Ovarian Biomarkers in OI

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Ovarian Biomarkers in OI

  1. 1. XVIII Annual Ob-Gyn Conference, Kuwait 2013 Ovarian Biomarkers in Ovulation Induction Sandro C. Esteves, MD, PhD Director, ANDROFERT Andrology & Human Reproduction Clinic Campinas, Brazil
  2. 2. Maximize beneficial effects of treatment Central Paradigm Individualization of Controlled Ovarian Stimulation (iCOS) Minimize complications and risks High-quality Gametes and Embryos Optimal Endometrial Receptivity ANDROFERT ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 2 2013 DECEMBER ANDROFERT androfert.com.br
  3. 3. Know the best biomarkers Understand how they work How to use biomarkers in Ovulation Induction ANDROFERT ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 3 2013 DECEMBER ANDROFERT androfert.com.br
  4. 4. Ovarian Biomarkers in Ovulation Induction Esteves SC – Kuwait’s XVIII Annual Ob-Gyn Conference, 2013 http://www.androfert.com.br/review ANDROFERT ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 4 2013 DECEMBER ANDROFERT androfert.com.br
  5. 5. Excessive Ovarian Response Avoid over-aggressive stimulation in ‘true’ high responders Diminished Ovarian Reserve (DOR) Why Predict Ovarian Response in OI? Avoid over-conservative stimulation in ‘true’ DOR Avoid over-conservative stimulation in ‘false’ high responders Avoid over-aggressive stimulation in ‘false’ DOR ANDROFERT ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 5 2013 DECEMBER ANDROFERT androfert.com.br
  6. 6. For Patients Prediction of Ovarian Response in OI Realistic Prognosis • Poor or Negligible Response • Cycle cancellation • Egg donation or adoption • Chances of Pregnancy and Live Birth ANDROFERT ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 6 2013 DECEMBER ANDROFERT androfert.com.br
  7. 7. Know the Biomarkers Hormonal Biomarkers FSH, Clomiphene citrate challenge test, Inhibin-B, Anti-Mullerian Hormone (AMH) Functional Biomarkers Antral Follicle Count (AFC) Genetic Biomarkers Single Nucleotide Polymorphisms for FSH, LH, E2 and AMH receptor genes ANDROFERT ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 7 2013 DECEMBER ANDROFERT androfert.com.br
  8. 8. A Valid Biomarker Should be Highly Sensitive and Highly Specific Diminished or Excessive Ovarian Response Specificity (D/B+D) Predictive Value (PPV=A/A+B; NPV=D/C+D) Accuracy (A+D/A+B+C+D) Esteves, 8 Biomarker Test Result Sensitivity (A/A+C) + - + True Positive (A) False Positive (B) - False Negative (C) True Negative (D) Adapted from: ASRM Practice Committee, Fertil Steril 2012;98:147
  9. 9. Evidence Level 1a Esteves, 9 Who is Who Before OI
  10. 10. Biomarkers in OI In a group of 131 women undergoing conventional COS after pituitary downregulation for IVF: Population AMH* ng/mL Cut-off Sensitivity Specificity Accuracy Highresponder1 2.1 85% 79% 0.82 Poor responder2 0.82 76% 86% 0.88 *Beckman-Couter generation II assay; 1>20 oocytes retrieved; 2≤4 oocytes retrieved Leão RBF, Nakano FY, Esteves SC. #O-51: ASRM 2013 ANDROFERT ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 10 2013 DECEMBER ANDROFERT androfert.com.br
  11. 11. AMH and AFC are not accurate for pregnancy prediction Evidence Level 1a Broer et al. Fertil Steril 2009 ; Broer et al. Hum Reprod Update, 17:46; 2011 Esteves, 11
  12. 12. How AMH and AFC Work ANDROFERT ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 12 2013 DECEMBER ANDROFERT androfert.com.br
  13. 13. AMH AFC Esteves, 13 Reflect No. pre-antral and small antral follicles (≤4-8mm) 2D-TVUS early follicular phase 2-10 mm (mean diameter) No. AF at a given time that can be stimulated by medication La Marca et al, Hum Reprod 2009;24:2264; Fleming et al, Fertil Steril 2012;98:1097; Broekmans et al. Fertil Steril, 2010; 94:1044-51; Scheffer et al. Hum Reprod 2003;18:700 .
