acceptable excipient” or “pharmaceutically acceptable carrier” refers to an excipient or carrier that does not produce an adverse, allergic or other untoward reaction when administered to a subject. It includes any and all solvents, such as, for example, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents. A pharmaceutically acceptable excipient or carrier refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. For human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by theregulatory offices such as the FDA (U.S. food and drug administration) or EMA (European medicines agency).

[0046] “Subject” refers to a mammal. Preferably, the subject is a human subject.

[0047] “Substituted cycle” is a menstrual cycle during which exogenous hormones, in particular exogenous estrogens, are administered. Typically, estrogens are first administered to stimulate endometrial growth, followed by administration of progesterone to stabilize the endometrium and prepare it for embryo implantation. Conversely a “natural cycle” is a menstrual cycle during which no exogenous estrogens are administrated (during a so-called “natural cycle”, exogenous progesterone may be administered to allow a better synchronization of the endometrium with the transferred embryo).DETAILED DESCRIPTION

[0048] A first object of the present invention is a method for determining a personalized assisted reproductive technology (ART) for decreasing the risk of miscarriage following frozen embryo transfer in a subject having a dysregulated endometrial immune profile, wherein said method comprises:a) determining the dysregulated endometrial immune profile of the subject prior to frozen embryo transfer by:measuring the expression levels of IL-18, TWEAK, CD56, IL-15 and Fnl4 in an endometrial sample of the subject,evaluating first IL-18 / TWEAK ratio, then CD56, and finally IL-15 / Fnl4 ratio; andb) determining a personalized ART adapted to the dysregulated endometrial immune profile of the subject.

[0049] As used herein, “endometrial sample” refers to a sample previously obtained from a subject. Accordingly, the methods described herein do not include obtaining an endometrial sample from a subject. Endometrial samples may be conserved in adequate conditions before being used in the methods as described herein. Preferably, theendometrial sample was obtained during the mid-luteal phase, for example about 9 days after the luteinizing hormone (LH) surge or after 7 days of progesterone supplementation.

[0050] The biomarkers to be measured in order to determine the endometrial immune profile of a subject, in particular the dysregulated endometrial immune profile of a subject, comprises or consists of IL-18, TWEAK, CD56, IL-15 and Fnl4 and, more specifically, IL- 18 / TWEAK ratio, CD56, and IL-15 / Fnl4 ratio.

[0051] Interleukin- 18 (IL- 18) is a pro-inflammatory cytokine crucial for immune regulation in reproduction, playing key roles in embryo implantation, trophoblast invasion, NK (natural killer) cell modulation, and placental vascularization. Interleukin-15 (IL- 15) supports embryo implantation and placentation by promoting uterine natural killer (uNK) cell maturation, function and cytokine production essential for reproductive processes. In the context of embryo implantation, TWEAK / Fnl4 signaling has been shown to regulate the cytotoxicity of uNK cells, which is important for controlling trophoblast invasion and preventing fetal rejection. The IL- 18 / TWEAK ratio is thus an indicator of both angiogenesis and the Thl / Th2 balance. Indeed, the IL- 18 / TWEAK ratio provides insights into the local immune environment and the potential presence of an immune deviation towards Thl cytokines, which can affect the implantation process. The IL-15 / Fnl4 is an indicator of the activation and maturation status of uNK cells, along with the evaluation CD56.

[0052] As used herein, the term “expression level” of a given biomarker (e.g., IL-18, TWEAK, CD56, IL-15 or Fnl4) refers to the transcription level (i.e., the level of mRNA or cDNA) or to the translation level (i.e., the level of protein) of said biomarker. In particular, the expression level may be the mRNA level. Thus, in some embodiments, by measuring the expression levels of IL-18, TWEAK, CD56, IL-15 and Fnl4 it is meant measuring the mRNA levels of IL-18, TWEAK, CD56, IL-15 and Fnl4.

[0053] The expression levels of IL-18, TWEAK, CD56, IL-15 and Fnl4 may be measured by any known method in the art. Methods for measuring an expression level are well-known to the skilled artisan. Commonly used methods for measuring a transcription level (i.e., a level of mRNA or cDNA) include PCR, qPCR, RT-PCR,RT-qPCR, northern blot, hybridization techniques such as, for example, use of microarrays, and combination thereof including hybridization of amplicons obtained by RT-PCR, sequencing such as, for example, next-generation DNA sequencing (NGS) or RNA-seq (also known as “Whole Transcriptome Shotgun Sequencing”). Commonly used methods for measuring a translation level (i.e., a level of protein) include immunohistochemistry, multiplex methods (such as Luminex), immunoassays, western blot, enzyme-linked immunosorbent assay (ELISA), flow cytometry (FACS), surface plasmon resonance (SPR), and mass spectrometry-based approaches.

[0054] In order to determine the endometrial immune profile of a subject, in particular the dysregulated endometrial immune profile of a subject, the measured expression levels of IL-18, TWEAK, CD56, IL-15 and Fnl4 are evaluated in a specific order: first IL-18 / TWEAK ratio, then CD56 and finally IL-15 / Fnl4 ratio. As used herein, by “evaluating” (a) given biomarker(s), it is meant analyzing or processing or taking into consideration said given biomarker(s). Thus, as described herein, the measured expression levels of IL-18, TWEAK, CD56, IL-15 and Fnl4 are analyzed or processed or taken into consideration in a specific order: first IL-18 / TWEAK ratio, then CD56 and finally IL-15 / Fnl4 ratio.

[0055] For example, evaluating (or analyzing or processing or taking into consideration) the IL-18 / TWEAK ratio may comprise calculating the IL-18 / TWEAK ratio (that is to say calculating the ratio of the measured expression level of IL- 18 and of the measured expression level of TWEAK) and comparing said ratio to a reference IL- 18 / TWEAK ratio obtained from a fertile female subject or from a population of fertile female subjects. A reference IL- 18 / TWEAK ratio may thus be a ratio of the expression level of IL- 18 and of the expression level of TWEAK measured in an endometrial sample obtained from a fertile female subject. Alternatively, a reference IL- 18 / TWEAK ratio may be a ratio of the mean expression level of IL- 18 and of the mean expression level of TWEAK measured in endometrial samples obtained from a population of fertile female subjects.

[0056] In comparison to the reference IL- 18 / TWEAK ratio obtained from a fertile female subject or from a population of fertile female subjects, the evaluated IL- 18 / TWEAK ratio may be high, low or normal. A high IL- 18 / TWEAK ratio means thatthe evaluated IL-18 / TWEAK ratio is substantially higher than the reference IL-18 / TWEAK ratio. A normal IL-18 / TWEAK ratio means that the evaluated IL-18 / TWEAK ratio is substantially similar to the reference IL-18 / TWEAK ratio. A low IL-18 / TWEAK ratio means that the evaluated IL-18 / TWEAK ratio is substantially lower than the reference IL-18 / TWEAK ratio.

[0057] In some embodiments, “substantially higher” means that the evaluated IL-18 / TWEAK ratio is at least 5% higher than the reference IL-18 / TWEAK ratio; “substantially lower” means that the evaluated IL-18 / TWEAK ratio is at least 5% lower than the reference IL-18 / TWEAK ratio; and “substantially similar” means that the evaluated IL-18 / TWEAK ratio is within 5% (i.e., less than 5% higher and less than 5% lower) of the reference IL-18 / TWEAK ratio. In some embodiments, “substantially higher” means that the evaluated IL-18 / TWEAK ratio is at least 10% higher than the reference IL-18 / TWEAK ratio; “substantially lower” means that the evaluated IL-18 / TWEAK ratio is at least 10% lower than the reference IL-18 / TWEAK ratio; and “substantially similar” means that the evaluated IL-18 / TWEAK ratio is within 10% (i.e., less than 10% higher and less than 10% lower) of the reference IL-18 / TWEAK ratio.

[0058] Evaluating (or analyzing or processing or taking into consideration) CD56 (that is to say the expression level of CD56) may comprise comparing the measured expression level of CD56 to a reference CD56 expression level obtained from a fertile female subject or from a population of fertile female subjects. A reference CD56 expression level may thus be a CD56 expression level measured in an endometrial sample obtained from a fertile female subject. Alternatively, a reference CD56 expression level may be a mean CD56 expression level measured in endometrial samples obtained from a population of fertile female subjects.

[0059] Evaluating (or analyzing or processing or taking into consideration) CD56 (that is to say the expression level of CD56) may also comprise, alternatively or additionally, comparing the number of CD56+ cells (in particular of CD56+ uNK cells) to a reference number of CD56+ cells (in particular of CD56+ uNK cells) obtained from a fertile female subject or from a population of fertile female subjects. A reference number of CD56+cells may be a mean number of CD56+ measured in endometrial samples obtained from a population of fertile female subjects.

[0060] In comparison to the reference CD56 expression level obtained from a fertile female subject or from a population of fertile female subjects, the evaluated CD56 expression level may be high, low or normal. A high CD56 expression level (or, in short, a high CD56) means that the evaluated CD56 expression level is substantially higher than the reference CD56 expression level. A normal CD56 expression level (or, in short, a normal CD56) means that the evaluated CD56 expression level is substantially similar to the reference CD56 expression level. A low CD56 expression level (or, in short, a low CD56) means that the evaluated IL-18 / TWEAK ratio is substantially lower than the reference CD56 expression level.

[0061] In some embodiments, “substantially higher” means that the evaluated CD56 expression level is at least 5% higher than the reference CD56 expression level; “substantially lower” means that the evaluated CD56 expression level is at least 5% lower than the reference CD56 expression level; and “substantially similar” means that the evaluated CD56 expression level is within 5% (i.e., less than 5% higher and less than 5% lower) of the reference CD56 expression level. In some embodiments, “substantially higher” means that the evaluated CD56 expression level is at least 10% higher than the reference CD56 expression level; “substantially lower” means that the evaluated CD56 expression level is at least 10% lower than the reference CD56 expression level ratio; and “substantially similar” means that the evaluated CD56 expression level is within 10% (i.e., less than 10% higher and less than 10% lower) of the reference CD56 expression level.

