Future Perspectives in the ART Lab
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The document summarizes a presentation on future perspectives in the ART (assisted reproductive technology) lab. It discusses how biomarkers can be used to: 1) predict ovarian response to controlled ovarian stimulation, 2) assess sperm and egg quality, and 3) select embryos. Current techniques for embryo selection include preimplantation genetic screening through biopsy and arrays. New techniques under study include metabolomic profiling and morphokinetic analysis through time-lapse imaging. The presentation envisions future ART labs using biomarkers to select sperm, eggs, and embryos through techniques like microfluidics that are validated, easy-to-use, replicable, low-cost and can improve outcomes.
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Future Perspectives in the ART Lab
- 1. Future Perspectives in the ART Lab Sandro Esteves, M.D., Ph.D. Director, ANDROFERT Center for Male Reproduction & Infertility Campinas, BRAZIL Fertility Experts Latinamerica Meeting 1 Mexico City, April 11-12, 2013
- 2. Esteves, 2 Maximize beneficial effects of treatment; Minimize complications and risks Redifinition of Success: SET leading to one healthy live birth Central Paradigm Individualization of ovarian stimulation Optimal endometrial receptivity High-quality gametes and embryos
- 3. Esteves, 3 Defining what is a “valid” biomarker in the context of reproductive medicine Overview of biomarkers with potential application in the ART lab Clinical translation: where are we and where are we going? What is in it for me?
- 4. A Valid Biomarker in Reproductive Medicine Provides Realistic Prognostic Information Esteves, 4 Adapted from: ASRM Practice Committee, Fertil Steril 2012;98:147 + - + - BiomarkerTestResult Outcome Assessed False Positive (B) False Negative (C) True Negative (D) True Positive (A) Sensitivity (A/A+C) Specificity (D/B+D) Predictive Value (A/A+B) Accuracy (Sens./1-Spec.= Area under the ROC curve)
- 5. Esteves, 5 Prediction of Ovarian Response Before COSBiomarkers Evidence Level 1a
- 6. Esteves, 6 • Avoid over-aggressive stimulation in ‘true’ high responders • Avoid over-conservative stimulation in ‘false’ high responders Excessive Ovarian Response • Avoid over-conservative stimulation in ‘true’ DOR • Avoid over-aggressive stimulation in ‘false’ DOR Diminished Ovarian Reserve (DOR) Why Do We Need Biomarkers to Predict Ovarian Response to COS?
- 7. Low-starting FSH dose (150 UI) AMH (ng/mL) >2.1¶ GnRH Agonist GnRH Antagonist Days of Stimulation 13 (12-14) 9 (8-11)* No. Oocytes (n) 14 (10-19) 10 (8.5-13.5)* OHSS requiring hospitalization 20 (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 to controlled ovarian stimulation for assisted conception. Hum Reprod. 2009; 24(4):867-75. *P ≤ 0.01 Esteves, 7 Evidence Level 2a RiskorExcessive Response
- 8. Intervention Meta-analytic Studies Population Effect on Pregnancy Rec-hLH supplementation to rec-hFSH Mochtar et al, 20071 Bosdou et al, 20122 Hill et al, 20123 Poor responders1,2 Age ≥35 yrs3 Higher OPR1 Higher LBR2 Higher CPR3 Growth Hormone Kyrou et al,20091 Kolibianakis et al, 20092 Duffy et al, 20103 Poor responders Higher LBR1,2,3 Higher PR2 Higher CPR3 Testosterone Bosdou et al , 2012 Poor responders Higher LBR Higher CPR Kolibianakis 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 Steril 2012;97:1108-4. Level 1a Esteves, 8 DiminishedOvarian Reserve
- 9. Esteves, 9 The ART Laboratory Today ART Lab Morphology-based Gametes and Embryos Selection FISH- based PGD/PGS Air Quality Control Vitrification Cleavage- stage and Blastocyst Culture
- 10. Esteves, 10 Embryo Freezing ● Five RCT: VITRI vs. Slow Freezing ●765 cycles ●Better outcomes with Vitrification: CPR = 39% x 33%; OR: 1.55; 95% CI: 1.03-2.32 OPR = 35% x 27%; OR: 1.82; 95% CI: 1.04-3.20 IR = 29% x 24%; OR: 1.49, 95% CI: 1.03-2.15 Vitrification In addition, Vitrification is simpler and faster than Slow Freezing AbdelFahez et al . Reproductive BioMedicine Online (2010) 20, 209– 222 Evidence Level 1a
- 11. Esteves, 11 Embryo 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: 1.10-1.76 CPR = 39% x 33%; OR: 1.27; 95% CI: 1.03-1.55 Blastocyst Transfer Improved ability to select embryos, but... 1. Risky for pts. with few embryos 2. Prolonged culture associated with imprinting, epigenetic disorders, pre-term birth 3. High rate of aneuploidy in blastocysts Manipalviratn et al, 2009; Kallen et al, 2009; Munné et al., 2012 Papanikolaou E et al. Hum Reprod 2008; 23: 91–99; Evidence Level 1a
