1. Premature Ovarian Failure Santiago, October 5th, 2007 Pr Philippe Touraine philippe.touraine@psl.aphp.fr Inserm Unit 845, Research Center Growth & Signaling, Faculty of Medicine Necker, Paris Dept Endocrinology & Reproductive Medicine, Hospital Pitié Salpetrière, Paris Hospital Pitié Salpetrière, Paris

2. 8th week : 600 000 oogonia 5 th Week of embryogenesis Endodermal stem cells Genital crest Ovogonia Ovogonia Oogonia Mitosis 28th Week Meiosis Primary Oocytes Atresia Maximal at 20thWeek

3. 6-7 Million germ cells at 20th Week  2/3 are oocytes in meiosis  1/3 are oogonia Atresia  < 20 % germ cells at birth la naissance  300 000 at puberty  400-500 follicles during genital life

4. Premature Ovarian Failure (POF)  WHO : Permanent Cessation of menstruations secondary to loss of follicular activity  Characterized by : Women < 40 yrs Women < 40 yrs Amenorrhea > 4 months Amenorrhea > 4 months Hypergonadotrophic Hypogonadism (FSH > 30 UI/L) Hypergonadotrophic Hypogonadism (FSH > 30 UI/L) Anasti JN, Fertil. Steril., 1998, 70 : 1-5. Anasti JN, Fertil. Steril., 1998, 70 : 1-5.  Incidence : # 1% Coulam CB et al., Obstet. Gynecol., 1986, 67 : 604-6

5. Premature Ovarian Failure 1% of 40-year-old-women Etiology most often remains unknown Autoimmunity FSHR anomalies Cx37, GDF9, BMP-15 Enzyme deficiencies Follicular Dysfunction Accelerated atresia Follicular Depletion Chemotherapy X chromosome anomalies Autoimmunity

6. Premature Ovarian Failure Accelerated atresia Follicular Depletion Chemotherapy X chromosome anomalies Autoimmunity

7. Schlessinger et al., Am J Med Genet, 2002 Total of cases with POF % cases with POF Primary amenoorhea Secondary amenorrhea

8. POF - FMR1  20% premutated and 14% grey zone develop POF ( Bretherick, 2005 )  13-21% familial forms POF are premutated  3-7% sporadic POF are premutated ( Shermann, 2000 )  Prevalence: 0.4% women with normal fertility

9. Radiotherapy : 6 Gy on ovaries : non reversible POF POF and chemotherapy Oligomenorrhea Normal Cycles Drugs Normal cycles Amenorrhea

10. Premature Ovarian Failure Autoimmunity FSHR anomalies Cx37, GDF9, BMP-15 Enzyme deficiencies Follicular Dysfunction

11. Genetics of POF  Is there any argument for a genetic compound  Genes and physiological menopause – Correlation between age of menopause in mother and girls De Bruin, Hum Reprod 2001 – Cohort of twins: POF described in up to 63% Snieder, J Clin Endocrinol Metab 1998 Gosden R, Hum Reprod, 2007 – Familial cases in 15 to 20%

12. Primordial Primary Ovulation Hsueh, Endocrine Review, 2000 Antral Secondary Atresia Blocade of follicular maturation

13. u 22 patients with primary amenorrhea u Pubertal development variable u Hypoplasic ovaries at histological examination u Presence of primordial and primary follicles FSH receptor gene mutation in the Finnish population Ala 189 Val

14. Setting up a POF Network Phenotyping of the patients (specificities of subgroups) Phenotyping of the patients (specificities of subgroups) Constitution of a DNA Bank Constitution of a DNA Bank  Identification of new mutations of genes involved in folliculogenesis Constitution of an ovarian tissue bank Constitution of an ovarian tissue bank Immunohistochemistry / steroidogenesis Immunohistochemistry / steroidogenesis Ovarian transcriptome Ovarian transcriptome Prospective follow-up of population Prospective follow-up of population

15. Current Evaluation Clinical Phenotype QuestionnaireKaryotype BiologicalPhenotype Hormonalevaluation MorphologicalPhenotype Ultrasonography Laparoscopy - Histology

16. 351 patients 50 excluded  40 yrs  FSH<30 mUI/l  Secondary POF  Karyotype “Turner-like” 301 patients 8 karyotypic anomalies 293 46,XX idiopathic 74 patients < 18 yrs (25%) Bachelot et al., 2007, submitted

17. Age at diagnosis (yrs) Mean age: 26.5 ± 7.8 Number of Patients 0 2 4 6 8 10 12 14 16 18 20 1112131415161718192021222324252627282930313233343536373839 Bachelot et al., 2007, submitted

18. Primary amenorrheaSecondary amenorrhea Number of Patients 0 50 100 150 200 250 absence partial normal Puberty Bachelot et al., 2007, submitted