  14. 14. Low Inter-cycle Fluctuations (Fanchin et al, Hum Reprod 2005;20:923) AMH ICC: 0.89; 95% IC: 0.83–0.94 Can be assessed at any cycle day with a single measurement Low Intra-cycle Fluctuations (Hehenkamp et al. JCEM 2006;91:4057) Max. Variation: 17.4% Esteves, 14 ICC: 0.55; 95% IC: 0.39–0.71 Max. Variation: 108%
  15. 15. Serum Levels: AMH Peak at age 25 and decrease with aging Early marker of diminished ovarian reserve Non-growing follicles (NGF) recruited per month Esteves, 15 Kelsey et al. Mol Hum Reprod 2012;18:79
  16. 16. AMH Accurate to Predict Ovarian Response Cut-off point 3.5 ng/mL* (Nardo et al, Fertil Steril 2009;92:1586) Ø  High sensitivity (88%), specificity (70%) and accuracy (0.81) to predict excessive response1 Cut-off point 1.4 ng/mL* (Kwee et al, Fertil Steril 2008;90:737) Ø  High sensitivity (76%) and specificity (86%) for DOR2 Caution to apply AMH cut-off points! Make sure the assay you rely on is the same used in the reference population Esteves, 16 *DSL assay; 1>20 oocytes retrieved; 2≤5 oocytes retrieved; Conversion: ng/mL to pmol/L = value in ng/mL X7.14
  17. 17. AMH ELISA assays with different performances: DSL and Immunotech Beckman-Couter gen II (AB DSL + Curves Im.) Fully automated ELISA (to be released) Lack international standardization and EQC Sample instability; measured levels altered by handling Collection in EDTA Storage at room temperature (up to 40% increase) No separation of serum from blood before postage Esteves, 17 Fleming et al. RBM online 2013;26:130; Nelson SM. Fertil Steril. 2013 Jan 8; Nelson & La Marca. RBM online 2011;23:411;
  18. 18. Moderate to Low Inter-cycle Fluctuations AFC van Disseldorp et al, Hum Reprod 2010;25:221 Esteves, 18 ICC: 0.71 (95% CI: 0.63–0.77); 29% individual cycle variation High Inter- and Intra-observer Reproducibility Scheffer et al. Ultrasound Obstet Gynecol 2002;20:270
  19. 19. Accurate to Predict Ovarian Response Cut-off point of 14 Kwee et al, Fertil Steril 2008;90:737 AFC High sensitivity (81%) and specificity (89%) to predict excessive response1 Cut-off point of 4 Bancsi et al, Fertil Steril 2002;77:328 Moderate sensitivity (61%) and High specificity (88%) and to predict DOR2 Caution to Apply Cut-off Points to Predict No. of Oocytes to be Retrieved For any given AFC there is a potential oocyte yield, but it can be altered by the stimulation strategy Esteves, 19 1>20 oocytes retrieved in conventional COS; 2≤4 oocytes retrieved
  20. 20. Lack of standardization1 • Inclusion criteria for antral follicles AFC Ø  e.g., 2–5 mm or 2–10 mm • Method for counting and measuring follicles • Variable scanning techniques • Image optimization Improved standardization proposed2 Three-dimensional automated follicular tracking3 •  Reduce intra- and inter-observer variability •  Requires offline analysis 1Nelson SM. Fertil Steril. 2013 Jan 8; •  Costly 2 Esteves, 20 Broekmans et al., Fertil Steril, 2010; 94(3):1044-51; 3Raine-Fenning et al., Fertil Steril 2009;91:1469.
