1. Fertility at midlife Prof. Duru Shah

2. The need for fertility at midlife Childhood cancer survivors  young women with low ovarian reserve Career oriented women Late marriages Delayed childbearing  rate of divorces Re-marriage

3. Infertility increases with age Courtesy American Society for Reproductive Medicine 2007 % infertility 9% 7% 15% 22% 29%

4. Causes of poor Results in older women  Follicular atresia  ↑ Spontaneous abortion rates due ↑ aneuploidy  ↓ Endometrial Receptivity  ↓ Oocyte Quality

5. The Rise And Fall Of Ovarian Reserve 5 th month in uterus7 million germ cells Birth2.6 to 4.7 million eggs/ovary Menarche5,00,000 eggs Reproductive age Follicles  @1000/ month Menopause Follicles  to < 100 Ref : N. S. Macklon Seminars in Reproductive Medicine 2005 7.0 5.0 3.0 1.0 0.3 3 6 9 5 10 30 50 3 6 9 5 10 30 50 Age (months) Age ( yrs ) Birth Prevalence of diminished OR is 10% in the general infertility practice

6. Quantitative & Qualitative decline of follicle pool Ref : Broekmans FJ et al Hum Reprod Update 12(6) 685-718, 2006

7. ART Success rates in different Ethnicities Ref. Karen Purcell, et.al. Fertility & Sterlity Vol. 87, Feb. 2007

8. The Ovarian Reserve  OR is currently defined as the no. & quality of follicles left in the ovary at any given time - impractical to actually count.

9.  Age related  in oocyte pool is associated with impaired fertility  Live births after ART ↓ after 35 years  Impl. Rate/embryo ↓ after 35 years of age  To identify women at young age with  reserve  to initiate early treatment  To identify the older woman with good ovarian reserve The need to assess ovarian reserve Ref : Broekmans FJ et al Hum Reprod Update 12(6) 685-718, 2006

10. Markers Of Ovarian Reserve Baseline hormones - FSH - Estradiol - Inhibin B- Antimullerian hormone Ultrasound parameters - Antral follicle count - Ovarian volume - Ovarian Stromal Blood Flow Ref : N. S. Macklon Seminars in Reproductive Medicine 2005 Contd..

11.  CCCT : Basal FSH on Day 3 - abnormal FSH on Day 10 following CC  EFFORT : change in E2 Level in response to 300 U of FSH administered on Day 3  GnRh Agonist Stimulation Test : Change in estradiol levels after 2 or 3 days in response to the flare effect of GnRH agonist administration  Hcg Theca Stimulation Test change Androstenedione levels on D3 of Hcg stim. LIMITED EVIDENCE ON VALIDITY OF THESE TESTS Challenge Tests For Ovarian Reserve Ref : N. S. Macklon Seminars in Reproductive Medicine 2005

12. Responses to therapy  Normal Responders When stimulated aggressively with injectable FSH will develop 5-8 mature follicles as well as several smaller ones.  Poor Responders (older age, poor ovarian reserve) Failure to produce an adequate no. of mature follicles(<4) & or a peak E2 levels < 500 pg/ml.  High Responders (PCOS) Defined as those woman with peak E2 levels > 4000 pg/ml on the day of hCG administration or > 15 retrieved oocytes. 3 main groups

13. Day 3 - FSH > 10 m IU /ml - Inhibin B < 45 pg/ml - AMH < 3 - Antral Follicle Count < 10 Age > 37 years of age - younger pts. may also have a poor response. Identifying Poor Responders Poor Ovarian Reserve

14. Ref : Kwee J et al Reprod Biology & Endocrinology 2007 5:9 Parameter Poor Responders (n=29) Normal responders (n=81) P Age at baseline (y) CD3 35.3+3.033.5+4.0.029 FSH(IU/L)12.0+1.56.6+1.8<.001 E2(pmol/L)124.1±54.1138.4±156.5.632 Inhibin B (ng/L)76.0±47.493.1±43.0.077 AMH(  g/L) 1.48±2.593.53±2.46<.001 Comparison of investigations