19. Clinical Phenotype 41 patients with a history of familial POF 41 patients with a history of familial POF  13.6% des POF 59 patients with autoimmunity biomarkers 59 patients with autoimmunity biomarkers  19.6 % of POF  37 with thyroid disorders  Diabetes, juvenile arthritis, vitiligo,….  6 patients with antiovarian antibodies (IFI) 41 patients with fluctuating POF 41 patients with fluctuating POF 5 pregnancies; 3 births 5 pregnancies; 3 births

20. Median (Range) Normal range FSH (IU/l) 82.6 (30.3–284) 3–9 LH (IU/l) 32.5 (5.9–109) 1–5 Estradiol (pg/ml) 25.8 (5–246) 20-350 Inhibin B (pg/ml) 10 (5–105) 60–200 134 woman have detectable E2 levels and 58 inhibin B 134 woman have detectable E2 levels and 58 inhibin B AMH detectable in 105 patients AMH detectable in 105 patients Bachelot et al., 2007, submitted

21. Available for 151 patients  Mean age 28.7 yrs (15-42)  Definition WHO 66 patients (42%) BMD normal 66 patients (42%) BMD normal 63 patients osteopenia (42%), 14 at neck and 25 at lumbar 63 patients osteopenia (42%), 14 at neck and 25 at lumbar 22 patients osteoporosis (15%), 7 at neck and 20 at lumbar 22 patients osteoporosis (15%), 7 at neck and 20 at lumbar Bone Mineral Density

22. 100µm 100µm N= 29 N = 75 Two Different Patterns Bachelot et al., 2007, submitted

23. Ovarian Histology 0 10 20 30 40 50 60 70 Secondary 70% secondary primordial primary Early antral Absence of foll antrum Follicular Histology Primary 25% Amenorrhea Bachelot et al., 2007, submitted

24. Histo - Histo + 5 10 15 20 25 30 35 40 Foll + Foll - Ultrasonogaphy Massin et al., 2004

25. FSH Receptor gene mutations Patient 1 Patient 2 Patient 3 Beau et al. JCI, 1998Touraine et al., Mol Endocrinol, 1999Meduri et al., JCEM, 2003

26. Phenotype-Genotype Correlation Patient 1 Patient 2 Patient 3 FinnishpopulationPubertyNormalNormal Lack of Delayed AmenorrheaSecondaryPrimaryPrimaryPrimary Histology Antral Follicles Early antral follicles Primordial Follicles Primary Follicles In vitro R-FSH activity 30% Arg573Cys 15% Leu601Val 0%Undetectable

27. Rec FSH Stimulation

28. Etiology actually possible in 30 patients (10%) 8 with karyotype anomalies 8 with karyotype anomalies 5 with FMR1 premutation 5 with FMR1 premutation 16 anomalies of genes involved in POF 16 anomalies of genes involved in POF 2 APS type 2, 1 with multiple AI diseases 2 APS type 2, 1 with multiple AI diseases

29. Clinical evaluation: Height Familial POF syndrome, Syndromic defects, Autoimmunity Karyotype Hormonal evaluation: FSH-E2; Androgens AMH, Inhibine B Ultrasonography: Surface, Presence of Follicles Evaluation of BMD DNA analysis: XFra FSHR analysis in patients with presence of follicles at ultrasonography Hormonal substitution Treatment of infertility What to do in our current practice? Bachelot et al., 2007, submitted

30. What to say?  Information +++  Psychological help  HRT  Explanation about pregnancy plans (oocyte donation…)  Discussion about “ovaries to be frozen”

31. Gross et al., Fertil Steril, 2005 Around POF Diagnosis

32. Department of Endocrinology and Reproductive Medicine and Reproductive Medicine Necker Hospital Necker Hospital Pr F. Kuttenn PathologyCochinCHICHEGP G Meduri Genetics M Misrahi, Bicêtre M Fellous, Cochin C Cotinot, INRA D Castrillon, Dallas, TX L.Messiaen, Gant, Belgium Surgery F Lecuru F Lecuru B Paniel Radiology C Matuchansky C Balleyguier Hormonal Biochemistry K Laborde Cytogenetics Nathalie Massin Anne Bachelot Yvette Le Rouzic Philippe Touraine National and International Collaboration

33. Team « Centre Maladies rares de la croissance » philippe.touraine@psl.aphp.fr Beau et al. J. Clin Invest, 1998;Touraine et al., Mol Endocrinol, 1999;Meduri et al., JCE&M, 2003; Massin et al., Hum Reprod, 2004; Bachelot et al., JCE&M, 2005; Massin et al., Horm Res, 2006 Meduri et al., Hum Reprod, 2006; Laissue et al., Eur J Endocrinol, 2006