The present invention relates to methods for predicting whether a pre-implanted embryo will be embedded into the endometrium of a female. The present invention further relates to a kit for predicting whether a pre-implanted embryo will be embedded into the endometrium of a female.

BACKGROUND OF THE INVENTION Worldwide, about 72.4 million couples suffer from sterility (1). Of these, about 40 million turn to Assisted Reproduction Techniques (ART) like in-vitro-fertilization (IVF) to overcome their undesired childlessness (2). Unfortunately, only about 20-30% of all IVF-cycles (controlled ovarian hyperstimulation followed by egg retrieval, fertilization and transfer of developed embryos) result in pregnancies (3). Unsuccessful treatment approaches do, however, cause considerable physical and psychological stress to the corresponding patients (4, 5). The chances that one specific IVF-cycle (after fresh stimulation/egg retrieval or after cryopreservation of two-pronuclear zygotes or embryos of earlier treatments) will induce a pregnancy was shown to depend on the fitness/viability and genetic health of the transferred embryo (6). Still, no more than 2-3 out of the about 9-15 embryos obtained on average in one treatment cycle (7) may be transferred (8). Otherwise, an excess of multiparous pregnancies would have to be expected (8). Considering the medical risks like preterm delivery, increased rate of surgically induced deliveries or twin-to-twin transfusion syndrome which are all associated with multiparous pregnancies (2, 9). This restriction appears to be absolutely reasonable - and it is also legally enforced in countries like Germany. The Goldstandard is the transfer of 1 embryo max.

Hence, to reduce the average number of potentially harmful treatment cycles, new diagnostic tools are desperately needed to identify the most capable embryo derived from the retrieved oocytes (7).

Beyond image-based approaches, molecular markers such as nucleic acids play an increasing role for reliable diagnosis of pathologies. Among nucleic acids, small non-coding RNAs, so-called microRNAs (miRNAs), have demonstrated a substantial diagnostic potential. The molecules regulate gene expression on a systematic scale (10). Many diseases as well as physiological conditions were already found to leave characteristic miRNA expression patterns in diverse biomaterials (11). After one decade of biomarker discovery, miRNA patterns are now more and more validated and have become a focus for improving the diagnostic accuracy for detecting pathologies. Most important is that the numerous studies reported miRNAs not only in cells but also in body fluids including semen or seminal plasma (12, 13). Aberrant expression of miRNAs has been observed in both gametes and early embryonic development of mammals (14, 15) and in fertility associated disorders of the human female partners (16), as well as male partners (17-23). In male partners with reduced fertility, the expression levels of several miRNAs were significantly altered between males with normal (normozoospermia) and impaired spermatogenesis (azoospermia, oligozoospermia, asthenozoospermia and oligoasthenozoospermia) (17-22). Similarly, the expression levels of several miRNAs were also altered in male partners showing different forms of non-obstructive azoospermia (NO A) compared to control males (24).

Only limited parameters/factors (Morphokentic and PGD) exist to determine the developmental potential of an embryo between day 0 and day 5/6 (implantation day) in order to select the best embryo for embryo transfer to establish pregnancy. For example, embryos which reach the day 3 cell stage can be tested for chromosomal or specific genetic defects prior to possible transfer by preimplantation genetic diagnosis (PGD). Alternatively, image-based approaches may be used.

The present inventors surprisingly found that the analysis of miRNAs, which secrete from an pre-implanted embryo into the extracellular environment, e.g. into the embryonic culture medium, allows to predict the developmental potential of the pre-implanted embryo and, thus, the selection of the embryo with the highest chance to result in a successful pregnancy. This, in turn, has the advantage that the average number of potentially harmful treatment cycles for the female partners can be reduced.

In particular, the present inventors performed, for the first time, a comprehensive analysis covering all 2,500 known human miRNAs and a reasonably large cohort. Additionally, samples have so far been investigated predominantly by RT-qPCR. Since a substantial bias in miRNA profiling dependent on the underlying experimental technique is known (25), the present inventors applied RT-qPCR only in the validation step, while the initial investigation has been carried out using microarray technology. Doing so, they found that the determination of the total number of miRNAs in the embryonic culture medium of (obtained from) a pre- implanted embryo and the evaluation whether specific miRNAs are present or absent in said medium allow the prediction whether a pre-implanted embryo will be embedded into the endometrium of a female or not. In addition, they identified specific miRNAs markers allowing the prediction whether a pre-implanted embryo will be embedded into the endometrium of a female or not with high sensitivity, specificity, and accuracy.

SUMMARY OF THE INVENTION

In a first aspect, the present invention relates to a method for predicting whether a pre- implanted embryo will be embedded into the endometrium of a female comprising the steps of: (i) determining the total number of miRNAs present in an embryonic culture medium of (obtained from) a pre-implanted embryo, and

(ii) comparing the total number of miRNAs to a reference total number of miRNAs,

wherein the comparison of said total number of miRNAs to the reference total number of miRNAs allows to predict whether the pre-implanted embryo will be embedded into the endometrium of a female.

In a second aspect, the present invention relates to a method for predicting whether a pre-implanted embryo will be embedded into the endometrium of a female comprising the steps of:

(i) determining the absence of at least one miRNA selected from the group consisting of SEQ ID NO: 102, SEQ ID NO: 104 to SEQ ID NO: 109, and a sequence having at least 80% sequence identity thereto in an embryonic culture medium of (obtained from) a pre-implanted embryo,

wherein the absence of said at least one miRNA indicates that the pre-implanted embryo will be embedded into the endometrium of a female, and/or

(ii) determining the presence of miRNA of SEQ ID NO: 103 or a sequence having at least 80% sequence identity thereto in an embryonic culture medium of (obtained from) a pre-implanted embryo,

wherein the presence of said miRNA indicates that the pre-implanted embryo will be embedded into the endometrium of a female.

In a third aspect, the present invention relates to a method for predicting whether a pre- implanted embryo will be embedded into the endometrium of a female comprising the steps of: (i) determining the level of at least one miRNA selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 101, and a sequence having at least 80%> sequence identity thereto present in an embryonic culture medium of (obtained from) a pre-implanted embryo, and

(ii) comparing the level of the at least one miRNA to a reference level of said at least one miRNA, wherein the comparison of the level of the at least one miRNA to the reference level of said at least one miRNA allows to predict whether the pre-implanted embryo will be embedded into the endometrium of a female.

In a fourth aspect, the present invention relates to a kit for predicting whether a pre- implanted embryo will be embedded into the endometrium of a female comprising

(i) means for

(ia) determining the total number of miRNAs,

(ib) determining the presence of at least one miRNA selected from the group consisting of SEQ ID NO: 102, SEQ ID NO: 104 to SEQ ID NO: 109, and a sequence having at least 80% sequence identity thereto and/or the absence of miRNA of SEQ ID NO: 103 or a sequence having at least 80% sequence identity thereto, and/or

(ic) determining the level of at least one miRNA selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 101, and a sequence having at least 80% sequence identity thereto

in an embryonic culture medium of (obtained from) a pre-implanted embryo, and

(ii) optionally at least one reference.

In a fifth aspect, the present invention relates to a method for predicting whether a pre- implanted embryo will be embedded into the endometrium of a female comprising the steps of:

(i) determining the total number of microvesicles present in an embryonic culture medium of (obtained from) a pre-implanted embryo, and

(ii) comparing the total number of microvesicles to a reference total number of microvesicles,

wherein the comparison of said total number of microvesicles to the reference total number of microvesicles allows to predict whether the pre-implanted embryo will be embedded into the endometrium of a female.

This summary of the invention does not necessarily describe all features of the present invention. Other embodiments will become apparent from a review of the ensuing detailed description. DETAILED DESCRIPTION OF THE INVENTION

Definitions

Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechno logical terms: (IUPAC Recommendations)", Leuenberger, H.G.W, Nagel, B. and Kolbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, GenBank Accession Number sequence submissions etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. In the event of a conflict between the definitions or teachings of such incorporated references and definitions or teachings recited in the present specification, the text of the present specification takes precedence.

The term "comprise" or variations such as "comprises" or "comprising" according to the present invention means the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. The term "consisting essentially of according to the present invention means the inclusion of a stated integer or group of integers, while excluding modifications or other integers which would materially affect or alter the stated integer. The term "consisting of or variations such as "consists of according to the present invention means the inclusion of a stated integer or group of integers and the exclusion of any other integer or group of integers.

The terms "a" and "an" and "the" and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "microR A" or "miRNA", as used herein, refer to single-stranded RNA molecules of at least 10 nucleotides and of not more than 35 nucleotides covalently linked together. Preferably, the polynucleotides of the present invention are molecules of 10 to 35 nucleotides or 15 to 35 nucleotides in length, more preferably of 16 to 28 nucleotides or 17 to 27 nucleotides in length, i.e. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides in length, not including optionally labels and/or elongated sequences (e.g. biotin stretches). The miRNAs regulate gene expression and are encoded by genes from whose DNA they are transcribed but miRNAs are not translated into protein (i.e. miRNAs are non-coding RNAs). The genes encoding miRNAs are longer than the processed mature miRNA molecules. The miRNAs are first transcribed as primary transcripts or pri-miRNAs with a cap and poly-A tail and processed to short, 70 nucleotide stem-loop structures known as pre-miRNAs in the cell nucleus. This processing is performed in animals by a protein complex known as the Microprocessor complex consisting of the nuclease Drosha and the double-stranded RNA binding protein Pasha. These pre-miRNAs are then processed to mature miRNAs in the cytoplasm by interaction with the endonuclease Dicer, which also initiates the formation of the RNA-induced silencing complex (RISC). When Dicer cleaves the pre-miRNA stem-loop, two complementary short RNA molecules are formed, but only one is integrated into the RISC. This strand is known as the guide strand and is selected by the argonaute protein, the catalytically active RNase in the RISC, on the basis of the stability of the 5' end. The remaining strand, known as the miRNA*, anti-guide (anti- strand), or passenger strand, is degraded as a RISC substrate. Therefore, the miRNA*s are derived from the same hairpin structure like the "normal" miRNAs. So if the "normal" miRNA is then later called the "mature miRNA" or "guide strand", the miRNA* is the "anti-guide strand" or "passenger strand".

The terms "microRNA*" or "miRNA*", as used herein, refer to single-stranded RNA molecules of at least 10 nucleotides and of not more than 35 nucleotides covalently linked together. Preferably, the polynucleotides of the present invention are molecules of 10 to 35 nucleotides or 15 to 35 nucleotides in length, more preferably of 16 to 28 nucleotides or 18 to 23 nucleotides in length, i.e. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides in length, not including optionally labels and/or elongated sequences (e.g. biotin stretches). The "miRNA*s", also known as the "anti-guide strands" or "passenger strands", are mostly complementary to the "mature miRNAs" or "guide strands", but have usually single-stranded overhangs on each end. There are usually one or more mispairs and there are sometimes extra or missing bases causing single-stranded "bubbles". The miRNA* s are likely to act in a regulatory fashion as the miR As (see also above). In the context of the present invention, the terms "miR A" and "miRNA*" are interchangeable used.