15. Ref : Kwee J et al Reprod Biology & Endocrinology 2007 5:9 Parameter Poor Responders (n=29) Normal responders (n=81) P Endpoints Total no. of follicles 4.6±2.617.7±9.7<.001 Total no. of oocytes 3.0±1.614.9±8.1<.001 Ongoing pregnancy n(%) 2(10)22(24).001 Comparison of investigations

16.  < 3 cc associated with ↑ cycle cancellation rates.  May exhibit inter cycle variability esp. in younger women with  AFC  Single best available predictor of response to ovarian stimulation with exogenous gonadotrophins  Poor predictive value to achieve pregnancy, since it cannot determine quality of oocytes Clinical value of Ultrasound evaluation of OR Clinical value of Ultrasound evaluation of OR

17. Treatment Options for Fertility at Midlife  COH + IUI  COH +IVF / ICSI  Donor Egg + Self Transfer.  Donor Egg + Surrogacy.

18. Ovarian Stimulation and IUI in women over 40 yrs. Study: Retrospective  247 women with unexplained infertility  Comparison of pregnancy rates with CC / Gn + IUI CC / Gn + IVF /IVM Ref. Amir Wiser et.al.; Reprod. Biomed Online 2012, 24,170-173

19. Ref. Amir Wiser et.al. Reprod Bio Med. 2012, 24,170-173 Values are n(%) a Yates’ P- value (comparison of all four groups) bP= 0.001 versus IVF cP= 0.001 versus IVF dP= 0.007 versus IVF Results: Outcome Clomiphene + IUI (n=46) Gonadotropin + IUI (n=39) IVM (n=38) IVF(n=124)P-value a Biochemical pregnancies 0(0) b 1(2.6)2 (5.2)28 (22.6)0.001 Clinical pregnancies 0(0) c 1(2.6)0(0)17(13.7)0.02 Live birth0(0) d 1(2.6)0(0)17(13.7)0.02 Ovarian Stimulation and IUI in women over 40 yrs.

20. COH Protocols for IVF  Pituitary down regulation  With Agonist  With Antagonist  Protocols  Minimal v/s Conventional stimulation  Modified Natural cycle

21. Pituitary Down reg. Antagonist v/s Agonist Meta analysis of 14 studies Ref. Danhua Pcc et.al. Human Reprod. Vol 26, 2011 AntagonistAgonist Mean duration of Stimulation9.9811.87 Mean number of oocytes retrieved5.1815.42 Mean no. of mature oocytes retrieved4.8754.69 Clinical pregnancy rate %21.73 %18.36 %

22. GnRh Antagonist v/s Agonist Protocol-in poor responders Results:- Gynaecworld Data Poor Ovarian Reserve (IVF) AntagonistAgonist No. of days stimulation1011 Mean gonadotropins dose used / cycle 5982.757125 Mean No. of mature eggs retrieved 4.173.5 Mean No. of eggs fertilized32.37 Clinical Pregnancy rate24.48%12.50%

23. Mild ovarian stimulation v/s conventional ovarian stimulation in poor responders  285 poor responders  119 cycles with mild ovarian stimulation  270 cycles with conventional ovarian stimulation  Both groups divided based on age above and below 37 yrs  Mild ovarian stimulation CC 100 mg/day or Letrozole 5 mg x 5 days. No medication + FSH / HMG 150-225 IU + rHCG 250 mg (dom. foll at 18mm) Ref. Gerald Pfetter et. al. Cochrane Data base, Reviews 2012

24. Mild ovarian stimulation with conventional ovarian stimulation in poor responders Mild ovarian stimulation with conventional ovarian stimulation in poor responders ≤ 37 yrs. old> 37 yrs. old Clinical pregnancy rate m-IVFc-IVFp-valuem-IVFc-IVF b P- value Per initiated cycle (%) 17.2 (10/58) 22.5 (34/151) NS6.6 4/61) 5.0 (6/119) NS Per transferred cycle(%) 23.3 (10/43) 33.0 (34/103) NS12.1 (4/33) 8.1 (6/74) NS Live birth rate Per initiated cycle (%) 10.3 (6/58) 19.2 (29/151) NS3.3 (2/61) 1.7 (2/119) NS Per transfer cycle (%) 14.0 (6/43) 28.2 (29/103) NS6.1 (2/33) 2.7 (2/74) NS Ref. Yoo JH et.al.; Comparison of mild Ovarian stimulation with conventional ovarian stimulation in poor responder. Clin Exp. Repnd Med 38; 159-163