[0062] Evaluating (or analyzing or processing or taking into consideration) the IL-15 / Fnl4 ratio may comprise calculating the IL-15 / Fnl4 ratio (that is to say calculating the ratio of the measured expression level of IL- 15 and of the measured expression level of Fnl4) and comparing said ratio to a reference IL-15 / Fnl4 ratio obtained from a fertile female subject or from a population of fertile female subjects. A reference IL-15 / Fnl4 ratio may thus be a ratio of the expression level of IL- 15 and of the expression level of Fnl4 measured in an endometrial sample obtained from a fertile female subject.Alternatively, a reference IL-15 / Fnl4 ratio may be a ratio of the mean expression level of IL-15 and of the mean expression level of Fnl4 measured in endometrial samples obtained from a population of fertile female subjects.

[0063] In comparison to the reference IL-15 / Fnl4 ratio obtained from a fertile female subject or from a population of fertile female subjects, the evaluated IL-15 / Fnl4 ratio may be high, low or normal. A high IL-15 / Fnl4 ratio means that the evaluated IL-15 / Fnl4 ratio is substantially higher than the reference IL-15 / Fnl4 ratio. A normal IL-15 / Fnl4 ratio means that the evaluated IL-15 / Fnl4 ratio is substantially similar to the reference IL-15 / Fnl4 ratio. A low IL-15 / Fnl4 ratio means that the evaluated IL-15 / Fnl4 ratio is substantially lower than the reference IL-15 / Fnl4 ratio.

[0064] In some embodiments, “substantially higher” means that the evaluated IL-15 / Fnl4 ratio is at least 5% higher than the reference IL-15 / Fnl4 ratio; “substantially lower” means that the evaluated IL-15 / Fnl4 ratio is at least 5% lower than the reference IL-15 / Fnl4 ratio; and “substantially similar” means that the evaluated IL-15 / Fnl4 ratio is within 5% i.e., less than 5% higher and less than 5% lower) of the reference IL-15 / Fnl4 ratio. In some embodiments, “substantially higher” means that the evaluated IL-15 / Fnl4 ratio is at least 10% higher than the reference IL-15 / Fnl4 ratio; “substantially lower” means that the evaluated IL-15 / Fnl4 ratio is at least 10% lower than the reference IL-15 / Fnl4 ratio; and “substantially similar” means that the evaluated IL-15 / Fnl4 ratio is within 10% (i.e., less than 10% higher and less than 10% lower) of the reference IL-15 / Fnl4 ratio.

[0065] Advantageously, the endometrial immune profile of a subject, in particular the dysregulated endometrial immune profile of a subject, may be determined at most about 12 months prior to frozen embryo transfer. For example, the endometrial immune profile of a subject, in particular the dysregulated endometrial immune profile of a subject, may be determined at most about 9 months, at most about 6 months, at most about 3 months or at most about 1 month prior to frozen embryo transfer. Preferably, frozen embryo transfer is carried out at most about 12 months, at most about 9 months, at most about 6 months, at most about 3 months or at most about 1 month after the determination of the endometrial immune profile of a subject, in particular the dysregulated endometrialimmune profile of a subject, if no pregnancy or gynecological surgery occurred since the determination of the endometrial immune profile. By gynecological surgery, it is meant surgery affecting the reproductive organs, such as, for example, the uterus and / or the ovaries.

[0066] The endometrial immune profile of a subject, in particular the dysregulated endometrial immune profile of a subject, may be determined at least about 1 month prior to frozen embryo transfer. For example, the endometrial immune profile of a subject, in particular the dysregulated endometrial immune profile of a subject, may be determined at least about 2 months, at least about 3 months, at least about 4 months, or at least about 5 months prior to frozen embryo transfer.

[0067] The endometrial immune profile of a subject, in particular the dysregulated endometrial immune profile of a subject, may thus be determined in a time period ranging from at least about 1 month to at most about 12 months prior to frozen embryo transfer, preferably from at least about 1 month to at most about 9 months prior to frozen embryo transfer.

[0068] The dysregulated endometrial immune profile may be an over-activated endometrial immune profile, a mixed endometrial immune profile, or an under-activated endometrial immune profile. Accordingly, in the method for determining a personalized ART for decreasing the risk of miscarriage following frozen embryo transfer as described herein, the subject may have an over-activated endometrial immune profile, a mixed endometrial immune profile, or an under-activated endometrial immune profile.

[0069] An under-activated endometrial immune profile is characterized by a low or normal IL-18 / TWEAK ratio, a low or normal CD56 expression level and a low or normal IL-15 / Fnl4 ratio, with at least one of IL-18 / TWEAK ratio, CD56 expression level and IL-15 / Fnl4 ratio being low.

[0070] For illustrative purposes, non-exhaustive examples of under-activated endometrial immune profiles include:an endometrial immune profile characterized by a low IL-18 / TWEAK ratio, a low CD56 expression level and a low IL-15 / Fnl4 ratio,an endometrial immune profile characterized by a low IL-18 / TWEAK ratio, a low CD56 expression level and a normal IL-15 / Fnl4 ratio,an endometrial immune profile characterized by a low IL- 18 / TWEAK ratio, a normal CD56 expression level and a low IL-15 / Fnl4 ratio,an endometrial immune profile characterized by a low IL- 18 / TWEAK ratio, a normal CD56 expression level and a normal IL-15 / Fnl4 ratio.

[0071] An under-activated endometrial immune profile may indicate that the endometrium of the subject is not fully effective for adhesion and for promoting adequate immunotrophism during initial placentation.

[0072] An over-activated endometrial immune profile is characterized by a high or normal IL-18 / TWEAK ratio and a high or normal IL-15 / Fnl4 ratio, with at least one of IL-18 / TWEAK ratio, CD56 expression level and IL-15 / Fnl4 ratio being high. In other words, an over-activated endometrial immune profile is characterized by a high or normal IL- 18 / TWEAK ratio, a high, normal or low CD56 expression, and a high or normal IL-15 / Fnl4 ratio, with at least one of IL-18 / TWEAK ratio, CD56 expression level and IL-15 / Fnl4 ratio being high.

[0073] For illustrative purposes, non-exhaustive examples of over-activated endometrial immune profiles include:an endometrial immune profile characterized by a high IL- 18 / TWEAK ratio, a high CD56 expression level and a high IL-15 / Fnl4 ratio,an endometrial immune profile characterized by a high IL- 18 / TWEAK ratio, a high CD56 expression level and a normal IL-15 / Fnl4 ratio,an endometrial immune profile characterized by a high IL- 18 / TWEAK ratio, a normal CD56 expression level and a high IL-15 / Fnl4 ratio,an endometrial immune profile characterized by a high IL- 18 / TWEAK ratio, a normal CD56 expression level and a normal IL-15 / Fnl4 ratio,an endometrial immune profile characterized by a high IL- 18 / TWEAK ratio, a low CD56 expression level and a normal IL-15 / Fnl4 ratio.

[0074] In some embodiments, an over-activated endometrial immune profile is characterized by a high IL- 18 / TWEAK ratio, with a high, normal or low CD56 expression level and with a high or normal IL-15 / Fnl4 ratio.

[0075] An over-activated endometrial immune profile may indicate that the endometrium of the subject is not prepared for the crucial step of trophoblast invasion and may be in a state that can reject the embryo because of a cytotoxic activation of uNK cells in LAKs (lymphocyte-activated killer cells).

[0076] A mixed endometrial immune profile is characterized by a high IL- 18 / TWEAK ratio and a low IL-15 / Fnl4 ratio or by a low IL- 18 / TWEAK ratio and a high IL-15 / Fnl4 ratio. In other words, a mixed endometrial immune profile is characterized by a high IL-18 / TWEAK ratio, a high, normal or low CD56 expression level and a low IL-15 / Fnl4 ratio, or by a low IL- 18 / TWEAK ratio, a high, normal or low CD56 expression level and a high IL-15 / Fnl4 ratio.

[0077] For illustrative purposes, non-exhaustive examples of mixed endometrial immune profiles include:an endometrial immune profile characterized by a high IL- 18 / TWEAK ratio, a normal CD56 expression level and a low IL-15 / Fnl4 ratio,an endometrial immune profile characterized by a low IL- 18 / TWEAK ratio, a normal CD56 expression level and a high IL-15 / Fnl4 ratio.

[0078] In some embodiments, a mixed endometrial immune profile is characterized by a high IL- 18 / TWEAK ratio, with a high, normal or low CD56 expression level and a low IL-15 / Fnl4 ratio.

[0079] A mixed endometrial immune profile may be considered as a subtype of an overactivated endometrial immune profile. A mixed endometrial immune profile may indicate that the endometrium of the subject is not prepared for the crucial step of trophoblast invasion and may be in a state that can reject the embryo because of a cytotoxic activation of uNK cells in LAKs (lymphocyte-activated killer cells). Additionally, a mixed endometrial immune profile may also indicate that the endometrium of the subject is not fully effective for promoting adequate immunotrophism during initial placentation.

[0080] As indicated above, a high IL-18 / TWEAK ratio characterizes either an over-activated or a mixed endometrial immune profile. In some embodiments, an over-activated or mixed endometrial immune profile is thus characterized by a high IL- 18 / TWEAK ratio.

[0081] In some embodiments, the subject has (i.e., is determined to have) an underactivated endometrial immune profile and the method as described herein comprises determining a personalized ART adapted to the under-activated endometrial immune profile of the subject.

[0082] In some embodiments, a personalized ART adapted to the under-activated endometrial immune profile comprises recommendations for stimulating mobilization of immune cells and expression of adhesion molecules.

[0083] For example, recommendations for stimulating mobilization of immune cells and expression of adhesion molecules may comprise:recommending an endometrial scratching in the luteal phase (e.g., mid luteal phase) of the cycle preceding the frozen embryo transfer to trigger the expression of adhesion molecules and IL- 15,recommending a supplementation of the luteal phase of the FET cycle (i.e., the cycle of the frozen embryo transfer) with chorionic gonadotropins to trigger local angiogenesis and uterine natural killer (uNK) cells mobilization, and / or advising to have sexual intercourse after the frozen embryo transfer to stimulate the local mobilization and expression of immune cells.

