WO2016011377A1 - Methods and systems for assessing infertility and related pathologies - Google Patents
Methods and systems for assessing infertility and related pathologies Download PDFInfo
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- G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
- G16B25/10—Gene or protein expression profiling; Expression-ratio estimation or normalisation
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- C12Q2600/00—Oligonucleotides characterized by their use
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- G—PHYSICS
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- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
Definitions
- Endometriosis affects 10%> to 15% of reproductive-age women. Symptoms of endometriosis may include infertility, chronic pelvic pain, irregular uterine bleeding, dysmenorrhea, and/or dyspareunia. Endometriosis is characterized by the abnormal growth of endometrial tissue, which normally lines the inside of one's uterus, on the outside of one's uterus. The displaced endometrial tissue may spread to one's ovaries, bowels, or pelvic tissue, and, in some cases, continues to act like normal intrauterine endometrial tissue during one's uterine cycle— by thickening, breaking down, and bleeding.
- the uterine cycle is regulated by hormones and has three major phases: the menstrual phase, the proliferative phase, and the secretory phase.
- the secretory phase is often further broken down into the early secretory stage, mid-secretory phase, and late secretory phase. Symptoms and severity vary by case along with the need for fertility treatment and the likelihood of success thereof.
- Retrograde menstruation occurs when menstrual blood containing endometrial cells flows back through the fallopian tubes and into the pelvic cavity, as opposed to flowing out the body.
- the endometrial cells present in the back flow are believed to stick to the pelvic walls and surfaces of the pelvic organs, where they continue to proliferate.
- Other proffered causes of endometriosis include embryonic cell growth, surgical scar implantation, endometrial cell transport, or an immune system disorder.
- Expression studies for examining genes associated with endometriosis have provided further understanding of its etiology. For example, the expression studies have indicated that misregulation of a number of molecular pathways are associated with endometriosis. While expression studies offer insight as to what genes correlate with endometriosis, there has yet to be a consistent approach that allows one to characterize endometriosis or inform treatment of endometriosis based on expression levels and regulation patterns.
- PCOS is a common endocrine system disorder with symptoms that may include irregular or no menstrual periods, heavy periods, excess body and facial hair, acne, pelvic pain, trouble getting pregnant, and patches of thick, darker, velvety skin. Impaired fecundity resulting from PCOS may be treated using a number of methods including diet adjustments, ovulation-inducing medications, surgical intervention, and assisted reproductive techniques such as in- vitro fertilization (IVF). For women with PCOS, like other disorders affecting fertility, success rates for these treatments vary on a case-by-case basis and are not generally predictable and understood.
- Aneuploidy is the presence of an abnormal number of chromosomes in a cell. High aneuploidy rates are often associated with poor oocyte and embryo quality, both of which decrease with age and often lead to unviable embryos and, accordingly, impaired fecundity. While aneuploidy rates appears to increase with a woman's age, the association has not been well characterized and the ability to predict aneuploidy rates for a given individual would be useful in informing family planning and possible fertility treatment.
- the invention relates to methods and systems for assessing infertility and related pathologies, including endometriosis, PCOS, and high aneuploidy rates.
- the invention includes systems and methods for assessing endometriosis and informing course of treatment. Aspects of the invention include identifying genetic signatures of endometriosis that correlate to the various phases of a woman's uterine cycle.
- a woman's phase-specific endometriosis signatures are identified by comparing the patient's genomic expression data to reference phase-specific expression patterns associated with endometriosis.
- the phase-specific endometriosis signatures are utilized to provide accurate diagnostics (e.g. determine phase of a patient's uterine cycle or determine type/severity of the endometriosis), tailor treatment based on the phase-specific endometriosis signature, and/or tailor treatment to coincide with a phase of interest.
- Systems and methods of the invention also relate to assessing risk of IVF failure in patients with PCOS.
- methods include identifying obese patients suffering from PCOS through a measure such as body mass index (BMI) and predicting likelihood of implantation, clinical pregnancy, and/or live birth outcomes in IVF treatment.
- BMI body mass index
- the invention includes systems and methods for assessing an individual's risk of producing an aneuploid embryo based on factors including age and follicle-stimulating hormone (FSH) levels.
- FSH follicle-stimulating hormone
- phase-specific genetic signatures for a patient are determined by identifying the patient's gene expression levels that correspond to a regulation pattern associated with a specific phase of the uterine cycle.
- the regulation pattern may be indicative of an endometriotic condition or a non-endometriotic condition.
- the regulation pattern specific to the uterine cycle may be obtained from a consensus data set that incorporates data from one or more sources, including a certain patient population, publications, studies, and data repositories (including protein-protein interactions and tissue expression patterns).
- the regulation pattern includes statistically-significant expression patterns associated with endometriosis obtained from the consensus data set.
- a meta-analysis is performed on the consensus data set to determine the regulation pattern.
- the meta-analysis may process and filter data based on a number a variables, such ectopic and/or eutopic tissue, the phases of the uterine cycle, particular patient populations, e.g. infertile/not infertile, positive/negative diagnosis for endometriosis, location of the ectopic tissue, pain and other endometriosis-associated symptoms.
- a variables such as ectopic and/or eutopic tissue, the phases of the uterine cycle, particular patient populations, e.g. infertile/not infertile, positive/negative diagnosis for endometriosis, location of the ectopic tissue, pain and other endometriosis-associated symptoms.
- the invention provides methods for assessing endometriosis that include conducting a laboratory procedure to determining levels of transcripts present in a sample obtained from a patient who is suspected of having endometriosis, and identifying transcript levels that correspond to a regulation pattern specific to a time -point in the patient's uterine cycle.
- the time -point of the regulation pattern is a phase of the uterine cycle.
- the identified transcript levels of the patient are then used to characterize endometriosis.
- the characterization may include determining the phase(s) of the subject's uterine cycle based on the identified transcripts. Additionally, the characterization may include determining the type/stage of the endometriosis based on the identified transcripts.
- the method may further include determining the type of treatment for the endometriosis (e.g. a drug or therapeutic that targets the gene or the biochemical pathways associated with the gene) or timing of the treatment based on the characterization (e.g., during a certain phase of the uterine cycle).
- the type of treatment for the endometriosis e.g. a drug or therapeutic that targets the gene or the biochemical pathways associated with the gene
- timing of the treatment based on the characterization (e.g., during a certain phase of the uterine cycle).
- Such methods include determining expression levels of one or more genes over different time-points during a subject's uterine cycle, identifying a time point during the uterine cycle in which expression levels are not synchronous or are dissimilar with respect to a non- endometriotic condition.
- the subject may have differentially expressed genes at a certain phase— in circumstances where a subject's genes are regulation pattern (i.e. up- regulated/de-regulated) during the proliferative phase is different from the non-endometriodic regulation patterns at the proliferative phase.
- a course of treatment may then be indicated to coincide with the phases where the misregulation is indicated.
- a course of treatment may be indicated that based on the misregulation, e.g. a drug or therapeutic that targets the gene or the biochemical pathways associated with the gene.
- Further embodiments involve determining genetic signatures of a patient across the various phases of the patient's uterine cycle in order to classify endometriosis.
- Such methods include determining expression levels of one or more transcripts in a sample obtained from a subject with endometriosis across different time-points of the subject's uterine cycle. The determined transcript levels are then compared to reference transcript levels corresponding to different time-points of the uterine cycle.
- the reference transcript level may be the consensus expression level of one or more transcripts obtained from a population of certain subjects.
- the subjects that make up the population for the reference level may be chosen based on certain phenotypic traits-e.g., positive for endometriosis, negative for endometriosis, infertile, fertile, certain age or weight, etc.
- differential transcripts at each time point of the uterine cycle are determined.
- the differential transcripts at each time point are considered the subject's genetic signature for the respective time points.
- the subject's genetic signature can then be used to classify endometriosis, e.g., determine the type/stage of the endometriosis, and to determine a course of treatment specific to the subject's genetic signatures.
- Certain aspects of the invention include an array for assessing endometriosis.
- the array includes a substrate and a plurality of oligonucleotides attached to the substrate at discrete addressable positions. At least one of the oligonucleotides hybridizes to a portion of one of the following genes: CCL3L1, CCL3, FAM180A, THBS2, PDGFRL, FN1, CLE 11 A, CCNA2, KIF20A, BUB IB, HSD17B6, HSD11B1, C7, C3, CXCL2, CXCL12, CXCL13, PDGFC, CXCL14, ACTA2, TAGLN, and SORBS 1.