  21. 21. How to Use AMH and AFC in OI ANDROFERT ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 21 2013 DECEMBER ANDROFERT androfert.com.br
  22. 22. Evidence Level 2b Biomarkers for iCOS in High Responders AMH (ng/mL) >2.1¶ GnRH Agonist Low-starting FSH dose (150 UI) (n=148) GnRH Antagonist (n=34) Days of Stimulation 13 (12-14) 9 (8-11)* No. Oocytes retrieved (n) 14 (10-19) 10 (8.5-13.5)* OHSS requiring hospitalization 20 (13.9%) 0 (0%)* 4 (2.7%) 1 (2.9%) 40.1% 63.6%* Cancellation CPR per transfer *P ≤ 0.01 Esteves, 22 ¶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.
  23. 23. Evidence Level GnRH Antagonists in High Responders 1a 9 RCT; 966 PCOS women GnRH Antagonist X Agonist Weight Mean Difference (WMD)1; Relative Risk (RR)2 Duration of OS -0.74 (95% CI: -1.12; -0.36)1 Gonadotropin dose -0.28 (95% CI: -0.43; -0.13)1 Oocytes retrieved 0.01 (95% CI: -0.24; 0.26)1 Risk of OHSS (Moderate & Severe) 20% vs 32% 0.59 (95% CI: 0.45-0.76)2 Clinical PR 1.01 (95% CI: 0.88; 1.15)2 Miscarriage rate 0.79 (95% CI: 0.49; 1.28)2 ~40% reduction in moderate/severe OHSS by using antagonists rather than agonists Esteves, 23 Pundir J et al. RBM Online 2012; 24:6-22.
  24. 24. GnRH Antagonist Protocol with Long-acting recFSH vs recFSH 4 RCT; 2377 pts. Clinical PR, Miscarriage, LBR Risk of OHSS Cancellation OR [95% CI] Not different 1.29 (0.78; 2.26) 5.67 (1.07; 30.13)* *p=0.04; risk of OHSS Mahmoud Youssef et al. van Fertil Steril 2012; 97(4): 876-85; Pouwer AW et al. Cochrane Database Syst Rev 2012; 6: CD009577. Esteves, 24
  25. 25. Biomarkers for iCOS in Poor Responders Up to 45% of Infertility Patients in ART Older patients (≥35 years) Poor responders Slow/Hypo-responders Deeply suppressed endogenous LH Marrs et al. Reprod Biomed Online 2004;8:175;Mochtar MH, Cochrane Database, 2007; Alviggi, et al. RBMOnline 2009; De Placido et al. Clin Endocrinol (Oxf) 2004;60:637 ANDROFERT ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 25 2013 DECEMBER ANDROFERT androfert.com.br
  26. 26. Ovarian Aging Impaired Oocyte Quality Reduced Fertilization Rate Reduced Embryo Quality Increased Miscarriage Rates Westergaard et al., 2000; Esposito et al., 2001; Humaidan et al., 2002 ANDROFERT ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 26 2013 DECEMBER ANDROFERT androfert.com.br
  27. 27. Normal LH “Window” Concept • Normal androgen and estrogen biosynthesis • Normal follicular growth and development • Normal oocyte maturation Reduced ovarian paracrine activity Androgen secretory capacity reduced Decreased numbers of functional LH receptors Reduced LH bioactivity Hurwitz & Santoro 2004 •  Piltonen et al., 2003 •  Vihko et al. 1996 •  Mitchell et al. 1995; Marama et al 1984 ANDROFERT ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 27 2013 DECEMBER ANDROFERT androfert.com.br
  28. 28. Level 1a LH Supplementation in DOR Regimen Mochtar et al, 2007 3 RCT (N=310) Poor responders Bosdou et al, 2012 7 RCT (N= 603) Poor responders Outcome Effect on Pregnancy r-hFSH+rLH vs. r-hFSH alone* OPR OR: 1.85 r-hFSH+rLH vs. r-hFSH alone* CPR LBR (only 1 RCT) Hill et al, 2012 7 RCT (N=902) Women advanced age ≥35 yrs. r-hFSH+rLH vs. r-hFSH alone CPR (95% CI: 1.10; 3.11) RD: +6%, (95% CI: -0.3; +13.0) RD: +19% (95% CI: +1.0; +36.0%) OR: 1.37 (95% CI: 1.03; 1.83) *long GnRH-a protocol; OR=odds-ratio; RD=risk difference Esteves, 28 Mochtar MH et al. Cochrane Database Syst Rev. 2007;2:CD005070; Bosdou JK et al, Hum Reprod Update 2012; 8(2):127-45. Hill MJ et al. Fertil Steril 2012; 97:1108-4.
