Future Perspectives in the ART Lab


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  • GHDuffy et al (Cochrane): Ten studies (440 subfertile couples) were included.In women who are not considered poor responders undergoing in IVF there is no evidence from randomised controlled trials to support the use of growth hormone. In women who are considered poor responders the use of growth hormone has been shown to significantly improve live birth (4 RCT- OR 5.39, 95% CI 1.89 to 15.35)and pregnancy rates (8 RCT - OR 3.28, 95% CI 1.74 to 6.20).  Quality of the evidence; differences in participant number, cause of subfertility, treatment protocol and outcomes measured all varied considerably between the trials. There was no uniformity of dose and timing of the intervention. A large scale trial with a standardised treatment protocol and intervention protocol is required. Kolibianakis: 6 RCT (169) –only poor responder: clinical pregnancy (rate difference: +16%, 95% CI: +4 to +28; fixed effects model) (number-needed-to-treat (NNT) = 6, 95% CI: 4-25) and live birth rates (rate difference: +17%, 95% CI: +5 to +30; fixed effects model) (NNT = 6; 95% CI: 3-20). Furthermore, GH addition was associated with a significantly higher proportion of patients reaching embryo transfer (rate difference: +22%, 95% CI: +7 to +36; fixed effects model). Kyrou: Five eligible RCTs, poor responder (n = 128). Odds ratio for live birth: 5.22, confidence interval: 95% 1.09–24.99Many different protocols of GH use: Owen et al. (1991) : 24 IU im/day on alternate days, starting simultaneously with hMG until the day of hCG administrationZhuang et al. (1994): 12 IU im/day on alternate daysSuikkari et al. (1996): 4 or 12 IU/day, starting on cycle day 3Tesarik et al (2005): 8IU of GH from day 7 of exogenous gonadotrophin administration till the day following the ovulation-triggering injection of hCGBergh et al. (1994): 0.1 IU/kg body weight/day sc, starting simultaneously with FSH until the day of hCG administrationDor et al. (1995) 18 IU sc on cycle day 2, 4, 6, 8
Kucuk et al. (2008): 4 mg (12 IU) sc, from day 21 of the preceding cycle and until the day of hCG administration The grounds for supplementing GH in ART are multiple. Insulin-like growth factors 1 (IGF-1) and 2 (IGF-2) are both present in follicular fluid and believed to play a crucial role in the cytoplasmic maturation. In several animal models of in vitro maturation exogenous administration of GH increased follicular IGF-1 and IGF-2 in as well as oocyte competence. Growth hormone could possibly increase the DNA repair capacity in oocytes as shown in liver cells. In support of this hypothesis, Mendoza et al. showed a positive correlation between the oocytes’ ability to evolve in morphologically normal embryos and GH levels in follicular fluid. Furthermore, several reports looking at the function of Granulosa cells in vitro indicated that IGF-1 improved the response to gonadotropin stimulation.Testosterone1 meta-analysis, Bosdou et al (2012): In two trials involving 163 patients, pretreatment with transdermal testosterone was associated with an increase in clinical preg- nancy [risk difference (RD): +15%, 95% confidence interval (CI): +3 to +26%] and live birth rates (RD: +11%, 95% CI: +0.3 to +22%) in poor responders undergoing ovarian stimulation for IVF.Only 2 trials:Massin 2006 (humanreproduction): prospective, randomized, double-blind, placebo-controlled study.The design was set up to perform a paired comparison of the ovarian parameters recorded in two consecutive cycles, each woman being used as her own control. And then, comparing testosterone to placebo. 