CHR Research Update

1. CHR Research Update David H Barad, MD1,2 Norbert Gleicher, MD1,3 1Center for Human Reproduction and Foundation for Reproductive Medicine; 2 Department of Obstetrics, Gynecology and Women’s Health, Albert Einstein College of Medicine; 3 Department of Obstetrics, Gynecology and Reproductive Sciences, Yale University School of Medicine

2. The speaker declares the potential conflicts of interest. Dr. Gleicher and Dr. Barad are listed as co-inventors on an already awarded and other pending patent applications which claim therapeutic benefits of DHEA in women with diminished ovarian reserve. Dr.s Gleicher and Barad are also listed as co-inventor son a pending patent application, which claims diagnostic benefits from evaluating the FMR1 gene in regards to ovarian reserve. Both doctors have received research support, speakers fees and travel funds from various pharmaceutical companies, none in any way related to the topic of this presentation.

3. The speakers declare that no brand-name medications and/or off-label, non-FDA-approved uses are discussed in the lecture.

4. The Aging Ovary

5. How to measure aging? How to treat aging?

6. How to measure aging • FSH • AMH • AFC • CCT • FMR1 Appears to regulate ovarian reserve

8. Fu et al. Cell,Vol. 67, 1047-1056, DECEMBER, 1991

11. Box and Whisker Plot, defining normal range of CGG repeats This box and whisker plot for the whole study population of 339 women reconfirmed the previously defined normal range of 26-32 CGG repeats (median 30). The graph in the right lower corner represents the frequency distribution of individual alleles, confirming the median CGG count at 30. Gleicher et al., RBN Online ; in press.

12. Triple CGG counts by box and whisker plots for individual racial/ethnic groups Gleicher et al RBM Online 2010;20:485-91.

13. FMR1 Genotypes • Normal: both alleles in 26 – 34 range • Heterozygous: one allele in 26 – 34 range one allele <26 or >34 • Het-norm/low • Het-norm/high • Homozygous: both alleles outside of 26 – 34 range

14. Linear regression of association between AMH levels and age based on FMR1 status The figure represents egg donors and infertility patients at all ages. Normal females at young ages have the highest, and women with homozygous CGG count abnormalities the lowest AMH levels. AMH levels, however, decline in normal women more rapidly than in heterozygous and homozygous patients. At approximately 35 years of age AMH levels in heterozygous women start to exceed those of normal women. AMH levels in homozygous women track those of normal women almost till age 50, when they start exceeding the latter. Gleicher et al. Reprod Biomed Online; In Press.

15. AMH levels in binned age groups based on FMR1 status The figure represents egg donors and infertility patients of all ages. Under age 30years AMH levels significantly differ amongst all three patient groups (p=0.009). Specifically, AMH in normal women is significantly higher between normal women and homozygous females (p<0.001) and between heterozygous and homozygous patients (p=0.002). By age 34.99, these statistical differences no longer are present. Gleicher et al. Reprod Biomed Online; In Press.

16. Conclusions Abnormal CGG counts on FMR1 denote risk for POA Women identified at risk can be monitored with AMH Continued POA→ Fertility preservation → Change in reproductive planning

17. Ovary, like thyroid and adrenals: Differentiation between function and autoimmunity

18. Thyroid/Adrenal • Function • Autoimmunity • Suppressing • Stimulating

19. We previously postulated the presence of autoimmune-induced ovarian hyperactivity (PCO, stimulating antibodies) and hypoactivity (POA, suppressiveautoantibodies) Gleicher et al, Autoimmune Rev, 2007;7:42-45.