25. Minimal Stimulation Protocol Advantages :-  Less expensive  Less office visits  ↓ incidence of OHSS  ↓ incidence of high order pregnancy Disadvantages :-  No excess oocytes/embryos for cryopreservation  Increased chance of cancelling blastocyst transfer.  ↓ no. of eggs from egg donors  Cannot be used if PGD required. Ref. Zarek Mild/minimal stimulation for IVF, Fertil Steril 2011

26. Modified Natural Cycle Ref. Isac – Jacques Kadoch et.al. Fertil Steril, Nov 2011 Cycle Day : 1 2 3 4 5 6 7 8 9 10 11 12 Indomethacin 50mg tds GnRh antagonist 0.25 SC ↓ HMG 150/ day Baseline Scan USG OPU EE-4mg/day OC pill Hcg 5000 IU Follicle > 14 mm ET > 6 mm Follicle > 17 No cysts ET < 5 mm

27. Modified Natural Cycle Advantages :- Advantages :-  Natural selection of single Oocyte of good quality.  ↓ expensive, stressful  ↓ OHSS, Multiple pregnancy  Healthier and receptive endometrium Disadvantages :- Disadvantages :-  ↓ pregnancy rates  ↑ cancellation rate Ref. Isaac – Jacques Kadoch et.al. Fertil Steril, Nov 2011

28. Adding Adjuvants to COH protocols Adjuvant therapy  DHEAS  Growth Hormone  Mitochondrial Nutrients  Aspirin  Acupuncture  Psychological Support. Ref. Gerald Pfetter et. al. Cochrane Data base, 2012 Contd….

29. Contd….. Rationale for use  Cholesterol → DHEA → Testosterone → E 2  Levels of DHEA ↓ with age.  DHEA → ↑ Follicular IGF-1 → Gonadotropin effect.  DHEA → promotes PCO environment in ovaries →  active oocytes ↓ atretic follicles DHEA Supplementation

30. DHEA Supplementation DHEA Supplementation  Recent studies have shown that DHEAS admin x 3 months in poor responders.  ↑ follicular microenvironment by ↑ oxygen levels in FF crucial for dev. of good quality oocytes Ref. Gerald Pfetter et. al. Cochrane Data base, 2012

31. Study:  To study the effect of DHEA supplementation on IVF treatment outcomes. Randomized controlled trial  133 pts. with prior poor ovarian response in IVF  Study group – DHEA group 25 mg 3 times a day.  Control group – COH without DHEA Ref. Ashraf Moawad et. al., Middle East Fertility Soc. Jou.2012 (17); 268-74 DHEA Supplementation DHEA Supplementation

32. Results Ref. Ashraf Moawad et. al., Middle East Fertility Soc. Jou.2012 (17); 268-74 Study group (no.= 67) Control group (no.= 66) P Value Retrieved oocytes5.9 ± 3.63.5 ± 2.9<0.001 MII oocytes(%)72.35 ± 11.8169.43 ± 12.75NS Fertilization rate66.7 ± 14.864.4 ± 19.6NS Cancellation rate9 (13.4%)19(28.8%)<0.01 Embryos transferred 2.8 ± 0.91.7 ± 1.1<0.001 DHEA Supplementation DHEA Supplementation

33. Ref. Ashraf Moawad et. al., Middle East Fertility Soc. Jou.2012 (17); 268-74 Study group (no.= 67) % Control group (no.= 66)(%) P Value Pregnancy rate (per transfer) 24.121.3NS Pregnancy rate (per cycle) 20.915.2<0.05 Chemical pregnancy rate2/58(3.4)2/47(4.3)NS Clinical pregnancy rate20.717.0 Ongoing pregnancy rate11/58(19.0)7/47(14.9)<0.05 Achievement of pregnancy. DHEA Supplementation DHEA Supplementation

