The Ovarian assessment Report™ (OAR)The New Standard for Ovulatory Egg Supply Testing

The vast majority of women have sufficient numbers of good quality eggs to have their own children before the age of 30,

but subsequently, it is highly variable when an individual’s supply of good quality eggs is depleted. The graph to the right shows data from the 2006 National Vital Statistics Report and demonstrates that between, 1990 and 2004, the largest increases in birth rates occurred in women with the highest chance of infertility (Vol. 55, No 1, Sept. 29, 2006). As this data only reflects the number of women able to achieve successful births (many requiring assisted reproductive technology), the number of women attempting un-successfully due to advanced maternal age is likely to be much higher. How age, egg quality and egg supply are related is not commonly understood. This results in many women waiting too long to ad-dress their inability to conceive, and missing their window for a successful treatment with reproductive technologies. Woman experiencing difficulty having children can benefit from proactive testing and education that can identify their risks for poor egg quality and supply, allowing these women to get help before it is too late.

A Growing Need for Proactive Education and Egg Supply Assessment

Indications: Any woman who isexperiencing infertility•being evaluated as a potential egg donor•considering egg preservation•

10-14 15-17 18-19 20-24 25-29 30-34 35-39 40-44 45-49 fertile infertile

-50% -41% -21% -13% -4% +18%+43% +62%

+150%

. . . when the risk of depleted egg supply is greatest

Women are having children later . . . % change in birth rate 1990-2004

age in years

ReproSource Fertility Diagnostics™ has performed extensive clinical studies validating the OAR™. The FDA recently issued a draft guidance which states that a diagnostic index derived from multiple laboratory values should be submitted to the FDA to deter-mine if the index and manner of reporting requires review and clearance. In compliance with this new guidance, the OAR™, which also utilizes for “Research Use Only” components for AMH and inhibin B detection, is being submitted to the FDA to determine if clearance is required. Diagnostic index reports that are determined by the FDA to require clearance should, until clearance is obtained, carry the label “For investigational use only. The performance characteristics of this product have not been established.”© ReproSource v 1 032809

The OAR provides the most accurate available assessment of a woman’s ovulatory egg supply using a single blood sample 10-11.

The OAR combines age with blood serum levels of various ovary related hormones such as AMH and FSH into a proprietary math-ematical algorithm to provide the Egg Retrieval Score (ERS) which has been calibrated to the most definitive available measure of ovulatory egg supply: number of eggs retrieved through ovarian stimulation.

The Source for Fertility Testing & Information ™

R e p r o S o u r c eF e r t i l i t y D i a g n o s t i c s ™

More babies…sooner through proactive diagnostics

ERSEgg Retrieval Score

AgeAMHFSHOther factors

OaRAlgorithm

The OAR: A New, More Accurate Diagnostic for Ovulatory Egg Supply Based Upon Two Scientific Breakthroughs

①Ovarian follicles discovered to produce hormones measurable in blood

② Mathematical algorithm developed to provide superior accuracy

AMHInhibin B

Egg

AMH (Anti-Müllerian) hormone and inhibin B are produced in the ovaries by the egg-bearing follicles 1-3. Several recent

clinical studies show that the blood levels of these hormones relate directly to the remaining egg supply 4-9. ReproSource, in collabo-ration with international leaders in fertility medicine, has helped solidify the link between these hormones and egg supply in the fertile and infertile populations.

Primordial follicle

Primary follicle

Pre-antral follicle

Antral follicle

Previously, FSH was the only widely available test in use for assessing egg supply,despite the fact that it has poor accuracy and usually is only abnormal once a woman has depleted her egg supply. FSH is produced by the brain and generally levels be-come elevated only after egg supply is very low. Recent discoveries have revealed that the hormones AMH (anti-Müllerian hormone) and inhibin B are secreted by the granulosa cells that surround each egg in the young follicles. For the first time, blood measurements that directly assess egg supply are now possible.