20. Evidence of autoimmune activation in women with different FMR1-genotypes Gleicher et al, ASRM, 2010.

21. Materials & Methods • 339 consecutive patients • 183 (50.0%) norm • 62 (18.3%) het – norm/high • 94 (27.7%) het – norm/low • 206 (60.8%) no autoimmunity • 133 (39.2%) autoimmune + • 75 PCO (≥12 oocytes / AMH >4.0ng/mL)

22. P=0.003 P<0.0001

23. Conclusions Het – norm/low defines an autoimmune-associated, quickly OR-depleting PCO phenotype, while het – norm/high defines low autoimmune risk Confirms involvement of FMR1 in recruitment and functional OR Establishes an association between het – norm/low genotype and female autoimmunity

24. Pregnancy chances based on FMR1 genotype Gleicher et al, ASRM, 2010.

25. Materials & Methods 455 consecutive IVF cycles Autoimmune profile: Y/N FMR1 genotype Multiple regression analyses adjusted for age, race, medication dosage, number of oocytes

26. Results

27. Results cont. Adjusted for age, het-norm/low maintained lowest rates (p=0.001), but significance was lost with adjustment for race, medication and oocyte numbers. Controlling for all covariates, autoimmunity almost reached significance (p=0.06).

28. Conclusions FMR1 genotype is predictive of pregnancy chances Loss of significance with adjustment for ovarian factors suggests/confirms a direct effect of FMR1 on the ovary In contrast, actual significance of autoimmunity independently may suggest an autoimmune effect not mediated by ovaries.

29. What connects between FMR1 and autoimmunity? Gleicher et al, ASRM, 2010.

30. Literature Search • PubMed, Medline • Keywords/phrases • Autoimmunity • X chromosome • FMR1 gene • Xq27 region

31. Results • Autoimmunity & POI/POF closely associated with X chromosome defects • Turner syndrome (XO)

32. Turner Syndrome (XO) Xq terminal deletions common Often large & characterized by 1o/2o amenorrhea Xq21 → 2o amenorrhea (FMR1 at Xq27) Fertile Turners → proximal Xq deletions POI: 4Mb locus exactly at Xq27-q28

33. Turner Syndrome (XO) cont. • Small Xq27-q28 deletions → variable phenotypes, some early menopause but able to reproduce • Increased autoimmunity • Diseases • Autoantibodies

34. Balanced Translocations Only Xq23-q27 deletions associated with POI/POF

35. Autoimmunity in general associated with more non-disjunctional events: Down Syndrome Klinefelter Syndrome Turner Syndrome

36. Autoimmunity high in practically all X-linked disorders • May, therefore, be caused by genes/mutations on the X chromosome

37. MHC-paralogue on long arm of X chromosome, if defective, reduces immunologic effectiveness

38. Summary The long arm of the X chromosome is of paramount importance for autoimmunity as well as POI/POF FMR1 maps to Xq27

39. FMR1, the crossroad for autoimmunity and ovarian function?

40. Non-disjunctional events and their consequences

41. Non-disjunctional events • ↓ • Chromosomal abnormalities • ↓ • Miscarriages

42. ?

43. DHEA

44. DHEA • Reduces (?) aneuploidy Gleicher et al, ASRM 2007 • Significantly reduces miscarriages Gleicher et al, ReprodBiol & Endocrinol 2009;7:108

45. Comparison of miscarriage rates between DHEA supplemented infertility patients and 2004 national US IVF outcome data. Gleicher et al. ReprodBiolEndocrinol 2009:108.

46. RESULTS When miscarriage rates were compared, equalized for number of patients, dhea supplementation reduced miscarriage rates even more significantly (p<0.0001), suggestive of a reduction of ~80%

47. Indirect proof But direct evidence still lacking

48. Materials & Methods 22 DOR patients on DHEA Each matched with 2 controls (no DHEA) based on age, years of cycle (mostly normal OR) Univariate general linear model to assess DHEA effects on % aneuploidy after adjustments for age, indications for PGS, stimulation, gonadotropin dosage

49. Results

50. Results cont. DHEA effects remained significant after adjustments (Exception: PGS for gender had lower aneuploidy; p<0.007) Greatest reduction in aneuploidy with short-term treatment (4-12 weeks) (21.6% 95%CI – 2.871 – 46.031)