[0084] Advantageously, recommendations for stimulating mobilization of immune cells and expression of adhesion molecules may comprise:recommending an endometrial scratching in the luteal phase (e.g., mid luteal phase) of the cycle preceding the frozen embryo transfer to trigger the expression of adhesion molecules and IL- 15, and / orrecommending a supplementation of the luteal phase of the FET cycle with chorionic gonadotropins to trigger local angiogenesis and uNK cells mobilization.

[0085] For example, a supplementation of the luteal phase with chorionic gonadotropins may be an administration of chorionic gonadotropins or choriogonadotropin alfa by subcutaneous route during the luteal phase. Said administration may be a single administration during the luteal phase or an administration repeated once, twice or more during the luteal phase. Preferably, a supplementation of the luteal phase with chorionic gonadotropins comprises three administrations of chorionic gonadotropins or choriogonadotropin alfa during the luteal phase. For example, chorionic gonadotropins or choriogonadotropin alfa may be administrated during the luteal phase 4, 6 and / or 8 days after the introduction of progesterone for luteal support (luteal support is commonly implemented in conventional ART procedures including an embryo transfer). An exemplary dose of chorionic gonadotropins is a dose of 2500 IU (international unit) of chorionic gonadotropins by subcutaneous injection.

[0086] In some embodiments, the subject has (z.e., is determined to have) an overactivated or a mixed endometrial immune profile and the method as described herein comprises determining a personalized ART adapted to the over-activated or mixed endometrial immune profile of the subject.

[0087] In some embodiments, a personalized ART adapted to the over-activated or mixed endometrial immune profile comprises recommendations for downregulating the local activity of local immune cells, in particular for controlling the dysregulated Th-l / Th-2 ratio reflected by a high IL-18 / TWEAK ratio.

[0088] For example, recommendations for downregulating the local activity of local immune cells may comprise recommending an immunomodulating therapy selected from glucocorticoids, intralipids, low-molecular-weight heparin (LMWH), and any combinations thereof.

[0089] For example, glucocorticoids such as prednisone may be administrated at a daily dose of 5 mg, 10 mg or 20 mg, preferably at a daily dose of 10 or 20 mg. For example, glucocorticoids may be administered orally. For example, glucocorticoids may be administered daily during the FET cycle, in particular from the third day of the FET cycle at least until the pregnancy test. An example of immunomodulating therapy may thus bethe daily administration of glucocorticoids (advantageously prednisone) at a dose of 20 mg, from the third day of the FET cycle at least until the pregnancy test. In case of a positive pregnancy test, the daily administration of glucocorticoids may be continued for about 8 weeks of pregnancy. Glucocorticoids may thus be gradually weaned off and stopped in case of negative pregnancy test or after about 10 weeks of pregnancy.

[0090] For example, intralipids (which are a fat emulsion comprising soybean oil, glycerin and egg phospholipids) may be administrated as a slow intravenous infusion. Typically, a slow intravenous infusion is an infusion at a rate ranging from 1 mL / minute to 3 mL / minute. For example, intralipids may be administered once during the FET cycle, for example around day 8 of the FET cycle. An exemplary dose of intralipids is a slow intravenous infusion of 20g / 100mL diluted in 400mL of an appropriate pharmaceutically acceptable buffer such as NaCl 0.9%. In case of a positive pregnancy test, a single administration of intralipids may be repeated once or twice, for example once at about 5 weeks of pregnancy and / or once at about 9 weeks of pregnancy.

[0091] For example, low-molecular- weight heparin (LMWH) such as enoxaparin (Lo venox) may be administrated as a daily subcutaneous injection. An exemplary dose of LMWH is a daily dose of 4000 IU of enoxaparin. For example, LMWH may be administered daily during the FET cycle, in particular from the start of the progesterone luteal supplementation at least until the pregnancy test. In case of a positive pregnancy test, the daily administration of LMWH may be continued during the first trimester of pregnancy.

[0092] The immunomodulating therapy as described herein may also comprise a combination of two or three of glucocorticoids, intralipids and low-molecular-weight heparin (LMWH). For example, the immunomodulating therapy may comprise a combination of glucocorticoids as described herein and intralipids as described herein; a combination of glucocorticoids as described herein and LMWH as described herein; or a combination of intralipids as described herein and LMWH as described herein.

[0093] Prior to frozen embryo transfer, a test cycle under immunomodulating therapy may be recommended to evaluate whether or not the tested immunomodulating therapy(e.g., glucocorticoids, intralipids, or low-molecular-weight heparin (LMWH)) is able to normalize the over-activated or mixed endometrial immune profile of the subject. A second endometrial immune profile of the subject is thus determined as described herein after immunomodulating therapy and prior to frozen embryo transfer. For example, a second endometrial immune profile of the subject is determined as described herein from an endometrial sample of the subject obtained during the luteal phase of a cycle under immunomodulating therapy. If the over-activated or mixed endometrial profile of the subject is normalized (i.e., if the endometrial immune profile of the subject is no longer over-activated or mixed), then the tested immunomodulating therapy is considered as efficient and implemented during the FET cycle, as described herein. If the overactivated or mixed endometrial profile is not normalized (i.e., if the endometrial immune profile of the subject is still over-activated or mixed), the immunomodulating therapy is adapted. For example, if administration of glucocorticoids was the tested immunomodulating therapy, administration of intralipids and / or LMWH may be recommended instead of administration of glucocorticoids or in addition to administration of glucocorticoids. Conversely, if administration of intralipids was the tested immunomodulating therapy, administration of glucocorticoids and / or LMWH may be recommended instead of administration of intralipids or in addition to administration of intralipids. Similarly, if administration of LMWH was the tested immunomodulating therapy, administration of glucocorticoids and / or intralipids may be recommended instead of administration of LMWH or in addition to administration of LMWH.

[0094] Recommendations for downregulating the local activity of local immune cells may also comprise recommending an increased supplementation of progesterone during the luteal phase of the LET cycle.

[0095] Luteal support is commonly implemented in conventional ART procedures including an embryo transfer. Luteal support is usually implemented through a supplementation of progesterone during the luteal phase of the cycle of the embryo transfer. Exogenous progesterone is thus administered to allow a better synchronization of the endometrium with the transferred embryo. In conventional ART procedures, exogenous progesterone is commonly supplemented by vaginal administration, at astandard daily dose ranging from 300 mg to 600 mg. For example, a standard dose of progesterone for luteal support is a daily dose of 600 mg of micronized progesterone administered vaginally (corresponding to a vaginal administration at a dose of 200 mg three times a day).

[0096] As used herein, recommending an increased supplementation of progesterone during the luteal phase means recommending a supplementation of progesterone during the luteal phase at a dose higher than the standard dose commonly prescribed in conventional ART procedures.

[0097] For example, an increased supplementation of progesterone during the luteal phase may be a supplementation of progesterone by vaginal administration at a daily dose of 1 200 mg (corresponding to a vaginal administration at a dose of 400 mg three times a day). Alternatively, an increased supplementation of progesterone during the luteal phase may be a supplementation of progesterone by vaginal administration at a standard daily dose (such as 600 mg of micronized progesterone administered vaginally three times a day at a dose of 200 mg) combined with a supplementation of progesterone by subcutaneous injection (for example at a daily dose of 25 mg, e.g., one subcutaneous injection of 25 mg per day).

[0098] In some embodiments, recommendations for downregulating the local activity of local immune cells comprises:recommending an immunomodulating therapy selected from glucocorticoids, intralipids, low-molecular-weight heparin (LMWH), and any combinations thereof, as described herein, andrecommending an increased supplementation of progesterone during the luteal phase of the FET cycle as described herein.

[0099] In some embodiments, the subject has (i.e., is determined to have) a mixed endometrial immune profile and the method as described herein comprises determining a personalized ART adapted to the mixed endometrial immune profile of the subject.

[0100] In some embodiments, a personalized ART adapted to the mixed endometrial immune profile comprises recommendations for downregulating the local activity of localimmune cells as described herein and recommendations for stimulating mobilization of immune cells and expression of adhesion molecules as described herein.

[0101] In some embodiments, a personalized ART adapted to the mixed endometrial immune profile thus comprises:recommending an immunomodulating therapy selected from glucocorticoids, intralipids, low-molecular-weight heparin (LMWH), and any combinations thereof, as described herein,recommending an increased supplementation of progesterone during the luteal phase of the FET cycle as described herein,recommending an endometrial scratching in the luteal phase (e.g., mid luteal phase) of the cycle preceding the frozen embryo transfer to trigger the expression of IL- 15 in order to enhance the maturation of immune cells, andrecommending a supplementation of the luteal phase of the FET cycle with chorionic gonadotropins as described herein.

[0102] Another object of the present invention is an immunomodulator for use in a method for decreasing the risk of miscarriage following frozen embryo transfer in a subject having an over-activated or mixed endometrial immune profile as described herein, wherein said immunomodulator is selected from glucocorticoids, intralipids, low-molecular-weight heparin (LMWH), and any combinations thereof, and wherein said over-activated or mixed endometrial immune profile is determined by:measuring the expression levels of IL-18, TWEAK, CD56, IL-15 and Fnl4 in an endometrial sample of the subject as described herein, andevaluating first IL-18 / TWEAK ratio, then CD56, and finally IL-15 / Fnl4 ratio as described herein.

[0103] Features, definitions and examples provided elsewhere in the present disclosure with regards to the endometrial sample, with regards to the measure and evaluation of the biomarkers IL-18, TWEAK, CD56, IL-15 and Fnl4, and with regards to the overactivated and mixed endometrial immune profiles apply mutatis mutandis.

[0104] In some embodiments, the over-activated or mixed endometrial immune profile is characterized by a high IL-18 / TWEAK ratio.

[0105] In some embodiments, the immunomodulator (i.e., the immunomodulating therapy) is glucocorticoids as described herein, intralipids as described herein, or LMWH as described herein. In some embodiments, the immunomodulator is a combination of two or three of glucocorticoids, intralipids and LMWH, as described herein.

[0106] Features, definitions and examples provided elsewhere in the present disclosure with regards to the administration of glucocorticoids, intralipids, LMWH, and any combinations thereof apply mutatis mutandis.

[0107] In some embodiments, the immunomodulator is for administration during the FET cycle (i.e., the cycle of the frozen embryo transfer).