The term "miRBase", as used herein, refers to a well established repository of validated miRNAs. The miRBase (www.mirbase.org) is a searchable database of published miRNA sequences and annotation. Each entry in the miRBase Sequence database represents a predicted hairpin portion of a miRNA transcript (termed mir in the database), with information on the location and sequence of the mature miRNA sequence (termed miR). Both hairpin and mature sequences are available for searching and browsing, and entries can also be retrieved by name, keyword, references and annotation. All sequence and annotation data are also available for download. The sequences of the miRNAs analysed herein are based on miRBase version v21.

The term "nucleotides", as used herein, refers to structural components, or building blocks, of DNA and RNA. Nucleotides consist of a base (one of four chemicals: adenine, thymine, guanine, and cytosine) plus a molecule of sugar and one of phosphoric acid. The term "nucleosides" refers to glycosylamine consisting of a nucleobase (often referred to simply base) bound to a ribose or deoxyribose sugar. Examples of nucleosides include cytidine, uridine, adenosine, guanosine, thymidine and inosine. Nucleosides can be phosphorylated by specific kinases in the cell on the sugar's primary alcohol group (-CH2-OH), producing nucleotides, which are the molecular building blocks of DNA and RNA.

The term "polynucleotide", as used herein, means a molecule of at least 10 nucleotides and of not more than 35 nucleotides covalently linked together. Preferably, the polynucleotides of the present invention are molecules of 10 to 35 nucleotides or 15 to 35 nucleotides in length, more preferably of 16 to 28 nucleotides or 17 to 27 nucleotides in length, i.e. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides in length, not including optionally spacer elements and/or elongation elements described below. The depiction of a single strand of a polynucleotide also defines the sequence of the complementary strand. Polynucleotides may be single stranded or double stranded, or may contain portions of both double stranded and single stranded sequences. The term "polynucleotide" means a polymer of deoxyribonucleotide or ribonucleotide bases and includes DNA and RNA molecules, both sense and anti-sense strands. In detail, the polynucleotide may be DNA, both cDNA and genomic DNA, RNA, cRNA or a hybrid, where the polynucleotide sequence may contain combinations of deoxyribonucleotide or ribonucleotide bases, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine and isoguanine. Polynucleotides may be obtained by chemical synthesis methods or by recombinant methods. In the context of the present invention, a polynucleotide as a single polynucleotide strand provides a probe (e.g. miRNA capture probe) that is capable of binding to, hybridizing with, or detecting a target of complementary sequence, such as a nucleotide sequence of a miRNA or miRNA*, through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation. Polynucleotides in their function as probes may bind target sequences, such as nucleotide sequences of miRNAs or miRNAs*, lacking complete complementarity with the polynucleotide sequences depending upon the stringency of the hybridization condition. There may be any number of base pair mismatches which will interfere with hybridization between the target sequence, such as a nucleotide sequence of a miRNA or miRNA*, and the single stranded polynucleotide described herein. However, if the number of mutations is so great that no hybridization can occur under even the least stringent hybridization conditions, the sequences are no complementary sequences. The polynucleotide variants including polynucleotide fragments or polynucleotide mutants and the miRNA variants including miRNA fragments or miRNA mutants are further defined below. Described herein are polynucleotides in form of single polynucleotide strands as probes for binding to, hybridizing with or detecting complementary sequences of miRNAs (targets), which are selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 619.

The polynucleotide, e.g. the polynucleotide used as a probe for detecting a miRNA or miRNA*, may be unlabeled, directly labeled, or indirectly labeled, such as with biotin to which a streptavidin complex may later bind. The polynucleotide, e.g. the polynucleotide used as a probe for detecting a miRNA or miRNA*, may also be modified, e.g. may comprise an elongation (EL) element. For use in a RAKE or MPEA assay, a polynucleotide with an elongation element may be used as a probe. The elongation element comprises a nucleotide sequence with 1 to 30 nucleotides chosen on the basis of showing low complementarity to potential target sequences, such as nucleotide sequences of miRNAs or miRNAs*, therefore resulting in not to low degree of cross-hybridization to a target mixture. Preferred is a homomeric sequence stretch N _n with n = 1 to 30, i.e. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, and N = A or C, or T or G. Particularly preferred is a homomeric sequence stretch N _n with n = 1 to 12, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, and N = A or C, or T or G. The polynucleotide, e.g. the polynucleotide used as a probe for detecting a miRNA or miRNA*, may be present in form of a tandem, i.e. in form of a polynucleotide hybrid of two different or identical polynucleotides, both in the same orientation, i.e. 5' to 3' or 3' to 5', or in different orientation, i.e. 5' to 3' and 3' to 5'. Said polynucleotide hybrid/tandem may comprise a spacer element. For use in a tandem hybridization assay, the polynucleotide hybrid/tandem as a probe may comprise a spacer (SP) element. The spacer element represents a nucleotide sequence with n = 0 to 12, i.e. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, nucleotides chosen on the basis of showing low complementarity to potential target sequences, such as nucleotide sequences of miR As or anti-miRNAs, therefore resulting in not to low degree of cross-hybridization to a target mixture. It is preferred that n is 0, i.e. that there is no spacer between the two miRNA sequence stretches.

The term "label", as used herein, means a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include 32P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and other entities which can be made detectable. A label may be incorporated into nucleic acids at any position, e.g. at the 3' or 5' end or internally. The polynucleotide for detecting a miRNA (polynucleotide probe) and/or the miRNA itself may be labeled.

The term "stringent hybridization conditions", as used herein, means conditions under which a first nucleic acid sequence (e.g. polynucleotide in its function as a probe for detecting a miRNA or miRNA*) will hybridize to a second nucleic acid sequence (e.g. target sequence such as nucleotide sequence of a miRNA or miRNA*), such as in a complex mixture of nucleic acids. Stringent conditions are sequence-dependent and will be different in different circumstances. Stringent conditions may be selected to be about 5 to 10°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm may be the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions may be those in which the salt concentration is less than about 1.0 M sodium ion, such as about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 20°C for short probes (e.g., about 10-35 nucleotides) and up to 60°C for long probes (e.g., greater than about 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal may be at least 2 to 10 times background hybridization. Exemplary stringent hybridization conditions include the following: 50%> formamide, 5x SSC, and 1%> SDS, incubating at 42°C, or, 5x SSC, 1% SDS, incubating at 65°C, with wash in 0.2x SSC, and 0.1% SDS at 65°C; or 6x SSPE, 10 % formamide, 0.01 %,Tween 20, 0.1 x TE buffer, 0.5 mg/ml BSA, 0.1 mg/ml herring sperm DNA, incubating at 42°C with wash in 05x SSPE and 6x SSPE at 45°C. The term "antisense", as used herein, refers to nucleotide sequences which are complementary to a specific DNA or R A sequence. The term "antisense strand" is used in reference to a nucleic acid strand that is complementary to the "sense" strand.

Residues in two or more polynucleotide s are said to "correspond" to each other if the residues occupy an analogous position in the polynucleotide structures. It is well known in the art that analogous positions in two or more polynucleotides can be determined by aligning the polynucleotide sequences based on nucleic acid sequence or structural similarities. Such alignment tools are well known to the person skilled in the art and can be, for example, obtained on the World Wide Web, for example, ClustalW (see www. ebi . ac .uk/clustalw) or Align (see http://www.ebi.ac.uk/emboss/align/index.html) using standard settings, preferably for Align EMBOSS ::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.

The term "fertilization", as used herein, refers to the event where the egg cell fuses with the male gamete, spermatozoon. After the point of fertilization, the fused product of the female and male gamete is referred to as a zygote or fertilized egg. The fusion of male and female gametes usually occurs following the act of sexual intercourse. Fertilization can also occur by assisted reproductive technology such as artificial insemination and in-vitro-fertilization (IVF).

The term "assisted reproductive technology (ART)", as used herein, encompasses clinical procedures including stimulation of ovulation via hormonal induction, intrauterine insemination (IUI), in-vitro-fertilization (IVF), and intracytoplasmic sperm injection (ICSI), a variation of IVF in which the sperm is injected directly into the oocyte cytoplasm.

The term "in-vitro-fertilization (IVF)", as used herein, refers to a process by which an egg (oocyte) is fertilized by sperm outside the body: in vitro ("in glass"). The process involves monitoring and stimulating a female's ovulatory process, removing an eggs or eggs (oocyte or oocytes) from the female's ovaries and letting sperm fertilize them in a liquid in a laboratory. The fertilized oocyte (zygote) is cultured for 2 to 6 days, preferably for 3 to 5 days, e.g. for 2, 3, 4, 5, or 6 days, in a growth/embryonic culture medium and is then transferred to the same or another female's uterus with the intention of establishing a successful pregnancy.

The term "pre-implanted embryo", as used herein, refers to an embryo from day 3 on after in-vitro-fertilization, but before embryo transfer into the uterus of a female. More particularly, the term "per-implanted embryo", as used herein, refers to an embryo between day 3 (4- to 8-cell stage) and day 6 (blastocyst stage), e.g. day 3, 4, 5, or 6, after in-vitro-fertilization, but before embryo transfer into the uterus of a female. A human oocyte is usually called embryo starting from day 3 after fertilization. This is the time point at which the embryonic genome is activated, i.e. when transcription is evident. This is usually the case at the 4- to 8-cell stage. The described process is called embryonic genome activation (EGA).

The term "oocyte", as used herein, refers to an egg between day 0 and 3 after (in-vitro- ) fertilization. After fertilization, the oocyte undergoes a series of cell divisions in which its net size remains the same, but following DNA synthesis mitosis results in cells of approximately equal, decreased size. In humans, there are three cleavage divisions from 1 cell to 2 cells (2- cell stage), 2 cells to 4 cells (4-cell stage) and 4 cells to 8 cells (8-cell stage). At around day 3 after fertilization, the oocyte transitions into an embryo. At this stage of development after fertilization, the molecular program of the oocyte is degraded and those of the embryo is activated.

The term "blastocyst", as used herein, refers to a structure formed in the early development of mammals. It possesses an inner cell mass (ICM) forming the embryo. The outer layer of the blastocyst consists of cells collectively called the trophoblast. This layer surrounds the inner cell mass and a fluid- filled cavity known as the blastocoele. The trophoblast gives rise to the placenta. In humans, blastocyst formation begins about 5 days after fertilization, when a fluid- filled cavity opens up in the morula, a ball consisting of a few dozen cells. The blastocyst has a diameter of about 0.1 to 0.2 mm and comprises 200 to 300 cells following rapid cleavage (cell division). 5 to 6 days post-fertilization, the blastocyst begins to embed itself into the endometrium of the uterine wall where it will undergo later developmental processes, including gastrulation. Embedding of the blastocyst into the endometrium requires that it hatches from the zona pellucida, which prevents it from adhering to the oviduct as it makes its way to the uterus. The blastocyst is completely embedded in the endometrium 11 to 12 days after fertilization.

The term "embryo transfer", as used herein, refers to a process of transferring embryos from in vitro culture to the uterus of a female. This is often done at day 3 (at the 4- to 8-cell stage), but is also increasingly performed at day 5 or 6 (blastocyst stage).

Generally, it is possible to predict with the methods of the present invention whether an oocyte at the 2-cell stage, 4-cell stage, 8-cell stage, or blastocyst stage will be embedded into the endometrium of a female or whether an oocyte at the 2-cell stage, 4-cell stage, 8-cell stage, or blastocyst stage will not lead to pregnancy in a female.

