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Current and potential biomarkers in clinical practice There is a continuous research for the identification and development of new molecular and genetic biomarkers that could improve the diagnostic accuracy or help the detection of various diseases; Currently the biomarkers used in clinical practice are limited, since most of the available tests do not have the sensitivity nor the specificity required.
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Needs of reliable biomarkers for use in diagnosis of reproductive defects: the use of robust assays is essential to qualify new molecules at the time of their identification. National Institutes of Health defined a serum biomarker as: "a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention" IN CLINICAL PRACTICE
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CLINICAL DEVELOPMENT OF A BIOMARKER 1.Preclinical exploration, identification of potential markers 2.Definition of their clinical relevance via the development of a clinical assay 3.Evaluation of the predictive ability through a retrospective study of a group or a case-control 4.Validation with a perspective approach
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Difficult to translate the results obtained in the laboratory to “bed", and the research efforts frequently stop at the early stages of the discovery of biomarkers, without any validation of promising preclinical targets; The phenotype of samples, collection and storage of materials, use of new assays and the over- interpretation of results ( which could lead to confusion) represent serious limitations for a biomarker to reach the final round of validation. LIMITS
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EVALUATION CRITERIA OF MARKERS
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ovarian reserve quality of the gametes embryo viability Euploidia endometrial receptivity pregnancy outcomes (miscarriage, ectopic pregnancy) obstetric complications (preeclampsia, preterm pregnancy) to discriminate between suitable and unsuitable embryos to be transferred in the programs of in vitro fertilization (IVF) Reproductive clinic medicine Biomarkers to better understand and predict:
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EARLY PREGNANCY BIOLOGICAL PATHWAYS The clinical utility of biochemical markers is the ability to provide objective results when measured with an accurate test.
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Human chorionic gonadotropin (hCG): once pregnancy is established, the INCREASE of hCG, produced by trophoblast cells, is indicative of embryo vitality vIUP viable intrauterine pregnancy: in 48h 53% hCG ratio: serum hCG a 48h/serum hCG a 0h es: <0,21 predictor of ectopic pregnancy MARKERS OF TROPHOBLAST FUNCTION
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Hyperglycosylated hCG (hCG-H): produced by extravillous cytotrophoblast cells and secreted at the time of implantation hCG-H/hCG= possible marker of early pregnancy failure (cutpoint: 13 µg/L) >50% to differentiate between viable pregnancies and early pregnancy failure MARKERS OF TROPHOBLAST FUNCTION Activin A: Dimeric glycoprotein,member of the family of transforming growth factor -β (TGF- β); produced by placenta, promotes the invasion of cytotrophoblasts. Abnormal values indicate problems in the implantation and in the vitality Level ≤ 0.37 ng/mL specific for the diagnosis of ectopic pregnancy Possible single marker of ectopic pregnancy (although activin A remains in Phase 3 of development)
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MARKERS OF TROPHOBLAST FUNCTION Pregnancy-associated plasma protein A (PAPP-A) produced by trophobasts Marker of early pregnancy failure and aneuploidia in combination of other potential markers: eg in association with P and the vascular endothelial growth factor (VEGF); marker of vitality but it is not predictive of ectopic pregnancies. A drop in PAPP-A levels is found in the first trimester of pregnancies with Down syndrome. It has been shown that low levels of PAPP-A in maternal serum are associated, in the absence of an abnormal karyotype, with an increased risk of preterm labor. Low levels of PAPP-A in maternal serum during the first trimester could indicate a lack of trophoblast invasion in the interface mother-fetus, that consequently induces also in this case a preterm labor. For abnormal, ectopic and extrauterine pregnancies under investigation: Glycoprotein-1 specific to pregnancy; hormone placental lactogen (HPL) or somatomammotrophin; Disintegrin and metalloproteinase-12 (ADAM-12); Markers of nucleic acids, mRNAs
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progesterone (P) estrogen (E2) inhibin A MARKERS OF CORPUS LUTEUM FUNCTION used in clinical practice to monitor the progress of a normal pregnancy, or to determine its complications
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PROGESTERONE AND ESTROGEN LEVELS P <5 ng / mL predictor for non-viable pregnancies In step 4 of development as a marker for EP and / or in combination