[0108] In some embodiments, the efficacy of the immunomodulator in normalizing the over-activated or mixed endometrial immune profile of the subject is verified prior to frozen embryo transfer. For example, the efficacy of the immunomodulator in normalizing the over-activated or mixed endometrial immune profile of the subject may be verified by determining as described herein a second endometrial immune profile of the subject after administration of the immunomodulator and prior to frozen embryo transfer. In particular, a second endometrial immune profile of the subject may be determined as described herein from an endometrial sample of the subject obtained during the luteal phase of a cycle under immunomodulating therapy.

[0109] In some embodiments, the efficacy of the immunomodulator in normalizing the over-activated or mixed endometrial immune profile of the subject is verified prior to frozen embryo transfer, by conducting a test cycle under immunomodulating therapy as described herein.

[0110] In some embodiments, the immunomodulator is for administration in combination with an increased supplementation of progesterone during the luteal phase of the FET cycle.[OHl] Features, definitions and examples provided elsewhere in the present disclosurewith regards to the increased supplementation of progesterone during the luteal phase of the FET cycle apply mutatis mutandis.

[0112] Another object of the present invention is a pharmaceutical composition for use in a method for decreasing the risk of miscarriage following frozen embryo transfer in a subject having an over-activated or mixed endometrial immune profile as described herein, wherein said pharmaceutical composition comprises an immunomodulator selected from glucocorticoids, intralipids, low-molecular-weight heparin (LMWH), and any combinations thereof, and wherein said over-activated or mixed endometrial immune profile is determined by:measuring the expression levels of IL-18, TWEAK, CD56, IL-15 and Fnl4 in an endometrial sample of the subject as described herein, andevaluating first IL-18 / TWEAK ratio, then CD56, and finally IL-15 / Fnl4 ratio as described herein.

[0113] In some embodiments, the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient.

[0114] Another object of the present invention is the use of an immunomodulator in the manufacture of a medicament for decreasing the risk of miscarriage following frozen embryo transfer (FET) in a subject having an over-activated or mixed endometrial immune profile as described herein, wherein said immunomodulator is selected from glucocorticoids, intralipids, low-molecular-weight heparin (LMWH), and any combinations thereof, and wherein said over-activated or mixed endometrial immune profile is determined by:measuring the expression levels of IL-18, TWEAK, CD56, IL-15 and Fnl4 in an endometrial sample of the subject as described herein, andevaluating first IL-18 / TWEAK ratio, then CD56, and finally IL-15 / Fnl4 ratio as described herein.

[0115] In some embodiments, the pharmaceutical composition or the medicament is for administration in combination with an increased supplementation of progesterone during the luteal phase of the FET cycle, as described herein.

[0116] Another object of the present invention is a method for decreasing the risk of miscarriage following frozen embryo transfer (FET) in a subject having a dysregulated endometrial immune profile, wherein said method comprises:a) determining the dysregulated endometrial immune profile of the subject prior to frozen embryo transfer by:measuring the expression levels of IL-18, TWEAK, CD56, IL-15 and Fnl4 in an endometrial sample of the subject as described herein,evaluating first IL-18 / TWEAK ratio, then CD56, and finally IL-15 / Fnl4 ratio as described herein; andb) providing a personalized ART adapted to the dysregulated endometrial immune profile of the subject.

[0117] Features, definitions, and examples provided elsewhere in the present disclosure with regards to the endometrial sample, with regards to the measure and evaluation of the biomarkers IL-18, TWEAK, CD56, IL-15 and Fnl4, and with regards to the dysregulated endometrial immune profiles apply mutatis mutandis.

[0118] In some embodiments, the method comprises a further step c) of proceeding with the frozen embryo transfer.

[0119] As indicated herein, the dysregulated endometrial immune profile may be an over-activated endometrial immune profile, a mixed endometrial immune profile, or an under-activated endometrial immune profile.

[0120] If the subject is determined to have an under-activated endometrial immune profile, the method comprises providing a personalized ART adapted to the under-activated endometrial immune profile of the subject in order to stimulate mobilization of immune cells and expression of adhesion molecules.

[0121] A personalized ART adapted to an under-activated endometrial immune profile may comprise:realizing an endometrial scratching in the luteal phase (e.g., mid luteal phase) of the cycle preceding the frozen embryo transfer to trigger the expression of adhesion molecules and IL- 15,administering a supplementation of the luteal phase of the FET cycle (i.e., the cycle of the frozen embryo transfer) with chorionic gonadotropins to trigger local angiogenesis and uterine natural killer (uNK) cells mobilization, and / or advising to have sexual intercourse after the frozen embryo transfer to stimulate the local mobilization and expression of immune cells.

[0122] In particular, a personalized ART adapted to an under-activated endometrial immune profile may comprise:realizing an endometrial scratching in the luteal phase (e.g., mid luteal phase) of the cycle preceding the frozen embryo transfer to trigger the expression of adhesion molecules and IL- 15, andadministering a supplementation of the luteal phase of the FET cycle with chorionic gonadotropins as described herein.

[0123] If the subject is determined to have an over-activated endometrial immune profile, the method comprises providing a personalized ART adapted to the overactivated endometrial immune profile of the subject in order to downregulate the local activity of local immune cells, in particular to control the dysregulated Th-l / Th-2 ratio reflected by a high IL-18 / TWEAK ratio.

[0124] A personalized ART adapted to an over-activated endometrial immune profile may comprise:administering an immunomodulating therapy selected from glucocorticoids, intralipids, low-molecular-weight heparin (LMWH), and any combinations thereof, , as described herein, and / oradministering an increased supplementation of progesterone during the luteal phase of the FET cycle, as described herein.

[0125] If the subject is determined to have a mixed endometrial immune profile, the method comprises providing a personalized ART adapted to the mixed endometrial immune profile of the subject in order to downregulate the local activity of local immune cells, in particular to control the dysregulated Th-l / Th-2 ratio reflected by a high IL-18 / TWEAK ratio and to stimulate mobilization of immune cells and expression ofadhesion molecules.

[0126] A personalized ART adapted to a mixed endometrial immune profile may comprise:administering an immunomodulator (i.e., immunomodulating therapy) selected from glucocorticoids, intralipids, low-molecular-weight heparin (LMWH), and any combinations thereof, as described herein,administering an increased supplementation of progesterone during the luteal phase of the FET cycle, as described herein,realizing an endometrial scratching in the luteal phase (e.g., mid luteal phase) of the cycle preceding the frozen embryo transfer to trigger the expression of adhesion molecules and IL- 15, and / oradministering a supplementation of the luteal phase of the FET cycle with chorionic gonadotropins to trigger local angiogenesis and uterine natural killer (uNK) cells mobilization.

[0127] Another object of the present invention is a method for decreasing the risk of miscarriage following frozen embryo transfer (FET) in a subject having over-activated or mixed endometrial immune profile, wherein said method comprises:a) determining the over-activated or mixed endometrial immune profile of the subject prior to frozen embryo transfer by:measuring the expression levels of IL-18, TWEAK, CD56, IL-15 and Fnl4 in an endometrial sample of the subject as described herein,evaluating first IL-18 / TWEAK ratio, then CD56, and finally IL-15 / Fnl4 ratio as described herein; andb) providing a personalized ART adapted to the over-activated or mixed endometrial immune profile of the subject.

[0128] Features, definitions and examples provided elsewhere in the present disclosure with regards to the endometrial sample, with regards to the measure and evaluation of the biomarkers IL-18, TWEAK, CD56, IL-15 and Fnl4, and with regards to the overactivated and mixed endometrial immune profiles apply mutatis mutandis.

[0129] In some embodiments, the method comprises a further step c) of proceeding with (i.e., carrying out) the frozen embryo transfer.

[0130] In some embodiments, the method comprises:a) determining the over-activated or mixed endometrial immune profile of the subject prior to frozen embryo transfer as described herein;b) administering an immunomodulator selected from glucocorticoids, intralipids, low- molecular-weight heparin (LMWH), and any combinations thereof; andc) proceeding with the frozen embryo transfer.In particular, the method may comprise:a) determining the over-activated or mixed endometrial immune profile of the subject prior to frozen embryo transfer as described herein;b) administering a therapeutically effective dose of an immunomodulator selected from glucocorticoids, intralipids, low-molecular-weight heparin (LMWH), and any combinations thereof; andc) proceeding with the frozen embryo transfer.

[0131] By “therapeutically effective dose (or amount)”, it is meant a dose (or amount) of immunomodulator that is aimed at, without causing significant negative or adverse side effects to the subject, normalizing the over-activated or mixed endometrial immune profile of the subject undergoing a frozen embryo transfer.

[0132] In some embodiments, the method comprises:a) determining the over-activated or mixed endometrial immune profile of the subject prior to frozen embryo transfer as described herein,b) administering an immunomodulator selected from glucocorticoids, intralipids, low- molecular-weight heparin (LMWH), and any combinations thereof,verifying the efficacy of the administered immunomodulator prior to frozen embryo transfer by determining a second endometrial immune profile of the subject as described herein; andc) proceeding with the frozen embryo transfer after administration of the same immunomodulator if the efficacy was verified or after administration of a different immunomodulator if the efficacy was not verified.

[0133] By “verifying the efficacy of the administered immunomodulator”, it is meant determining whether administering the immunomodulator resulted in a normalization of the over-activated or mixed endometrial immune profile of the subject. In other words, by “verifying the efficacy of the administered immunomodulator”, it is meant determining whether the endometrial immune profile of the subject is no longer overactivated or mixed after administering the immunomodulator.

[0134] Features, definitions and examples provided elsewhere in the present disclosure with regards to the test under therapy apply mutatis mutandis to the step of verifying the efficacy of the administered immunomodulator.

[0135] By a “different immunomodulator”, it is meant an immunomodulator other than the immunomodulator whose efficacy was not verified or an immunomodulator in addition to the immunomodulator whose efficacy was not verified. For example, intralipids and / or LMWH may be administered instead of glucocorticoids or in addition to glucocorticoids. Conversely, glucocorticoids and / or LMWH may be administered instead of intralipids or in addition to intralipids. Similarly, glucocorticoids and / or intralipids may be administered instead of LMWH or in addition to LMWH.