  29. 29. Rationale of LH supplementation Action of LH at the follicular level in a dose dependent manner increases androgen production; Androgens are later aromatized to estrogens and may help restore the follicular milieu; LH has also a direct positive effect on final follicular maturation; Altogether, positive effect in oocyte quality and, therefore, embryo quality and implantation. Esteves, 29
  30. 30. Sources of LH Activity Purity (LH content) hCG content (IU/vial) LH activity (IU/vial) Specific activity (LH/mg protein) Rec-hLH >99% 0 75 22,000 IU hMG-HP* 3% ~70 75* ≥ 60 IU *derives from hCG Adapted from ASRM Practice Committee. Fertil Steril. 2008; 90:S13-20. ANDROFERT ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 30 2013 DECEMBER ANDROFERT androfert.com.br
  31. 31. Sources of LH Activity Sources of LH Activity Beta unit hCG Longer in hCG; (Higher receptor affinity) Carboxyl terminal segment Absent in LH and present in hCG (Longer Half-life) LH ANDROFERT ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 31 2013 DECEMBER ANDROFERT androfert.com.br
  32. 32. hMG Grondal et al. 2009: r-FSH Sources of LH Activity GCs gene expression in pts. treated with hMG and rec-hFSH q  Lower expression of LH/hCG receptor gene and other genes involved in steroids biosynthesis in hMG group Down-regulation of receptors owed to constant ligand exposure to hCG (Menon et al. 2004) CYP11A activity decreased by 2.4 fold Lower steroids synthesis and P levels q  Higher potency of rec-hFSH inducing more LH/hCG receptors Grondal ML et al. Fertil Steril 2009; 91: 1820-1830. Menon KM et al. Biol Reprod 2004; 70:861-866 Esteves, 32
  33. 33. Sources of LH Activity Matched case-control study; N=4,719 IVF pts. 35 30 25 P=0.02 31 26 20 15 25 19 14 10 14 5 Duration of Stimulation (days) Mean No. oocytes retrieved IR (%) CPR per transfer (%) 0 Fixed 2:1 r-hFSH (150IU)/r-hLH (75IU) HMG rec-hFSH + HMG Buhler KF, Fisher R. Gynecol Endocrinol 2011;1-6. ANDROFERT ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 33 2013 DECEMBER ANDROFERT androfert.com.br
  34. 34. Individualization of OI with AMH AMH cut-off points used to individualize COS in 118 women undergoing IVF; Outcome compared with a group of 131 women who received conventional stimulation High Responders AMH >2.1 Poor Responders AMH ≤ 0.82 Esteves, 34 rec-hFSH FbM 112.5 to 150 IU daily + GnRH antagonist rec-hFSH FbM + 75 IU rec-hLH + GnRH antagonist • Total daily dose: 262.5 to 375 IU Leão RBF, Nakano FY, Esteves SC. #O-51: ASRM 2013
  35. 35. iCOS Using AMH High Responders 60 50 40 57.0 56.0 p=0.03 cCOS 39.3 p=0.04 30 20 p=0.92 18.5 14.3 14.7 10 iCOS p=0.38 14.0 4.8 0 Observed Excessive Response (%) Oocytes retrieved (N) OHSS (%) Pregnancy (%) 1Excessive response: >20 oocytes retrieved; *Pts. received GnRH-a trigger + embryo vitrification; Mild/severe OHSS reported Leão RBF, Nakano FY, Esteves SC. #O-51: ASRM 2013 Esteves, 35