34. StudyGH protocol Owen et.al. (1991) 24 IU im/day on alternate day starting simultaneously with hMG until the day of hCG Zhuang et.al. (1994) 12 IU im/day on alternate days Bergh et.al. (1994) 0.1 IU /kg body weight /day sc, starting simultaneously with FSH until the day of hCG administration Dor et.al. (1995) 18 IU sc on cycle day 2,4,6,8 Suikkari et.al (1996) 4 to 12 IU, starting on cycle day 3 Kucuk et.al. (2008) 12 IU sc, from day 21 of the preceding cycle and until the day of hCG adminstration Growth Hormone- No Standardized dose Ref. Kolibianakis et.al. Hum. Reprod. Update 2009; 15(6);613-22

35. Growth Hormone Rationale  GH in foll. fluid is assoc. with rapid cleavage, good cleaving embryo morphology and a high potential for embryo implantation.  GH concern is ↓ in older women as compared to younger women.  GH admin. Improves intra foll. estradriol levels → better oocyte quality  ? Improves uterine receptivity. Contd….

36. Meta analysis:  Ten RCT’s with 440 sub fertile couples included to study the effect of GH in IVF protocol in poor responders. Results:  No difference in outcome measures and adverse events in routine use of GH.  Statiscally significant ↑ in live birth rates & pregnancy rates with use of GH in poor responders without ↑ adverse events.  Which subgroup of poor responders would benefit, has not been identified. Ref. Duffy JM N et al, Cochrane Reviews 2010 Growth Hormone

37. Mitochondrial Nutrients Rationale for use  Selection of highest quality oocytes during early reproductive years → leaves less favorable oocytes at advanced age.  Process of aging exerts an unfavorable influence on dormant oocytes. Ref. :Yaakov B et.al., Fertility & Sterility Vol 93 (1) 2010;272-275

38. Mitochondrial Function  Mitochondria in every cell → produce energy  Oocyte has the largest number of mitochondrial DNA copies v/s any other cell in the body - 2 x 200 thousand copies.  Women with poor ovarian reserve are found to have a lower number of mt DNA copies v/s women with normal ovarian function.

39. Process of Aging Hall mark of aging :  Accumulation of point mutations & deletions of mitochondrial DNA leads to Respiratory Chain disorders in tissues where energy Consumption is high. ie. eye, heart, liver, kidney.  Mitochondrial Respiratory Chain disorders are the most prevalent group of inherited neuro metabolic disorders. Ref. :Yaakov B et.al., Fertility & Sterility Vol 93 (1) 2010;272-275 Contd.

40.  Mitochondrial Respiratory Disorders→ ↑ production of Reactive oxygen speries (ROS)  ↑ ROS prod → ↓ O2 levels. → ↑ mt DNA damage → ↑ oxidant prod. → ↑↑ mt DNA damage  Important role to play in the aging of oocytes. Contd. Ref. Cooke M.et.al. J Int. soc. Sports Nutr. 2008;5:8 Process of Aging

41. Can Mitochondrial function be improved?  COQ is a lipophilic molecule that is produced within the mitochondria and is essential for antioxidant defense. (COQ-Co-enzyme Q)  In Vitro Studies: CoQ supplementation has shown oxidation balance in the fibroblasts of CoQ deficient patients. Contd. Ref. :Yaakov B et.al., Fertility & Sterility Vol 93 (1) 2010;272-275

42. Mitochondrial Nutrients Rationale of Use  Mitochondrial function could be improved by supplementation of CoQ 10 and Alpha Lipoic Acid (ACA) →↓ risk of Trisomy + other chromosomal aneuploidies  Addition to culture media of embryos for better oocyte and embryo quality → healthy pregnancy outcome. Ref. Chew GT et.al; Qim 2004; 97: 537-48

43. Current Evidence for supplement of MN 2011. Ref. Gerald Pfetter et. al. Cochrane Data base, 2012  12 RCT’s fulfilled criteria out of 1335 abstracts – low comparability amongst studies.  8 out of 12 trials above were new trials  Various interventions studied. Outcome:- Currently, no clear evidence supporting the use of any intervention in mitochondrial disorders.  Further research needed

44. AspirinAspirin  Low dose Aspirin does not improve ovarian or uterine blood flow or Ovarian responsiveness  May ↑ ovarian Antral follicular count.  May improve COH parameters  No beneficial result. Ref. Frattarelli JL, Mc Williams DG, Hill MJ et al. Fertil Steril. 2007 Jul 3

45. AcupunctureAcupuncture Meta analysis  13 Randomized Controlled Trials with Acupuncture V/s no Acupuncture / Sham Acupuncture  2500 women  5 trials with acupuncture at OPU  8 trials with acupuncture at ET Conclusion: Not enough evidence that acupuncture improves ART results.