[0136] In some embodiments, the method further comprises administering an increased supplementation of progesterone during the luteal phase of the FET cycle as described herein.

[0137] In some embodiments, the method comprises:a) determining the over-activated or mixed endometrial immune profile of the subject prior to frozen embryo transfer as described herein;b) administering a therapeutically effective dose of an immunomodulator selected from glucocorticoids, intralipids, low-molecular-weight heparin (LMWH), and any combinations thereof;administering an increased supplementation of progesterone during the luteal phase of the FET cycle as described herein; andc) proceeding with the frozen embryo transfer.

[0138] In some embodiments, the method comprises:a) determining the over-activated or mixed endometrial immune profile of the subject prior to frozen embryo transfer as described herein,b) administering an immunomodulator selected from glucocorticoids, intralipids, low- molecular-weight heparin (LMWH), and any combinations thereof,verifying the efficacy of the administered immunomodulator prior to frozen embryo transfer by determining a second endometrial immune profile of the subject as described herein; andc) proceeding with the frozen embryo transfer after administration of the same immunomodulator if the efficacy was verified or after administration of a different immunomodulator if the efficacy was not verified and with administration of an increased supplementation of progesterone during the luteal phase of the FET cycle as described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0139] Figure 1 is a flow-chart describing the randomized controlled open two-arm trial conducted to evaluate the interest of the pre-conceptional endometrial immune profiling to increase birth rates through personalized care before IVF.

[0140] Figure 2 is a forest plot identifying the particular subgroups of patients who experienced substantial benefits from precision care, including in particular the subgroup of patients who received a frozen embryo transfer (also referred to as frozen-thawed embryo transfer).

[0141] Figure 3 is a graph comparing the live birth rate (LBR) in the case group of patients and in the control group of patients, depending on the type of embryo transfer (fresh embryo transfer vs. frozen embryo transfer (FET)).

[0142] Figure 4 is a graph comparing the miscarriage rate per initiated pregnancy in the case group of patients and in the control group of patients, depending on the type of embryo transfer (fresh embryo transfer vs. frozen embryo transfer (FET)).

[0143] Figure 5 is a graph comparing the live birth rate (LBR) in the case group of patients and in the control group of patients following frozen embryo transfer.

[0144] Figure 6 is a graph comparing the miscarriage rate per initiated pregnancy in the case group of patients and in the control group of patients following frozen embryo transfer.EXAMPLES

[0145] The present invention is further illustrated by the following examples.Example 1:

[0146] A randomized controlled open two-arm trial (clinicaltrials.gov identifier NCT02262117) was completed, to evaluate the interest of the pre-conceptional endometrial immune profiling to increase birth rates through personalized care before IVF. 493 patients were enrolled from October 2015 to February 2023. Endometrial biopsies were collected during the midluteal phase. Endometrial immune profiling was carried out through the analysis of cytokine biomarkers in the endometrium, as detailed below. As shown on the flow-chart of Figure 1, patients with a diagnosed immune endometrial dysregulation were randomized: half of the patients received conventional medical care (disregarding the immune profile) while the other half received precision medical care (according to their immune endometrial profile). The primary analysis was based on the modified intention-to-treat population (excluding patients who did not receive an embryo transfer) and the primary efficacy endpoint was the live birth rate (LBR).Materials and MethodsInclusion criteria

[0147] The inclusion criteria were infertile patients with an indication to perform either an IVF with or without ICSI (intracytoplasmic sperm injection). The indication for IVF were tubal infertility, endometriosis, ovarian dysovulation or idiopathic infertility afterIUI (intrauterine insemination) failure. Patients were younger than 38 years old (age lower than or equal to 38 years at the time of inclusion), with no ovarian insufficiency. The number of previous oocyte pick-up for IVF attempts were strictly lower than 3. If a previous live birth had occurred in the past by IVF, the rank of the new attempt was 1.Endometrial biopsy, collection and measurements

[0148] In order to target the mid- luteal phase and avoid problems associated with cycle fluctuations, 90% of patients were prepared in a substituted cycle (i.e., a cycle during which exogenous hormones are administered) and samples were taken exactly 7 days after the introduction of progesterone. 10% were evaluated on a monitored natural cycle (i.e., a cycle during which no hormone is administered) and samples were taken 9 days after the luteinizing hormone (LH) surge with progesterone dosed 48 hours before sampling. The endometrial fragment was gently aspirated by rotating a Cornier pipelle within the endometrial cavity. The pipelle content was divided into two parts: the first part was placed in 4% formaldehyde (QPath Formol 4% buffered, VWR Chemicals, Fontenay-sous-Bois, France) for endometrial datation, by a histological test to determine the phase of the cycle, and CD-56 immuno-labelling. The second part was placed in RNAlater stabilization solution for immunological analysis (MatriceLab Innove, France).

[0149] After confirmation of the histological dating, RNA was extracted from the biopsy sample conserved in RNAlater (Qiagen). RNA extraction was performed on Biomekl.5 using Kit RNAdvance Tissue (Beckman-Coulter). The RNA was reverse-transcribed into cDNA with the First Strand cDNA Synthesis Kit for RT-PCR (Roche, Meylan, France), according to the manufacturer’s instructions. The cDNAs were stored at -20°C until use.

[0150] Quantitative RT-PCR was performed with a Light Cycler 480 instrument (Roche Diagnostic) and the Light Cycler 480 SYBR Green I Master mix (Roche Diagnostic). Final concentrations for reaction set-up were 0.5 p of sense and anti-sense primers and 1 / 20 of diluted cDNA. Cycling conditions were as follows: denaturation (95°C for 5 min), amplification and quantitation (95°C for 10 sec, 60°C for 10 s and 72°C for 15 sec) repeated 40 times, a melting curve program (65-95°C with a ramp rate of 2.2°C / sec) and a cooling step to 4°C. Each quantitative RT-PCR assay included a solution without cDNAand inter-run calibrator (IRC) samples as negative and positive controls. The IRC for all the primers (IL- 18, IL-15, TWEAK, Fnl4 and CD56) was obtained from pools of RNA endometrial samples. The IRC cDNA, after dilution by a factor of 20, underwent the same quantitative RT-PCR protocol as the unknown samples. PCR efficiency for each quantified target and reference was calculated with known serial dilutions of each specific cDNA. LightCycler®480 Software release 1.5.0 was used to analyze data, and each specific target transcription level was normalized to the geometric mean of the transcription level of the reference gene, with the software’s advanced relative quantification workflow. Gene amplification efficiency was specifically determined. For each sample, the results were expressed as the ratio of target / reference cDNA.

[0151] Immunohistochemistry (IHC) was performed on the biopsy sample tissue conserved in 4% formol on 5 -pm thick slides, with an automated streptavidin-biotin method (Benchmark GX, Ventana Medical Systems). The prediluted anti-CD56 (clone 123C3) murine monoclonal primary antibody (Ventana Medical Systems®, Roche Diagnostics) was applied according to the manufacturer’s instructions. Briefly, after deparaffinization of the slides, antigen retrieval was performed for 60 minutes in a pH 8.4 Cell Conditioning 1 solution. The CD56 primary antibody was then applied for 32 min. Slides for negative controls were prepared by replacing the primary antiserum with nonimmune IgG. Slides were then incubated for 8 min with a biotinylated anti-mouse secondary antibody. Diaminobenzidine or 3-amino-9-ethylcarbazole was used as the chromogen (iVIEW DAB detection kit, Ventana Medical Systems) and slides were counterstained with hematoxylin for 2 min, incubated in bluing reagent (for 2 min), and mounted. Between each step, slides were rinsed with reaction buffer. The uNK cell count was measured as the mean of CD56+ cells in 4 representative fields at x400 magnification.Determination of the endometrial immune profile

[0152] To establish the endometrial immune profile, a step-by-step procedure first considered the IE-18 / TWEAK mRNA ratio (reflecting local angiogenesis and possibly a Thl deviation), then the CD56 expression level (reflecting uNK cell mobilization), and finally the IE-15 / Fn-14 mRNA ratio (indicative of uNK cell maturation and uNKcytotoxic activation).

[0153] Using standardized RT-qPCR method, the expression norms of the biomarkers were previously established in a fertile cohort. In particular, it was documented that an immune profile was reproducible from one cycle to the next over a six-month period if no surgery or pregnancy had occurred in the interim.

[0154] Endometrial immune profiles was classified into four types.

[0155] A balanced endometrial immune activation profile, which is characterized by IL-18 / TWEAK and IL-15 / Fnl4 mRNA ratios and a CD56 expression level within the same range as previously defined in a fertile cohort. This profile suggests that the endometrium is ready to go through the following steps of implantation, including apposition, adhesion, and invasion. Patients presenting with this endotype were not randomized and were excluded from the study.

[0156] The three other subgroups represented patients with immune dysregulation who were randomized via the electronic server (Cleanweb-APHP).An under-activated endometrial immune profile was characterized by at least one of the three biomarkers (IL-18 / TWEAK ratio, CD56 expression level, and / or IL-15 / Fnl4 ratio) being low and the other two biomarkers either normal or low. This profile suggests that the endometrium is not fully effective for adhesion and promoting adequate immunotrophism during initial placentation.An over-activated endometrial immune profile was characterized by at least one of the three biomarkers (IE-18 / TWEAK ratio, CD56 expression level, and / or IE-15 / Fnl4 ratio) being high, with IE-18 / TWEAK ratio and IE-15 / Fnl4 ratio being either normal or high. A mixed endometrial immune profile was distinguished by a high IE-18 / TWEAK ratio (excess Th-1 cytokines) and a low IE-15 / Fnl4 mRNA ratio (reflecting immature NK cells).The over-activated and mixed profiles suggest that the endometrium is not prepared for the crucial step of trophoblast invasion and may be in a state that can reject the embryo because of a cytotoxic activation of uNK cells in EAKs (lymphocyte-activated killer cells). A test under therapy (glucocorticoids or intralipids) was proposed if the patientwas randomized in the personalized arm.Randomization

[0157] Randomization by blocks was made using the electronic server (Cleanweb-APHP) which allocated patients in a 1:1 ratio to the groups “dysregulated - conventional care” or “dysregulated - precision care” once histological and immune results confirmed the mid-luteal phase and the validity of the endometrial immune profile. Only patients with diagnosed endometrial immune dysregulation were randomized.Conventional care

[0158] The patient had a standard fresh or frozen embryo transfer without scratching, or adjunction of glucocorticoids, intralipids, chorionic gonadotropins or double sequential embryo transfer. If the attempt failed, the clinician could decide to personalize the patient’s attempt at the second embryo transfer when the patient ended her participation in the present study.Precision care

[0159] Once randomized to the precision care group, the patients received a treatment depending on their individual immune profile.