25 women with placebo ans24 withtrasndermictestosterone ( testosterone 1%), Women applied once-daily 1 g of gel (10 mg of testosterone) on the external side of the thigh. Testosterone absorption with the gel is approximately 10%. Either testosterone or placebo gels were applied for 15–20 days in the period preceding the second stimulation for IVF or ICSI, i.e. during the period of pituitary desensitization in women treated with a long GnRH agonist protocol or during pill administration in women treated with another analogue protocol. Comparingtopreviouscycle, thebothgroupstherewasanincrease in thenumberofoocytesonthesecond. Placebo group: 3,6 to 5 (p<0,02) / testosterone: 3 to 5,31(p<0,02). Placebo x testo: p=0,8, no difference: numbers of pre-ovulatory follicles, total and mature oocytes and embryos did not significantly differ between testosterone and placebo-treated patients.. Kim, 2011 (FertilityandSterility): poorresponders, 55 with placebo and 55 withtrasndermictestosterone ( testosterone 1%), with a 1.25 mg/d nominal delivery rate of testosterone was started from sixth day of E-P pretreatment and continued for 21 days. All antagonist cycle. The numbers of oocytes retrieved, mature oocytes, fertilized oocytes, and good-quality embryos were significantly higher in the TTG pretreatment group. Embryo implantation rate and clinical pregnancy rate per cycle initiated also were significantly higher in the women pretreated with TTG.Explanations to use: It has been suggested that the accumulation of androgens in the micro milieu of the primate ovary, plays a critical role in early follicular development and granulosa cell proliferation. Androgen excess has been shown to stimulate early stages of follicular growth and increase the number of preantral and antral follicles. In addition, increased intraovarian concentration of androgens seems to augment follicle stimulating hormone (FSH) re- ceptor expression in granulosa cells and thus, potentially lead to enhanced responsiveness of ovaries to FSH. Besides these experimental data, further clinical observa- tions on women with polycystic ovary syndrome or testosterone-treated female transsexuals, suggest that exposure to exogenous androgens may lead to increased number of developing follicles, regardless of gonadotrophin stimulation Furthermore, it has been reported that inadequate levels of endogenous androgens are associated with decreased ovarian sensitivity to FSH and low pregnancy rates after IVF.Bosdou et al alsoevaluatedotherinterventionssuch as use ofaromataseinhibitors, androgens, etc.
  • Future Perspectives in the ART Lab

    1. 1. Future Perspectives inthe ART LabSandro Esteves, M.D., Ph.D.Director, ANDROFERTCenter for Male Reproduction & InfertilityCampinas, BRAZILFertility Experts Latinamerica Meeting 1Mexico City, April 11-12, 2013
    2. 2. Esteves, 2Maximize beneficialeffects of treatment;Minimize complicationsand risksRedifinition ofSuccess:SET leading to onehealthy live birthCentralParadigmIndividualizationof ovarianstimulationOptimalendometrial receptivityHigh-qualitygametes andembryos
    3. 3. Esteves, 3Defining what is a “valid” biomarker in thecontext of reproductive medicineOverview of biomarkers with potentialapplication in the ART labClinical translation: where are we and whereare we going?What is in it for me?
    4. 4. A Valid Biomarker in ReproductiveMedicine Provides RealisticPrognostic InformationEsteves, 4 Adapted from: ASRM Practice Committee, Fertil Steril 2012;98:147+-+ -BiomarkerTestResultOutcome AssessedFalsePositive(B)FalseNegative(C)TrueNegative(D)TruePositive(A)Sensitivity (A/A+C)Specificity (D/B+D)Predictive Value(A/A+B)Accuracy(Sens./1-Spec.= Areaunder the ROC curve)
    5. 5. Esteves, 5Prediction of Ovarian ResponseBefore COSBiomarkersEvidenceLevel1a
    6. 6. Esteves, 6• Avoid over-aggressive stimulation in‘true’ high responders• Avoid over-conservative stimulation in‘false’ high respondersExcessiveOvarianResponse• Avoid over-conservative stimulation in‘true’ DOR• Avoid over-aggressive stimulation in‘false’ DORDiminishedOvarianReserve(DOR)Why Do We Need Biomarkers toPredict Ovarian Response toCOS?