The implantation stages of the embryo into the uterine endometrium can be seen as taking place in three phases: (i) apposition, (ii) adhesion and (iii) the embedding into the endometrium. With the embedding of the embryo into the uterine endometrium, the implantation process is completed. In the context of the present invention, embedding of a pre-implanted embryo into the endometrium of a female equals pregnancy. This means that the methods of the present invention which are directed to the prediction whether a pre-implanted will be embedded into the endometrium of a female can also be worded as predicting whether a pre-implanted embryo will lead to pregnancy in a female.

The term "pregnancy," as used herein, means the time during which one or more offspring develops inside a female. There are multiple definitions of the beginning of a pregnancy. Healthcare providers normally count the initiation of pregnancy from the first day of the female's last menstrual period. Using this date, the resulting fetal age is called the gestational age. This choice was a result of inability to discern the point in time when the actual conception happened. In in-vitro-fertilization, the gestational age is calculated by days from oocyte retrieval + 14 days (the 14 days before the known time of conception).

Pregnancy detection can be accomplished using one or more various pregnancy tests, which detect hormones generated by the newly formed placenta, serving as biomarkers of pregnancy. Human chorionic gonadotropin (hCG) is, for example, a hormone produced by the placenta after implantation. The presence of hCG is detected in in some pregnancy tests (hCG pregnancy strip tests). Blood and urine tests can detect pregnancy 12 days after implantation. Blood pregnancy tests are more sensitive than urine tests (giving fewer false negatives). Home pregnancy tests are urine tests, and normally detect a pregnancy 12 to 15 days after fertilization. A quantitative blood test can determine approximately the date the embryo was conceived because hCG doubles every 36 to 48 hours. A single test of progesterone levels can also help determine how likely a fetus will survive in those with a threatened miscarriage (bleeding in early pregnancy).

If the pre-implanted embryo will not lead to pregnancy, the above described tests give a negative result. In this cases, the hormone human chorionic gonadotropin (hCG) level is, for example, not increased.

The term "female", as used herein, may be a human female or another mammal.

The term "embryonic culture", as used herein, refer to a component of in-vitro- fertilization wherein resultant embryos (pre-implanted embryos) are allowed to grow for some time in an artificial medium. Any embryonic culture medium may be used which allows growing of the resultant embryos (pre-implanted embryos) for some time. The duration of embryonic culture can be varied, conferring different stages of embryogenesis at embryo transfer. The main stages at which embryo transfer is performed are cleavage stage (day 2 to 4 after in-vitro-fertilization) and blastocyst stage (day 5 or 6 after in-vitro-fertilization). The term "being about the same", as used herein, means plus or minus 5%, preferably plus or minus 2%, of the numerical value of the number with which it is used. Therefore, about 50% means in the range of 45 to 55%, preferably in the range of 48 to 52%

The term "level", as used herein, refers to an amount (e.g. in grams, mole, or ion counts) or concentration (e.g. absolute or relative concentration) of the miR A measured herein. The term "level", as used herein, also comprises scaled, normalized, or scaled and normalized amounts or values. Preferably, the level determined herein is the expression level.

The term "sensitivity", as used herein, refers to the number of true positive patients (%) with regard to the number of all patients (100%). The sensitivity is calculated by the following formula: Sensitivity= TP/(TP+FN) (TP= true positives; FN=false negatives).

The term "specificity", as used herein, relates to the number of true negative patients (%) with regard to the number of all patients (100%). The specificity is calculated by the following formula: Specificity= TN/(TN+FP) (TN= true negatives; FP=false positives).

The term "accuracy", as used herein, means a statistical measure for the correctness of classification or identification of sample types. The accuracy is the proportion of true results (both true positives and true negatives).

The result of each analysis group is usually calculated from a plurality of isolated samples, i.e. from at least 2 isolated samples, preferably from between 2 and 20, more preferably from between 10 and 60, and even more preferably from between 50 and 100 isolated samples. The methods of the present invention can be carried out in combination with other methods for predicting whether a pre-implanted embryo will be embedded into the endometrium of a female or whether a pre-implanted embryo will not lead to pregnancy in a female.

The term "AUC", as used herein, relates to an abbreviation for the area under a curve. In particular, it refers to the area under a Receiver Operating Characteristic (ROC) curve. The term "Receiver Operating Characteristic (ROC) curve", as used herein, refers to a plot of the true positive rate against the false positive rate for the different possible cut points of a diagnostic test. It shows the trade-off between sensitivity and specificity depending on the selected cut point (any increase in sensitivity will be accompanied by a decrease in specificity). The area under an ROC curve is a measure for the accuracy of a diagnostic test (the larger the area the better, optimum is 1 , a random test would have a ROC curve lying on the diagonal with an area of 0.5 (see, for reference, for example, JP. Egan. Signal Detection Theory and ROC Analysis). In the context of the present invention, the term "kit of parts (in short: kit)" is understood to be any combination of at least some of the components identified herein, which are combined, coexisting spatially, to a functional unit, and which can contain further components.

The term "point-of-care testing (POCT)", as used herein", refers to a medical diagnostic testing at or near the point of care that is the time and place of patient care. This contrasts with the historical pattern in which testing was wholly or mostly confined to the medical laboratory, which entailed sending off specimens away from the point of care and then waiting hours or days to learn the results, during which time care must continue without the desired information. Point-of-care tests are simple medical tests that can be performed at the bedside. The driving notion behind POCT is to bring the test conveniently and immediately to the patient. This increases the likelihood that the patient, physician, and care team will receive the results quicker, which allows for immediate clinical management decisions to be made. POCT is often accomplished through the use of transportable, portable, and handheld instruments and test kits. Small bench analyzers or fixed equipment can also be used when a handheld device is not available— the goal is to collect the specimen and obtain the results in a very short period of time at or near the location of the patient so that the treatment plan can be adjusted as necessary before the patient leaves.

The term "microvesicle", as used herein, refers to fragments of plasma membrane ranging from 100 nm to 1000 nm shed from almost all cell types. Thus, microvesicles are also shed from the cells of a pre-implanted embryo into the embryonic culture medium. Microvesicles play a role in intercellular communication and can transport mR A, miR A, and proteins between cells. Microvesicles originate directly from the plasma membrane of the cell. They remove misfolded proteins, cytotoxic agents and metabolic waste from the cell. Embodiments of the invention

The present inventors surprisingly found that the analysis of miR As, which secrete from an pre-implanted embryo into the extracellular environment, e.g. into the embryonic culture medium, allows to predict the developmental potential of the pre-implanted embryo and, thus, the selection of the embryo with the highest chance to result in a successful pregnancy. This, in turn, has the advantage that the average number of potentially harmful treatment cycles for the female partners can be reduced.

In particular, the present inventors performed, for the first time, a comprehensive analysis covering all 2,500 known human miRNAs and a reasonably large cohort. Additionally, samples have so far been investigated predominantly by RT-qPCR. Since a substantial bias in miR A profiling dependent on the underlying experimental technique is known (25), the present inventors applied RT-qPCR only in the validation step, while the initial investigation has been carried out using microarray technology. Doing so, they found that the determination of the total number of miRNAs in the embryonic culture medium of (obtained from) a pre- implanted embryo and the evaluation whether specific miRNAs are present or absent in said medium allow the prediction whether a pre-implanted embryo will be embedded into the endometrium of a female or not. In addition, they identified specific miRNAs markers allowing the prediction whether a pre-implanted embryo will be embedded into the endometrium of a female or not with high sensitivity, specificity, and accuracy.

Thus, in a first aspect, the present invention relates to a method for predicting whether a pre-implanted embryo will be embedded into the endometrium of a female comprising the steps of:

(i) determining the total number of miRNAs present in an embryonic culture medium of (obtained from) a pre-implanted embryo, and

(ii) comparing the total number of miRNAs to a reference total number of miRNAs.

More specifically, the present invention relates to a method for predicting whether a pre- implanted embryo will be embedded into the endometrium of a female comprising the steps of: (i) determining the total number of miRNAs present in an embryonic culture medium of (obtained from) a pre-implanted embryo, and

(ii) comparing the total number of miRNAs to a reference total number of miRNAs,

wherein the comparison of said total number of miRNAs to the reference total number of miRNAs allows to predict whether the pre-implanted embryo will be embedded into the endometrium of a female.

The present inventors surprisingly found that miRNAs secrete from a pre-implanted embryo into the extracellular environment, e.g. into the embryonic culture medium, and that the analysis of their total number allows the prediction whether a pre-implanted embryo will be embedded into the endometrium of a female or not. In particular, the present inventors found that the total number of miRNAs is lower in embryonic culture medium obtained from embryos which embedded into the endometrium of a female compared to the total number of miRNAs determined in the embryonic culture medium obtained from embryos which not embedded into the endometrium of a female. Thus, the determination of the total number of miRNAs is highly suitable to predict, whether a pre-implanted embryo in question will be embedded into the endometrium of a female or not. The total number of miRNAs is preferably determined in the embryonic culture medium by taking a sample of said medium, e.g. using a pipette. The sample size may vary between 0.5 and 2 ml, e.g. 0.5, 1. 1.5, or 2 ml. The sampling is preferably performed around the time point of embryo transfer, e.g. at day 3, 4, 5, or 6 after in-vitro-fertilization, into the uterus of a female. The above analysis allows the prediction, if the pre-implanted embryo of a female or which of the pre-implanted embryos of a female will be embedded into the endometrium of a female and, thus, should be used for embryo transfer.

The present inventors detected that a total of 619 different miRNAs are comprised in the embryonic culture medium. Thus, the miRNAs are preferably selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 619, e.g. SEQ ID NO: 1 to SEQ ID NO: 101, and a sequence having at least 80%, preferably at least 85%, more preferably at least 90%>, and most preferably at least 95% or 99%, e.g. at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity thereto.

The reference total number of miRNAs may be any number which allows to predict whether a pre-implanted embryo will be embedded into the endometrium of a female or not.

In one embodiment, the reference total number of miRNAs is the total number of miRNAs determined by measuring at least one reference embryonic culture medium of (obtained from) a pre-implanted embryo for which it is known that said embryo not embedded into an endometrium of a female. In this case, it is preferred that the total number of miRNAs is below the reference total number of miRNAs. This indicates that the pre-implanted embryo will be embedded into the endometrium of a female.

Preferably, said reference embryonic culture media are at least 2 reference embryonic culture media, more preferably at least 2 to 100 reference embryonic culture media, even more preferably at least 10 to 500 reference embryonic culture media, and most preferably at least 50 to 10.000 reference embryonic culture media, e.g. at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 150, 200, 250, 300, 400, 500, 1.000, 2.000, 3.000, 4.000, 5.000, or 10.000 reference embryonic culture media.

Preferably, the embryonic culture medium and the reference embryonic culture medium (media) have the same composition and are cultured under identical conditions. This applies also to the other aspects of the present invention. Preferably, the total number of miRNAs is at least 5%, more preferably at least 8%, e.g. at least 5, 5.5, 6, 6.5, 7, 7.5, or 8%, below the reference total number of miRNAs.