[0160] For patients diagnosed with under-active immune profile, the treatment strategy was directed to stimulate mobilization of immune cells and expression of adhesion molecules. The precision care was characterized:by endometrial scratching in the mild luteal phase of the cycle preceding the embryo transfer with the objective to trigger the expression of adhesion molecules and interleukin-15,by supplementing the luteal phase with chorionic gonadotropins, to trigger local angiogenesis and uNK cells mobilization, andby advising to have sexual intercourse after the embryo transfer to stimulate the local mobilization and expression of immune cells.The chorionic gonadotropins mentioned above were usually administered as 250pg / 0.5 ml of choriogonadotropin alfa (Ovitrelle) by subcutaneous route in the lutealphase, 4, 6 and 8 days after the egg collection or the introduction of progesterone.For patients over 35 years old with at least one previous ET failure, a double sequential transfer of one embryo on day 3 and one embryo on day 5 was proposed to stimulate the local embryo-endometrium dialogue before implantation.Micronized progesterone for luteal support, which is part of the conventional care, was usually prescribed at the standard dose of 200 mg three times a day.

[0161] For patients diagnosed with an over-active immune profile or a mixed profile, the strategy aimed to down-regulate the local activity of local immune cells. In this subgroup, immunomodulating therapy was introduced, aiming at controlling the dysregulated Th-l / Th-2 ratio reflected by a high IL-18 / TWEAK ratio. Glucocorticoids were prescribed as a first line of treatment, and slow intravenous infusion of intralipids as a second line of treatment in case of resistance to glucocorticoids. Glucocorticoid tablets were taken by the patient from the third day of the cycle until the pregnancy test and continued for 2 months if pregnant (from 21 days to 3 months) after the endometrial immune analysis. Glucocorticoids were gradually weaned off and stopped in case of negative pregnancy test or after 10 weeks of pregnancy. Intralipids (intralipid 20g / 100mL diluted in 400mL of NaCl 0.9%) were administered by slow intravenous infusion during ovarian stimulation (around day 8 of the cycle) and repeated if pregnant, at 5 weeks and 9 weeks. Glucocorticoids and / or intralipids were administered with a variable dosage, the most common dose of glucocorticoids being 20 mg daily.The dose of micronized progesterone for luteal support was increased from the conventional dose to 400 mg three times a day for its documented immunosuppressive properties.

[0162] Prior to any embryo transfer, a cycle test under therapy was proposed to evaluate whether or not glucocorticoids or intralipids were able to normalize the endometrial profile. If the endometrial profile was normalized, then the therapy tested was considered as efficient and added for the next embryo transfer. If the endometrial profile was not normalized under glucocorticoids, intralipids were used. If the endometrial profile was not normalized under intralipids, glucocorticoids were used.

[0163] If the over-activated profile was associated with a low uNK cell mobilization(uNK cells < 10 / field) or immature uNK cells (mixed profile), endometrial scratching was added to the cycle preceding embryo transfer and chorionic gonadotropins were used in the luteal phase after the embryo transfer.Embryo transfer

[0164] The delay between the last endometrial immune analysis and the embryo transfer did not exceed 9 months for the mITT analysis. If a spontaneous pregnancy or a gynecological surgery occurred between the biopsy and embryo transfer, patients were excluded from the mITT analysis.

[0165] IVF after a monitored ovarian hyper- stimulation for a fresh embryo transfer as well as endometrial preparation for frozen embryos were conducted as per common protocols. Endometrial immune profiling did not impact the standard embryo transfer policy except for patients with under-active immune profiles. For these patients, a specific policy of transfer was in place, if they were over 35 years old or previously failed with standard embryo transfer. In such a profile, a sequential double transfer was proposed, with a first embryo transferred on day 2-3 and a second embryo on day 5-6.

[0166] To evaluate the impact of embryo quality on subsequent pregnancy rate, embryo transfers were classified into 2 classes (“top” transfer or “no top” transfer) according to the embryologist’s observations on the day of transfer. Each embryo transfer included in the study was classified anonymously by two embryologists. On days 2-3, the standard BLEFCO (Biologistes des Laboratoires d Etude de la Fecondation et de la Conservation de I’Oeuf) classification was used to evaluate the embryos (Boyer P, Boyer M. Non-invasive evaluation of the embryo: morphology of preimplantation embryos. Gynecol Obstet Fertil. (2009) 37:908-16) and on day 5 the Gardner classification was used for the evaluation of blastocyst quality (Gardner DK et al. Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer. Fertil Steril. (2000) 73:1155-8). On day 2-3, “top” grade A high quality embryo was defined as an embryo with typically equal-sized blastomeres or unequal-sized blastomeres according to the number of cells with less than 10% fragmentation. On day 2 the embryo should have 2 to 4 cells, and 6 to 10 on day 3. On day 5, “top” grade A excellent quality blastocyst wasdefined as a blastocyst with large, fully expanded blastocoel, inner cell mass, and trophectoderm tightly packed and clearly defined [B5AA-B5AB-B5BA-B4AA-B4AB-B4BA], Top transfers were defined as the transfer of top quality embryos. If two embryos were transferred, the two embryos were evaluated as “top”. All the other combinations were classified as “no top” transfer.Outcome analysis

[0167] The live birth rate was defined as the birth of a living baby and was the primary outcome of the mITT analysis. Secondary outcomes were the ongoing pregnancy rate, the clinical pregnancy rate and the miscarriage rate. The ongoing pregnancy rate was defined by a scan attesting the presence of a gestational sac with an embryonic cardiac activity, which had progressed beyond the first trimester (12 weeks) and was continuing. The clinical pregnancy rate was defined by a BhCG over 100 IU / L in the serum 12 to 10 days after the embryo transfer. Miscarriage referred to the loss of a pregnancy that had occurred after embryo transfer, at any stage of pregnancy, from implantation to the end of the first trimester (12 weeks gestation). Miscarriage did not include biochemical pregnancies (corresponding to an unconfirmed single pregnancy test) that were considered as no pregnancy in the outcome analysis but included early pregnancy losses (gestational sac seen on ultrasound but no heartbeat).Statistical methodology

[0168] Categorical data are presented as numbers (percentages). Continuous variables are presented as means with standard deviations (SDs) and medians with interquartile ranges (interquartile range (IQR) described as 25th and 75th percentile) for normal and skewed distributions, respectively.The primary analysis was based on the modified intention-to-treat (mITT) population (excluding patients without ET) and the primary efficacy endpoint was the live birth rate. The primary efficacy endpoint was compared between the personalized and conventional care using a binary logistic regression.

[0169] A logistic regression model was performed including prior known risk factors as covariates (age class, embryo quality, embryo transfer and endometrial immune profile).The 95% two-sided confidence interval for odds ratio (OR) was computed using the bias-corrected and accelerated (BCa) bootstrap interval. OR was presented together with a two-sided 95% BCa confidence interval and associated p-values.

[0170] All secondary analyses were based on the mITT population. The secondary binary endpoints were analyzed using the same methods as the primary endpoint. For secondary continuous endpoints that were normally distributed with a homogeneity of variance across groups, a t-test was used. For secondary continuous endpoints that were normally or asymptotically normally distributed and heteroscedastic, the Welch t-test was used. For secondary continuous endpoints that are heavy-tailed and skewed, the Mann-Whitney U test was used.

[0171] Pre-specified subgroup analyses to evaluate variations in treatment effect were done using logistic regression models, with terms for treatment, subgroup, and interaction of treatment with subgroup. All reported subgroup analyses were pre-specified.

[0172] Assuming a 25% birth rate per embryo transfer with conventional care and a 40% relative increase in birth rate with precision care, a sample size of 152 patients per group was needed to achieve 80% power to detect this difference using a chi-square test at a two-sided 5% significance level. Given an anticipated 25% exclusion rate postrandomization, a total of 380 dysregulated patients (190 per group) had to be randomized. To reach this target, approximately 500 patients were screened since 20% were expected not to be dysregulated. All statistical tests were two-sided and were performed at the 0.05 level. All tests were performed using SAS version 9.4 or later.Results

[0173] In total, 493 patients were included in the study. The immune profiling analysis was successfully performed for 484 patients. Out of the 484 patients, 78% (378) had a dysregulated endometrial immune profile. Among deregulated patients, 190 were randomized to receive conventional care, and 188 were randomized to receive precision care according to their type of immune dysregulation. An outcome was available for 295 dysregulated patients: 155 dysregulated with conventional care and 140 dysregulated with precision care.

[0174] 240 patients were scheduled for a fresh embryo transfer, and among those, 3 were converted to intra-uterine insemination. 55 patients were scheduled for a frozen embryo transfer. For fresh embryo transfer, 80% of the patients were stimulated using an antagonist protocol and 20% using a long agonist protocol. For frozen transfers, 42% of the patients were prepared through natural cycles, 10% with FSH mild stimulation and 48% were prepared with a substituted cycle.

[0175] Table 1 below summarizes the clinical and demographic data of patients randomized in conventional versus precision care.Table 1: Descriptive clinical data of patients included in the study><>>*A H stands for anti-Mullerian hormone

[0176] 106 patients had a balanced endometrial immune profile, comprising 22% of the cohort (484 patients for whom the immune profiling analysis was successfully performed), while 378 patients had dysregulated profile, making up 78% of the cohort. No significant differences were observed between the conventional and precision groups with regard to age, previous embryo transfers, fresh or frozen transfers, protocols used,transfer quality, or the distribution of different immune profiles (Tables 1 & 2). Nor did they differ between dysregulated and non-dysregulated women.