    7. 7. Low-startingFSH dose (150 UI)AMH (ng/mL) >2.1¶GnRH Agonist GnRHAntagonistDays of Stimulation 13 (12-14) 9 (8-11)*No. Oocytes (n) 14 (10-19) 10 (8.5-13.5)*OHSS requiringhospitalization20 (13.9%) 0 (0%)*Cancellation 4 (2.7%) 1 (2.9%)CPR per transfer 40.1% 63.6%*¶DSL assay; Adapted from Nelson SM et al . Anti-Müllerian hormone-based approach tocontrolled ovarian stimulation for assisted conception. Hum Reprod. 2009; 24(4):867-75.*P ≤ 0.01Esteves, 7EvidenceLevel2aRiskorExcessiveResponse
    8. 8. InterventionMeta-analyticStudiesPopulationEffect onPregnancyRec-hLHsupplementationto rec-hFSHMochtar et al, 20071Bosdou et al, 20122Hill et al, 20123Poorresponders1,2Age ≥35 yrs3Higher OPR1Higher LBR2Higher CPR3Growth HormoneKyrou et al,20091Kolibianakis et al, 20092Duffy et al, 20103PoorrespondersHigher LBR1,2,3Higher PR2Higher CPR3Testosterone Bosdou et al , 2012PoorrespondersHigher LBRHigher CPRKolibianakis et al, Hum Reprod Update 2009,15:613-22; Kyrou et al, 2009;91: 749–66; Duffy et al,Cochrane Database Syst Rev 2010;1:CD000099; Mochtar MH et al. Cochrane Database Syst Rev.2007,2:CD005070; Bosdou JK et al, Hum Reprod Update 2012;8:127-45; Hill MJ et al. Fertil Steril2012;97:1108-4.Level1aEsteves, 8DiminishedOvarianReserve
    9. 9. Esteves, 9The ART LaboratoryTodayARTLabMorphology-basedGametes andEmbryos SelectionFISH-basedPGD/PGSAir QualityControlVitrificationCleavage-stage andBlastocystCulture
    10. 10. Esteves, 10Embryo Freezing● Five RCT: VITRI vs. Slow Freezing●765 cycles●Better outcomes with Vitrification:CPR = 39% x 33%; OR: 1.55; 95% CI: 1.03-2.32OPR = 35% x 27%; OR: 1.82; 95% CI: 1.04-3.20IR = 29% x 24%; OR: 1.49, 95% CI: 1.03-2.15VitrificationIn addition, Vitrification is simpler andfaster than Slow FreezingAbdelFahez et al . Reproductive BioMedicine Online (2010) 20, 209– 222EvidenceLevel1a
    11. 11. Esteves, 11Embryo Culture● Eight RCT: Blastocyst vs Cleavage-stage Transfers; 1,654 patients●Better outcomes with Blastocyst ET:LBR = 35% x 28%; OR: 1.39; 95% CI: 1.10-1.76CPR = 39% x 33%; OR: 1.27; 95% CI: 1.03-1.55BlastocystTransferImproved ability to select embryos, but...1. Risky for pts. with few embryos2. Prolonged culture associated with imprinting, epigeneticdisorders, pre-term birth3. High rate of aneuploidy in blastocystsManipalviratn et al, 2009; Kallen et al, 2009; Munné et al., 2012Papanikolaou E et al. Hum Reprod 2008; 23: 91–99;EvidenceLevel1a
    12. 12. Esteves, 12BiomarkersintheARTLabGametesandEmbryosMorphologicalbiomarkersMetabolicbased-biomarkersGenetic-basedbiomarkersProteomic-basedbiomarkersThe ART LaboratoryTomorrow
    13. 13. Esteves, 13Sperm Quality Biomarkers• Hyaruronic Acid Binding• Polarization Microscopy• MSOME• Electroforetic Sperm Isolation• Magnetic-activated Cell Sorting• Microfluids• Microarray Technology• ProteomicsSpermSelectionTechniquesNormalcy of SpermChromatin Content
    14. 14. Esteves, 14DNA Integrity is the Key SpermBiomarkerCurrent non-invasive sperm selection techniquescannot directly assess sperm DNA fragmentationDyes are used to reach the nucleus,using fixed specimensIn general, labor-intensive techniquesRecent progress (Enciso et al, 2012):• New synthetic peptide (DWI)• Derived from p53 protein• Affinity to various DNA lesions• Rapid and inexpensive• Still unable to penetrate intactsperm membranesSpermSelectionTechniques
    15. 15. Alternative has beenTESA-ICSISperm % TUNEL + % CPR %IREjaculated (N=18) 23.6 ± 5.1 6 2Testicular (N=18) 4.8 ± 3.6 44 21P value <0.001 <0.05 <0.01Greco E et al. Hum Reprod 2005;20:226–30*Absolute differences between two specimens ranging from -3.3% to -56.3%.Moskovtsev et al. Fertil Steril 2010; 93(4): 1142–6.DNA damage in Testicular Spermatozoa (13.3%)is three-fold lower compared with EjaculatedSpermatozoa (39.7%)*Esteves, 15
