David H Barad, MD 1,2 Norbert Gleicher, MD 1,3 1 Center 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

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.

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

 How to measure aging?  How to treat aging?

 FSH  AMH  AFC  CCT  FMR1 Appears to regulate ovarian reserve

Fu et al. Cell,Vol. 67, , DECEMBER, 1991

This box and whisker plot for the whole study population of 339 women reconfirmed the previously defined normal range of 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.

Gleicher et al RBM Online 2010;20:

 Normal: both alleles in 26 – 34 range  Heterozygous: one allele in 26 – 34 range one allele 34 Het-norm/low Het-norm/high  Homozygous: both alleles outside of 26 – 34 range

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.

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.

 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

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

 Function  Autoimmunity  Suppressing  Stimulating

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

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

 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)

P< P=0.003

 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

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

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

Genotype Pregnancies (%) P Normal38.6 p=0.001 Het-norm/high31.7 Het-norm/low22.2

 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).

 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.

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

 PubMed, Medline  Keywords/phrases  Autoimmunity  X chromosome  FMR1 gene  Xq27 region

 Autoimmunity & POI/POF closely associated with X chromosome defects  Turner syndrome (XO)

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

 Small Xq27-q28 deletions → variable phenotypes, some early menopause but able to reproduce  Increased autoimmunity  Diseases  Autoantibodies

 Only Xq23-q27 deletions associated with POI/POF

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

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

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

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

FMR1, the crossroad for autoimmunity and ovarian function?

Non-disjunctional events and their consequences

Non-disjunctional events ↓ Chromosomal abnormalities ↓ Miscarriages

FMR1 Gene Miscarriages Non-Disjunctional Events Autoimmunity ?

 Reduces (?) aneuploidy Gleicher et al, ASRM 2007  Significantly reduces miscarriages Gleicher et al, Reprod Biol & Endocrinol 2009;7:108

Gleicher et al. Reprod Biol Endocrinol 2009:108.

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%

 Indirect proof  But direct evidence still lacking

 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

DHEANo DHEAP-value Oocytes9.6± ±6.3N.S. Ets1.5±0.71.4±0.9N.S. Cryo0.6±1.20.7±1.6N.S. Grade3.5±0.33.4±0.4N.S. Biopsies6.6±3.67.3±3.9N.S. Aneuploid n2.8±2.54.5± %38.2± ±22.4<0.001

 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 – – )

 First direct evidence that DHEA positively affects non-disjunctional events (aneuploidy)

 Positively affects autoimmunity  Autoimmunity increases non- disjunctional events  Does DHEA affect ovaries by reducing autoimmune effects?

DHEA FMR1 Gene Miscarriages Non-disjunctional Events Autoimmunity ? ?