[0177] Among dysregulated patients, 30% had under-active profile, 47% had over-active profile, and 13.8% had mixed profile, as detailed in Table 2 below.Table 2: Summary of type of immune imbalance and type of embryo transfer*DSET stands for double sequential embryo transfer

[0178] Comparing the live birth rate (LBR) between dysregulated patients randomized to conventional versus personalized care constituted the primary endpoint of this study. The modified intention-to-treat (mITT) analysis revealed a significant increase in LBR with precision medical care, rising from 29.7% to 41.4%. The unadjusted odds ratio (OR) was 1.68 [1.04-2.73], p=0.036. Notably, the OR adjusted for age class, embryo quality attransfer, fresh or frozen transfer, and endometrial immune profile type was 1.75 [1.04-2.92], p=0.03).

[0179] In terms of the secondary endpoints assessed in the mITT analysis, both clinical pregnancy and ongoing pregnancy rates were consistently elevated with precision care (50.7% and 41.4%, p=0.04) when compared to conventional care (39.4% and 30.4%), as demonstrated through both unadjusted and adjusted analyses. No difference was observed regarding the miscarriage rate between conventional and precision groups. mITT analysis with primary and secondary endpoints are summarized in Table 3 below.

[0180] Subgroup analyses unveiled particular subgroups which experienced substantial benefits from precision care (Figure 2), such as patients with morphologically sub-optimal embryos for transfer, patients who had previously undergone two or more embryo transfers and experienced failures.

[0181] A particular subgroup of patients who experienced substantial benefits from precision care is the subgroup of patients who were transferred frozen embryos. The modified intention-to-treat (mITT) analysis thus revealed a significant increase in live birth rate when a frozen embryo was transferred, rising from 14.7% (conventional care) to 48.6% (precision care), OR: 5.48 (1.72-17.43]). By contrast, in the subgroup of patients who were transferred fresh embryos, precision care had little impact on the live birth rate (37.9% with precision care vs. 34.5% with conventional care, OR: 1.16 (0.67-2.01)).Table 3: OutcomeValues are number (%) or number / total number (%). CI stands for confidence interval.*Unadjusted model** Adjusted model for age class, embryos quality, embryo transfer and endometrial immune profile£Unadjusted p-value from Type 3 analysis££Adjusted p-value from Type 3 analysisbBias-Corrected and Accelerated bootstrap (BCa) confidence interval from 10,000 replications

[0182] As compared to fresh embryo transfers, frozen embryo transfers are associated with advantages such as:a decrease in the risk of ovarian hyperstimulation syndrome as the ovarian stimulation is uncoupled from embryo transfer,an increased receptivity of the endometrium as the hormone levels during the transfer cycle are closer to physiological levels,the possibility to conduct preimplantation genetic testing, if relevant and where authorized.

[0183] Further post-hoc statistical analyzes were thus carried out to better understand the substantial benefit seen in the subgroup of patients who received precision care and a frozen embryo transfer.Example 2:

[0184] The study presented in Example 1 showed that the endometrial immune environment is a crucial element in improving the success of assisted reproductive technology (ART). However, further investigation is needed to better characterize the influence of the endometrial environment depending on the type of embryo transfer (fresh embryo transfer vs. frozen embryo transfer).

[0185] Additional post-hoc pair-matched analyzes were thus carried out, using pair-matching to limit the possibility that variables other than the type of embryo transfer may skew the analysis.Materials and Methods

[0186] A retrospective matched-pair analysis was conducted to study the impact of regulating the immune environment on the performance of a subsequent embryo transfer as a function of the type of transfer (fresh embryo transfer vs. frozen embryo transfer, also referred to as frozen-thawed embryo transfer).

[0187] The PRECONCEPTIO software was used to extract all the endometrial immune profiles established between January 1st, 2020 and June 30th, 2023 for patients beingfollowed for an ART procedure either at Hopital Pierre Rouques "Les Bluets" (Paris, France) or at Groupe Hospitalier Diaconesses Croix Saint-Simon (Paris, France) - both these hospitals work with the same laboratory of embryology. In parallel, data was extracted from the MEDIFIRST software to select all the patients who were transferred a single embryo at the blastocyte stage (day 5 / 6) between January 2020 and March 2024 for the case group and between January 2018 and March 2024 for the matched control group.Inclusion criteria

[0188] A case group (“treatment group”) was thus compiled, corresponding to the patients (1) who had an endometrial immune profile identified as dysregulated and subsequently benefited from precision care adapted to their endometrial immune profile and (2) who were transferred a single day-5 / 6 embryo within 9 months of the endometrial immune profiling.

[0189] Each patient from the case group was matched to a “control” patient who were transferred a single day-5 / 6 embryo without having an endometrial immune profile established within the 9 months preceding the embryo transfer (and thus without benefiting from precision care adapted to their endometrial immune profile).

[0190] The criteria matched between the case group and the control group were the following:maternal age (+ / -1 year),- number of previous egg retrievals (including the egg retrievals not followed by a transfer),number of previous embryo transfers,- type of ART (FIV, ICSI),type of embryo transfer (fresh embryo transfer, frozen embryo transfer), degree of expansion of the blastocoel (B1-B2 / B3-B4 / B5-B6) according to the Gardner classification.Endometrial immune profiling

[0191] Endometrial immune profiling was carried out through the analysis of cytokine biomarkers in the endometrium biopsy samples, as detailed in Example 1 (section “Endometrial biopsy, collection and measurements” and section “Determination of the endometrial immune profile”).Conventional care

[0192] The patients of the control group received conventional care, adapted to the type of embryo transfer (fresh embryo transfer vs. frozen embryo transfer).Precision care

[0193] The patients of the case group, having an identified dysregulated endometrial immune profile, received a personalized care adapted to their individual endometrial immune profile (i.e., under-activated endometrial immune profile, over-activated endometrial immune profile or mixed endometrial immune profile), as detailed in Example 1 (section “Precision care”).

[0194] Of note, for patients with an over-activated or mixed endometrial immune profile, the immunomodulating therapy included glucocorticoids, intralipids and / or low-molecular-weight heparin. The patients with an over-activated or mixed endometrial immune profile thus received glucocorticoids, intralipids, or low-molecular-weight heparin, either alone or in combination.Embryo transfer

[0195] IVF after a monitored ovarian hyper-stimulation for a fresh embryo transfer as well as endometrial preparation for frozen embryos were conducted as per common protocols. Endometrial immune profiling did not impact the standard embryo transfer policy.Outcome analysis

[0196] The live birth rate was defined as the birth of a living baby and was the primaryoutcome of the retrospective pair-matched analysis. The secondary outcome was the miscarriage rate per initiated pregnancy. Miscarriage is defined as a loss of a pregnancy that had occurred after embryo transfer, at any stage of pregnancy, from implantation to the end of the first trimester (12 weeks gestation). Initiated pregnancy is defined as two positive pregnancy tests at 48h hours of interval. Miscarriage did not include biochemical pregnancies (corresponding to an unconfirmed single pregnancy test) that were considered as no pregnancy in the outcome analysis but included early pregnancy losses (gestational sac seen on ultrasound but no heartbeat).Statistical methodology

[0197] Categorical data are presented as numbers (percentages). Continuous variables are presented as means with standard deviations (SDs) and medians with interquartile ranges (interquartile range (IQR) described as 25th and 75th percentile) for normal and skewed distributions, respectively.The primary efficacy (live birth rate) endpoint was compared between the case group and the control group using a one-way analysis of variance (Kruskal-Wallis test) or a two-way analysis of variance (ANOVA). The secondary endpoint was analyzed using the same methods as the primary endpoint.One-way analysis of variance is used to test the difference between the means of several subgroups (e.g., case group and matched control group) of a variable (e.g., primary outcome and secondary outcome). The two-way analysis of variance is an extension to the one-way analysis of variance. There are two qualitative factors (factor A: case group / control group and factor B fresh embryo / frozen embryo) on one dependent continuous variable (primary and secondary outcome).Three null hypotheses are tested in this procedure:factor A does not influence variable Yfactor B does not influence variable Ythe effect of factor A on variable Y does not depend on factor B (i.e., there is no interaction of factors A and B).Two-way analysis of variance requires that data for each combination of the two qualitative factors A and B. If the Levene test is positive (p<0.05) then the variances inthe different groups are different (the groups are not homogeneous) and a non-parametric test is used (Kruskal-Wallis test). If the p-value is less than 0.05 (or another preselected significance level), then it can be concluded that the means of at least two of the subgroups differ significantly.Results

[0198] In total, 268 patients were included in the retrospective pair-matched study: 134 case patients (patients who benefited from precision care adapted to their dysregulated endometrial immune profile and who were transferred a single day-5 / 6 embryo within 9 months of the endometrial immune profiling) paired with 134 matched control patients.

[0199] As shown in Table 4 below and in Figure 3, a comparison between the matched control patients and the case patients confirmed a significant increase in live birth rate (LBR) in the patients who benefited from precision care (case group) and who received a frozen embryo transfer (FET), from 16.67% (matched control group) to 46.43% (case group). By contrast, no significant increase in LBR was observed in the patients who benefited from precision care (case group) and who received a fresh embryo transfer, as compared to their matched control patients who received a fresh embryo transfer (36% vs. 36%).Table 4: LBR in case group vs. control group as a function of ET type

[0200] Using the two-way analysis of variance, the LBR was significantly different between the case group and the control group, as the difference was related to the increase in LBR of frozen embryos in the case group (p=0.01).

[0201] Furthermore, as shown in Table 5 below and in Figure 4, a comparison between the case patients and the control patients indicated that the increased LBR observed in thepatients who benefited from precision care (case group) and who received a frozen embryo transfer (FET) was associated with a decrease in the miscarriage rate per initiated pregnancy, from 48% (control group) to 18.75% (case group). By contrast, no decrease in the miscarriage rate was observed in the patients who benefited from precision care and who received a fresh embryo transfer, as compared to the control patients who received a fresh embryo transfer (17.39% vs. 22.73%, respectively). Of note, the analysis pertaining to the miscarriage rate was restricted to patients who initiated a pregnancy (71 patients in the case group and 47 patients in the control group).Table 5: Miscarriage rate in case group vs. control group as a function of ET type

[0202] Using the two-way analysis of variance, the miscarriage per initiated pregnancy was significantly different between the case group and the control group, as the difference was related to the decrease of miscarriage of frozen embryos in the case group (p=0.038).