In a particular embodiment, the reference total number of miRNAs, which is the total number of miRNAs determined by measuring at least one reference embryonic culture medium of (obtained from) a pre-implanted embryo for which it is known that said embryo not embedded into an endometrium of a female, is 163. Thus, in a preferred embodiment, the total number of miRNAs is at least 5%, more preferably at least 8%, e.g. at least 5, 5.5, 6, 6.5, 7, 7.5, or 8%, below 163 (as reference total number of miRNAs) indicating that the pre-implanted embryo will be embedded into the endometrium of a female.

In one alternative embodiment, the reference total number of miRNAs is the total number of miRNAs determined by measuring at least one reference embryonic culture medium of (obtained from) a pre-implanted embryo for which it is known that said embryo embedded into an endometrium of a female. In this case, it is preferred that the total number of miRNAs being about the same as the reference total number of miRNAs. This indicates that the pre- implanted embryo will be embedded into the endometrium of a female.

Preferably, said reference embryonic culture media are at least 2 reference embryonic culture media, more preferably at least 2 to 100 reference embryonic culture media, even more preferably at least 10 to 500 reference embryonic culture media, and most preferably at least 50 to 10.000 reference embryonic culture media, e.g. at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 150, 200, 250, 300, 400, 500, 1.000, 2.000, 3.000, 4.000, 5.000, or 10.000 reference embryonic culture media.

Preferably, the embryonic culture medium and the reference embryonic culture medium (media) have the same composition and are cultured under identical conditions. This applies also to the other aspects of the present invention.

In a particular embodiment, the reference total number of miRNAs, which is the total number of miRNAs determined by measuring at least one reference embryonic culture medium of (obtained from) a pre-implanted embryo for which it is known that said embryo embedded into an endometrium of a female, is 149. Thus, in a preferred embodiment, the total number of miRNAs being about 149 indicates that the pre-implanted embryo will be embedded into the endometrium of a female. The term "being about the same'V'about" is defined above. The skilled person knows how to determine the total number of miR As. For example, the total number of miRNA may be determined by nucleic acid hybridization, nucleic acid amplification, polymerase extension, sequencing, mass spectroscopy, a immunochemical method, or any combination thereof. Preferably,

(i) the nucleic acid hybridization is performed using a microarray/biochip, or using in situ hybridization,

(ii) the nucleic acid amplification is performed using real-time PCR (RT-PCR) or quantitative real-time PCR (qPCR),

(iii) the sequencing is next generation sequencing, or

(iv) the immunochemical method is an enzyme linked immunosorbent assay (ELISA).

In a second aspect, the present invention relates to a method for predicting whether a pre-implanted embryo will be embedded into the endometrium of a female comprising the steps of:

(i) determining the absence of at least one miRNA, e.g. at least 1, 2, 3, 4, 5, 6, or 7 miRNA(s), selected from the group consisting of SEQ ID NO: 102, SEQ ID NO: 104 to SEQ ID NO: 109, and a sequence having at least 80%, more preferably at least 85%, even more preferably at least 90%>, and most preferably at least 95%>, e.g. at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity thereto in an embryonic culture medium of (obtained from) a pre-implanted embryo, wherein the absence of said at least one miRNA indicates that the pre-implanted embryo will be embedded into the endometrium of a female, and/or

(ii) determining the presence of miRNA of SEQ ID NO: 103 or a sequence having at least 80%), more preferably at least 85%>, even more preferably at least 90%>, and most preferably at least 95%, e.g. at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%>, sequence identity thereto in an embryonic culture medium of (obtained from) a pre-implanted embryo,

wherein the presence of said miRNA indicates that the pre-implanted embryo will be embedded into the endometrium of a female.

All preferred combinations of 2, 3, 4, 5, 6, or 7 miRNAs selected from the group consisting of SEQ ID NO: 102 and SEQ ID NO: 104 to SEQ ID NO: 109 as well as the single miRNAs referred to in item (i) are comprised in Figure 7. See also Figure 3.

The present inventors surprisingly found that miRNAs secrete from a pre-implanted embryo into the extracellular environment, e.g. into the embryonic culture medium, and that the analysis of their presence and/or absence allows the prediction whether a pre-implanted embryo will be embedded into the endometrium of a female or not. In particular, the present inventors found that specific miRNAs can be found in embryonic culture medium obtained from embryos which not embedded into the endometrium of a female. Other specific miRNAs can be found in embryonic culture medium obtained from embryos which embedded into the endometrium of a female. Thus, the determination of the presence and/or absence of specific miRNAs is highly suitable for a prediction, whether a pre-implanted embryo in question will be embedded into the endometrium of a female or not.

The presence and/or absence of miRNAs is preferably determined in the embryonic culture medium by taking a sample of said medium, e.g. using a pipette. The sample size may vary between 0.5 and 2 ml, e.g. 0.5, 1. 1.5, or 2 ml. The sampling is preferably performed around the time point of embryo transfer, e.g. at day 3, 4, 5, or 6 after in-vitro-fertilization, into the uterus of a female. The above analysis allows the prediction, if the pre-implanted embryo of a female or which of the pre-implanted embryos of a female will be embedded into the endometrium of a female and, thus, should be used for embryo transfer.

The present inventors found that the best separation was obtained for miRNA of SEQ

ID NO: 104 (miR-634). The absence of this single miRNA predicted with accuracy of 71% at a sensitivity of 85% whether embryo transfer would lead to pregnancy.Thus, the absence of miRNA of SEQ ID NO: 104 (miR-634) or a sequence having at least 80%, more preferably at least 85%), even more preferably at least 90%>, and most preferably at least 95%, e.g. at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity thereto in the embryonic culture medium preferably indicates that the pre-implanted embryo will be embedded into the endometrium of a female.

If the presence/absence of more than 1 miRNA is determined, e.g. of 2 or more miRNAs, it is referred herein to a set comprising at least 2 miRNAs.

The skilled person knows how to determine the presence and/or absence of miRNAs.

For example, the presence and/or absence of miRNAs may be determined by nucleic acid hybridization, nucleic acid amplification, polymerase extension, sequencing, mass spectroscopy, a immunochemical method, or any combination thereof. Preferably,

(i) the nucleic acid hybridization is performed using a microarray/biochip, or using in situ hybridization,

(ii) the nucleic acid amplification is performed using real-time PCR (RT-PCR) or quantitative real-time PCR (qPCR),

(iii) the sequencing is next generation sequencing, or

(iv) the immunochemical method is an enzyme linked immunosorbent assay (ELISA). In a third aspect, the present invention relates to a method for predicting whether a pre- implanted embryo will be embedded into the endometrium of a female comprising the steps of:

(i) determining the level of at least one miRNA, e.g. at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or 101 miRNA(s), selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 101, and a sequence having at least 80%, more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95%, e.g. at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity thereto present in an embryonic culture medium of (obtained from) a pre-implanted embryo, and

(ii) comparing the level of the at least one miRNA to a reference level of said at least one miRNA.

More specifically, the present invention relates to a method for predicting whether a pre- implanted embryo will be embedded into the endometrium of a female comprising the steps of:

(i) determining the level of at least one miRNA, e.g. at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or 101 miRNA(s), selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 101, and a sequence having at least 80%, more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95%, e.g. at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity thereto present in an embryonic culture medium of (obtained from) a pre-implanted embryo, and

(ii) comparing the level of the at least one miRNA to a reference level of said at least one miRNA,

wherein the comparison of the level of the at least one miRNA to the reference level of said at least one miRNA allows to predict whether the pre-implanted embryo will be embedded into the endometrium of a female.

The present inventors surprisingly found that miRNAs secrete from a pre-implanted embryo into the extracellular environment, e.g. into the embryonic culture medium, and that the analysis of their level allows the prediction whether a pre-implanted embryo will be embedded into the endometrium of a female or not. In particular, the miRNA(s) selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 101 were found by the present inventors as being significantly deregulated in embryonic culture medium obtained from embryos which embedded into the endometrium of a female and embryos which not embedded into the endometrium of a female. Thus, these miRNAs are highly suitable for a prediction, whether a pre-implanted embryo in question will be embedded into the endometrium of a female or not. The level of miRNAs is preferably determined in the embryonic culture medium by taking a sample of said medium, e.g. using a pipette. The sample size may vary between 0.5 and 2 ml, e.g. 0.5, 1. 1.5, or 2 ml. The sampling is preferably performed around the time point of embryo transfer, e.g. at day 3, 4, 5, or 6 after in-vitro-fertilization, into the uterus of a female. The above analysis allows the prediction, if the pre-implanted embryo of a female or which of the pre- implanted embryos of a female will be embedded into the endometrium of a female and, thus, should be used for embryo transfer.

Preferably, the at least one miRNA, e.g. the at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 miRNA(s), is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 50, and a sequence having at least 80%, more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95%, e.g. at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity thereto. Said miRNAs were found by the present inventors as being most significantly deregulated in embryonic culture medium obtained from embryos which embedded into the endometrium of a female and embryos which not embedded into the endometrium of a female (see Figure 5).

More preferably, the at least one miRNA, e.g. the at least 1, 2, 3, 4, 5, or 6 miRNA(s), is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 6, and a sequence having at least 80%, more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95%, e.g. at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%>, sequence identity thereto. Said miRNAs have been validated using single RT-qPCR assays. All preferred combinations of 2, 3, 4, 5, or 6 miRNAs selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 6 as well as the single miRNAs are comprised in Figure 6. The determination of the level of the miRNA of SEQ ID NO: 1 (miR-29c-3p) is particularly preferred. Said miRNA correlated with an AUC of 0.83 to positive pregnancy.

If the level of more than 1 miRNA is determined, e.g. of 2 or more miRNAs, it is referred herein to a set comprising at least 2 miRNAs. The reference level may be any level which allows to predict whether a pre-implanted embryo will be embedded into the endometrium of a female or not.

In one embodiment, the reference level is the level determined by measuring at least one reference embryonic culture medium of (obtained from) a pre-implanted embryo for which it is known that said embryo embedded into an endometrium of a female.

In this case, it is preferred that

the level of the at least one miRNA selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3 to SEQ ID NO: 7, SEQ ID NO: 9 to SEQ ID NO: 12, SEQ ID NO: 14 to SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30 to SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 40 to SEQ ID NO: 44, SEQ ID NO: 46 to SEQ ID NO: 48, and a sequence having at least 80% sequence identity thereto being about the same as the reference level, which indicates that the pre-implanted embryo will be embedded into the endometrium of a female, and/or the level of the at least one miRNA selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO: 21 to SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 50, and a sequence having at least 80% sequence identity thereto being about the same as the reference level, which indicates that the pre- implanted embryo will be embedded into the endometrium of a female.

Preferably, said reference embryonic culture media are at least 2 reference embryonic culture media, more preferably at least 2 to 100 reference embryonic culture media, even more preferably at least 10 to 500 reference embryonic culture media, and most preferably at least 50 to 10.000 reference embryonic culture media, e.g. at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 150, 200, 250, 300, 400, 500, 1.000, 2.000, 3.000, 4.000, 5.000, or 10.000 reference embryonic culture media.

In one alternative embodiment, the reference level is the level determined by measuring at least one reference embryonic culture medium of (obtained from) a pre-implanted embryo for which is it known that said embryo not embedded into an endometrium of a female.