- systems and methods of the invention relate to determining that a patient has a decreased probability of successful IVF treatment where the patient is diagnosed with PCOS and the patient's BMI is greater than or equal to a threshold level.
- the threshold level may be 30 kg/m .
- methods of the invention relate to assessing future aneuploidy rates.
- the methods includes conducting a laboratory procedure to determine a follicle stimulating hormone (FSH) level in a sample obtained from an individual and matching the FSH level with the individual's age.
- the method also includes the steps of identifying a prospective risk of producing an aneuploid embryo at a given age based upon said matching step.
- FSH follicle stimulating hormone
- the sample may include blood or urine obtained from the individual.
- the matching step may include comparing the FSH level to a threshold level.
- prospective risk may be identified by taking an initial risk of producing an aneuploidy embryo and increasing that risk by about 10% for each year of the individual's age above puberty.
- prospective risk may be identified by taking an initial risk of producing an aneuploidy embryo and increasing that risk by about 15% for each year of the individual's age above puberty.
- the threshold level may be about 13 mUI/mL.
- Methods may include preparing a written report recommending an accelerated course of treatment for the individual or preparing a written report recommending oocyte retrieval and cryopreservation.
- methods may include retrieving and cryopreserving an oocyte from the individual where the FSH level is greater than the threshold level.
- FIG. 1 illustrates the thickness changes of the endometrial lining as the uterine cycle progresses from the proliferative stage to the late secretory stage.
- FIG. 2 provides a schematic illustration of the meta-analysis process.
- FIG. 3 illustrates retrograde menstruation
- FIG. 4 illustrates conditions associated with endometriosis.
- FIG. 5 illustrates various parameters examined in the meta-analysis of data and associated with endometriosis.
- FIG. 6A illustrates gene expression of the eutopic endometrium of the samples across the proliferative, early secretory, mid-secretory, and late secretory phases.
- FIG. 6B illustrates gene expression of the ectopic endometrium of endometriosis positive samples across the proliferative, early secretory, mid-secretory, and late secretory phases.
- FIG. 7 illustrates the phase-specific genetic signature differences between endometriosis and normal populations.
- FIG. 8 illustrates up-regulated and de -regulated genes associated with endometriosis at the proliferative stage.
- FIG. 9 illustrates up-regulated and de -regulated genes associated with endometriosis at the early secretory stage.
- FIG. 10 illustrates up-regulated genes associated with endometriosis at the mid- to late secretory phase.
- FIG. 11 illustrates the percentage of cohort for several diagnosis groups.
- FIG. 12 illustrates methods for analyzing multi-cycle IVF data.
- FIG. 13 illustrates the impact of obesity on oocyte retrieval, number of viable embryos, implantation rate, and live birth outcomes for several diagnosis groups.
- FIG. 14 illustrates a system for performing methods of the invention. Detailed Description
- the invention generally relates to methods and systems for assessing endometriosis in a subject and informing course of treatment. Aspects of the invention include identifying genomic signatures for endometriosis that correlate to the various phases of a woman's uterine cycle. The phase-specific endometriosis signatures are utilized to provide accurate diagnostics (e.g.
- phase of a patient's uterine cycle or determine type/severity of the endometriosis tailor treatment based on the phase-specific endometriosis signature, and/or tailor treatment to coincide with a particular phase.
- Endometriosis is the abnormal proliferation of endometrial tissue outside of the uterine.
- the endometrial tissue outside of the uterine is often referred to as ectopic tissue;
- the ectopic endometrial tissue behaves in a similar manner as the eutopic tissue, i.e. thickening and bleeding over the course of the uterine (or menstrual) cycle.
- the menstrual fluid generated from the ectopic tissue unlike the eutopic tissue, has no direct route of discharge.
- cysts often form at sites of endometriotic adhesion and the surrounding area may become chronically inflamed, which elicits cellular responses relating to immunity and tissue remodeling.
- stage of the endometriosis is based on the location, amount, depth, and size of the ectopic tissue. Specific criteria include the extent and spread of the tissue, the involvement of pelvic structures in the disease, the extent of pelvic adhesions, and the blockage of fallopian tubes.
- Stage I (subtle stage) involves minimal ectopic tissue, i.e. subtle cyst-like growths from 1 to 3 mm.
- Stage II (typical stage) includes mild ectopic tissue, including cysts and fibrous growths that may span 1 to 2 cm.
- Stage III cystic ovarian stage
- Stage IV severe stage
- Stage involves wide-spread solid tumors covering a majority of the pelvic structures.
- the uterine cycle governing endometrial tissue has several different phases. The different phases are characterized by hormone changes, and thus the phases vary from person to person.
- the uterine cycle begins with the menstrual or menstruation phase.
- the menstrual phase is the phase during which the endometrium is shed as menstrual flow.
- the first day of menstrual flow is defined as the first day of the menstrual cycle.
- the menstrual phase lasts about 3 to 7 days.
- the pituitary glands begin to secrete follicle-stimulating hormone (FSH).
- FSH follicle-stimulating hormone
- the rise in FSH triggers the proliferation phase (Follicular).
- the proliferation phase is the part of the uterine cycle during which follicles inside the ovaries develop and mature in preparation for ovulation.
- the levels of FSH increase in the bloodstream during the proliferation phase, stimulating the maturation of follicles.
- the follicles each contain an egg, and usually only one will reach full growth and will be released at ovulation.
- the ovaries produce estrogen, which causes endometrium tissue to thicken. Once estrogen levels peak, the pituitary glands slow the secretion of FSH in favor of secreting luteinizing hormone (LH). Increased levels of LH cause the mature follicle to rupture and release the egg. The released egg will travel to the fallopian tubes. The releasing of the egg is called ovulation, and it usually occurs about 14 days from the beginning of the next uterine cycle.
- the end of ovulation marks the beginning of the secretory (Luteal) phase.
- LH and FSH decrease.
- the ruptured follicle closes after releasing the egg and forms a corpus luteum, which produces progesterone.
- Estrogen levels are high during the secretory phase, and progesterone and estrogen cause the lining of the uterus to thicken more in order to prepare for possible fertilization. If the egg is not fertilized, the corpus luteum degenerates, progesterone production stops and estrogen levels decrease. Eventually, the top layers of the endometrial lining break down and shed, starting a new uterine cycle.
- FIG. 1 illustrates the changing of the thickness of the endometrial lining as the uterine cycle progresses from the proliferative stage to the late secretory stage.
- aspects of the invention determine and analyze gene expression patterns during different time-points over the uterine cycle.
- the different time-points are the various phases of the uterine cycle.
- expression levels of one or more genes may be determined during the menstrual phase, proliferation phase, or the secretory phase (early, mid or late).
- Methods of the invention involve obtaining a sample, e.g. a tissue or body fluid, which is suspected to include an endometrial-associated gene or gene product.
- the sample may be collected in any clinically acceptable manner.
- a tissue is a mass of connected cells and/or extracellular matrix material, e.g.
- a body fluid is a liquid material derived from, for example, a human or other mammal.
- Such body fluids include, but are not limited to, mucous, blood, plasma, serum, serum derivatives, bile, blood, maternal blood, phlegm, saliva, sweat, amniotic fluid, menstrual fluid, mammary fluid, follicular fluid of the ovary, fallopian tube fluid, peritoneal fluid, urine, and cerebrospinal fluid (CSF), such as lumbar or ventricular CSF.
- a sample may also be a fine needle aspirate or biopsied tissue.
- a sample also may be media containing cells or biological material.
- infertility-associated genes or gene products may be found in reproductive cells or tissues, such as gametic cells, gonadal tissue, fertilized embryos, and placenta.
- the sample is drawn maternal blood or saliva.
- the sample is obtained from endometrial tissue.
- the endometrial tissue may be eutopic (e.g. normal intrauterine endometrial tissue), or ectopic, (e.g., misplaced endometrial tissue).
- the endometrial tissue samples may be obtained over different time-points across the uterine cycle.
- a genomic sample is collected from a subject followed by enrichment for genetic regions or genetic fragments of interest, for example by hybridization to a nucleotide array comprising endometrial-related genes or gene fragments of interest.
- the sample may be enriched for genes of interest (e.g., endometrial-associated genes) using methods known in the art, such as hybrid capture. See for examples, Lapidus (U.S. patent number 7,666,593), the content of which is incorporated by reference herein in its entirety.
- R A may be isolated from eukaryotic cells by procedures that involve lysis of the cells and denaturation of the proteins contained therein.
- Tissue of interest includes gametic cells, gonadal tissue, endometrial tissue, fertilized embryos, and placenta.