Biology Behind The BreakthroughAMH and inhibin B are directly related to egg supply, unlike FSH

AMH and inhibin B provide different information

The OaR™ Algorithm Greater Accuracy, Greater Reliability

The ERs: a validated, standardized referenceCalibrated to a definitive measure of Ovulatory Egg Supply

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AMH Inhibin B

Expression of AMH and Inhibin B

During Follicle Development

AMH, also known as Müllerian Inhibiting Substance (MIS), has long been known as a key hormone in embryonic development where it prevents the female system from de-veloping in males. Research has shown that AMH is also first produced by ovarian fol-licles from the single layer of flattened pre-granulosa cells surrounding the egg during the primordial follicle stage. AMH production is thought to be fairly constant cells in each follicle until ceasing when a follicle reaches the pre-antral stage 12.

Inhibin B expression is believed to begin later at the primary follicle stage when the flattened granulosa cells become more cube-like 13. Inhibin B is thought to provide a more functional assessment of the egg pool since it is produced by follicles in response to FSH, and therefore may demonstrate the potential of the eggs to perform as expected.

Science and Physiology Clinical Research Behind the OAR™

The Egg Retrieval Score (ERS), compared with other tests such as FSH, AMH or inhibin B, has superior accuracy in both fertile and infertile

women for predicting “ovulatory egg supply” (the supply of eggs available for ovulation). Using the number of eggs per retrieval from 454 egg retrievals

from 244 women (including 79 egg donors, 26 female partners of males requiring TESA, and 139 infertile women), a receiver op-erating characteristic curve was constructed (graph to left) that demonstrated superior sensitiv-ity and specificity of the ERS in identifying women with 4 eggs or fewer retrieved. The ERS had the highest AUROC (see table to right) and highly correlated with eggs retrieved in both the fertile and infertile population. By combining multiple analyte values together, the OAR provides a safeguard against incorrect clinical interpretations that could occur due to errors in any single analyte measurement.

One of the biggest challenges today in the fertility field is standardization of diagnostic value ranges and calibration to clinical outcomes 14. Although

various methods of estimating egg supply exist, experts generally agree that the number of eggs obtained through ovarian stimulation followed by egg retrieval is the most definitive available measures of a wom-an’s ovulatory egg supply. The graph to the right demonstrates that the ERS correlates strongly to eggs retrieved in 454 egg retrievals.

the ers standard range

1 to 5 reduced 6 to 10 fair 11 to 15 good 16 to 20 excellent

0.0

0.2 0.4 0.6 0.8 1.0

FSH

ORI

ERS Out Performs FSH

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outcoMe: ≤ 4 eggs retrieved454 retrievals, 244 women

ERS

FSH

Egg Retrieval Score By Category

OutcOmes Of 454 egg RetRievals

80706050403020100

% o

f R

etri

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48%0 to 4 43%

9%

15%5 to 8 63%

22%

1.9%9 to 11 50.5%

47.6%

0

10

20

30

40

50

60

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100

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series 3

series 2

v

12+9 to 115 to 80 to 4

Eggs retrieved:

12+ eggs0.35 0.15

0.126760.457750.81579

Excellentn=145

Goodn=193

Fairn=89

Reducedn=27

0-4 5 -11 12+

One of the best ways to compare sensitiv-ity amongst tests is to use cut points such that all tests have the same low false positive rate (eg 10%). To compare false positive rates, cut points can be used such that all tests have a high sensitivity (eg 90%). The above table reflects performance of ERS, AMH, and FSH in detecting 4 eggs or less at retrieval (n=454 retrievals) and demonstrates that the ERS has a superior sensitivity and specificity, including statistically significant superiority to AMH (p=0.05)

eRs is mORe sensitive and specific than fsh OR amh

ERS 0.90 36% 75%

AMH 0.85 45% 63%

FSH 0.77 64% 38%

AUROCArea under ROC curve

False Positives

@ 90% sensitivity

Sensitivity@10% false

positives• 95% more sensitive than FSH• 20% more sensitive than AMH

• 75% more false positives with FSH• 25% more false positives with AMH

0.0

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1.0

1.5

2.0

22-25

26-30

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Egg Retrieval Score (ERS) Predicts Ovulatory Egg Supply