In this case, it is preferred that

the level of the at least one miRNA selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3 to SEQ ID NO: 7, SEQ ID NO: 9 to SEQ ID NO: 12, SEQ ID NO: 14 to SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30 to SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 40 to SEQ ID NO: 44, SEQ ID NO: 46 to SEQ ID NO: 48, and a sequence having at least 80% sequence identity thereto is below the reference level, which indicates that the pre- implanted embryo will be embedded into the endometrium of a female, and/or

the level of the at least one miRNA selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO: 21 to SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 50, and a sequence having at least 80% sequence identity thereto is above as the reference level, which indicates that the pre-implanted embryo will be embedded into the endometrium of a female.

Preferably, the level of the at least one miRNA is at least 0.4 fold or at least 0.6 fold above/below the reference level, more preferably at least 1.1 -fold or at least 1.2-fold above/below the reference level, even more preferably at least 1.5-fold or at least 2-fold above/below the reference level, and most preferably at least 2.5-fold above/below the reference level.

If the level of more than 1 miRNA is determined, e.g. of 2 or more miRNAs, it is referred herein to a set comprising at least 2 miRNAs.

The determination of the level of the at least one miRNA may be carried out by any convenient means for determining the level of a nucleotide sequence such as miRNA. For this purpose, qualitative, semi-quantitative and quantitative detection methods can be used. Quantitative detection methods are preferred. A variety of techniques are well known to the person skilled in the art.

The skilled person knows how to determine the presence and/or absence of miRNAs. For example, the presence and/or absence of miRNAs may be determined by nucleic acid hybridization, nucleic acid amplification, polymerase extension, sequencing, mass spectroscopy, a immunochemical method, or any combination thereof. Preferably,

(i) the nucleic acid hybridization is performed using a microarray/biochip, or using in situ hybridization,

(ii) the nucleic acid amplification is performed using real-time PCR (RT-PCR) or quantitative real-time PCR (qPCR),

(iii) the sequencing is next generation sequencing, or

(iv) the immunochemical method is an enzyme linked immunosorbent assay (ELISA). The aforesaid real time polymerase chain reaction (RT-PCR) may include the following steps: (i) extracting total RNA from the embryonic culture medium, (ii) obtaining cDNA samples by RNA reverse transcription (RT) reaction using miRNA-specific primers, (iii) designing miRNA-specific cDNA forward primers and providing universal reverse primers to amplify the cDNA via polymerase chain reaction (PCR), (iv) adding a fluorescent probe to conduct PCR, and (v) detecting and comparing the variation in levels of miRNAs in the embryonic culture medium and in the reference embryonic culture medium.

A variety of kits and protocols to determine the miRNA level by real time polymerase chain reaction (RT-PCR) such as real time quantitative PCR (RT qPCR) are available. For example, reverse transcription of miRNAs may be performed using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) according to manufacturer's recommendations. Briefly, miRNA may be combined with dNTPs, MultiScribe reverse transcriptase and the primer specific for the target miRNA. The resulting cDNA may be diluted and may be used for PCR reaction. The PCR may be performed according to the manufacturer's recommendation (Applied Biosystems). Briefly, cDNA may be combined with the TaqMan assay specific for the target miRNA and PCR reaction may be performed using ABI7300.

Nucleic acid hybridization, for example, may be performed using a microarray/biochip or in situ hybridization. The microarray/biochip allows the analysis of a single miRNA as well as multiple miRNAs comprised in an embryonic culture medium. For nucleic acid hybridization, for example, the polynucleotides (probes) described herein with complementarity to the corresponding miRNAs to be detected are attached to a solid phase to generate a microarray/biochip. Said microarray/biochip is then incubated with miRNAs, isolated (e.g. extracted) from the embryonic culture medium, which may be labelled or unlabelled. Upon hybridization of the labelled miRNAs to the complementary polynucleotide sequences on the microarray/biochip, the success of hybridisation may be controlled and the intensity of hybridization may be determined via the hybridisation signal of the label in order to determine the level of each tested miRNA in said embryonic culture medium.

Alternatively, the miRNA level may be determined using an immunochemical method, e.g. using an ELISA. Said method may include the following steps: (i) isolating miRNAs from an embryonic culture medium, (ii) hybridizing polynucleotide probes (complementary) to the miRNAs to obtain hybrids of said polynucleotides probes and said miRNAs, and (iii) binding said hybrids to antibodies capable of specifically binding hybrids of said polynucleotide probes and said miRNAs, and (iv) detecting the antibody-bound hybrids. The above described techniques/methods may also be used in order to determine the total number of miRNAs (see first aspect of the present invention) or the presence and/or absence of miRNAs (see second aspect of the present invention).

In a fourth aspect, the present invention relates to (the use, e.g. the in vitro use, of) a kit for predicting whether a pre-implanted embryo will be embedded into the endometrium of a female comprising

(i) means for

(ia) determining the total number of miRNAs,

(ib) determining the presence of at least one miRNA, e.g. at least 1, 2, 3, 4, 5, 6, or 7 miRNA(s), selected from the group consisting of SEQ ID NO: 102, SEQ ID NO:

104 to SEQ ID NO: 109, and a sequence having at least 80%, preferably at least 85%, more preferably at least 90%>, and most preferably at least 95% or 99%, e.g. at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99%), sequence identity thereto and/or the absence of miRNA of SEQ ID NO: 103 or a sequence having at least 80%>, preferably at least 85%, more preferably at least 90%>, and most preferably at least 95% or 99%, e.g. at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity thereto, and/or

(ic) determining the level of at least one miRNA, e.g. at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,

31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or 101 miRNA(s), selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 101, and a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, e.g. at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity thereto

in an embryonic culture medium of (obtained from) a pre-implanted embryo, and (ii) optionally at least one reference.

The reference may be any reference which allows the prediction whether a pre- implanted embryo will be embedded into the endometrium of a female or not. For example, said reference may be a reference level and/or a reference total number of miRNAs (see first to third aspect of the present invention). The kit may further comprise a container and/or a data carrier. The data carrier may be a non-electronical data carrier, e.g. a graphical data carrier such as an information leaflet, an information sheet, a bar code or an access code, or an electronical data carrier such as a floppy disk, a compact disk (CD), a digital versatile disk (DVD), a microchip or another semiconductor-based electronical data carrier. The access code may allow the access to a database, e.g. an internet database, a centralized, or a decentralized database. The access code may also allow access to an application software that causes a computer to perform tasks for computer users or a mobile app which is a software designed to run on smartphones and other mobile devices.

Said data carrier may further comprise the at least one reference, e.g. the reference level of the level of the at least one miR A determined herein and/or the reference total number of miR As determined herein. In case that the data carrier comprises an access code which allows the access to a database, said at least one reference, e.g. said reference level and/or said reference total number of miRNAs, may be deposited in this database.

Preferably, the means in (ia), (ib), and/or (ic) comprise

at least one polynucleotide (probe),

at least one primer pair, and/or

at least one polynucleotide (probe) and at least one antibody capable of binding a hybrid of said at least one polynucleotide (probe) and said at least one miRNA.

Said means allow to determine the total number of miRNAs, the presence/absence of the above- mentioned miRNAs and/or the level of the above-mentioned miRNAs.

It is further preferred that

(i) the at least one polynucleotide is complementary to the at least one miRNA mentioned above, or

(ii) the at least one polynucleotide has at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, e.g. at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity to the polynucleotide according to (i).

It is particularly preferred that the polynucleotide as defined in (ii) has at least 80%, preferably at least 85%, more preferably at least 90%>, and most preferably at least 95% or 99%, e.g. at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity over a continuous stretch of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more nucleotides, preferably over the whole length, to the polynucleotide according to (i). In addition, the polynucleotide as defined in (ii) (i.e. polynucleotide variant) is only regarded as a polynucleotide as defined in (ii) (i.e. polynucleotide variant) within the context of the present invention, if it is still capable of binding to, hybridizing with, or detecting the respective target miR A, i.e. the target miR A according to SEQ ID NO: 1 to SEQ ID NO: 619, preferably SEQ ID NO: 1 to SEQ ID NO: 101 , through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation under stringent hybridization conditions. The skilled person can readily assess whether a polynucleotide as defined in (ii) (i.e. polynucleotide variant) is still capable of binding to, hybridizing with, recognizing or detecting the respective target miRNA, i.e. the target miRNA according to SEQ ID NO: 1 to SEQ ID NO: 619, preferably SEQ ID NO: 1 to SEQ ID NO: 101. Suitable assays to determine whether hybridization under stringent conditions still occurs are well known in the art. However, as an example, a suitable assay to determine whether hybridization still occurs comprises the steps of: (a) incubating the polynucleotide as defined in (ii) attached onto a biochip with the respective target miRNA, i.e. the target miRNA according to SEQ ID NO: 1 to SEQ ID NO: 619, preferably SEQ ID NO: 1 to SEQ ID NO: 101, (b) washing the biochip to remove unspecific bindings, (c) subjecting the biochip to a detection system, and (c) analyzing whether the polynucleotide can still hybridize with the respective target miRNA. As a positive control, the respective non-mutated polynucleotide as defined in (i) may be used. Preferably stringent hybridization conditions include the following: 50% formamide, 5x SSC, and 1% SDS, incubating at 42°C, or, 5x SSC, 1% SDS, incubating at 65°C, with wash in 0.2x SSC, and 0.1% SDS at 65°C; or 6x SSPE, 10 % formamide, 0.01 %,Tween 20, 0.1 x TE buffer, 0.5 mg/ml BSA, 0.1 mg/ml herring sperm DNA, incubating at 42°C with wash in 05x SSPE and 6x SSPE at 45°C.

The above means may also comprise a microarray/biochip, a RT-PCT system, a PCR- system, a flow cytometer, a bead-based multiplex system or a next generation sequencing system. The at least one polynucleotide may be part of the microarray/biochip or may be attached to the beads of the beads-based multiplex system.

Said kit may also comprise materials desirable from a commercial and user standpoint including a buffer(s), a reagent(s) and/or a diluent(s) for determining the level mentioned above.

Preferably, the miRNAs in (ia) are selected from the group consisting of SEQ ID NO:

1 to SEQ ID NO: 619, and a sequence having at least 80%>, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, e.g. at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity thereto. Preferably, the miRNA(s) in (ic) is (are) selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 50, and a sequence having at least 80%, preferably at least 85%, more preferably at least 90%>, and most preferably at least 95% or 99%, e.g. at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity thereto. More preferably, the miRNA(s) in (ic) is (are) selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 6, and a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, e.g. at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity thereto.

It is particularly preferred that the kit is useful for conducting the methods according to the first to third aspect.

It is also particularly preferred that the kit allows a point-of-care testing (POCT).

The first aspect of the present invention may alternatively be worded as follows: A method for predicting whether a pre-implanted embryo will not lead to pregnancy in a female comprising the steps of:

(i) determining the total number of miRNAs present in an embryonic culture medium of

(obtained from) a pre-implanted embryo, and

(ii) comparing the total number of miRNAs to a reference total number of miRNAs.