- RNA may be isolated from fluids of interest by procedures that involve denaturation of the proteins contained therein. Fluids of interest include blood, menstrual fluid, mammary fluid, follicular fluid of the ovary, peritoneal fluid, or culture medium. Additional steps may be employed to remove DNA. Cell lysis may be accomplished with a nonionic detergent, followed by microcentrifugation to remove the nuclei and hence the bulk of the cellular DNA.
- RNA is extracted from cells of the various types of interest using guanidinium thiocyanate lysis followed by CsCl centrifugation to separate the RNA from DNA (Chirgwin et al, Biochemistry 18:5294-5299 (1979)).
- Poly(A)+ RNA is selected by selection with oligo-dT cellulose (see Sambrook et al, MOLECULAR CLONING—A LABORATORY MANUAL (2ND ED.), Vols. 1-3, Cold Spring Harbor
- RNA from DNA can be accomplished by organic extraction, for example, with hot phenol or
- RNase inhibitors may be added to the lysis buffer.
- mRNA For many applications, it is desirable to preferentially enrich mRNA with respect to other cellular RNAs, such as transfer RNA (tRNA) and ribosomal RNA (rRNA).
- tRNA transfer RNA
- rRNA ribosomal RNA
- Most mRNAs contain a poly(A) tail at their 3' end. This allows them to be enriched by affinity
- poly(A)+ mRNA is eluted from the affinity column using 2 mM EDTA/0.1% SDS.
- expression levels of a patient are compared to a reference data specific to phase in the uterine cycle.
- the reference data may comprise phase- specific endometriosis signatures (ectopic signatures) or phase-specific normal signatures (eutopics).
- the signatures may be determined by conducting a meta-analysis on one or more sources of expression data obtained from normal patients, endometriosis patients, or both. A meta-analysis suitable for use in accordance with the invention is described hereinafter.
- the phase-specific signatures are typically regulation pattern exhibited by either the healthy or the diseased tissue. Regulation patterns associated with endometriosis typically include up-regulated or de-regulated genes and the misregulation changes across the various phases of the uterine cycle.
- Up-regulation is a process that occurs within a cell triggered by a signal (originating internal or external to the cell), which results in increased expression of one or more genes and as a result the protein(s) encoded by those genes. Conversely, de-regulation is a process resulting in decreased gene and corresponding protein expression.
- the reference data may include a consensus expression levels associated with a particular patient population (e.g. endometriosis population or normal population).
- genes whose expression levels correlate significantly with endometriosis include CCL3L1, CCL3, FAM180A, THBS2, PDGFRL, FN1, CLE 11 A, CCNA2, KIF20A, BUB IB, HSD17B6, HSD11B1, C7, C3, CXCL2, CXCL12, CXCL13, PDGFC, CXCL14, ACTA2, TAGLN, and SORBS 1.
- de-regulated genes associated with the proliferative phase include CCNA2, KIF20A, BUB1B.
- Up-regulated genes associated with the proliferative phase include HSD17B6, HSDl 1B1, C7, C3, CXCL2, CXCL12, CXCL14.
- de-regulated genes associated with the early secretory phase include CXCL13.
- Up-regulated genes associated with the early secretory phase include CCNA2, KIF20A, BUB1B.
- up-regulated genes associated with the mid- to late include ACTA2, TAGLN, and SORBS 1.
- methods of the invention provide for obtaining phase- specific genetic reference data (i.e. signature or regulation pattern) based on data obtained from a number of endometriosis related sources.
- the data sources may include public and private endometriosis related databases.
- the reference endometriosis data set may include data obtained from a multitude of patients of similar or diverse background, a variety of sample types, and samples taken over different time points.
- parameters associated with the data set include age, negative/positive diagnosis of endometriosis, stage/type of the disease, pain associated with endometriosis, gravidity/parity, endometrioma position, tissue sampling method, phase of the uterine cycle, and ethnicity.
- FIG. 2 provides a schematic illustration of the meta-analysis process.
- microarray data is obtained from several studies that examine gene expression differences between the tissue of patients with endometriosis and the tissue of normal patients (e.g. normal patients). The association between gene expression and endometriosis may be analyzed within each case or by comparing cases and controls using analysis of variance. According to certain embodiments, the micro-array data between studies may differ due to patient variability, tissue type, uterine phase during sample, experimental technique, etc.
- the micro-array data is entered into the system, processed to normalize the expression data and then subjected to a statistical analysis to identify endometriosis-related expression patterns of statistical significance.
- Statistical parameters may be chosen to identify gene expression patterns that are statistically significant. Based on the data, the system can also identify statistically significant expression patterns associated with specific phases of the uterine cycle.
- MART additive regression tree
- Each tree is a recursive graph of decisions the possible consequences of which partition patient parameters; each node represents a question (e.g., is the FSH level greater than x?) and the branch taken from that node represents the decision made (e.g. yes or no).
- the choice of question corresponding to each node is automated.
- a MART model is the weighted sum of iteratively produced regression trees. At each iteration, a regression tree is fitted according to a criterion in which the samples more involved in the prediction error are given priority. This tree is added to the existing trees, the prediction error is recalculated, and the cycle continues, leading to a progressive refinement of the prediction.
- the strengths of this method include analysis of many variables without knowledge of their complex interactions beforehand.
- a different approach called the generalized linear model expresses the outcome as a weighted sum of functions of the predictor variables.
- the weights are calculated based on least squares or Bayesian methods to minimize the prediction error on the training set.
- a predictor's weight reveals the effect of changing that predictor, while holding the others constant, on the outcome.
- the relative values of their weights are less meaningful; steps must be taken to remove that collinearity, such as by excluding the nearly redundant variables from the model.
- the weights express the relative importance of the predictors.
- Less general formulations of the generalized linear model include linear regression, multiple regression, and multifactor logistic regression models, and are highly used in the medical community as clinical predictors.
- a series of logistic regression models may be used.
- the p-values and odds ratio can be used for statistical inference.
- Logistic regression models are common statistical classification models.
- the endometriotic expression patterns across the different phases that are statistically significant are considered biomarkers or signatures for the disease.
- the reference phase-specific endometriotic signatures can then be used to identify a patient's phase-specific endometriotic signatures, classify the patient's endometriosis and tailor treatment of the same.
- the patient's genetic signatures are identified by comparing the patient's expression data across one or more time-points in the uterine cycle to reference phase- specific expression levels.
- the patient's phase specific genetic signature for endometriosis may include expression levels that are the same as or dissimilar from the reference phase-specific reference data.
- the reference phase-specific pattern or expression data may represent expression levels of subjects having endometriosis. In such instance, similarities between the patient's expression levels and the reference may be indicative of the patient's phase-specific genetic signature.
- the reference phase-specific pattern or expression data may represent expression levels of subjects without endometriosis. In such instance, dissimilarities between the patient's expression levels and the reference may be indicative of the patient's phase-specific genetic signature.
- a treatment regimen can be prescribed or set forth in an informative report that is targeted to the patient's signature.
- a drug or therapeutic that targets the gene or the biochemical pathways associated with the gene may be prescribed.
- the course of treatment is tailored to the patient's expression signatures in each phase. For example, treatment may only be indicated in one of the phases (such as the proliferative phase) or different treatments may be indicated for two or more of the phases. As such, methods of the inventions advantageous inform both timing and type of treatment.
- the invention provides methods for assessing endometriosis that include determining levels of transcripts present a patient's sample, who is suspected of having endometriosis, identifying those transcript levels that correspond to a regulation pattern specific to a time-point in a uterine cycle and characterizing endometriosis based upon the identified transcript levels.
- the time -point of the regulation pattern is a phase of the uterine cycle.
- the characterization may include determining the phase(s) of the subject's uterine cycle based on the identified transcripts. Additionally, the characterization may include determining the type/stage of the endometriosis based on the identified transcripts.
- the method may further include determining the timing or type of treatment for the endometriosis based on the characterization.
- Some embodiments for targeting the treatment of endometriosis include determining expression levels of one or more genes over different time-points during a subject's uterine cycle, identifying a time point during the uterine cycle in which expression levels are
- the subject may have differentially expressed genes at a certain phase, in circumstances where a subject's genes are regulation pattern (i.e. upregulated/deregulated) during the proliferative phase is different from the non-endometriodic regulation patterns at the proliferative phase.
- Treatments may involve a variety of known methods such as hormone therapies (e.g., hormonal contraceptives, gonadotropin-releasing hormone (Gn-RH) agonists and antagonists, Medroxyprogesterone, and Danazol), surgery to remove endometrial tissue, or even
- Further embodiments involve determining phase-specific genetic signatures of a patient across the various phases of the patient's uterine cycle to classify endometriosis.