The Source for Fertility Testing & Information ™

R e p r o S o u r c eF e r t i l i t y D i a g n o s t i c s ™ Science and Physiology Clinical Research Behind the OAR™

Patient’sScore

Objective: Establish the Egg Retrieval Score™as a useful predictor of ovulatory egg supply.Methods: Under IRB approval, frozen menstrual cycle day three serum samples along with retrospectively collected medical information were obtained from 244 women undergoing 454 egg retrievals, between March 2002 and August 2007 at an academic, international referral center for fertility care. Exclusion criteria: cancelled cycles for social reasons; Day 3 estradiol levels greater than 100 pg/ml (confounder of FSH and inhibin B levels). The test population included women likely to have excellent egg supply (79 egg donors and 26 female partners of men with azoospermia) as well as women with likely poor egg supply (139 infertile women). Samples were de-identified and sent to ReproSource for determination of AMH, inhibin B, FSH, LH, and estradiol levels. Laboratory data was combined with age to calculate the Egg Retrieval Score and corresponding clinical outcomes data were sent to a third party for unblinding and analysis.Results/Conclusions: The Egg Retrieval Score™ is highly correlated with the number of eggs obtained through ovarian stimulation and can be a useful tool for initial ovarian assessment of women being considered for this procedure 10,11 .

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Egg Retrieval Score™The Egg Retrieval Score™ is an index value with a standard range from 1 to 20 generated from combining age with menstrual cycle “Day 3” blood levels of AMH, inhibin B, FSH, and estradiol. The bar to the left indicates the strong cor-relation of the Egg Retrieval Score to number of eggs obtained through retrieval after ovarian stimulation: red= reduced; yellow= fair; light green=good; and green=excellent.

egg RetRieval scORe™

Age Specific Comparison: In the graph to the left, the distribution of Egg Retrieval Scores™ in women being evaluated in fertility clinics is displayed by age group. Within each age group, the frequency (y-axis) of a particular Egg Retrieval Score™ (x-axis) is shown and can be used to compare an individual to her own age group as well as to other age groups.

Probability Plots: In the graph to the left, estimated probability plots for 3 categories of egg retrievals were generated using the raw data from the graph below. The percent probability (y-axis) of obtaining 4 or fewer eggs at retrieval (red), 5 to 11 eggs (light green), and 12 or more eggs (green) is displayed against the Egg Retrieval Score (x-axis).

Raw Data: In the graph to the left, the number of eggs obtained from 244 women under-going 454 egg retrievals (y-axis) is plotted against the associated Egg Retrieval Score (x-axis). The coloring of the graph background groups the data points into the following categories for number of eggs retrieved: red=0 to 4, light green=5to 11, and green=12+.

reduced(fewer eggs)

excellent(more eggs)

The bar serves as the common X-axis for the above three graphs. The portions of the above graphs that correspond to the patient’s Egg Retrieval Score are emphasized with the thin blue bar that extends vertically through all three graphs.