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E3 :derived in nature from estrogen hormone. A peak of salivary estriol is found several weeks before the onset of spontaneous preterm labor, possibility of using the salivary estriol as a predictor of risk of preterm labor. SalEST™ laboratory technique for measuring the levels of salivary estriol however the method is not satisfactory [the ACOG(American College of Obstetricians and Gynecologists, Istituto americano degli ostetrici e dei ginecologi) does not recommend the test of salivary estriol as a screening test of premature labor because it generates a high percentage of false positive results and it could add significant costs to prenatal care and unnecessary interventions]. The positive predictive value of the salivary estriol test is just 20% (negative predictive value is approximately 98%). In addition, the salivary estriol predicts relatively well the birth at term, but it is not particularly useful for the prediction of preterm more precocious births. ESTRIOL E3
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Multiple Marker Test Performance for Prediction of Ectopic Pregnancy
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In an initial phase and before the placental production P is secreted by the corpus luteum and is a critical signal for the establishment of a normal pregnancy; P serum: single specific biomarker to determine non-viable pregnancy in early gestation; hCG, in combination with transvaginal ultrasound, is useful for the diagnosis of ectopic pregnancy; E2 levels give important information on ovarian function and / or diseases that affect the testes, ovaries or adrenal glands; E2 levels may allow doctors to monitor the progression of the pregnancy, the efficacy of fertility treatments, or to evaluate menopause symptoms; Inhibin A and hCG beta-core, the major metabolite of hCG in mother urine have been studied as potential biomarkers for determining ectopic pregnancies against normal pregnancies. Found….. in clinical practice
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Detectable only in women with the arrival of menarche, is secreted by the corpus luteum, and has a negative feedback action on the pituitary production of FSH: inhibin A is consequently detectable in the late luteal phase of the menstrual cycle. INHIBIN A Conflicting data on cutpoint: 50 pg / mL and 28.67 pg / mL for EP and ectopic pregnancies Marker of vitality in Phase 3 of development
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The AFP is produced by the liver of the fetus and it can be measured in maternal blood. Normally its levels increases during the weeks of pregnancy, then they decrease rapidly after childbirth, returning to normal levels. In some cases the AFP concentration is present in higher values already in the early stages of pregnancy. Alpha Fetoprotein Normal values of alpha feto protein Adults< 5ng/mL 1° trimester of pregnancy< 50ng/mL 2° trimester of pregnancy < 140ng/mL 3° trimester of pregnancy < 450ng/mL Factors that could elevate AFP values: Incorrect dating of pregnancy Twin pregnancy Spina bifida, anencephaly Rare anomalies No anomaly but just a value above normal
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A glycoprotein that acts as a glue between the maternal decidua and amniotic membranes, is normally present in vaginal secretions before the 16th-18th week and usually reappears at the beginning of labor. The test is based on the detection of the presence of fibronectin in maternal secretions using a vaginal swab in the second trimester. The test is positive when the level of fibronectin exceeds the threshold of 50 ng / ml. The sensitivity was from 13 to 55% and the specificity between 83 and 99%. Fetal Fibronectin
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It is an immunoassay diagnostic test that can be done after sample collection with a swab during vaginal speculum. The fetal fibronectin test is not able to confirm if a woman is in labor, but it can determine if it is not. A negative result with the fFN test indicates a high improbability that the woman give birth within a week or two. A positive result, however, is not so useful because it indicates that the woman is at high risk of giving birth prematurely, but it does not provide a guarantee of an imminent birth. LIMIT: The inadequate positive predictive value of the examination. This is the reason for which the fetal fibronectin test is not recommended by ACOG for routine screening, but it is just used for the screening of symptomatic women. The result of fFN test can be compromised by the urine, blood or seminal plasma and it is not indicated in case of membranes rupture. Fetal fibronectin test
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BIOCHEMICAL PROFILE OF REPRODUCTIVE BIOMARKERS IN FEMALE CLINICAL PRACTICE
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MARKERS OF ANGIOGENESIS VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF): It supports the vascular development of foetoplacental unit in combination or for EP Phase 2 of clinical development GROWTH FACTOR PLACENTA-LIKE: It is produced by trophoblasts in implantation sites Phase 3 of clinical development for EP
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MARKERS OF ENDOMETRIAL FUNCTION Leukemia inhibitory factor (LIF) belonging to the family of interleukin IL -6; it is important in inflammation and in the implantation. GLICODELIN protein found in the endometrium and in the fallopian tubes associated with immunomodulation during implantation; Marker of extrauterine pregnancy in Phase 3 of development. Mucin-1 glycoprotein of apical epithelial surface expressed in the endometrium and fallopian tubes; it is involved in the interaction blastocyst-endometrium during implantation; phase 1-2 of the development. Adrenomedullin peptide hormone that promotes angiogenesis endometrial; phase 1 of development.