- Such methods include determining expression levels of one or more transcripts in a sample obtained from a subject with endometriosis across different time-points of the subject's uterine cycle. The determined transcript levels are then compared to reference transcript levels corresponding to different time-points of the uterine cycle.
- the reference transcript level may be the consensus expression level of one or more transcripts obtained from a patient population.
- the patient population chosen for the reference level may be chosen based on certain phenotypic traits-e.g., positive for endometriosis, negative for endometriosis, infertile, fertile, certain age or weight, etc.
- differential transcripts at each time point of the uterine cycle are determined.
- the differential transcripts at each time point are considered the subject's genetic signature for the respective time points.
- the subject's genetic signature can then be used to classify endometriosis, e.g., determine the type/stage of the endometriosis and, and to determine a course of treatment specific to the subject's genetic signatures.
- the invention involves assessing transcripts present in a biological sample.
- Such methods may involve preparing amplified cDNA from total R A. cDNA is prepared and indiscriminately amplified without diluting the isolated RNA sample or distributing the mixture of genetic material in the isolated RNA into discrete reaction samples. Preferably, amplification is initiated at the 3' end as well as randomly throughout the whole transcriptome in the sample to allow for amplification of both mRNA and non-polyadenylated transcripts.
- the double-stranded cDNA amplification products are thus optimized for the generation of sequencing libraries for Next Generation Sequencing platforms.
- Suitable kits for amplifying cDNA in accordance with the methods of the invention include, for example, the Ovation ® RNA- Seq System.
- Methods of the invention also involve sequencing the amplified cDNA. While any known sequencing method can be used to sequence the amplified cDNA mixture, single molecule sequencing methods are preferred.
- the amplified cDNA is sequenced by whole transcriptome shotgun sequencing (also referred to herein as (“RNA-Seq").
- Whole transcriptome shotgun sequencing (RNA-Seq) can be accomplished using a variety of next- generation sequencing platforms such as the Illumina Genome Analyzer platform, ABI Solid Sequencing platform, or Life Science's 454 Sequencing platform.
- Differential transcript levels within the biological sample can also be analyzed using via microarray techniques.
- the amplified cDNA can be used to probe a microarray containing gene transcripts associated with one or conditions or diseases, such as any prenatal condition, or any type of cancer, inflammatory, or autoimmune disease.
- the invention provides a microarray including a plurality of
- oligonucleotides attached to a substrate at discrete addressable positions, in which at least one of the oligonucleotides hybridizes to a portion of a gene selected from CCL3L1, CCL3, FAM180A, THBS2, PDGFRL, FN1, CLE 11 A, CCNA2, KIF20A, BUB IB, HSD17B6, HSD11B1, C7, C3, CXCL2, CXCL12, CXCL13, PDGFC, CXCL14, ACTA2, TAGLN, and SORBS1.
- microarrays Methods of constructing microarrays are known in the art. See for example Yeatman et al. (U.S. patent application number 2006/0195269), the content of which is hereby incorporated by reference in its entirety.
- Microarrays are prepared by selecting probes that include a polynucleotide sequence, and then immobilizing such probes to a solid support or surface.
- the probes may comprise DNA sequences, RNA sequences, or copolymer sequences of DNA and RNA.
- the polynucleotide sequences of the probes may also comprise DNA and/or RNA analogues, or combinations thereof.
- the polynucleotide sequences of the probes may be full or partial fragments of genomic DNA.
- the polynucleotide sequences of the probes may also be synthesized nucleotide sequences, such as synthetic oligonucleotide sequences.
- the probe sequences can be synthesized either enzymatically in vivo, enzymatically in vitro (e.g., by PCR), or non-enzymatically in vitro.
- the probe or probes used in the methods of the invention are preferably immobilized to a solid support which may be either porous or non-porous.
- the probes of the invention may be polynucleotide sequences which are attached to a nitrocellulose or nylon membrane or filter covalently at either the 3' or the 5' end of the polynucleotide.
- hybridization probes are well known in the art (see, e.g., Sambrook et al., MOLECULAR CLONING—A LABORATORY MANUAL (2ND ED.), Vols. 1-3, Cold Spring Harbor
- the solid support or surface may be a glass or plastic surface.
- hybridization levels are measured to microarrays of probes consisting of a solid phase on the surface of which are immobilized a population of polynucleotides, such as a population of DNA or DNA mimics, or, alternatively, a population of RNA or RNA mimics.
- the solid phase may be a nonporous or, optionally, a porous material such as a gel.
- a microarray comprises a support or surface with an ordered array of binding (e.g., hybridization) sites or "probes" each representing one of the genes described herein, particularly one of CCL3L1, CCL3, FAM180A, THBS2, PDGFRL, FN1, CLE11A, CCNA2, KIF20A, BUB IB, HSD17B6, HSD11B1, C7, C3, CXCL2, CXCL12, CXCL13, PDGFC, CXCL14, ACTA2, TAGLN, and SORBS1.
- the microarrays are addressable arrays, and more preferably positionally addressable arrays.
- each probe of the array is preferably located at a known, predetermined position on the solid support such that the identity (i.e., the sequence) of each probe can be determined from its position in the array (i.e., on the support or surface).
- each probe is covalently attached to the solid support at a single site.
- Microarrays can be made in a number of ways, of which several are described below. However produced, microarrays share certain characteristics. The arrays are reproducible, allowing multiple copies of a given array to be produced and easily compared with each other. Preferably, microarrays are made from materials that are stable under binding (e.g., nucleic acid hybridization) conditions. The microarrays are preferably small, e.g., between 1 cm 2 and 25 cm 2 ,
- a given binding site or unique set of binding sites in the microarray will specifically bind (e.g., hybridize) to the product of a single gene in a cell (e.g., to a specific mR A, or to a specific cDNA derived therefrom).
- a binding site or unique set of binding sites in the microarray will specifically bind (e.g., hybridize) to the product of a single gene in a cell (e.g., to a specific mR A, or to a specific cDNA derived therefrom).
- other related or similar sequences will cross hybridize to a given binding site.
- the microarrays of the present invention include one or more test probes, each of which has a polynucleotide sequence that is complementary to a subsequence of RNA or DNA to be detected.
- the position of each probe on the solid surface is known.
- the microarrays are preferably positionally addressable arrays.
- each probe of the array is preferably located at a known, predetermined position on the solid support such that the identity (i.e., the sequence) of each probe can be determined from its position on the array (i.e., on the support or surface).
- the microarray is an array (i.e., a matrix) in which each position represents one of the biomarkers described herein.
- each position can contain a DNA or DNA analogue based on genomic DNA to which a particular RNA or cDNA transcribed from that genetic marker can specifically hybridize.
- the DNA or DNA analogue can be, e.g., a synthetic oligomer or a gene fragment.
- probes representing each of the markers are present on the array.
- the array comprises probes for genes known to be associated with endometriosis.
- the array probes may be specific to genes known to be associated with endometriosis at a certain phase of the uterine cycle.
- the probe to which a particular polynucleotide molecule specifically hybridizes according to the invention contains a complementary genomic polynucleotide sequence.
- the probes of the microarray preferably consist of nucleotide sequences of no more than 1,000 nucleotides. In some embodiments, the probes of the array consist of nucleotide sequences of 10 to 1,000 nucleotides.
- the nucleotide sequences of the probes are in the range of 10-200 nucleotides in length and are genomic sequences of a species of organism, such that a plurality of different probes is present, with sequences complementary and thus capable of hybridizing to the genome of such a species of organism, sequentially tiled across all or a portion of such genome.
- the probes are in the range of 10-30 nucleotides in length, in the range of 10-40 nucleotides in length, in the range of 20-50 nucleotides in length, in the range of 40-80 nucleotides in length, in the range of 50-150 nucleotides in length, in the range of 80-120 nucleotides in length, and most preferably are 60 nucleotides in length.
- the probes may comprise DNA or DNA "mimics" (e.g., derivatives and analogues) corresponding to a portion of an organism's genome.
- the probes of the microarray are complementary RNA or RNA mimics.
- DNA mimics are polymers composed of subunits capable of specific, Watson-Crick-like hybridization with DNA, or of specific hybridization with RNA.
- the nucleic acids can be modified at the base moiety, at the sugar moiety, or at the phosphate backbone.
- Exemplary DNA mimics include, e.g., phosphorothioates.
- DNA can be obtained, e.g., by polymerase chain reaction (PCR) amplification of genomic DNA or cloned sequences.