[0203] The above observations were confirmed when taking into consideration only the patients who received a frozen embryo transfer. As shown in Table 6 below and in Figure 5, a comparison between the control patients and the case patients confirmed a significant increase in live birth rate (LBR) from 16.67% (control group) to 46.43% (case group) with a p-value of 0.000035 using the Kruskal-Wallis test.Table 6: LBR of frozen embryos in case group vs. control group

[0204] As shown in Table 7 below and in Figure 6, a comparison between the control patients and the case patients confirmed that the increased LBR was associated with a significant decrease in the miscarriage rate, from 48% (control group) to 18.75% (casegroup) with a p-value = 0.0097 using the Kruskal- Wallis test.Table 7: Miscarriage rate of frozen embryos in case group vs. control group

[0205] These results confirm that endometrial immune profiling and the subsequent precision care adapted to the individual endometrial immune profile offer substantial benefits as they increase the chances of a live birth following a frozen embryo transfer. Strikingly, the results indicate that the substantial benefits translate in particular as a decrease in the miscarriage rate in patients who initiated a pregnancy following a frozen embryo transfer. Considering the reported advantages of frozen embryo transfer over fresh embryo transfer, the benefits are of particular relevance. Of note, such benefits were not observed in patients who received a fresh embryo transfer.Example 3

[0206] Similar matched-pair analyses were conducted after selecting a case group of patients having an over-activated or mixed endometrial immune profile.Materials and Methods

[0207] A case group (“treatment group”) was thus compiled, corresponding to the patients (1) who had an over-activated or mixed endometrial immune profile and subsequently benefited from precision care adapted to their endometrial immune profile and (2) who were transferred a single day-5 / 6 embryo within 9 months of the endometrial immune profiling.

[0208] Each patient from the case group was matched to a “control” patient who were transferred a single day-5 / 6 embryo without having an endometrial immune profile established within the 9 months preceding the embryo transfer (and thus without benefiting from precision care adapted to their endometrial immune profile).

[0209] The criteria matched between the case group and the control group were thefollowing:maternal age (+ / -1 year),- number of previous egg retrievals (including the egg retrievals not followed by a transfer),number of previous embryo transfers,- type of ART (FIV, ICSI),type of embryo transfer (fresh embryo transfer, frozen embryo transfer), degree of expansion of the blastocoel (B1-B2 / B3-B4 / B5-B6) according to the Gardner classification.

[0210] Endometrial immune profiling was carried out through the analysis of cytokine biomarkers in the endometrium biopsy samples, as detailed in Example 1 (section “Endometrial biopsy, collection and measurements” and section “Determination of the endometrial immune profile”).

[0211] The patients of the control group received conventional care, adapted to the type of embryo transfer (fresh embryo transfer vs. frozen embryo transfer). The patients of the case group, having an over-activated or mixed endometrial immune profile, received a personalized care adapted to their individual endometrial immune as indicated in Example 2.

[0212] Embryo transfers were conducted as indicated in Example 2.

[0213] The outcome analysis and statistical methodology were the same as in Example 2.Results

[0214] In total, 216 patients were included in the retrospective pair-matched study: 108 case patients (patients who benefited from precision care adapted to their overactivated or mixed endometrial immune profile and who were transferred a single day-5 / 6 embryo within 9 months of the endometrial immune profiling) paired with 108 matched control patients.

[0215] As shown in Table 8 below, a comparison between the control patients and thecase patients showed a significant increase in live birth rate (LBR) in the patients who benefited from precision care (case group) and who received a frozen embryo transfer (FET), from 12.12% (control group) to 45.45% (case group). By contrast, no significant increase in LBR was observed in the patients who benefited from precision care (case group) and who received a fresh embryo transfer, as compared to the control patients who received a fresh embryo transfer (33% vs. 33%).Table 8: LBR in case group vs. control group as a function of ET type

[0216] Using the two-way analysis of variance, LBR was significantly different between the case group and the control group, as the difference was related to the increase in LBR of frozen embryos in the case group (p=0.008).

[0217] Furthermore, as shown in Table 9 below, a comparison between the case patients and the control patients indicated that the increased LBR observed in the patients who benefited from precision care (case group) and who received a frozen embryo transfer (FET) was associated with a decrease in the miscarriage rate per initiated pregnancy, from 50% (control group) to 16.67% (case group). By contrast, no decrease in the miscarriage rate was observed in the patients who benefited from precision care and who received a fresh embryo transfer, as compared to the control patients who received a fresh embryo transfer (16.67% vs. 22.22%, respectively). Of note, the analysis pertaining to the miscarriage rate was restricted to patients who initiated a pregnancy (54 patients in the case group and 32 patients in the control group).Table 9: Miscarriage rate in case group vs. control group as a function of ET type

[0218] Using the two-way analysis of variance, the miscarriage per initiated pregnancy was significantly different between the case group and the control group, as the difference was related to the decrease of miscarriage of frozen embryos in the case group (p=0.044).

[0219] The above observation was confirmed when taking into consideration only the patients who received a frozen embryo transfer. As shown in Table 10 below, a comparison between the control patients and the case patients confirmed that the increased LBR was associated with a significant decrease in the miscarriage rate, from 50% (control group) to 16.67% (case group) as indicated above, with a p-value = 0.0169 using the Kruskal- Wallis test. No significant decrease in the miscarriage rate was observed following a fresh embryo transfer.Table 10: Miscarriage rate of frozen embryos in case group vs. control group

[0220] These results indicate that endometrial immune profiling and the subsequent precision care adapted to the individual endometrial immune profile offer substantial benefits to patients identified and treated for an over-activated or mixed endometrial immune profile who received a frozen embryo transfer. Strikingly, the results indicate that the substantial benefits translate in particular as a decrease in the miscarriage rate in patients who initiated a pregnancy following a frozen embryo transfer.

Claims

CLAIMS1. An immunomodulator for use in a method for decreasing the risk of miscarriage following frozen embryo transfer (FET) in a subject having an over-activated or mixed endometrial immune profile, wherein said immunomodulator is selected from glucocorticoids, intralipids, low-molecular-weight heparin, and any combinations thereof, and wherein said over-activated or mixed endometrial immune profile is determined by:- measuring the expression levels of IL- 18 (interleukin- 18), TWEAK (TNF weakly inducer of apoptosis), CD56 (cluster of differentiation 56), IL-15 (interleukin- 15) and Fnl4 (fibroblast growth factor-inducible 14) in an endometrial sample of the subject, and- evaluating first IL-18 / TWEAK ratio, then CD56, and finally IL-15 / Fnl4 ratio.

2. The immunomodulator for use according to claim 1, wherein evaluating the IL-18 / TWEAK ratio comprises calculating the IL-18 / TWEAK ratio and comparing said ratio to a reference IL-18 / TWEAK ratio obtained from a fertile female subject.

3. The immunomodulator for use according to claim 1 or 2, wherein evaluating the CD56 expression level comprises comparing said expression level to a reference CD56 expression level obtained from a fertile female subject.

4. The immunomodulator for use according to any one of claims 1 to 3, wherein evaluating the IL-15 / Fnl4 ratio comprises calculating the IL-15 / Fnl4 ratio and comparing said ratio to a reference IL-15 / Fnl4 ratio obtained from a fertile female subject.

5. The immunomodulator for use according to any one of claims 1 to 4, wherein the over-activated endometrial immune profile or the mixed endometrial immune profile is characterized by a high IL-18 / TWEAK ratio.

6. The immunomodulator for use according to any one of claims 1 to 5, wherein said immunomodulator is for administration in combination with an increased supplementation of progesterone during the luteal phase of the FET cycle.

7. A method for determining a personalized assisted reproductive technology (ART) for decreasing the risk of miscarriage following frozen embryo transfer (FET) in a subject having a dysregulated endometrial immune profile, wherein said method comprises:a) determining the dysregulated endometrial immune profile of the subject prior to frozen embryo transfer by:measuring the expression levels of IL 18 (interleukin- 18), TWEAK (TNF weakly inducer of apoptosis), CD56 (cluster of differentiation 56), IL-15 (interleukin- 15) and Fnl4 (fibroblast growth factor-inducible 14) in an endometrial sample of the subject,evaluating first IL- 18 / TWEAK ratio, then CD56, and finally IL- 15 / Fn 14 ratio; andb) determining a personalized ART adapted to the dysregulated endometrial immune profile of the subject.

8. The method according to claim 7, wherein evaluating the IL- 18 / TWEAK ratio comprises calculating the IL- 18 / TWEAK ratio and comparing said ratio to a reference IL- 18 / TWEAK ratio obtained from a fertile female subject.

9. The method according to claim 7 or 8, wherein evaluating the CD56 expression level comprises comparing said expression level to a reference CD56 expression level obtained from a fertile female subject.

10. The method according to any one of claims 7 to 9, wherein evaluating the IL- 15 / Fn 14 ratio comprises calculating the IL- 15 / Fn 14 ratio and comparing said ratio to a reference IL- 15 / Fn 14 ratio obtained from a fertile female subject.

11. The method according to any one of claims 7 to 10, wherein the dysregulated endometrial immune profile is determined at most 12 months prior to frozen embryo transfer.

12. The method according to any one of claims 7 to 11, wherein the dysregulated endometrial immune profile determined at step a) is an over-activated endometrial immune profile characterized by a high or normal IL-18 / TWEAK ratio and a high or normal IL-15 / Fnl4 ratio, with at least one of IL-18 / TWEAK ratio, CD56 expression level and IL-15 / Fnl4 ratio being high.

13. The method according to any one of claims 7 to 12, wherein the dysregulated endometrial immune profile determined at step a) is a mixed endometrial immune profile characterized by a high IL-18 / TWEAK ratio and a low IL-15 / Fnl4 ratio.

14. The method according to claim 12 or 13, wherein the personalized ART determined at step b) is adapted to the over-activated and mixed endometrial immune profile and comprises recommendations for downregulating the local activity of local immune cells.

15. The method according to claim 14, wherein recommendations for downregulating the local activity of local immune cells comprise recommending an immunomodulating therapy, preferably glucocorticoids, intralipids and / or low- molecular-weight heparin.

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