Do you have what it takes – from follicle to healthy baby? Scott Nelson Muirhead Chair in Obstetrics & Gynaecology

The big challenge of assessing variability Jane Oocyte number Melisa Age Wallace and Kelsey PLOS One 2010

Aim to demonstrate that: Technical issues with AMH measurement are now resolved by Roche Randomised controlled trials now confirm AMH better than all other biomarkers for ovarian response prediction Optimising ovarian response is critical for optimal outcome

Measuring AMH

We had developed the global reference range DSL assay Gen II assay 25,000 women 10,984 women Nelson et al Fertil Steril 2011 Nelson et al RBMOnline 2012 Nelson et al Fertil Steril 2013

Different labs gave different results Bias from mean (%) Average AMH concentration(pmol/L) Zuvela, et al Reprod Biol 2013

Different labs gave different results Bias from sample mean (%) Laboratory Bias from mean (%) Average AMH concentration(pmol/L) Data from 10 laboratories for all samples analysed by that laboratory (each laboratory returned between 4 and 20 results) Zuvela, et al Reprod Biol 2013

Our previous AMH assay options Iliodromiti, Anderson and Nelson Hum Reprod Update 2015

Manual assays show huge day to day variability 1.7 0.6

Is your lab ensuring you get reproducible results?

Beckman Coulter release an automated AMH assay Beckman Coulter Access 2 AMH Iliodromiti, Anderson and Nelson Hum Reprod Update 2015

Supposed to give almost identical values to Gen II Y = 0.968(x) + 0.11 Beckman Coulter Access AMH assay documentation

Beckman Coulter new assay does not behave as expected – again! Slope: 0.781 (95% CI 0.758, 0.805) Intercept: 0.128 (95% CI 0.070, 0.198) Nelson et al Fertil Steril (in press)

Roche release their automated assay Elecsys/cobas Iliodromiti, Anderson and Nelson Hum Reprod Update 2015

Multicentre study confirms Roche automated AMH assay reproducible AMH (ng/ml) Age (years) Anderson et al Fertil Steril 2015 Nelson et al Fertil Steril (in press)

Multicentre study confirms Roche automated AMH assay reproducible AFC AMH (ng/ml) Age (years) Age (years) Anderson et al Fertil Steril 2015 Nelson et al Fertil Steril (in press)

Roche: sensitive robust automated AMH assay Elecsys AMH serum (ng/ml) Elecsys AMH Li Heparin (ng/mL) Robust to short and long-term storage Elecsys AMH Li Heparin stressed Elecsys AMH serum fresh Robust to sample storage temperature Elecsys AMH serum stressed Elecsys AMH serum fresh Robust to type of collection Gassner and Jung Clin Chem Lab Med 2014

Factors that affect AMH

GWAS identified 3 major SNPs for AMH Illumina HumanHap550 quad 113 SNPs 0.8% in girls Perry and Nelson submitted

AMH is dynamic across the lifecourse Dewailly et al Hum Repro Update 2014

We can measure AMH on any day of the cycle 4.0 Menses Follicular Ovulation Luteal AMH (ng/mL) 5.0 ≤20 years 21–25 26–30 31–35 >35 AMH Oestradiol Progesterone 4.5 3.5 4.0 3.5 3.0 3.0 AMH (ng/mL) 2.5 2.5 2.0 1.5 2.0 1.0 0.5 1.5 0.0 Menses Follicular Ovulation Luteal Kissell et al Hum Reprod 2014

Ethnic differences may not exist in ovarian reserve Geometric Mean AMH Although initial comparison showed South Asian women to have a higher serum AMH, compared with white European and Afro-Caribbean women (F = 3.817; P < 0.005), South Asian women attending the clinic were significantly younger and less likely to be smokers than women from other ethnic groups. The prevalence of polycystic ovary syndrome (PCOS) was significantly higher in South Asian and South East Asian women than in other ethnic groups. Differences in serum AMH were no longer significant after controlling for confounding factors: age, body mass index (BMI), and smoking status with (P = 0.869) and without (P = 0.215) controlling for PCOS. Age (years) Bleil et al Fertil Steril 2013 Bhide et al BJOG 2014

GnRHa alters AMH in time dependent manner Months Su et al JCEM 2013 Anderson et al Hum Repro 2006