- PCR primers are preferably chosen based on a known sequence of the genome that will result in amplification of specific fragments of genomic DNA.
- Computer programs that are well known in the art are useful in the design of primers with the required specificity and optimal amplification properties, such as Oligo version 5.0 (National Biosciences).
- each probe on the microarray will be between 10 bases and 50,000 bases, usually between 300 bases and 1,000 bases in length.
- PCR methods are well known in the art, and are described, for example, in Innis et al, eds., PCR PROTOCOLS: A GUIDE TO METHODS AND APPLICATIONS, Academic Press Inc., San Diego, Calif. (1990). It will be apparent to one skilled in the art that controlled robotic systems are useful for isolating and amplifying nucleic acids.
- An alternative, preferred means for generating the polynucleotide probes of the microarray is by synthesis of synthetic polynucleotides or oligonucleotides, e.g., using N- phosphonate or phosphoramidite chemistries (Froehler et al., Nucleic Acid Res. 14:5399-5407 (1986); McBride et al, Tetrahedron Lett. 24:246-248 (1983)). Synthetic sequences are typically between about 10 and about 500 bases in length, more typically between about 20 and about 100 bases, and most preferably between about 40 and about 70 bases in length. In some
- synthetic nucleic acids include non-natural bases, such as, but by no means limited to, inosine.
- nucleic acid analogues may be used as binding sites for
- nucleic acid analogue is peptide nucleic acid (see, e.g., Egholm et al, Nature 363:566-568 (1993); U.S. Pat. No. 5,539,083).
- Probes are preferably selected using an algorithm that takes into account binding energies, base composition, sequence complexity, cross-hybridization binding energies, and secondary structure. See Friend et al, International Patent Publication WO 01/05935, published Jan. 25, 2001; Hughes et al, Nat. Biotech. 19:342-7 (2001).
- positive control probes e.g., probes known to be complementary and hybridizable to sequences in the target polynucleotide molecules
- negative control probes e.g., probes known to not be complementary and hybridizable to sequences in the target polynucleotide molecules
- positive controls are synthesized along the perimeter of the array.
- positive controls are synthesized in diagonal stripes across the array.
- the reverse complement for each probe is synthesized next to the position of the probe to serve as a negative control.
- sequences from other species of organism are used as negative controls or as "spike-in" controls.
- the probes are attached to a solid support or surface, which may be made, e.g., from glass, plastic (e.g., polypropylene, nylon), polyacrylamide, nitrocellulose, gel, or other porous or nonporous material.
- a preferred method for attaching the nucleic acids to a surface is by printing on glass plates, as is described generally by Schena et al, Science 270:467-470 (1995). This method is especially useful for preparing microarrays of cDNA (See also, DeRisi et al, Nature Genetics 14:457-460 (1996); Shalon et al, Genome Res. 6:639-645 (1996); and Schena et al, Proc. Natl. Acad. Sci. U.S.A. 93: 10539-11286 (1995)).
- a second preferred method for making microarrays is by making high-density
- oligonucleotide arrays Techniques are known for producing arrays containing thousands of oligonucleotides complementary to defined sequences, at defined locations on a surface using photolithographic techniques for synthesis in situ (see, Fodor et al, 1991, Science 251 :767-773; Pease et al, 1994, Proc. Natl. Acad. Sci. U.S.A. 91 :5022-5026; Lockhart et al, 1996, Nature Biotechnology 14: 1675; U.S. Pat. Nos.
- oligonucleotides e.g., 60-mers
- the array produced is redundant, with several oligonucleotide molecules per RNA.
- microarrays e.g., by masking
- any type of array for example, dot blots on a nylon hybridization membrane (see Sambrook et al., MOLECULAR CLONING--A LABORATORY MANUAL (2ND ED.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989)) could be used.
- very small arrays will frequently be preferred because hybridization volumes will be smaller.
- the arrays of the present invention are prepared by synthesizing polynucleotide probes on a support.
- polynucleotide probes are attached to the support covalently at either the 3' or the 5' end of the polynucleotide.
- microarrays of the invention are manufactured by means of an ink jet printing device for oligonucleotide synthesis, e.g., using the methods and systems described by Blanchard in U.S. Pat. No. 6,028,189; Blanchard et al, 1996, Biosensors and Bioelectronics 11 :687-690; Blanchard, 1998, in Synthetic DNA Arrays in Genetic
- the oligonucleotide probes in such microarrays are preferably synthesized in arrays, e.g., on a glass slide, by serially depositing individual nucleotide bases in "microdroplets" of a high surface tension solvent such as propylene carbonate.
- the microdroplets have small volumes (e.g., 100 pL or less, more preferably 50 pL or less) and are separated from each other on the microarray (e.g., by hydrophobic domains) to form circular surface tension wells, which define the locations of the array elements (i.e., the different probes).
- Microarrays manufactured by this ink-jet method are typically of high density, preferably having a density of at least about 2,500 different probes per 1 cm.sup.2.
- the polynucleotide probes are attached to the support covalently at either the 3' or the 5' end of the polynucleotide.
- the polynucleotide molecules which may be analyzed by the present invention are DNA, RNA, or protein.
- the target polynucleotides are detectably labeled at one or more nucleotides. Any method known in the art may be used to detectably label the target polynucleotides.
- this labeling incorporates the label uniformly along the length of the DNA or RNA, and more preferably, the labeling is carried out at a high degree of efficiency.
- the detectable label is a luminescent label.
- fluorescent labels such as a fluorescein, a phosphor, a rhodamine, or a polymethine dye derivative.
- fluorescent labels include, for example, fluorescent phosphoramidites such as FluorePrime (Amersham Pharmacia, Piscataway, N.J.), Fluoredite (Millipore, Bedford, Mass.), FAM (ABI, Foster City, Calif), and Cy3 or Cy5 (Amersham Pharmacia, Piscataway, N.J.).
- the detectable label is a radiolabeled nucleotide.
- target polynucleotide molecules from a patient sample are labeled differentially from target polynucleotide molecules of a reference sample.
- the reference can comprise target polynucleotide molecules from normal tissue samples.
- polynucleotide molecules specifically bind or specifically hybridize to the complementary polynucleotide sequences of the array, preferably to a specific array site, wherein its
- Arrays containing double-stranded probe DNA situated thereon are preferably subjected to denaturing conditions to render the DNA single-stranded prior to contacting with the target polynucleotide molecules.
- Arrays containing single-stranded probe DNA may need to be denatured prior to contacting with the target polynucleotide molecules, e.g., to remove hairpins or dimers which form due to self
- Optimal hybridization conditions will depend on the length (e.g., oligomer versus polynucleotide greater than 200 bases) and type (e.g., RNA, or DNA) of probe and target nucleic acids.
- length e.g., oligomer versus polynucleotide greater than 200 bases
- type e.g., RNA, or DNA
- oligonucleotides As the oligonucleotides become shorter, it may become necessary to adjust their length to achieve a relatively uniform melting temperature for satisfactory hybridization results.
- General parameters for specific (i.e., stringent) hybridization conditions for nucleic acids are described in Sambrook et al., MOLECULAR CLONING—A LABORATORY MANUAL (2ND ED.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
- Typical hybridization conditions for the cDNA microarrays of Schena et al. are hybridization in 5 x SSC plus 0.2% SDS at 65°C for four hours, followed by washes at 25° C in low stringency wash buffer (1 x SSC plus 0.2% SDS), followed by 10 minutes at 25°C in higher stringency wash buffer (0.1 x SSC plus 0.2% SDS) (Schena et al, Proc. Natl. Acad. Sci. U.S.A. 93: 10614 (1993)).
- Useful hybridization conditions are also provided in, e.g., Tijessen, 1993, HYBRIDIZATION WITH NUCLEIC ACID PROBES, Elsevier Science Publishers B.V.; and Kricka, 1992, NONISOTOPIC DNA PROBE TECHNIQUES, Academic Press, San Diego, Calif.
- Particularly preferred hybridization conditions include hybridization at a temperature at or near the mean melting temperature of the probes (e.g., within 51°C, more preferably within 2FC.) in 1 M NaCl, 50 mM MES buffer (pH 6.5), 0.5% sodium sarcosine and 30% formamide.
- the fluorescence emissions at each site of a microarray may be, preferably, detected by scanning confocal laser microscopy.
- a separate scan, using the appropriate excitation line, is carried out for each of the two fluorophores used.
