DRG INTERNATIONAL, INC. Where medical needs meet innovation Note: Unless otherwise stated in our catalog or other company documentation accompanying DRG International, Inc. product(s), these assay kits are intended for research use only (RUO) in the United States. 1

03/04/2014 AMH BEYOND IVF: RAISING THE BAR IN REPRODUCTIVE HEALTH DIAGNOSTICS 2

What is AMH? 140 kDa glycoprotein produced during normal embryogenesis by the Sertoli cells of the embryonic testis causes involution of the Müllerian duct in developing males. It inhibits female gonadogenesis by inducing apoptosis of target gonadal cells TGF-β super family hormone with paracrine control functions in folliculogenesis 3

AMH: Direct Measure of Ovarian Reserve Antonio Lamarca chart 4

Research on AMH, including potential clinical utility, is increasing rapidly IVF / Ovarian response and OHSS POF POA POI Oncofertility Ovarian surgery Gonadotoxicity Menopause PCOS Granulosa cell tumors Hypogonadotropic hypogonadism Non-obstructive azoospermia Neonatal gender determination What’s next? 5

Primary Research Areas Reproductive function and pregnancy complications Comprehensive line of assays to TGF-beta superfamily hormones that play key roles in folliculogenesis Classical and novel maternal screening markers for chromosomal anomalies, preeclampsia, IUGR, and gestational diabetes Reproductive Function AMH Inhibin A Inhibin B Total Inhibin Follistatin Activin A Pregnancy Inhibin A AFP I-hCG Unconjugated Estriol (UE3) PAPP-A PAPP-A2 Coming soon! IGFs / BPs Activin B Activin AB 6

7

Predictability of AMH 8

9

AMH is not cycle-day dependent 10

FSH indirect, cycle day dependent 11

FSH is a Surrogate Marker of Ovarian Reserve 12

Simple utility of direct ovarian markers 13

Introduction to PCOS Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders among females. PCOS affects 6-12% of women of reproductive age. In a population like India, that number represents >50 million females with PCOS in varying degrees of severity, largely influenced by obesity. PCOS is comprised of a number of different medical conditions which can sometimes make definitive diagnosis difficult. The one common finding that determines PCOS is polycystic ovaries. This makes AMH an important biochemical feature of PCOS. 14

Learning Objectives After this session participants will be able to: 1. explain polycystic ovary syndrome, 2. explain the most common criteria for diagnosing PCOS, 3. discuss AMH as a marker of PCOS, and 4. describe the benefits of assessing AMH for the diagnosis of PCOS. 15

What is Polycystic Ovary Syndrome (PCOS) A condition in which a woman has an imbalance of female (and male) sex hormones. menstrual cycle changes, cysts in the ovaries, difficulty getting pregnant / infertility, and other health changes. Ovaries unable to release mature, competent oocyte. PCOS is typically diagnosed in women in their 20s or 30s; it also affects teenage girls. The symptoms often begin when a girl's periods start. Enlarged ovaries containing numerous small cysts can be detected by ultrasound, but polycystic ovaries alone do not confirm the diagnosis though. 16

Symptoms Most common includes changes in the menstrual cycle: amenorrhea or irregular periods (MCL and heaviness) PCOS can cause virilization Body hair on chest, belly, and face Decreased breast size Enlargement of the clitoris (rare) Thinning of the hair on the head, i.e., male-pattern baldness Voice gets deeper Skin changes: Acne that gets worse Dark or thick skin markings and creases around the armpits, groin, neck, and breasts Obese 17

Polycystic Ovary Morphology Antral follicles can be seen lining the periphery of the ovary, appearing as a 'string of pearls' on ultrasound examination. In PCOS, these antral follicles are in a state of arrested development due to a disruption of normal folliculogenesis. These follicles 2-8mm in size produce the highest concentrations of AMH. PCO defined by one or both ovaries having 12 or more 2- 8 mm follicles and/or an ovarian volume of >10ml 18