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activin-A; C-reactive protein; placental growth factor and its soluble receptor tyrosine kinase (s-1Flt); leptin; transforming growth factor-α1; plasminogen activator inhibitor. POTENTIAL BLOOD BIOMARKERS IN PRESYMPTOMATIC AND PREDICTIVE PREECLAMPSIA LIMITS: 1. these biomarkers have a limited clinical application, because of the low sensitivity and specificity of the available methods for their detection; 2. there is no single indicator that allows a precocious prediction of pre- eclampsia.
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test of ovarian reserve includes hormone markers (FSH, E2, inhibin-B, antimullerian hormone [AMH]), and ultrasound markers (ovarian volume, antral follicle count [AFC]); all of them have major limitations in terms of accuracy, invasiveness and cost that must be considered carefully to make reliable predictions; most of the potential biomarkers studied in reproductive medicine, or those already in use, are aimed at the diagnosis of diseases, such as infertility, polycystic ovarian syndrome, endometriosis, or the viability of a pregnancy; the aim is to identify individual markers for each disease, while multivariable diagnostic markers have been reported until now. CONSIDERATIONS
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The use of two or more markers can provide a diagnostic test with a better sensitivity and specificity; Combinations of biomarkers have been shown to support an initial diagnosis of premature pregnancy or ectopic, suggesting that a strategy of multiple biomarkers could help to identify non-viable pregnancies. BIOMARKERS IN COMBINATION
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The conventional analysis of sperm provides important parameters such as sperm concentration, vitality, motility and morphology, but it is considered an indicator of low reproductive potential and it is not predictive for the outcome of pregnancy Different assays have been developed and used in research in order to assess the damage to sperm DNA, which is clinically more informative and relevant. LIMITS: 1. Semen analysis provides useful information for the initial evaluation of male infertility, but it does not measure precisely the fertility; 2. It does not consider functional aspects of sperm cells such as the ability to fertilize the egg. IN MALE
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CYTOKINES AND REPRODUCTION ● Important autocrine and paracrine regulators of ovarian function since they are produced in the ovary and follicular fluid; ● The cytokine production is stimulated by LH; ● Pro-inflammatory cytokines stimulate the NO pathway in the regulation of ovulation; ● They control the ovarian proliferation, apoptosis, folliculogenesis, the luteum genesis, the oogenesis and the release of hormones; ● They regulate the upstream response of hormonal regulators, the fertility and in some cases the development of ovarian disorders.
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INTERLEUKINS
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INTERLEUKIN EFFECTS
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IL-1
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IL-1FUNCTIONS
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POSSIBLE INTERACTION BETWEEN EMBRYO AND ENDOMETRIUM The thyroid system is involved in the regulation of LIF (leukemia inhibitory factor) and IL, key factors for the success of the implantation
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PRO- AND ANTI-INFLAMMATORY PROCESSES IN LABOUR
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Role of NK cells in pregnancy Decidual NK cells are a subgroup derived from peripheral NK, which promote the success of pregnancy. In pathological conditions, an altered ratio of NK cells and NK killer regulators can damage the trophoblastic tissue, resulting in abortion. Immunological mechanism of tolerance and rejection of the trophoblast. Tr Cell: regulatory T cell; MO: macrophage; + P: progesterone receptor. NK: NK cell Decidual; PIBF: progesterone induced blocking factor.
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