- a laser may be used that allows simultaneous specimen illumination at wavelengths specific to the two fluorophores and emissions from the two fluorophores can be analyzed simultaneously (see Shalon et al., 1996, "A DNA microarray system for analyzing complex DNA samples using two-color fluorescent probe hybridization," Genome Research 6:639-645, which is incorporated by reference in its entirety for all purposes).
- the arrays are scanned with a laser fluorescent scanner with a computer controlled X-Y stage and a microscope objective. Sequential excitation of the two fluorophores is achieved with a multi-line, mixed gas laser and the emitted light is split by wavelength and detected with two photomultipher tubes. Fluorescence laser scanning devices are described in Schena et al, Genome Res. 6:639-645 (1996), and in other references cited herein. Alternatively, the fiber-optic bundle described by Ferguson et al., Nature Biotech. 14: 1681-1684 (1996), may be used to monitor mRNA abundance levels at a large number of sites
- FIG. 11 illustrates the percentage of cohort for patient groups in the study.
- FIG. 12 illustrates methods for analyzing multi-cycle IVF data including the method used in the study discussed below where generalized estimation equation (GEE) is used to calculate patient-level distributions using all available patient-specific IVF cycle data.
- GEE generalized estimation equation
- FIG. 13 illustrates the impact of obesity on oocyte retrieval, number of viable embryos, implantation rate, and live birth outcomes for patient groups including patients suffering from diminished ovarian reserve, endometriosis, idiopathic, male factor, PCOS, and tubal factor showing a significant decrease in implantation rate and number of live birth outcomes for obese PCOS patients.
- Body fat may be indicated by weight, waist circumference (e.g., the circumference of the abdomen, measured at the natural waist (in between the lowest rib and the top of the hip bone), the umbilicus (belly button), or at the narrowest point of the midsection), waist-to-hip ratio (e.g., calculated by measuring the waist and the hip (at the widest diameter of the buttocks), and then dividing the waist measurement by the hip measurement), skinfold thickness (e.g., using a special caliper to measure the thickness of a "pinch" of skin and the fat beneath it in specific areas of the body and using equations to predict body fat percentage based on these measurements), bioelectrical impedance (see, Hu F.
- body fat is indicated by body mass index (BMI).
- BMI body mass index
- Body fat as measured by one of the methods above, can then be compared to a reference number to determine if the individual is obese. Diminished success rates for PCOS diagnosed individuals may be indicated where, for instance, BMI is determined to be greater than 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 kg/m . In a preferred embodiment, a BMI greater than 30 kg/m is considered to obese and at higher risk of IVF failure.
- systems and methods of the invention include receiving a PCOS diagnosis for a patient.
- systems and methods may include diagnosing PCOS in an individual through, for example, one of the methods described in Sheehan, Polycystic Ovarian Syndrome: Diagnosis and Management, Clin Med Res. 2004 Feb; 2(1): 13- 27.
- methods of the invention may include determining a likelihood of IVF success for the individual (e.g., represented by a % score indicating a likelihood of live birth after IVF treatment, a % reduction from average success rate, or an estimated number of cycles to achieve live birth).
- the likelihood of IVF success rate may be reported to the individual or the individual's physician alone or in combination with other fertility information
- the PCOS and obesity information may be combined with other factors to determine an overall likelihood of IVF success for the patient or to provide treatment recommendations for the patient.
- Methods of the invention include determining a woman's relative risk of producing an aneuploid embryo based upon her age and her FSH level.
- FSH level may be determined from a body fluid such as urine or blood. A sample may be obtained directly from the patient or may be received. Because urine levels of FSH vary throughout the day, in certain embodiments, urine may be collected over a 24-hour period before FSH levels are determined. FSH levels in the sample may be determined using a laboratory procedure such as an immunofluorometric assay. See Kesner JS, Knecht EA, Krieg EF., Jr Time-resolved immunofluorometric assays for urinary luteinizing hormone and follicle stimulating hormone. Anal Chim Acta. 1994;285: 13-22 incorporated herein by reference in its entirety.
- a greater increase, by age, of aneuploidy rates may be indicated where the FSH level for a woman is greater than as threshold level such as, for example, 10, 11, 12, 13, 14, or 15 mUI/ML.
- threshold level such as, for example, 10, 11, 12, 13, 14, or 15 mUI/ML.
- a woman's FSH level is above 13mUI/mL, she may be at an increased risk of producing aneuploid embryos as she ages.
- the woman's risk of producing an aneuploid embryo may be determined from her FSH levels and her age above puberty or fertility.
- the risk may be increased by 8%, 9%, 10%, 11%, or 12% (from an initial or base risk level) for each year of her reproductive lifespan (e.g., time from beginning of puberty to menopause, or from beginning of regular ovulation to menopause).
- the risk may be increased by 13%, 14%, 15%, 16%, or 17% (from an initial or base risk level) for each year of her reproductive lifespan.
- reproductive lifespan may be determined for an individual based on the actual age they reached puberty or began regular ovulation (as determined, for example, by a detailed patient history) or may be assumed to have begun at standard age such as 12, 13, 14, 15, 16, or 17.
- An initial or base risk of producing an aneuploid embryo may be determined from an average rate among the population or taken from known studies such as Franasiak, et al., The nature of aneuploidy with increasing age of the female partner: a review of 15,169 consecutive trophectoderm biopsies evaluated with comprehensive chromosomal screening, Fertil Steril. 2014 Mar;101(3):656-663; incorporated herein in its entirety.
- systems and methods of the invention may include reporting to this increased risk to the patient, physician, or other individual, where the patient's FSH level is greater than 13 mUI/mL.
- Various embodiments may include recommending or performing a treatment for the patient including avoiding certain assistive reproductive technologies, beginning treatments earlier, or, in some cases, harvesting eggs or embryos and storing for later use in assistive reproductive technologies such as IVF.
- Methods for retrieving and/or storing eggs and embryos are known. See Cil, et al, Current trends and progress in clinical applications of oocyte cryopreservation, Curr Opin Obstet Gynecol. 2013 June ; 25(3); Killick, S (2006). "Ultrasound and fertility". In Bates, J. Practical gynaecological ultrasound (2nd ed.). Cambridge, England: Cambridge University Press, pp. 120-5; the contents of which are incorporated herein in their entirety.
- Reports as referred to herein may be produced in written form on paper or in a computer file and may be prepared by a computing device and sent to a user (e.g., patient, physician or other individual) through an input/output device such as a monitor, interactive display, or printer, for example.
- a user e.g., patient, physician or other individual
- an input/output device such as a monitor, interactive display, or printer, for example.
- Methods of the invention may be performed using any type of computing device, such as a computer, that includes a processor, e.g., a central processing unit, or any combination of computing devices where each device performs at least part of the process or method.
- a processor e.g., a central processing unit
- systems and methods described herein may be performed with a handheld device, e.g., a smart tablet, or a smart phone, or a specialty device produced for the system.
- Methods of the invention can be performed using software, hardware, firmware, hardwiring, or combinations of any of these.
- Features implementing functions can also be physically located at various positions, including being distributed such that portions of functions are implemented at different physical locations (e.g., imaging apparatus in one room and host workstation in another, or in separate buildings, for example, with wireless or wired
- processors suitable for the execution of computer program include, by way of example, both general and special purpose microprocessors, and any one or more processor of any kind of digital computer.
- a processor will receive instructions and data from a read-only memory or a random access memory or both.
- the essential elements of computer are a processor for executing instructions and one or more memory devices for storing instructions and data.
- a computer will also include, or be operatively coupled to receive data from or transfer data to, or both, one or more mass storage devices for storing data, e.g., magnetic, magneto-optical disks, or optical disks.
- Information carriers suitable for embodying computer program instructions and data include all forms of non- volatile memory, including by way of example semiconductor memory devices, (e.g., EPROM, EEPROM, solid state drive (SSD), and flash memory devices); magnetic disks, (e.g., internal hard disks or removable disks); magneto- optical disks; and optical disks (e.g., CD and DVD disks).
- semiconductor memory devices e.g., EPROM, EEPROM, solid state drive (SSD), and flash memory devices
- magnetic disks e.g., internal hard disks or removable disks
- magneto- optical disks e.g., CD and DVD disks
- optical disks e.g., CD and DVD disks.
- the processor and the memory can be supplemented by, or incorporated in, special purpose logic circuitry.
- the subject matter described herein can be implemented in a computing system that includes a back-end component (e.g., a data server), a middleware component (e.g., an application server), or a front-end component (e.g., a client computer having a graphical user interface or a web browser through which a user can interact with an implementation of the subject matter described herein), or any combination of such back-end, middleware, and front- end components.
- the components of the system can be interconnected through network by any form or medium of digital data communication, e.g., a communication network.