Combined contraception reduces AMH combined OCs (ethinyl E2 [EE] and desogestrel), transdermal patches (EE and norelgestromin), or vaginal rings (EE and etonogestrel) Kallio et al Fertil Steril 2013

Smoking can reduce AMH independent of COCP Median AMH values in subgroups AMH (ng/ml) Age (years) Dolleman et al JCEM 2013

PCOS women have higher AMH AFC Webber et al Lancet 2003 Bhide et al Fertil Steril 2014 Iliodromiti et al JCEM 2013 PCO ovary has x6 the density of pre-antral follicles compared with normal ovary

PCOS women have higher AMH 4.7ng/ml Webber et al Lancet 2003 Bhide et al Fertil Steril 2014 Iliodromiti et al JCEM 2013 PCO ovary has x6 the density of pre-antral follicles compared with normal ovary

Disease can temporarily reduce your AMH AMH at initial diagnosis relative to age matched controls Van Dorp et al Hum Repro 2013

How does AMH compare to its competitors

The major competitor - AFC Image from 2001 Image from 2009 Dewailly, et al. Hum Reprod Update 2013

We will always be improving US resolution 18 Follicle number per ovary 16 14 12 10 Max Transducer Freq (MHz) 8 6 6 7 7.5 8 8.5 9 12 4 2 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012 Year of data collection Dewailly, et al Hum Reprod Update 2013

AFC normal ranges are just being established Infertility population Oocyte donor population 5,724 women 9,978 women Iliodromiti et al submitted

MRI determined AFC – the next step in resolution Hagen et al JCEM 2014

MRI determined AFC strongly correlates with AMH Hagen et al JCEM 2014

Are AMH and AFC equivalent?

We thought AMH and AFC were interchangeable Poor Excessive IMPORT Consortia Hum Repro Update 2013 Export Consortia Fertil Steril 2014

Only AMH predicted oocyte yield in antagonist RCT AFC performed poorly Arce et al Fertil Steril 2013

At each clinic AMH better than AFC Nelson et al Fertil Steril 2015

At each clinic AMH better than AFC Nelson et al Fertil Steril 2015

AMH is consistently better in both RCTs GnRH agonist RCT DSL AMH assay GnRH antagonist RCT Gen II AMH assay Nelson et al ASRM 2014

AFC adds little to prediction of oocyte yield GnRH antagonist RCT GnRH agonist RCT AMH Panels illustrating the capability of AMH and AFC to predict the number of oocytes retrieved for the long GnRH agonist (left panels) and GnRH antagonist (right panels) trial, respectively. The upper panels (A and B) show the partial residuals from the model including study cohort, AFC and ‘Study Center’ plotted against AMH. The lower panels (C and D) show the partial residuals from the model including study cohort, AMH and ‘Study Center’ plotted against AFC. AFC Nelson et al Fertil Steril 2015

Only AMH required in Xpect trial Screening assessment of candidate predictors Baseline assessment of candidate predictors Prediction of high response AUROC AMH 0.77 AMH & AFC & FSH 0.80 Cycle 1 Stimulation day 1 assessment of candidate predictors Smoking added to poor response prediction Cycle 2 Nyboe Andersen, et al Hum Reprod 2011

Prediction of high response (>18 oocytes) Only AMH required in the PURSUE trial Prediction of high response (>18 oocytes) AUROC AMH 0.86 AMH & AFC 0.88 AMH, AFC, FSH & age 0.89 Corifollitropin alfa arm of the Pursue Study (n=686) Women aged 35 to 42 years, Body weight ≥50 kg, BMI ≥18 and ≤32 kg/m2 Oehninger, Nelson et al RBOnline (in press)

No added value of additional predictors Only AMH required in novel recFSH Phase II trial Oocytes retrieved rhFSH (fixed daily dose, ug/day) No added value of additional predictors R2 AFC 26% AMH 35% AMH & AFC 38% Arce et al Fertil Steril 2014

Why is AFC so bad – the variability is huge Difference between paired measurements Mean of two counts   Intraobserver variability Deb, et al Ultrasound Obstet Gynecol 2009

Why is AFC so bad – the variability is huge Difference between paired measurements Mean of two counts Interobserver variability Mean of two counts Difference between paired measurements   Intraobserver variability Deb, et al Ultrasound Obstet Gynecol 2009