More specifically, the present invention alternatively relates to a method for predicting whether a pre-implanted embryo will not lead to pregnancy in a female comprising the steps of:

(i) determining the total number of miRNAs present in an embryonic culture medium of

(obtained from) a pre-implanted embryo, and

(ii) comparing the total number of miRNAs to a reference total number of miRNAs,

wherein the comparison of said total number of miRNAs to the reference total number of miRNAs allows to predict whether the pre-implanted embryo will not lead to pregnancy in a female.

The miRNAs are preferably selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 619, and a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, e.g. at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity thereto.

The reference total number of miRNAs may be any number which allows to predict whether a pre-implanted embryo will not lead to pregnancy in a female.

In one embodiment, the reference total number of miRNAs is the total number of miRNAs determined by measuring at least one reference embryonic culture medium of (obtained from) a pre-implanted embryo known to lead to pregnancy in a female. In this case, it is preferred that the total number of miRNAs is above the reference total number of miRNAs. This indicates that the pre-implanted embryo will not lead to pregnancy in a female.

Preferably, said reference embryonic culture media are at least 2 reference embryonic culture media, more preferably at least 2 to 100 reference embryonic culture media, even more preferably at least 10 to 500 reference embryonic culture media, and most preferably at least 50 to 10.000 reference embryonic culture media, e.g. at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 150, 200, 250, 300, 400, 500, 1.000, 2.000, 3.000, 4.000, 5.000, or 10.000 reference embryonic culture media.

Preferably, the total number of miRNAs is at least 5%, more preferably at least 8%, e.g. at least

5, 5.5, 6, 6.5, 7, 7.5, or 8%, above the reference total number of miRNAs.

In a particular embodiment, the reference total number of miRNAs, which is the total number of miRNAs determined by measuring at least one reference embryonic culture medium of (obtained from) a pre-implanted embryo known to lead to pregnancy in a female, is 149. Thus, in a preferred embodiment, the total number of miRNAs is at least 5%, more preferably at least 8%, e.g. at least 5, 5.5, 6, 6.5, 7, 7.5, or 8%, above 149 (as reference total number of miRNAs) indicating that the pre-implanted embryo will not lead to pregnancy in a female.

In one alternative embodiment, the reference total number of miRNAs is the total number of miRNAs determined by measuring at least one reference embryonic culture medium of (obtained from) a pre-implanted embryo known to not lead to pregnancy in a female. In this case, it is preferred that the total number of miRNAs being about the same as the reference total number of miRNAs. This indicates that the pre-implanted embryo will not lead to pregnancy in a female.

Preferably, said reference embryonic culture media are at least 2 reference embryonic culture media, more preferably at least 2 to 100 reference embryonic culture media, even more preferably at least 10 to 500 reference embryonic culture media, and most preferably at least 50 to 10.000 reference embryonic culture media, e.g. at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 150, 200, 250, 300, 400, 500, 1.000, 2.000, 3.000, 4.000, 5.000, or 10.000 reference embryonic culture media. In a particular embodiment, the reference total number of miRNAs, which is the total number of miRNAs determined by measuring at least one reference embryonic culture medium of (obtained from) a pre-implanted embryo known to not lead to pregnancy in a female, is 163. Thus, in a preferred embodiment, the total number of miRNAs being about 163 indicates that the pre-implanted embryo will not lead to pregnancy in a female.

The skilled person knows how to determine the total number of miRNAs. For example, the total number of miRNA may be determined by nucleic acid hybridization, nucleic acid amplification, polymerase extension, sequencing, mass spectroscopy, a immunochemical method, or any combination thereof. Preferably,

(i) the nucleic acid hybridization is performed using a microarray/biochip, or using in situ hybridization,

(ii) the nucleic acid amplification is performed using real-time PCR (RT-PCR) or quantitative real-time PCR (qPCR),

(iii) the sequencing is next generation sequencing, or

(iv) the immunochemical method is an enzyme linked immunosorbent assay (ELISA).

The second aspect of the present invention may alternatively be worded as follows: A method for predicting whether a pre-implanted embryo will not lead to pregnancy in a female comprising the steps of:

(i) determining the presence of at least one miRNA, e.g. at least 1, 2, 3, 4, 5, 6, or 7 miRNA(s), selected from the group consisting of SEQ ID NO: 102, SEQ ID NO: 104 to SEQ ID NO: 109, and a sequence having at least 80%, preferably at least 85%, more preferably at least 90%>, and most preferably at least 95%> or 99%>, e.g. at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity thereto in an embryonic culture medium of (obtained from) a pre-implanted embryo, wherein the presence of said at least one miRNA indicates that the pre-implanted embryo will not lead to pregnancy in a female, and/or

(ii) determining the absence of miRNA of SEQ ID NO: 103 or a sequence having at least 80% sequence identity thereto in an embryonic culture medium of (obtained from) a pre-implanted embryo,

wherein the absence of said miRNA indicates that the pre-implanted embryo will not lead to pregnancy in a female.

All preferred combinations of 2, 3, 4, 5, 6, or 7 miRNAs selected from the group consisting of SEQ ID NO: 102 and SEQ ID NO: 104 to SEQ ID NO: 109 as well as the single miRNAs referred to in item (i) are comprised in Figure 7. See also Figure 3. The presence of miRNA of SEQ ID NO: 104 or a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, e.g. at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity thereto in the embryonic culture medium preferably indicates that the pre- implanted embryo will not lead to pregnancy in a female.

The skilled person knows how to determine the total number of miRNAs. For example, the total number of miRNA may be determined by nucleic acid hybridization, nucleic acid amplification, polymerase extension, sequencing, mass spectroscopy, a immunochemical method, or any combination thereof. Preferably,

(i) the nucleic acid hybridization is performed using a microarray/biochip, or using in situ hybridization,

(ii) the nucleic acid amplification is performed using real-time PCR (RT-PCR) or quantitative real-time PCR (qPCR),

(iii) the sequencing is next generation sequencing, or

(iv) the immunochemical method is an enzyme linked immunosorbent assay (ELISA).

The third aspect of the present invention may alternatively be worded as follows: A method for predicting whether a pre-implanted embryo will not lead to pregnancy in a female comprising the steps of:

(i) determining the level of at least one miRNA, e.g. at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or 101 miRNA(s), selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 101, and a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, e.g. at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity thereto present in an embryonic culture medium of (obtained from) a pre-implanted embryo, and

(ii) comparing the level of the at least one miRNA to a reference level of said at least one miRNA.

More specifically, the present invention alternatively relates to a method for predicting whether a pre-implanted embryo will not lead to pregnancy in a female comprising the steps of:

(i) determining the level of at least one miRNA, e.g. at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or 101 miRNA(s), selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 101, and a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, e.g. at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity thereto present in an embryonic culture medium of (obtained from) a pre-implanted embryo, and (ii) comparing the level of the at least one miRNA to a reference level of said at least one miRNA,

wherein the comparison of the level of the at least one miRNA to the reference level of said at least one miRNA allows to predict whether the pre-implanted embryo will not lead to pregnancy in a female.

Preferably, the at least one miRNA, e.g. at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 miRNA(s), is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 50, and a sequence having at least 80%, preferably at least 85%o, more preferably at least 90%>, and most preferably at least 95%> or 99%>, e.g. at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity thereto (see Figure 5).

More preferably, the at least one miRNA, e.g. 1, 2, 3, 4, 5, or 6 miRNA(s), is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 6, and a sequence having at least 80%), preferably at least 85%>, more preferably at least 90%>, and most preferably at least 95%> or 99%, e.g. at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%), sequence identity thereto. All preferred combinations of 2, 3, 4, 5, or 6 miRNAs selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 6 as well as the single miRNAs are comprised in Figure 6.

The reference level may be any level which allows to predict whether a pre-implanted embryo will not lead to pregnancy in a female.

In one embodiment, the the reference level is the level determined by measuring at least one reference embryonic culture medium of (obtained from) a pre-implanted embryo known to lead to pregnancy in a female.

In this case, it is preferred that the level of the at least one miRNA selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3 to SEQ ID NO: 7, SEQ ID NO: 9 to SEQ ID NO: 12, SEQ ID NO: 14 to SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30 to SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 40 to SEQ ID NO: 44, SEQ ID NO: 46 to SEQ ID NO: 48, and a sequence having at least 80% sequence identity thereto is above the reference level, which indicates that the pre- implanted embryo will not lead to pregnancy in a female, and/or

the level of the at least one miRNA selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO: 21 to SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 50, and a sequence having at least 80% sequence identity thereto is below the reference level, which indicates that the pre-implanted embryo will not lead to pregnancy in a female.

Preferably, the level of the at least one miRNA is at least 0.4 fold or at least 0.6 fold above/below the reference level, more preferably at least 1.1 -fold or at least 1.2-fold above/below the reference level, even more preferably at least 1.5-fold or at least 2-fold above/below the reference level, and most preferably at least 2.5-fold above/below the reference level.

In one alternative embodiment, the reference level is the level determined by measuring at least one reference embryonic culture medium of (obtained from) a pre-implanted embryo known to not lead to pregnancy in a female.

In this case, it is preferred that

the level of the at least one miRNA selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3 to SEQ ID NO: 7, SEQ ID NO: 9 to SEQ ID NO: 12, SEQ ID NO: 14 to SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30 to SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 40 to SEQ ID NO: 44, SEQ ID NO: 46 to SEQ ID NO: 48, and a sequence having at least 80% sequence identity thereto being about the same as the reference level, which indicates that the pre-implanted embryo will not lead to pregnancy in a female, and/or

the level of the at least one miRNA selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO: 21 to SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 50, and a sequence having at least 80% sequence identity thereto being about the same as the reference level, which indicates that the pre- implanted embryo will not lead to pregnancy in a female.

The skilled person knows how to determine the total number of miR As. For example, the total number of miRNA may be determined by nucleic acid hybridization, nucleic acid amplification, polymerase extension, sequencing, mass spectroscopy, a immunochemical method, or any combination thereof. Preferably,

(i) the nucleic acid hybridization is performed using a microarray/biochip, or using in situ hybridization,

(ii) the nucleic acid amplification is performed using real-time PCR (RT-PCR) or quantitative real-time PCR (qPCR),

(iii) the sequencing is next generation sequencing, or

(iv) the immunochemical method is an enzyme linked immunosorbent assay (ELISA).

The fourth aspect of the present invention may alternatively be worded as follows: (Use, e.g. in vitro use of) a kit for predicting whether a pre-implanted embryo will not lead to pregnancy in a female comprising

(i) means for

(ia) determining the total number of miRNAs,

(ib) determining the presence of at least one miRNA, e.g. 1, 2, 3, 4, 5, 6, or 7 miRNA(s), selected from the group consisting of SEQ ID NO: 102, SEQ ID NO: 104 to SEQ ID NO: 109, and a sequence having at least 80%, preferably at least

85%, more preferably at least 90%>, and most preferably at least 95% or 99%, e.g. at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%), sequence identity thereto and/or the absence of miRNA of SEQ ID NO: 103 or a sequence having at least 80%>, preferably at least 85%, more preferably at least 90%>, and most preferably at least 95% or 99%, e.g. at least

80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity thereto, and/or

(ic) determining the level of at least one miRNA, e.g. at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,

52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or 101 miRNA(s), selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 101, and a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, e.g. at least 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity thereto

in an embryonic culture medium of (obtained from) a pre-implanted embryo, and (ii) optionally a reference.