- the reference set of data may be stored at a remote location and the computer communicates across a network to access a reference set of data for all patients along with clinical outcomes (e.g., IVF success rates) to compare data derived from the female subject to the reference set.
- the reference set is stored locally within the computer and the computer accesses the reference set within the CPU to compare subject data to the reference set.
- Examples of communication networks include cell network (e.g., 3G or 4G), a local area network (LAN), and a wide area network (WAN), e.g., the Internet.
- the subject matter described herein can be implemented as one or more computer program products, such as one or more computer programs tangibly embodied in an information carrier (e.g., in a non-transitory computer-readable medium) for execution by, or to control the operation of, data processing apparatus (e.g., a programmable processor, a computer, or multiple computers).
- a computer program also known as a program, software, software application, app, macro, or code
- Systems and methods of the invention can include instructions written in any suitable programming language known in the art, including, without limitation, C, C++, Perl, Java, ActiveX, HTML5, Visual Basic, or JavaScript.
- a computer program does not necessarily correspond to a file.
- a program can be stored in a file or a portion of file that holds other programs or data, in a single file dedicated to the program in question, or in multiple coordinated files (e.g., files that store one or more modules, sub-programs, or portions of code).
- a computer program can be deployed to be executed on one computer or on multiple computers at one site or distributed across multiple sites and
- a file can be a digital file, for example, stored on a hard drive, SSD, CD, or other tangible, non-transitory medium.
- a file can be sent from one device to another over a network (e.g., as packets being sent from a server to a client, for example, through a Network Interface Card, modem, wireless card, or similar).
- Writing a file involves transforming a tangible, non-transitory computer-readable medium, for example, by adding, removing, or rearranging particles (e.g., with a net charge or dipole moment into patterns of magnetization by read/write heads), the patterns then representing new collocations of information about objective physical phenomena desired by, and useful to, the user.
- writing involves a physical transformation of material in tangible, non-transitory computer readable media (e.g., with certain optical properties so that optical read/write devices can then read the new and useful collocation of information, e.g., burning a CD-ROM).
- writing a file includes transforming a physical flash memory apparatus such as NAND flash memory device and storing information by transforming physical elements in an array of memory cells made from floating- gate transistors.
- Methods of writing a file are well-known in the art and, for example, can be invoked manually or automatically by a program or by a save command from software or a write command from a programming language.
- Suitable computing devices typically include mass memory, at least one graphical user interface, at least one display device, and typically include communication between devices.
- the mass memory illustrates a type of computer-readable media, namely computer storage media.
- Computer storage media may include volatile, nonvolatile, removable, and non-removable media implemented in any method or technology for storage of information, such as computer readable instructions, data structures, program modules, or other data. Examples of computer storage media include RAM, ROM, EEPROM, flash memory, or other memory technology, CD-ROM, digital versatile disks (DVD) or other optical storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, Radio frequency Identification tags or chips, or any other medium which can be used to store the desired information and which can be accessed by a computing device.
- a computer system or machines of the invention include one or more processors (e.g., a central processing unit (CPU) a graphics processing unit (GPU) or both), a main memory and a static memory, which communicate with each other via a bus.
- system 200 capable of carrying out methods of the invention, can include a computer 249 (e.g., laptop, desktop, or tablet).
- the computer 249 may be configured to communicate across a network 209.
- Computer 249 includes one or more processor 259 and memory 263 as well as an input/output mechanism 254.
- server 213, which includes one or more of processor 221 and memory 229, capable of obtaining data, instructions, etc., or providing results via interface module 225 or providing results as a file 217.
- Server 213 may be engaged over network 209 through computer 249 or terminal 267, or server 213 may be directly connected to terminal 267, including one or more processor 275 and memory 279, as well as input/output mechanism 271.
- Systems 200 or machines according to the invention may further include, for any of I/O 259 or 237, or interface module 225, a video display unit (e.g., a liquid crystal display (LCD) or a cathode ray tube (CRT)).
- a video display unit e.g., a liquid crystal display (LCD) or a cathode ray tube (CRT)
- Computer systems or machines according to the invention can also include an alphanumeric input device (e.g., a keyboard), a cursor control device (e.g., a mouse), a disk drive unit, a signal generation device (e.g., a speaker), a touchscreen, an accelerometer, a microphone, a cellular radio frequency antenna, and a network interface device, which can be, for example, a network interface card (NIC), Wi-Fi card, or cellular modem.
- NIC network interface card
- Wi-Fi card Wireless Fidelity
- Memory 263, 279, or 229 can include a machine-readable medium on which is stored one or more sets of instructions (e.g., software) embodying any one or more of the methodologies or functions described herein.
- the software may also reside, completely or at least partially, within the main memory and/or within the processor during execution thereof by the computer system, the main memory and the processor also constituting machine-readable media.
- the software may further be transmitted or received over a network via the network interface device.
- any portion of the systems and methods disclosed herein, can be implemented by computer, including the devices described above.
- Information is collected from a female subject. This data is then inputted into the central processing unit (CPU) of a computer.
- the CPU is coupled to a storage or memory for storing instructions for implementing methods of the present invention.
- the instructions when executed by the CPU, cause the CPU to provide a probability of successful in vitro fertilization in a selected cycle of in vitro fertilization.
- the CPU provides this determination by inputting the subject data into an algorithm trained on a reference set of data from a plurality of women for whom fertility-associated phenotypic traits and pregnancy outcomes for each cycle of IVF is known.
- the reference set of data may be stored locally within the computer, such as within the computer memory.
- the reference set may be stored in a location that is remote from the computer, such as a server.
- the computer communicates across a network to access the reference set of data.
- the CPU then provides a probability of achieving pregnancy at a selected point in time based on the data entered into the algorithm.
- GSE7846, GSE6364 (Sha et al, 2007; Burney et al, 2007): eutopic endometrium from endometriosis patients vs separate group of normal patients.
- a normalized expression matrix was computed for each study: For Affymetrix data, RMA normalization was used (Rafael et al., 2003). For Illumina, the log2 normalized values reported in the original publications were used.
- QC metrics were calculated using Bioconductor packages (Gentleman et al, 2004) such as arrayQualityMetrics and affyQCReport.
- Pathway analysis was performed using SPIA (signaling pathway impact analysis).
- Gene ontology analysis was performed using both i) a Fisher's Exact test on the counts of significant genes (p ⁇ 0.005) in and not in a given GO term, and ii) a Wilcoxon Rank Sum test on the gene specific t scores comparing genes in and not in a given GO term.
- pathway categories with the highest magnitude include chemokine/cytokine signaling and other immune response mechanisms, focal adhesion, extra-cellular matrix interactions, and angiogenesis (See 'Pathways' list.)
- genes see below
- the upregulation of CCL3L1, CCL3, FAM180A, THBS2, PDGFRL, FNl, CLECl lA all reflect the invasive tissue remodeling and immune-response associated with the development of endometriosis.
- ACTG1, Claudin-4 and EZR have all been implicated in the mediation of cell motility in different cancer types.
- MMP23B matrix metalloproteinase 23B
- THBS2 thrombospondin 2
- MMP23 has been found to be predominantly expressed in reproductive tissues (Velasco et al., 1999). Degradation of ECM is essential for cells to invade the peritoneum. MMPs are involved in the breakdown of extracellular matrix during embryonic development and reproduction, as well as arthritis and metastasis (reviewed by Christiane et al, 2014). THBS2 modulates cell-matrix interactions, and interestingly, is involved with integrin aVB3 in the modulation of cell spreading and migration of endothelial cells (Bornstein et al, 2000).
- TGF-betal -mediated downregulation of this transcription factor is associated with the induction of cell migration (Kawai et al., 2012).
- TGFB1 is actually enriched at sites of ectopic endometrium (Komiyana et al., 2007; Medina and Lebovic, 2010).
- Biological neuronal markers indicate specific types of nerve fibers present within endometrial layers (Tokushige et al, 2006).
- ROB03 which competes with ROBOl/2, representing an anti-repulsion mechanism
- chemokine binding protein 2 and chemokine (C-C motif) receptor 5 (CCR5).
- CCR5 chemokine (C-C motif) receptor 5
- This locus represents a small inducible cytokine.
- the encoded protein also known as macrophage inflammatory protein 1 alpha, plays a role in inflammatory responses through binding to the receptors CCR1, CCR4 and CCR5.