46.  Psychol distress of inability to conserve → self esteem. ↑ anxiety, depression, somaization ↑ sexual disturbances  Psychological support and psychosexual counselling & androgen suppl. → extremely important. Ref. Vander Stege et.al. Menopause 15: 23-31 Psychological and Psychosexual support support

47. Egg Donation – Global results Ref. National summary & Fertility Clinic reports – CDC 2010

48. Egg Donation – Gynaecworld Experience Ref. Shah Duru et.al. unpublished data 2007 - 2013 70 60 50 40 30 20 10 Donor Egg + Self Transfer Donor Egg + Surrogate Transfer 41% 66% Self Egg Self Transfer 28%

49.  During treatment for pelvic pathology  During treatment of cancer in adolescents and reproductive age group. Preserving Ovarian Reserve

50.  Salpingectomy for ectopic pregnancy → ↓ AFC → ↓ oocytes obtained during ART.  Excise hydrosalpinx close to the tube - ↓ compromise to ovarian blood supply. Preferable instead of electrocoagulation. Ref. Gelbaya et. al. Fertil Steril 2006 Pelvic Pathology Contd….

51.  Laparoscopic ovarian Cystectomy with endometriomas.  If the purpose of removing endometrioma is to improve fertility, then :-  Presence of Endometrioma does not hamper IVF results.  Remove endometrioma only if > 4 cms. and prevents OPU.  Fenestration and selective ablation of endometrioma bed, if plane of cleavage poor. Ref. Togas Tulandi et.al. Elsevier December 16, 2011 Pelvic Pathology

52. AMH 6 5 4 3 2 1 5.99 ± 3.36 ng/ml AFC + Ov. Vol. 4.6 ± 3.1ng /ml P= >.005 P >.005 FSH Results PCOS and Ovarian Drilling (OD) Ref. Weerakiet S, et.al. Gynecol Endocrinol 2007;23(8): 455-60 PCOS with OD PCOS without OD P= NS

53. During treatment for cancer in young women  Reduce impact of chemotherapy on ovaries – Ovarian suppression with GnRh Analogs  Remove & preserve ovarian tissue prior to starting radio therapy with transposition of ovaries out of field of radiation.  Cryopreservation of ovarian cortex/oocytes/ embryos Strategies for Fertility Preservation Preservation Strategies for Fertility Preservation Preservation Ref. Federica Tomao et.al. Reproduction Fertility Breast Cancer 2010

54. Study  Randomized, prospective triple blind, single center, controlled clinical trial.  No. of patients – 600  Randomized into 2 groups. - 300 vitrified oocytes - 300 fresh oocytes. Preserving Fertility - fresh v/s vitrified oocytes Preserving Fertility - fresh v/s vitrified oocytes Ref. Ana Co et.al, Human Reprod. 2010, 25, (Antonio Pellicer’s group, Valencia Spain)

55. Ongoing Pregnancy Clinical Pregnancy rate /cycle 50 40 30 20 10 Vitrified eggs Fresh eggs 41.7 Pregnancy Rate Implantation Rate Preserving Fertility 43.7 50.2 49.8 40.9 39.9 Results Ref. Ana Co et.al, Human Reprod. 2010, 25, (Antonio Pellicer’s group, Valencia Spain)

56. ConclusionsConclusions  With women delaying pregnancies, poor ovarian reserve and Premature Ovarian Failure (POF) leading to infertility is on the rise.  Important to determine declining ovarian reserve before POF sets in.  Ovarian Reserve varies in different ethnicities at the same age. Contd…

57.  Important to customize the protocol of COH to optimize fertility potential  Preventive action to preserve fertility is essential whilst treating childhood cancer patients and during pelvic surgery. Contd… ConclusionsConclusions

58.  Oocyte cryopreservation allows women at risk for POF to preserve their reproductive potential.  Despite advance in ART, egg donation seems to be the only option with good results at midlife.  Adequate counselling and emotional support is highly recommended. ConclusionsConclusions