How AMH can inform clinical practice

Low AMH does not reduce short term fecundability AMH quintiles, middle 3 combined Hagen et al Fertil Steril 2012

AMH can individualize fertility prognosis for oncology patents Courtesy of RA Anderson 2015

We can use AMH to individualize fertility preservation Age FSH Inhibin B ** * pmol/L Years IU/L pg/ml Anderson and Cameron JCEM 2011 Anderson and Nelson Maturitas 2012 Anderson et al Eur J Cancer 2013

We can use AMH to predict the menopause Zoe Chloe Dolleman et al JCEM 2013

We can use AMH for family planning OK to delay family Ellie Have a family or work for Apple/Google Dawn Anderson and Nelson Hum Repro 2012

AMH should not be used to exclude from treatment Sensitivity Specificity 1.0 0.8 0.6 0.4 0.2 live birth DOR 2.39 (95%CI 1.85 – 3.08) Iliodromiti et al Hum Reprod Update 2014

With Univfy we showed that AMH enhanced prediction Validation Parameters AMH-PM AFC-PM AMH-AFC-PM AUC of Receiver-Operating Characteristic Analysis 0.716 0.706 Control AUC 0.674 AUC Improvement 6.3% 4.8% PLORA – log scale 29.1 22.5 28.3 PLORA Improvement 76.2% 59.0% 73.3% % Reclassified to have higher LB rate 62% 71% 67% % Reclassified to have lower LB rate 14% 8% 12% Tier-specific prediction error – See next slide for details ≤ 4% ≤ 8% Nelson et al Fertil Steril 2015

We now know 15 is an optimal oocyte yield 40 30 Live birth rate (%) 20 10 1 5 10 15 20 25 30 35 40 Oocyte yield Sunkara, et al Hum Reprod 2011 Steward et al Fertil Steril 2014

Plateau even in the US after 15 but OHSS increases 256,381 in vitro fertilization cycles Steward et al Fertl Steril 2014

AMH can optimise stimulation Optimal Population % Inadequate gonadotrophin exposure Iatrogenic Poor response Excessive gonadotrophin exposure Iatrogenic OHSS Oocyte yield

We can use AMH to stratify care AMH (pmol/L) Antagonist hCG/GnRHa trigger 40 20 Standard treatment 7 Maximise oocyte yield 1 Nelson et al Hum Reprod 2009 Yates et al Hum Reprod 2011

We can use AMH to stratify care Excessive response AMH (pmol/L) P < 0.001 10 20 30 Antagonist % Cycles HighAMH >15 Pmol/L Antagonist hCG/GnRHa trigger 40 20 Standard treatment 7 Maximise oocyte yield Reduced OHSS 1 Nelson et al Hum Reprod 2007 Nelson et al Hum Reprod 2009

We can use AMH to stratify care Excessive response Increased live births AMH (pmol/L) High AMH >15 Pmol/L Antagonist Agonist P < 0.001 P < 0.001 80 10 20 30 Antagonist % Cycles HighAMH >15 Pmol/L Antagonist hCG/GnRHa trigger 60 40 Live Birth % 40 20 20 Standard treatment 7 Maximise oocyte yield Reduced OHSS 1 Nelson et al Hum Reprod 2007 Nelson et al Hum Reprod 2009

We can use AMH to stratify care AMH (pmol/L) 30 Antagonist hCG/GnRHa trigger 25 40 Live birth rate (%) 20 20 Standard treatment 15 7 10 Maximise oocyte yield Pre-AMH Post-AMH 1 Nelson, et al Hum Reprod 2009 Yates, et al Hum Reprod 2011

AMH may allow truly individualised dosing AMH (pmol/L) 5 10 15 20 25 30 35 40 100 80 60 Optimum range (8–14 oocytes) ≥20 oocytes 15–19 oocytes 4–7 oocytes ≤3 FE 999049 (ug/kg) targeting 11 oocytes Proportion of subjects % AMH pmol/L Ferring ESTHER-1 RCT ongoing

AMH will be the biomarker of choice for individualising stimulation Iliodromiti, Anderson & Nelson Hum Repro Update 2014

Conclusions: We now have a robust automated AMH assay from Roche RCTs confirm that AMH is superior to all other biomarkers AMH can be used to personalise reproductive potential and therapy