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WO2018096084A1 - Micro-arn en tant que biomarqueur en fécondation in vitro - Google Patents

Micro-arn en tant que biomarqueur en fécondation in vitro Download PDF

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Publication number
WO2018096084A1
WO2018096084A1 PCT/EP2017/080312 EP2017080312W WO2018096084A1 WO 2018096084 A1 WO2018096084 A1 WO 2018096084A1 EP 2017080312 W EP2017080312 W EP 2017080312W WO 2018096084 A1 WO2018096084 A1 WO 2018096084A1
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seq
mirnas
female
mirna
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Andreas Keller
Eckart Meese
Jan Kirsten
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Universitaet des Saarlandes
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Universitaet des Saarlandes
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • 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.
  • miRNAs small non-coding RNAs
  • microRNAs small non-coding RNAs
  • 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).
  • miRNAs 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).
  • NO A non-obstructive azoospermia
  • 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.
  • 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.
  • 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
  • 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:
  • 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
  • the present invention relates to a kit for predicting whether a pre- implanted embryo will be embedded into the endometrium of a female comprising
  • 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:
  • 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).
  • 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.
  • 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.
  • 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.
  • RNA-induced silencing complex RNA-induced silencing complex
  • 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 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”.
  • microRNA* refers to single-stranded RNA molecules of at least 10 nucleotides and of not more than 35 nucleotides covalently linked together.
  • 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).
  • 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.
  • miRBase 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.
  • nucleotides 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.
  • 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.
  • -CH2-OH primary alcohol group
  • polynucleotide means a molecule of at least 10 nucleotides and of not more than 35 nucleotides covalently linked together.
  • 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.
  • polynucleotide means a polymer of deoxyribonucleotide or ribonucleotide bases and includes DNA and RNA molecules, both sense and anti-sense strands.
  • 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.
  • 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.
  • a target of complementary sequence such as a nucleotide sequence of a miRNA or miRNA*
  • 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.
  • nucleotide sequence of a miRNA or miRNA* 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*
  • EL elongation
  • 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.
  • 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.
  • the polynucleotide hybrid/tandem as a probe may comprise a spacer (SP) element.
  • label means a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means.
  • 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.
  • stringent hybridization conditions 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.
  • 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.
  • antisense refers to nucleotide sequences which are complementary to a specific DNA or R A sequence.
  • 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 .
  • fertilization 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).
  • assisted reproductive technology such as artificial insemination and in-vitro-fertilization (IVF).
  • ART assisted reproductive technology
  • IUI intrauterine insemination
  • IVF in-vitro-fertilization
  • ICSI intracytoplasmic sperm injection
  • in-vitro-fertilization 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.
  • pre-implanted embryo 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).
  • oocyte 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.
  • blastocyst 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.
  • 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).
  • 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.
  • embryo transfer 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).
  • 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.
  • 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.
  • gestational age 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).
  • the hormone human chorionic gonadotropin (hCG) level is, for example, not increased.
  • female may be a human female or another mammal.
  • embryonic culture refers 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).
  • level 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.
  • concentration e.g. absolute or relative concentration
  • level also comprises scaled, normalized, or scaled and normalized amounts or values.
  • the level determined herein is the expression level.
  • sensitivity refers to the number of true positive patients (%) with regard to the number of all patients (100%).
  • accuracy 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.
  • AUC relates to an abbreviation for the area under a curve. In particular, it refers to the area under a Receiver Operating Characteristic (ROC) curve.
  • ROC Receiver Operating Characteristic
  • ROC Receiver Operating Characteristic
  • 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.
  • POCT point-of-care testing
  • microvesicle 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.
  • 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.
  • 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:
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • the total number of miRNA may be determined by nucleic acid hybridization, nucleic acid amplification, polymerase extension
  • nucleic acid hybridization is performed using a microarray/biochip, or using in situ hybridization
  • the nucleic acid amplification is performed using real-time PCR (RT-PCR) or quantitative real-time PCR (qPCR),
  • the sequencing is next generation sequencing, or
  • the immunochemical method is an enzyme linked immunosorbent assay (ELISA).
  • 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:
  • 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.
  • 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.
  • 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.
  • 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.
  • sequence identity thereto in the embryonic culture medium preferably indicates that the pre-implanted embryo will be embedded into the endometrium of a female.
  • 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 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.
  • nucleic acid hybridization nucleic acid amplification
  • polymerase extension polymerase extension
  • sequencing mass spectroscopy
  • mass spectroscopy a immunochemical method, or any combination thereof.
  • nucleic acid hybridization is performed using a microarray/biochip, or using in situ hybridization
  • the nucleic acid amplification is performed using real-time PCR (RT-PCR) or quantitative real-time PCR (qPCR),
  • the sequencing is next generation sequencing, or
  • the immunochemical method is an enzyme linked immunosorbent assay (ELISA).
  • ELISA enzyme linked immunosorbent assay
  • 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
  • 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:
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • nucleic acid hybridization is performed using a microarray/biochip, or using in situ hybridization
  • the nucleic acid amplification is performed using real-time PCR (RT-PCR) or quantitative real-time PCR (qPCR),
  • the sequencing is next generation sequencing, or
  • the immunochemical method is an enzyme linked immunosorbent assay (ELISA).
  • the aforesaid real time polymerase chain reaction 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.
  • PCR polymerase chain reaction
  • RT-PCR real time polymerase chain reaction
  • RT qPCR real time quantitative PCR
  • 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 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.
  • 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.
  • 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.
  • 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).
  • 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
  • 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:
  • SEQ ID NO: 109 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.
  • 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,
  • 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.
  • 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.
  • said at least one reference e.g. said reference level and/or said reference total number of miRNAs, may be deposited in this database.
  • the means in (ia), (ib), and/or (ic) comprise
  • At least one polynucleotide (probe),
  • At least one primer pair at least one primer pair, and/or
  • 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.
  • the at least one polynucleotide is complementary to the at least one miRNA mentioned above, or
  • 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 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).
  • polynucleotide as defined in (ii) 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.
  • a polynucleotide as defined in (ii) i.e. polynucleotide variant
  • 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.
  • 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.
  • the miRNAs in (ia) are selected from the group consisting of SEQ ID NO:
  • 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.
  • 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.
  • kit is useful for conducting the methods according to the first to third aspect.
  • kit allows a point-of-care testing (POCT).
  • POCT point-of-care testing
  • 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:
  • 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:
  • 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.
  • 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.
  • 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.
  • the total number of miRNAs is at least 5%, more preferably at least 8%, e.g. at least
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the total number of miRNAs being about 163 indicates that the pre-implanted embryo will not lead to pregnancy in a female.
  • the total number of miRNAs may be determined by nucleic acid hybridization, nucleic acid amplification, polymerase extension, sequencing, mass spectroscopy, a immunochemical method, or any combination thereof.
  • 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.
  • nucleic acid hybridization is performed using a microarray/biochip, or using in situ hybridization
  • the nucleic acid amplification is performed using real-time PCR (RT-PCR) or quantitative real-time PCR (qPCR),
  • the sequencing is next generation sequencing, or
  • 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:
  • 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.
  • sequence identity thereto in the embryonic culture medium preferably indicates that the pre- implanted embryo will not lead to pregnancy in a female.
  • the total number of miRNAs may be determined by nucleic acid hybridization, nucleic acid amplification, polymerase extension, sequencing, mass spectroscopy, a immunochemical method, or any combination thereof.
  • 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.
  • nucleic acid hybridization is performed using a microarray/biochip, or using in situ hybridization
  • the nucleic acid amplification is performed using real-time PCR (RT-PCR) or quantitative real-time PCR (qPCR),
  • the sequencing is next generation sequencing, or
  • 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:
  • 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
  • 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:
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • nucleic acid hybridization is performed using a microarray/biochip, or using in situ hybridization
  • the nucleic acid amplification is performed using real-time PCR (RT-PCR) or quantitative real-time PCR (qPCR),
  • the sequencing is next generation sequencing, or
  • 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
  • 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
  • RNA 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,
  • 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.
  • 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.
  • said at least one reference e.g. said reference level and/or said reference total number of miRNAs, may be deposited in this database.
  • the means in (ia), (ib), and/or (ic) comprise
  • At least one polynucleotide (probe),
  • At least one primer pair at least one primer pair, and/or
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • kit is useful for conducting the methods according to the alternative first to third aspect.
  • kit allows a point-of-care testing (POCT).
  • POCT point-of-care testing
  • 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:
  • 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
  • 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.
  • 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.
  • microvesicles play a role in intercellular communication and can transport mRNA, miR A, and proteins between cells.
  • 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.
  • 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.
  • 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.
  • the embryonic culture medium and the reference embryonic culture medium are preferably, the embryonic culture medium and the reference embryonic culture medium
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the embryonic culture medium and the reference embryonic culture medium (media) have the same composition and are cultured under identical conditions.
  • 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.
  • 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.
  • the present invention elates to a kit for predicting whether a pre- implanted embryo will be embedded into the endometrium of a female comprising
  • 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.
  • said reference may be a reference total number of microparticles (see fifth aspect of the present invention).
  • the further specific embodiments of the kit it is referred to the fourth aspect of the present invention.
  • FIG. 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.
  • FIG. 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.
  • 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.
  • 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 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.
  • 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).
  • CIP Calf Intestinal Alkaline Phosphatase
  • RNA molecules 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 45 ⁇ 1 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 5 ⁇ resolution. Data were extracted using the Agilent Feature Extraction Software (Agilent Technologies). Reverse Transcription and qRT-PCR of miRNA
  • RNA 50 ng was reverse transcribed using the miScript Reverse Transcription kit (Qiagen) according to the manufacturer's recommendations.
  • cDNA was then diluted 1 :5 and 1 of cDNA was mixed with 10 ⁇ , ⁇ 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.
  • 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.
  • quantile normalization was carried out and not detected miRNAs were subsequently eliminated.
  • 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.
  • 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.
  • 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.
  • the resulting heat map with dendrograms on top and bottom can be found in Figure 5.
  • 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.
  • miRNAs were tested and miR-16 and miR-39 were evaluated as endogenous controls.
  • 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.
  • 149 and 163 miRNAs were detected on average in each female with positive and negative pregnancies, respectively.
  • the embryonic culture media from embryos leading to a pregnancy following implantation have a decreased repertoire of miRNAs.
  • miR-634 SEQ ID NO: 104 with an accuracy of 71% and a sensitivity of 85% was correlated to a successful outcome.
  • miR-29c-3p SEQ ID NO: 1
  • the results founded on the identification of miRNAs which contained in the embryonic culture media of couples undergoing fertility treatment.
  • 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.
  • Virant-Klun I Stahlberg A, Kubista M, Skutella T. MicroRNAs: From Female Fertility, Germ Cells, and Stem Cells to Cancer in Humans. Stem Cells Int 2016;2016:3984937.

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Abstract

La présente invention concerne des méthodes permettant de prédire si un embryon pré-implanté sera incorporé dans l'endomètre d'une femme. La présente invention concerne en outre un kit permettant de prédire si un embryon pré-implanté sera incorporé dans l'endomètre d'une femme.
PCT/EP2017/080312 2016-11-28 2017-11-24 Micro-arn en tant que biomarqueur en fécondation in vitro Ceased WO2018096084A1 (fr)

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