The reference may be any reference which allows the prediction whether a pre- implanted embryo will not lead to pregnancy in a female. For example, said reference may be a reference level and/or a reference total number of miR As (see first to third aspect of the present invention).

The kit may further comprise a container and/or a data carrier. The data carrier may be a non-electronical data carrier, e.g. a graphical data carrier such as an information leaflet, an information sheet, a bar code or an access code, or an electronical data carrier such as a floppy disk, a compact disk (CD), a digital versatile disk (DVD), a microchip or another semiconductor-based electronical data carrier. The access code may allow the access to a database, e.g. an internet database, a centralized, or a decentralized database. The access code may also allow access to an application software that causes a computer to perform tasks for computer users or a mobile app which is a software designed to run on smartphones and other mobile devices.

Said data carrier may further comprise the at least one reference, e.g. the reference level of the level of the at least one miR A determined herein and/or the reference total number of miRNAs determined herein. In case that the data carrier comprises an access code which allows the access to a database, said at least one reference, e.g. said reference level and/or said reference total number of miRNAs, may be deposited in this database.

Preferably, the means in (ia), (ib), and/or (ic) comprise

at least one polynucleotide (probe),

at least one primer pair, and/or

at least one polynucleotide (probe) and at least one antibody capable of binding a hybrid of said at least one polynucleotide (probe) and said at least one miRNA.

Said means allow to determine the total number of miRNAs, the presence/absence of the above- mentioned miRNAs and/or the level of the above-mentioned miRNAs.

It is further preferred that

(i) the at least one polynucleotide is complementary to the at least one miRNA mentioned above, or (ii) the at least one polynucleotide has at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, e.g. at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity to the polynucleotide according to (i).

The above means may also comprise a microarray/biochip, a RT-PCT system, a PCR- system, a flow cytometer, a bead-based multiplex system or a next generation sequencing system. The at least one polynucleotide may be part of the microarray/biochip or may be attached to the beads of the beads-based multiplex system.

Said kit may also comprise materials desirable from a commercial and user standpoint including a buffer(s), a reagent(s) and/or a diluent(s) for determining the level mentioned above.

Preferably, the miRNAs in (ia) are selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 619, and a sequence having at least 80%>, preferably at least 85%, more preferably at least 90%>, and most preferably at least 95% or 99%, e.g. at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity thereto.

Preferably, the miRNA(s) in (ic) is (are) selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 50, and a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, e.g. at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity thereto. More preferably, the miRNA(s) in (ic) is (are) selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 6, and a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, e.g. at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity thereto.

It is particularly preferred that the kit is useful for conducting the methods according to the alternative first to third aspect.

It is also particularly preferred that the kit allows a point-of-care testing (POCT).

In a fifth aspect, the present invention relates to a method for predicting whether a pre- implanted embryo will be embedded into the endometrium of a female comprising the steps of:

(i) determining the total number of microvesicles present in an embryonic culture medium of (obtained from) a pre-implanted embryo, and

(ii) comparing the total number of microvesicles to a reference total number of microvesicles.

More particularly, the fifth aspect of the present invention relates to a method for predicting whether a pre-implanted embryo will be embedded into the endometrium of a female comprising the steps of: (i) determining the total number of microvesicles present in an embryonic culture medium of (obtained from) a pre-implanted embryo, and

(ii) comparing the total number of microvesicles to a reference total number of microvesicles,

wherein the comparison of said total number of microvesicles to the reference total number of microvesicles allows to predict whether the pre-implanted embryo will be embedded into the endometrium of a female.

The present inventors surprisingly found that already the determination of the total number of microvesicles shed from the cells of a pre-implanted embryo into an embryonic culture medium allows the prediction whether a pre-implanted embryo will be embedded into the endometrium of a female or not. In particular, the present inventors found that the total number of microvesicles is lower in embryonic culture medium obtained from embryos which embedded into the endometrium of a female compared to the total number of microvesicles determined in the embryonic culture medium obtained from embryos which not embedded into the endometrium of a female. As mentioned above, microvesicles play a role in intercellular communication and can transport mRNA, miR A, and proteins between cells. Thus, without being bound to any theory, their appears to be a correlation between the total number of miR As and the number of microvesicles found. Accordingly, already the determination of the total number of microvesicles is highly suitable to predict, whether a pre-implanted embryo in question will be embedded into the endometrium of a female or not.

The total number of microvesicles is preferably determined in the embryonic culture medium by taking a sample of said medium, e.g. using a pipette. The sample size may vary between 0.5 and 2 ml, e.g. 0.5, 1. 1.5, or 2 ml. The sampling is preferably performed around the time point of embryo transfer, e.g. at day 3, 4, 5, or 6 after in-vitro-fertilization, into the uterus of a female. The above analysis allows the prediction, if the pre-implanted embryo of a female or which of the pre-implanted embryos of a female will be embedded into the endometrium of a female and, thus, should be used for embryo transfer. The determination of the number of microvesicles can be performed, for example, using visually means.

The reference total number of microvesicles may be any number which allows to predict whether a pre-implanted embryo will be embedded into the endometrium of a female or not.

In one embodiment, the reference total number of microvesicles is the total number of microvesicles determined by measuring at least one reference embryonic culture medium of (obtained from) a pre-implanted embryo for which it is known that said embryo not embedded into an endometrium of a female. In this case, it is preferred that the total number of microvesicles is below the reference total number of micro vesicles. This indicates that the pre- implanted embryo will be embedded into the endometrium of a female.

Preferably, said reference embryonic culture media are at least 2 reference embryonic culture media, more preferably at least 2 to 100 reference embryonic culture media, even more preferably at least 10 to 500 reference embryonic culture media, and most preferably at least 50 to 10.000 reference embryonic culture media, e.g. at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 150, 200, 250, 300, 400, 500, 1.000, 2.000, 3.000, 4.000, 5.000, or 10.000 reference embryonic culture media.

Preferably, the embryonic culture medium and the reference embryonic culture medium

(media) have the same composition and are cultured under identical conditions.

Preferably, the total number of microvesicles is at least 0.5-fold, more preferably at least 1-fold, even more preferably at least 1.5-fold, e.g. at least 0.5-fold, 0.6-fold, 0.7-fold, 0.8-fold, 0.9- fold, 1-fold, 1.1 -fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, or 1.9 fold, below the reference total number of microvesicles.

In a particular embodiment, the reference total number of microvesicles, which is the total number of microvesicles determined by measuring at least one reference embryonic culture medium of (obtained from) a pre-implanted embryo for which it is known that said embryo not embedded into an endometrium of a female, is 7.35 billion microparticles per ml. Thus, in a preferred embodiment, the total number of microvesicles is at least 0.5-fold, more preferably at least 1-fold, even more preferably at least 1.5-fold, e.g. at least 0.5-fold, 0.6-fold, 0.7-fold, 0.8-fold, 0.9-fold, 1-fold, 1.1 -fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7- fold, or 1.9 fold, below 7.35 billion microparticles per ml (as reference total number of microvesicles) indicating that the pre-implanted embryo will be embedded into the endometrium of a female.

In one alternative embodiment, the reference total number of microvesicles is the total number of microvesicles determined by measuring at least one reference embryonic culture medium of (obtained from) a pre-implanted embryo for which it is known that said embryo embedded into an endometrium of a female. In this case, the total number of microvesicles being about the same as the reference total number of microvesicles. This indicates that the pre- implanted embryo will be embedded into the endometrium of a female. Preferably, said reference embryonic culture media are at least 2 reference embryonic culture media, more preferably at least 2 to 100 reference embryonic culture media, even more preferably at least 10 to 500 reference embryonic culture media, and most preferably at least 50 to 10.000 reference embryonic culture media, e.g. at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 150, 200, 250, 300, 400, 500, 1.000, 2.000, 3.000, 4.000, 5.000, or 10.000 reference embryonic culture media.

Preferably, the embryonic culture medium and the reference embryonic culture medium (media) have the same composition and are cultured under identical conditions.

In a particular embodiment, the reference total number of micro vesicles, which is the total number of microvesicles determined by measuring at least one reference embryonic culture medium of (obtained from) a pre-implanted embryo for which it is known that said embryo embedded into an endometrium of a female, is 3.8 billion microparticles per ml. Thus, in a preferred embodiment, the total number of microparticles being about 3.8 billion microparticles per ml indicates that the pre-implanted embryo will be embedded into the endometrium of a female. The term "being about the same'V'about" is defined above.

In a further aspect, the present invention elates to a kit for predicting whether a pre- implanted embryo will be embedded into the endometrium of a female comprising

(i) means for determining the total number of microparticles in an embryonic culture medium of (obtained from) a pre-implanted embryo, and

(ii) optionally at least one reference.

The reference may be any reference which allows the prediction whether a pre- implanted embryo will be embedded into the endometrium of a female or not. For example, said reference may be a reference total number of microparticles (see fifth aspect of the present invention). Regarding the further specific embodiments of the kit, it is referred to the fourth aspect of the present invention.

All methods of the present invention may also be designated as in vitro methods.

Various modifications and variations of the invention will be apparent to those skilled in the art without departing from the scope of invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art in the relevant fields are intended to be covered by the present invention.

BRIEF DESCRIPTION OF THE FIGURES The following figures and examples are merely illustrative of the present invention and should not be construed to limit the scope of the invention as indicated by the appended claims in any way.

Figure 1: List of all miRNAs. The miRNA name as annotated in miRBase V21, the respective SEQ ID NO and the sequence for this miRNA according to miRBase V21 is listed.

Figure 2: Experimental results for all miRNAs. Listed are the miRNA name, the SEQ ID NO, the median for this miRNA in group 1 (not fertile), the median expression in group 2 (fertile), the standard deviation in group 1, the standard deviation in group 2, the fold change, the raw significance value of a 2-tailed un-paired t-test, the adjusted p-value of the t-test.

Figure 3 : Percentage of detected miRNAs (dark) that versus not detected miRNAs (tale) in negative samples (left bar each) and positive samples (right bar each). All results are significant according to fishers test p-value (alpha level of 0.05).

Figure 4: Histograms of raw p-values, adjusted p-values and the AUC values for the comparison of 619 miRNAs in embryonic culture media where oocytes did not lead to pregnancy versus oocytes leading to pregnancy. Solid black lines in the first two histograms denote the 0.05 alpha level, in the right histogram AUC of 0.25 to 0.75. Black dotted line represents miRNAs with AUC of 0.5.

Figure 5: Heat map showing miRNA abundance in positive samples (bar above the heat map in dark) and negative samples (bar above the heat map in tale).

Figure 6: Summary of all combinations of 2, 3, 4, 5, or 6 miRNAs selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 6 as well as the single miRNAs which level can be determined.

Figure 7: Summary of all combinations of 2, 3, 4, 5, 6, or 7 miRNAs selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4 to SEQ ID NO: 9 as well as the single miRNAs which presence/absence can be determined. EXAMPLES

The examples given below are for illustrative purposes only and do not limit the invention described above in any way.