Health conditions associated with PCOS Women with PCOS are more likely to develop: Infertility Insulin resistance Type 2 diabetes Weight gain and obesity Metabolic Syndrome High blood pressure High cholesterol Cardiovascular diseases Inflammation Endometrial cancer Breast cancer (slightly increased risk) 19

Elevated AMH in PCOS FIG. 1. Box-and-whisker plots showing the values of serum AMH (1ng/ml 7.14 pmol/liter) and 2- to 5-mm follicle number in patients with PCOS (n 59) and in controls (n 45). Horizontal small bars represent the 10–90th percentile range, and the boxes indicate the 25th- 75th percentile range. The horizontal line in each box corresponds to the median. PASCAL PIGNYET.AL The Journal of Clinical Endocrinology & Metabolism 88(12):5957–

AMH in PCOS Anti-Müllerian hormone (AMH) before and 5 years after in anovulatory and ovulatory group and control subjects Fertility and Sterility Volume 98, Issue 4, Pages , October 2012 Volume 98, Issue 4 21

Literature Suggested Patient Stratification Individualization of treatment based on subjective ovarian response categories Treatment strategies should be designed to minimize risk while maximizing oocyte yield The suggested AMH / antral follicle count (AFC) thresholds are based on the correlation of AMH and AFC and the associated response obtained from literature. These are not population specific, and this should be an important consideration. Adapted from: Nelson. Biomarkers of ovarian response. Fertil Steril

Differences between commercial assays FeatureDRG International, Inc.Other Commercially Available Assay Capture Abspecific for a linear epitope on the pro region not impacted by cleavage directed against the mature (or is it Pro?) region, which appears to be a conformational epitope impacted by different sample collection and handling conditions and/or naturally occurring protein processing Detection Abspecific for a linear epitope on the mature region not impacted by cleavage directed against the mature region and a conformational epitope, which appears to be impacted by changes in sample conditions and cleavage, or both SpecificityHuman specificAntibodies derived against rec human AMH, but have significant cross-reactivity with many species (especially bovine, chicken, rat, mouse, pig, etc.) Calibration (Ag)rec human AMH, mammalian (CHO cell)- derived Bovine serum AMH InterferenceNone knownComplements, and heterophilic antibodies (animal IgG, especially bovine) DilutionDilutes linearly from >75ng/mL to Limit of Detection Do not dilute. Company reports to not dilute over 10 ng/mL because the samples will over recover. Affects PCOS and neonatal gender determinations. 23

Summary of PCOS Polycystic ovary syndrome affects between 6 and 12% of all women of reproductive age worldwide. Some geographies report as high as 18% prevalence! Over 70% of PCO / PCOS women are undiagnosed! PCOS can be treated, if diagnosed. Periodic checks, repeat diagnostic testing is prescribed. According to the Revised Rotterdam Criteria (most common criteria used for assessment of PCOS) a patient must demonstrate two out of the three listed criteria: Oligo- or amenorrhea hyperandrogenism, and evidence of polycystic ovary (usually determined by ultrasound). Common finding that determines PCOS is polycystic ovaries, which makes AMH an important biochemical feature of PCOS. Because AMH is produced by the granulosa cells of ovary, elevated AMH levels indicate a polycystic ovary even when an accurate antral follicle count cannot be obtained, and in the absence of hyperandrogenism and/or anovulation. AMH is consistently >2x greater in PCOS than in non-PCO age-matched controls 24

Conclusion The DRG International, Inc. assay methods offer clear analytical and clinical performance advantages over other commercially available assays. The DRG International, Inc. assay addresses the challenges raised regarding the reliability of AMH testing due to interferences and lab-to-lab variability presumably due to sample handling. The increased sensitivity and accuracy throughout the wide- physiological range of AMH makes the DRG International, Inc. assay useful for broader applications in gynecological assessment of patients. 25