- 12. Esteves, 12 Biomarkers intheARTLab Gametes and Embryos Morphological biomarkers Metabolic based- biomarkers Genetic- based biomarkers Proteomic- based biomarkers The ART Laboratory Tomorrow
- 13. Esteves, 13 Sperm Quality Biomarkers • Hyaruronic Acid Binding • Polarization Microscopy • MSOME • Electroforetic Sperm Isolation • Magnetic-activated Cell Sorting • Microfluids • Microarray Technology • Proteomics SpermSelection Techniques Normalcy of Sperm Chromatin Content
- 14. Esteves, 14 DNA Integrity is the Key Sperm Biomarker Current non-invasive sperm selection techniques cannot directly assess sperm DNA fragmentation Dyes are used to reach the nucleus, using fixed specimens In general, labor-intensive techniques Recent 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 intact sperm membranes SpermSelection Techniques
- 15. Alternative has been TESA-ICSI Sperm % TUNEL + % CPR %IR Ejaculated (N=18) 23.6 ± 5.1 6 2 Testicular (N=18) 4.8 ± 3.6 44 21 P value <0.001 <0.05 <0.01 Greco 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 Ejaculated Spermatozoa (39.7%)* Esteves, 15
- 16. Esteves, 16 Oocyte Quality Biomarkers OocyteSelection Techniques • Polarization Microscopy (Polscope) • Oxygen Consumption (Embryoscope) • Microarray Technology Cumulus cells gene expression (mRNA transcripts) • Molecular Mining of Follicular Fluid Amino acids, metabolites, peptides, proteins Mass Spectroscopy, Raman Spectroscopy Nuclear Magnetic Resonance
- 17. Esteves, 17 Embryo Quality Biomarkers Rationale: >50% in vitro-produced embryos are abnormal Developmental Stage: • Cleavage-stage Embryo Biopsy (most used) • Polar Body (single allelic copy) • Blastocyst (trophectoderm cells) EmbryoSelection Techniques InvasiveBiopsy: 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. Esteves, 18 EmbryoSelection Techniques Biopsy: Polar body, Day-3 embryo and Trophectoderm cells OPR: 59% vs 38% controls (p<0.001) Munné et al, Fertil Steril 2010 PGS sure™ (Blue Genome, UK) Micro-array CGH solution to count all chromosomes in <12h Micro-array lab hardware Embryo Quality Biomarkers
- 19. Esteves, 19 Embryo Quality Biomarkers Metabolic Profile: • Glucose and pyruvate uptake • Amino acid turnover • Oxygen consumption Proteomics • Mass Spectroscopy • Raman Spectroscopy • Nuclear Magnetic Resonance Morphokinetics • Time-lapse microscopy EmbryoSelection Techniques Non-Invasive Katz-Jaffe & McReynolds, Fertil Steril 2013;99:1073-77
- 20. Esteves, 20 Embryo Quality Biomarkers Metabolic Profile (2010): • Via-Metrics™ (Molecular Biometrics, USA) EmbryoSelection Techniques Non-Invasive Advocated as a Highly Sensitive Method of Metabolomics Analysis by NIR Spectroscopy Market withdrawal due to instrument inability to perform accurate measurements
- 21. Esteves, 21 Embryo Quality Biomarkers Morphokinetics (2010): Image capture over time Combination of morphological, dynamic and quantitative information about developmental events EmbryoSelection Techniques Non-Invasive Principle: 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. Esteves, 22 Embryo Quality Biomarkers Stage-top Incubator Tokai-Hit, Japan InCu-Cell Live™ Sanyo, Japan BioStation™ Nikon, Japan Several Time-lapse Technologies Available: Time-lapse Technologies
- 23. Esteves, 23 Embryo Quality BiomarkersTime-lapse Technologies Primo-Vision™ Cryo-InnovationLtd., Hungary EmbryoScope version C™ Nanorespirometer + Time-lapse videomicrography Unisense Fertilitech, Denmark
- 24. Esteves, 24 Embryo Quality BiomarkersTime-lapse Technologies Eeva™ (Videomicrography + Computer Vision Software) Auxogyn, USA Cell tracking and prediction software (measure of time embryo takes to achieve specific milestones):
- 25. Esteves, 25 Clinical Translation: Where we are Biomarkersin theARTLab Time-lapse Technology Micro-array CGH
- 26. Esteves, 26 Clinical Translation: Where we are going Biomarkersin theARTLab Sperm and Oocyte Selection Using Biomarkers Embryo Selection by Real-time Secretome + Morphokinetics Analysis Microfluidic Platform for Embryo Culture
- 27. Esteves, 27 The Biomarkers Era has arrived. Several markers under investigation and some already translated Valid biomarkers are highly sensitive and specific, and have high predictive value For application at a global level, ART lab’s biomarkers/ technologies should be VEELI: Validated Easy to use Easy to replicate Low cost Improve outcomes Future Perspectives in the ART Lab Conclusions
- 28. Esteves, 28
Notes de l'éditeur
- 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.