Materials and Methods

Study Population and Sample Collection

The study was approved by the Institutional Review Board (Nr. 183/14) of the University Hospital of Saarland. Informed consent was obtained from each of the participants. Embryonic culture media were collected from 56 reproductive-age female partners of selected couples undergoing oocyte retrieval for assisted reproduction techniques and infertility treatment, in addition to control media (CSC-Medium, Irvine Scientific, USA) that were used for comparison analysis.

Total RNA, Including miRNAs Isolation

Total RNA, including miRNAs was purified from embryonic collected culture media using miRNeasy Micro Kit (Qiagen) according to the manufacturer's instructions. The aenorhabditis elegans (C. elegans) miR-39 mimic was added to each isolation as an internal spike-in control using Spike-In Control (Qiagen). Small RNA Analysis Kit on the Bioanalyzer 2100 (Agilent Technologies) was used to resolve and quantify the small nucleic acid fraction of extracted RNAs and concentration and purity were measured using NanoDrop 2000 (Thermo Scientific). To remove any DNA contamination, DNase I (Ambion) treatment was carried out according to the manufacturer's instructions. Conventional PCR with exon spanning primers for GAPDH (forward: 5 '-CGACC ACTTTGTC AAGCTC A-3 ' (SEQ ID NO: 620); reverse: 5 ^* - AGGGGTCT AC ATGGC AACTG-3 ' (SEQ ID NO: 621)) was performed to exclude residual DNA in the samples (26).

MiRNA Microarray Analysis

MiRNA expression profiles of 56 embryonic collected culture media samples were established by applying human miRNA microarrays (Agilent Technologies), containing probes corresponding to 2549 human miRNAs. These microarrays contain about 40 replicates for each probe complement to each of the 2549 mature miRNAs of miRBase v21. All procedures were carried out according to the manufacturer's instructions. Microarray hybridizations were done following the manufacture protocol. Briefly, lOOng of total RNA from each embryonic sample was dephosphorylated with Calf Intestinal Alkaline Phosphatase (CIP) at 37°C for 30 minutes following a denaturalization and then a ligation for 2 hours at 16°C using Agilent miRNA Complete Labeling and Hyb Kit (Agilent Technologies). In this step, a molecule of Cyanine 3- pCp is incorporated into the 3 '-end of RNA molecules. Labeled RNA will be dried and resuspended with Hybridization Buffer and Blocking Agent, incubated 10 minutes at 100°C and transferred to an ice water bath for 5 minutes. Samples were hybridized in a volume of 45μ1 to the SurePrint G3 Human v21 miRNA Array (Agilent Technologies) for 20 hours at 55°C and 20rpm. Microarrays were then washed at room temperature for 5 minutes in Gene Expression Wash Buffer 1 and 5 minutes at 37°C in Gene Expression Wash Buffer 2. Arrays were scanned on an Agilent G2565BA microarray scanner under the default settings recommended by Agilent Technologies for miRNA microarrays with 100% PMT and 5μιη resolution. Data were extracted using the Agilent Feature Extraction Software (Agilent Technologies). Reverse Transcription and qRT-PCR of miRNA

The expression level of miRNAs in embryonic culture media was determined for all 56 samples using the miScript SYBR Green Kit (Qiagen) by RT-qPCR. RNA (50 ng) was reverse transcribed using the miScript Reverse Transcription kit (Qiagen) according to the manufacturer's recommendations. The resulted cDNA was then diluted 1 :5 and 1 of cDNA was mixed with 10 \, ΊΧ QuantiTect SYBR Green PCR Master Mix, 2 10X miScript Universal Primer, 2 10X miScript Primer Assay for 6 selected miRNAs (hsa-miR-29c-3p (SEQ ID NO: 1), hsa-miR-566 (SEQ ID NO: 2), hsa-miR-22-5p (SEQ ID NO: 3), hsa-miR- 6812-5p (SEQ ID NO: 4), hsa-let-7c-5p (SEQ ID NO: 5) and hsa-miR-6076 (SEQ ID NO: 6)) in a total volume of 20 Hsa-miR-16-2 and Ce miR-39 1 miScript Primer Assays were used as an endogenous control (Qiagen) for normalization analysis. Reactions were run on a StepOnePlus Real-Time PCR System (Applied Bio systems) with the following thermal cycling parameters: initial activation step 95°C for 15 minutes followed by 40 cycles at 94°C for 15 seconds (denaturation), 55°C for 30 seconds (annealing), and 70°C for 30 seconds (extension). Then final dissociation curves (melting curves) were made.

Statistical Analysis

The raw microarray data have been processed using the freely available R statistical programming environment (version 3.2.4. for MacOS). MiRNAs were considered as detected according to the detection flag provided by Agilent's detection flag, relying to a significant intensity signal above the background. These are denominated as present calls. To account for global variations between microarrays, quantile normalization was carried out and not detected miRNAs were subsequently eliminated. To find miRNAs that are more frequently present in the one or the other group (only the binary signal 'present' versus 'absent'), Fishers Exact test was applied to the 2x2 contingency table, containing present calls for each miRNA in both groups. For comparing groups, hypothesis tests were performed. Since not all miRNAs were normally distributed, significance values not only for the parametric t-test but also for the non- parametric Wilcoxon-Mann Whitney test were calculated. If not mentioned explicitly, p-values were subjected to adjustment for multiple testing applying the Benjamini-Hochberg approach. Besides hypothesis tests, the area under the receiver operator characteristic curve (AUC value) was also calculated. As cluster approach, hierarchical clustering on the Euclidian distance has been done. Before calculating the distances, miRNAs have been normalized to z-scores.

Results

MiRNA counts in culture media following embryo transfer

The miRNA repertoire of 56 embryonic culture media after embryos were transferred has been profiled. In 39 cases the embryo transfer did not lead to a pregnancy while in 17 cases it lead to positive pregnancy. First, the total number of miRNAs that are found in embryonic culture media was determined. According to the analysis, a total of 619 different human miRNAs were present in the embryonic culture media samples (see Figure 2). Since this number is above the estimation and even within the range of miRNome complexity in most human organs and larger than in most body fluids (27), the question was raised whether the complexity of the miRNome differs between samples leading to pregnancy as compared to negative samples. Per patient with negative result, 163 miRNAs were detected on average while only 149 miRNAs were found per sample with positive pregnancy result. Since these results indicate an overall decreased repertoire of miRNAs in patients with positive result, the further question was raised whether specific miRNAs are present to a different percentage in both groups. Specifically, 2x2 contingency tables for each miRNA were calculated, containing the number of positive and negative samples with the miRNA being present or absent. Significance values were obtained by Fishers Exact test. The best separation was obtained for miR-634 (SEQ ID NO: 104). The absence of this single miRNA predicted with accuracy of 71% at a sensitivity of 85% whether embryo transfer would lead to a positive pregnancy. The results for eight markers with p-values below 0.05 prior to adjustment are presented in Figure 3. In line with the previous finding that culture media from embryos leading to a pregnancy following implantation have a decreased repertoire of miRNAs, seven of the eight markers presented in Figure 3 are present to a statistically significant lower percentage in such samples. Already the presence of single miRNAs was thus correlated to successful outcome. MiRNA abundance in embryonic culture media following embryo transfer

Next, the question was raised, beyond the consideration of present and absent miRNAs, whether the miRNA expression intensity is likewise correlated to positive pregnancy. By performing t- test (un-paired twotailed), 101 of 619 miRNAs (16.4%) were significant at an alpha level of 0.05. Even following adjustment for multiple testing, miR-29c-3p (SEQ ID NO: 1) remained significant. Of these 101, 69 (68.6%) had significantly lower expression intensity in samples from embryos leading to pregnancy, corresponding to the findings in the previous section. Figure 4 presents histograms showing the distribution of raw- and adjusted p-values as well as the AUC values. To generate a graphical representation, the most significantly dysregulated miRNAs were clusted. The resulting heat map with dendrograms on top and bottom can be found in Figure 5. On the right hand side, three clusters of miRNAs were observed, two containing down-regulated miRNAs and one up-regulated miRNAs in embryonic culture media of embryos leading to pregnancy. On the bottom, two clusters of samples were found. The left cluster contains 27 samples of which 25 come from negative samples while in the right cluster, a mixture between negative and positive samples was observed. The best marker was miR-29c- 3p (SEQ ID NO: 1) with raw- and adjusted p-value of 3.1x10-5 and 0.019 and an AUC value of 0.83. Expression values for all miRNAs are presented in Figure 2.

Validation of selected miRNAs by RT-qPCR

Since microarray data - as well as other high-throughput methods such as next generation sequencing or high-throughput RT-PCR - likely lead to false positive findings, a subset of miRNAs by using single RT-qPCR assays was validated. Six miRNAs (let-7c-5p (SEQ ID NO: 5), miR-22-5p (SEQ ID NO: 3), miR-29c-3p (SEQ ID NO: 1), miR-566 (SEQ ID NO: 2), miR- 6076 (SEQ ID NO: 6) and miR-6812-5p (SEQ ID NO: 4)) were tested and miR-16 and miR-39 were evaluated as endogenous controls.

In comparing CT values (without endogenous control) and delta CT values for the 6 miRNAs with both endogenous controls we observed very concordant results. The effects for all miRNAs were strongest by using miR-39 as endogenous control. In sum, RT-qPCR matched generally well to microarray results, most importantly, the general pattern of lower expression in samples leading to pregnancy was verified. Microvesicle distribution

In a further approach to understand the source of miRNAs that are present or over-expressed to a larger or lower fraction in fertile, dynamic light scattering was applied to measure the number and size distribution of microvesicles in embryonic culture media. On eight samples it was observed that the size distribution between fertile and non- fertile samples showed already slight variations. The most striking differences were observed in the total number of microvesicles. For fertile samples a median of 3.8 billion particles per ml was measured, for infertile the number was 1.93-fold increased (7.35 billion particle per ml). This indicates that microvesicles are a potential source of the miRNAs that are found to a significant fraction to be higher expressed in embryonic culture media. Thus, measuring the repertoire of microvesicles represents an alternative to determining the miRNAs as described above.

Table 1 : Microvesicle distribution

Discussion

In the present study, miRNA microarray and real-time qRT-PCR validation analyses were used to identify the miRNA which were contained in the embryonic culture media of couples undergoing fertility treatment. A total of 619 miRNAs were identified in a total 56 samples. Of which, 149 and 163 miRNAs were detected on average in each female with positive and negative pregnancies, respectively. In the line of these detected miRNAs, the embryonic culture media from embryos leading to a pregnancy following implantation have a decreased repertoire of miRNAs. In addition, in particular miR-634 (SEQ ID NO: 104) with an accuracy of 71% and a sensitivity of 85% was correlated to a successful outcome. Based on the miRNA expression intensity of the detected 619 miR As, in particular miR-29c-3p (SEQ ID NO: 1) with an AUC value of 0.83 was correlated to positive pregnancy.

Summary

In summary, the results founded on the identification of miRNAs which contained in the embryonic culture media of couples undergoing fertility treatment. Although, the embryonic culture media from embryos leading to a pregnancy following implantation have a decreased repertoire of miRNAs, in particular miR-634 (SEQ ID NO: 104) and miR-29c-3p (SEQ ID NO: 1), were correlated to the successful pregnancy outcome (accuracy of 71% and sensitivity of 85%)) and positive pregnancy (AUC value of 0.83), respectively.

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