0 2 4 6 8 10 12 14 16 18 20

22-25

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41-45

0 5 10 15 20 25

0.000000

4.583330

9.166659

13.749989

18.333319

22.916648

27.499978

32.083308

36.666637

41.249967

45.833297

50.416626

54.999956

0-4 eggs

5 -11 eggs

12+ eggs

pRO

ba

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ity

16

de Vet, A., et al., Antimüllerian hormone serum levels: a putative marker for ovarian aging. 1. Fertil Steril , 2002; 77(2): p. 357-62.Fanchin, R., et al., Serum anti-Müllerian hormone is more strongly related to ovarian 2. follicular status than serum inhibin B, estradiol, FSH and LH on day 3. Hum Reprod, 2003; 18(2): p. 323-7.van Rooij, I.A., et al., Serum anti-Müllerian hormone levels: a novel measure of ovarian 3. reserve. Hum Reprod, 2002; 17(12): p. 3065-71.Bancsi, L.F., et al., Predictors of poor ovarian response in in vitro fertilization: a prospective 4. study comparing basal markers of ovarian reserve. Fertil Steril, 2002; 77(2): p. 328-36.Erdem, M., et al., Comparison of basal and clomiphene citrate induced FSH and inhibin 5. B, ovarian volume and antral follicle counts as ovarian reserve tests and predictors of poor ovarian response in IVF. J Assist Reprod Genet, 2004; 21(2): p. 37-45.Fabregues, F., et al., Ovarian reserve test with human menopausal gonadotropin as a 6. predictor of in vitro fertilization outcome. J Assist Reprod Genet, 2000; 17(1): p. 13-9.Ficicioglu, C., et al., The role of inhibin B as a basal determinant of ovarian reserve. 7. Gynecol Endocrinol, 2003; 17(4): p. 287-93.Riggs R, Bocca S, Yin L, Bourque V, Leader B, Oehninger S, Stadtmauer L, Anti-Müllerian 8. hormone serum levels predict response to controlled ovarian hyperstimulation in oocyte donors. European Society for Human Reproductive Biology (ESHRE), 2008; AbstractRiggs RM, Duran EH, Baker MW, Kimble TD, Hobeika E, Yin L, Matos-Bodden L, Leader B, 9. Stadtmauer L. Assessment of ovarian reserve with anti-Müllerian hormone: a comparison

of the predictive value of anti-Müllerian hormone, follicle-stimulating hormone, inhibin B, and age. Am J Obstet Gynec, 2008; Aug;199(2):202.Leader, B, Baca QJ, Stadtmauer L, Riggs R, Rivnay B, L Yin. Ovarian Reserve Index (ORI) 10. predicts number of oocytes at retrieval in fertile and infertile women. European Society for Human Reproductive Biology (ESHRE), 2008; Oral PresentationLeader B, Quinn E, Sullivan L, Yin L, Riggs R, Stadtmauer L Ovarian Reserve Index out 11. performs AMH, inhibin B, and FSH in predicting poor egg supply. American Society for Reproductive Medicine (ASRM), 2008; AbstractFeyereisen, E., et al., Anti-Müllerian hormone: clinical insights into a promising biomarker of ovarian 12. follicular status. Reprod Biomed Online 2006; 12(6): p. 695-703.Wang, Y., et al., Gonadotropin control of inhibin secretion and the relationship to follicle 13. type and number in the hpg mouse. Biol Reprod, 2005; 73(4): p. 610-8.Scott RT, Elkind-Hirsch KE, Styne-14. Gross A, Miller KA, Frattarelli JL The predictive value for in vitro fertility delivery rates is greatly impacted by the method used to select the threshold between normal and elevated basal follicle-stimulating hormone. Fertil Steril 2008; 89:868–78.

References

The Ovarian Assessment Report™The Complexity of Ovarian Reserve Made Patient Friendly

Payment Options

The payment options for the OAR include submitting to insurance, account billing or patient billing.

Specimen Preparation 8.5ml serum tube

For complete specimen requirements see the OAR Sample Preparation Protocol or contact Client Services at 1.800.667.8893

A Convenient Two Module ReportOne patient friendly module (4 page booklet format) with clear, simple explanations of:

Egg Supply and hormone testing• Age and its effect on egg quality• Summary comments• Resources for patients (references, websites)• Logos/contact info for the fertility center•

One clinician module (single page):Individual hormone values plus Egg Retrieval • ScoreFull explanation of data supporting test with each • report, conveniently available for review

In addition to having the most accurate ovulatory egg supply test available using a single blood sample, clinicians enjoy the opportunity of having materials to facilitate the interaction with patients. By hav-ing two modules, clinicians can easily customize their encounter with patients of any background to achieve the best possible interaction.

Ovarian Assessment Report™1.2 Patient Copy

Patient: Jane Doe Physician: Dr. J. Smith Date of Report: 10/12/08

private and confidential

Dr. J. smith fertility clinic

14 Crosby Drive Bedford MA 01730 email: clientservices@reprosource.com

web: www.reprosource.comtel: 800.667.8893 781.687.5899

fax: 781.935.3068