    16. 16. Esteves, 16Oocyte Quality BiomarkersOocyteSelectionTechniques• Polarization Microscopy (Polscope)• Oxygen Consumption (Embryoscope)• Microarray TechnologyCumulus cells gene expression (mRNA transcripts)• Molecular Mining of Follicular FluidAmino acids, metabolites, peptides, proteinsMass Spectroscopy, Raman SpectroscopyNuclear Magnetic Resonance
    17. 17. Esteves, 17Embryo Quality BiomarkersRationale: >50% in vitro-produced embryos are abnormalDevelopmental Stage:• Cleavage-stage Embryo Biopsy (most used)• Polar Body (single allelic copy)• Blastocyst (trophectoderm cells)EmbryoSelectionTechniquesInvasiveBiopsy:Techniques:• FISH (single-cell test; technical limitations)• PCR (DNA amplification-based approach)• CGH (combination of molecular and cytogenetic)• Single-nucleotide Polymorphism Micro-array• Next-generation Sequencing (single gene)• Quantitative Real-time PCR (qPCR)
    18. 18. Esteves, 18EmbryoSelectionTechniquesBiopsy:Polar body, Day-3 embryo andTrophectoderm cellsOPR: 59% vs 38% controls (p<0.001)Munné et al, Fertil Steril 2010PGS sure™ (Blue Genome, UK)Micro-array CGH solution tocount all chromosomes in <12hMicro-array labhardwareEmbryo Quality Biomarkers
    19. 19. Esteves, 19Embryo Quality BiomarkersMetabolic Profile:• Glucose and pyruvate uptake• Amino acid turnover• Oxygen consumptionProteomics• Mass Spectroscopy• Raman Spectroscopy• Nuclear Magnetic ResonanceMorphokinetics• Time-lapse microscopyEmbryoSelectionTechniquesNon-InvasiveKatz-Jaffe & McReynolds, Fertil Steril 2013;99:1073-77
    20. 20. Esteves, 20Embryo Quality BiomarkersMetabolic Profile (2010):• Via-Metrics™ (Molecular Biometrics, USA)EmbryoSelectionTechniquesNon-InvasiveAdvocated as a HighlySensitive Method ofMetabolomics Analysisby NIR SpectroscopyMarket withdrawal due toinstrument inability toperform accuratemeasurements
    21. 21. Esteves, 21Embryo Quality BiomarkersMorphokinetics (2010):Image capture over timeCombination of morphological, dynamic and quantitativeinformation about developmental eventsEmbryoSelectionTechniquesNon-InvasivePrinciple:1st cytokynesis (within 14 6 min)Time between 1st and 2nd mitosis (11.1 2.2 h)Time between 2nd and 3rd mitosis (1.0 1.6 h)Payne et al, 1997; Lemmen et al, 2008; Wong et al, 2010*; Meseguer et al, 2011;Hashimoto et al, 2012
    22. 22. Esteves, 22Embryo Quality BiomarkersStage-top IncubatorTokai-Hit, JapanInCu-Cell Live™Sanyo, JapanBioStation™Nikon, JapanSeveral Time-lapse Technologies Available:Time-lapseTechnologies
    23. 23. Esteves, 23Embryo Quality BiomarkersTime-lapseTechnologiesPrimo-Vision™Cryo-InnovationLtd., HungaryEmbryoScope version C™Nanorespirometer + Time-lapse videomicrographyUnisense Fertilitech, Denmark
    24. 24. Esteves, 24Embryo Quality BiomarkersTime-lapseTechnologiesEeva™ (Videomicrography +Computer Vision Software)Auxogyn, USACell tracking and prediction software (measure of time embryo takesto achieve specific milestones):
    25. 25. Esteves, 25Clinical Translation:Where we areBiomarkersintheARTLabTime-lapseTechnologyMicro-arrayCGH
    26. 26. Esteves, 26Clinical Translation:Where we are goingBiomarkersintheARTLabSperm andOocyteSelectionUsingBiomarkersEmbryoSelection byReal-timeSecretome +MorphokineticsAnalysisMicrofluidic Platformfor Embryo Culture
    27. 27. Esteves, 27The Biomarkers Era has arrived. Several markers underinvestigation and some already translatedValid biomarkers are highly sensitive and specific, andhave high predictive valueFor application at a global level, ART lab’s biomarkers/technologies should be VEELI:ValidatedEasy to useEasy to replicateLow costImprove outcomesFuture Perspectives inthe ART LabConclusions
    28. 28. Esteves, 28