  36. 36. iCOS Using AMH Poor Responders 80 60 72.0 cCOS p=0.02 46.6 45.0 40 20 iCOS p=0.03 3.5 p=0.06 23.3 p=0.51 20.0 26.8 4.8 0 Expected Poor Oocytes retrieved Cancellation (%) Pregnancy/cycle Response (%) (N) (%) Poor response <5 oocytes retrieved Leão RBF, Nakano FY, Esteves SC. #O-51: ASRM 2013 Esteves, 36
  37. 37. Take Home Messages AMH and AFC are currently the best biomarkers to predict ovarian response to COS. AMH and AFC are direct biomarkers of ovarian reserve. Both markers have similar accuracy to predict who is at risk of excessive and poor response in COS. After identifying ‘Who is Who’, mild stimulation and GnRH antagonists in pts. at risk of excessive response, and rec-hLH supplementation in DOR, maximize treatment benefits and minimize risks. ANDROFERT ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 37 2013 DECEMBER ANDROFERT androfert.com.br
  38. 38. Thank you
  39. 39. iCOS Using AMH Excessive1 Oocytes retrieved OHSS Pregnancy Poor2 Oocytes retrieved Cancellation Pregnancy/ET 1Excessive Esteves, 39 iCOS (n=118) P value 39.3% Response to COS Conventional COS (n=131) 14.3% 18.5 ± 6.7 14.3% 57.1% 14.7± 6.2 4.8%* 55.6% 0.03 0.04 0.38 0.92 72.0% 3.5 ± 3.1 45.0% 20.0% 46.6% 4.8 ± 3.5 23.3% 26.8% 0.02 0.03 0.06 0.51 response: >20 oocytes retrieved; 2Poor response: <5 oocytes retrieved; *Pts. received GnRH-a trigger + embryo vitrification; No severe OHSS reported
  40. 40. Progesterone Rise What we have learned… Number of oocytes Estradiol levels on hCG day FSH dose Rec-hFSH vs. hMG positively associated with P levels P levels not associated with oocyte and embryo quality, nor with fertilization and cleavage rates Bosch et al. 2008, 2010; Xu et al, 2012; Kolibianakis et al 2012; Venetis et al. 2012; Griesinger et al 2013 Esteves, 40 ANDROFERT, Referral Center for Male Reproduction
  41. 41. No CYP17 LH Esteves, 41 FSH LH
  42. 42. Progesterone thresholds affecting PR controversial Bosch et al. 2010 (N=4,032) Irrespective of GnRH analogue; CUT-OFF = 1.5 ng/mL Xu et al, 2012 (N=11,055) GnRH agonist Ovarian response Number of oocytes Serum P threshold (ng/mL) Poor ≤4 1.5 Intermediate 5-19 1.75 High ≥20 ■  Fresh ■  FET 2.25 Esteves, 42 ANDROFERT, Referral Center for Male Reproduction
  43. 43. Effect of progesterone levels on day of hCG administration on pregnancy Griesinger et al, 2013 (6 RCT, N=1866; Antagonist cycles) P4 cut-off: 1.5 ng/mL P4 rise related to ovarian response: Low-responder: 4.5% High-responder: 19% Overall: 8.4% OPR not impaired in high responders with P elevation Ongoing PR: OR = 0.55 (0.37–0.81) Griesinger et al. Fertil Steril 2013 Esteves, 43 ANDROFERT, Referral Center for Male Reproduction

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