- FAMl 80 FAMl 2.898 6.725 Expression is TGF-B A 80A dependent in mammalian systems (Kosla et al, 2013)
- ITLN2 ITLN2 2.122 5.107 Possibly involved in
- ECM Error-Coupled Cell
- RCA Complement Activation
- SCR short consensus repeat
- FN1 FN1 1.668 16 Fibr Cell adhesion , migration, onec embryogenesis, wound tin healing, blood
- CNN3 CNN3 1.326 6.538
- ARPC2 ARPC 1.172 13.069
- H2AFJ H2AFX
- HIST1H2AC HIST1H2AC, AD, AE, BD,
- HIST1H3E HIST1H4A;
- HIST2H2AB AC, BE, BF;
- LILRB1 LILRB1; MAPK8; NFATC1;
- PIK3CG PIK3R1, R3;
- FIG. 4 The accepted mechanism for endometriosis is retrograde menstruation, which is the backflow of menstrual fluid and associated endometrial cells through the fallopian tubes. See FIG. 3. As shown in FIG. 4, retrograde menstruation causes adhesion and proliferation of endometrial cells outside of the uterine. The ectopic endometrial growth causes inflammation, immune system evasions, and EP dependence/P4 resistances. FIG. 4 also sets forth other potential contributory factors of endometriosis, including the presence of Mullerian rests, endometrial stem cells, and metaplasia.
- Methods of the invention rely on genetics and bioinformatics in order to identify clinically significant genetic signatures of endometriosis.
- the genetic signatures determined via methods of the invention, can be used to classify a subject's clinical condition (e.g. uterine phase or grade of endometriosis) and can be used to target treatment.
- FIG. 5 illustrates the clinical parameters that were assessed for the micro-array studies: age, presence/absence of endometriosis, stage of the disease, presence of pain, gravidty/parity, endometrioma position, tissue sampling method, phase of the uterine cycle, ethnicity, and leiomyata.
- FIG. 6A illustrates gene expression of the eutopic endometrium of the samples across the proliferative, early secretory, mid-secretory, and late secretory phases.
- FIG. 6B illustrates gene expression of the ectopic endometrium across the proliferative, early secretory, mid-secretory, and late secretory phases. K-means clustering analysis of the micro-array data indicated showed that genetic signatures that ectopic and eutopic endometrial tissue were phased dependent in different ways.
- the endometriosis phase-specific expression patterns were compared to the normal phase-specific expression patterns in order to identify genetic expression signatures specific to endometriosis and specific to a certain phase of the uterine cycle.
- FIG. 7 illustrates the phase- specific genetic signature differences between endometriosis and normal populations.
- the proliferative phase showed 430 genes with a fold-change greater than 2.0 (P -value less than 0.0005)
- the early secretory phase showed 151 genes with a fold-change greater than 2.0 (P -value less than 0.0005)
- the mid-late secretory phase showed 3 genes with a fold-change greater than 2.0 (P-value less than 0.0005).
- FIGS. 8-10 the meta-analysis revealed that certain genes of the endometriosis samples are up-regulated and down-regulated as compared to the normal.
- the misregulation of genes of the endometriosis sample varied across the different phases.
- the phase-specific regulation pattern of genes associated with endometriosis can be used as a regulation signature of the disease.
- FIG. 8 illustrates up-regulated and de-regulated genes associated with endometriosis at the proliferative stage.
- FIG. 9 illustrates up-regulated and deregulated genes associated with endometriosis at the early secretory stage.
- FIG. 10 illustrates up- regulated genes associated with endometriosis at the mid to late secretory phase.
- phase-specific endometriosis signatures identified using methods of the invention can be used as biomarkers for the disease and to guide course of treatment. Additional information can be correlated into the meta-analysis to obtain phase-specific endometriosis signatures associated with particular parameters, for example, age, stage of endometriosis, infertility, and other phenotypic traits. The clinical applications of the phase-specific endometriosis signatures are discussed hereinafter.
- the phase-specific endometriosis signatures can be utilized to target diagnosis of endometriosis.
- the expression levels of transcripts in one or more samples obtained from a patient suspected of having endometriosis can be compared to known phase-specific endometriosis signatures.
- the samples can be obtained at a particular phase or across several time -points of the patient's uterine cycle.
- the expression levels can be compared to signatures corresponding to one phase or diverse group of signatures from the various phases of the uterine cycle. Similarities between the patient's expression level and the phase-specific endometriosis signatures are the patient's phase-specific endometriosis signature and indicate that the patient has endometriosis.
- a course of treatment can be chosen that is tailored to the patient's phase-specific endometriosis signatures.
- drugs may be recommended or prescribed to the patient to coincide with the phase in which the patient has endometriosis signatures.
- drugs may be recommended or prescribed to the patients that are known to target the gene or the biochemical pathways associated with the gene.
- the comparison between the patient's expression pattern and the endometriosis signatures may be indicative of the grade of the patient's endometriosis.
- phase-specific genetic signatures can also be applied to identify and chart the patient's specific uterine cycle.
- the uterine cycle is very individual specific-ranging between 21 days to 35 days, with the norm being 28 days.
- the length of the phases of the uterine cycle likewise varies among individuals. Since treatment of endometriosis may be implicated for certain phases, the ability to genetically confirm the phase of an individual to direct the timing of treatment is advantageous.
- the patient's expression levels across different time -points can be compared phase-specific endometriosis signatures to determine the timing of the patient's uterine cycle. For instances, correlations between the patient's expression levels and signatures of a particular phase are indicative of the phase of the patient.
- Utilizing genetics to determine the timing of a patient's uterine cycle provides benefits such as being able to tailor treatment of a variety of reproductive conditions, including the treatment of infertility, premenstrual dysphoric disorder, and endometriosis. In various scenarios, a better understanding of the timing of one's uterine cycle provides greater insight into the hormonal state of the patient, which may guide hormone treatment regimens.
- LLP dataset of 5208 cycles was used for the analysis. Logistic regression models were created and controlled for age, day 3 follicle stimulating hormone (FSH), peak estradiol level, number of oocytes retrieved, number of embryos transferred, and whether intra-cytoplasmic sperm injection (ICSI) procedure was performed).
- FSH follicle stimulating hormone
- ICSI intra-cytoplasmic sperm injection
- the infertility diagnoses included in the analysis were diminished ovarian reserve, endometriosis, idiopathic, male factor, PCOS, and tubal factor (Table 1).
- Table 1 Sample information for diagnoses included
- NB p-value is obtained from a proportion-test, which checks if the proportion of obese patients for a given diagnosis is greater than in the data combining all six diagnoses.
- Idiopathic 0.71 0.22 As a secondary analysis, a specific point along an IVF cycle was determined where the effects of obesity become significant. To do this, 'landmarks' such as number of oocytes retrieved, rates of embryo development, number of embryos transferred and implantation rate were correlated with obesity. This analysis was repeated using data subset for different common infertility diagnoses, to determine what parts of the cycle are most affected by obesity, for each diagnosis.
- Obesity was not correlated significantly with any IVF cycle 'landmarks' between oocyte retrieval and embryo transfer, for any diagnosis. This result indicates that the effect of obesity on IVF outcome occurs after embryo transfer takes place.
- Table 5 Effects of both 'implantation rate less than 50%' and obesity on live birth outcome for PCOS patients.
- EmbryosTransferred 0 binary CountofDeg-obese + Age + FSHMax + PeakE2 + Retrieved+icsi diagnosis effect(obese) p-value(obese) lwr upr n (cycles) n (obese)
- obese obese % obese p- diagnosis cycles cycles patients patients patients value all 5208 648 2738 344 0.124
- p-value is from proportion test: proportion of obese overall to proportion of obese in diagnosis
- Patients analyzed included those with partners of normal karyotype, who underwent fresh in vitro fertilization (IVF) cycles in which 1 oocyte was retrieved, PGS was performed, and day 3 FSH levels were known for the cycle.
- IVF fresh in vitro fertilization
- PGS was performed
- day 3 FSH levels were known for the cycle.
- the effects of patients' age and FSH levels were correlated with aneuploidy status using generalized estimation equation (GEE) models.
Abstract
Description
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EP3169811A4 (en) | 2017-12-27 |
EP3169811A1 (en) | 2017-05-24 |
KR20170063519A (en) | 2017-06-08 |
US20160017426A1 (en) | 2016-01-21 |
US20200017912A1 (en) | 2020-01-16 |
SG11201700303UA (en) | 2017-02-27 |
CA2954895A1 (en) | 2016-01-21 |
JP2017526345A (en) | 2017-09-14 |
US10208350B2 (en) | 2019-02-19 |
AU2015289464A1 (en) | 2017-02-02 |
IL249984A0 (en) | 2017-03-30 |
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