Hananel Holzer, MD Medical Director, MUHC Reproductive Center McGill University Health Center

1. McGill University McGill University Health Centre Hananel Holzer, MD Medical Director, MUHC Reproductive Center McGill University Health Center Director, REI Division, Dept. of Obstetrics & Gynecology McGill University

2. A comprehensive approachfor fertility preservation

3. Fertility preservation • 713,220 new cases of female cancer were diagnosed in 2009 in the U.S. (Jemal 2009) • Childhood cancers 1%. 122 new cases per million children (Skinner 2006) • By 2010, one in 250 people will be a cancer survivor (Blatt 1999) • 80% of children and adolescents diagnosed with cancer become long-term survivors • One of long-term side-effects of cancer therapy is premature gonadal failure – infertility

4. Ovarian reserve • Females born with a finite pool of oocytes ( Zukerman 1951) • Challenged by Johnson, Tilly et al; 2004, 2005;Germ line stem cells

5. Ovarian reserve W. Hamish B. Wallace 2010

6. Ovarian reserve W. Hamish B. Wallace 2010

7. Ovarian reserve W. Hamish B. Wallace 2010

8. Chemotherapy • Destruction of growing follicles • loss of primordial follicles • ovarian atrophy

9. Early Menopause and Infertility Primordial Follicle Count ⇩⇩⇩ Ovarian Reserve ⇩⇩⇩

10. 2500 2000 1500 Number of PMF’s 1000 500 0 0 2050 75 100mg/kg Dose of Cyclophosphamide • The effect of chemotherapy on the ovary is not “all or none” • No. of surviving primordial follicles following exposure correlates inversely to dose of chemotherapy (p = 0.0001) (Meirow et.al. Hum Reprod 1999)

11. Cortical fibrosis • Focal ovarian cortical fibrosis • No follicles in zone of fibrosis • “Wedge shaped”- corresponds to the blood supply (Meirow et al. Hum Reprod 2007)

12. Chemotherapy: Direct and indirect damage Vascular injury Cortical fibrosis Apoptosis medulla cortex (Meirow et al. Hum Reprod 2007)

13. Loss of regulatory mechanisms • Loss of local regulatory factors (AMH?) • Accelerated recruitment of primordial follicles. • Decreasing ovarian reserve

14. Chemotherapy • Alkylating agents: • Cyclophosphamide,ifosfamide, nitrosoureas, chlorambucil, melphalan, and Busulphan • not cell-cycle specific, highest risk of ovarian failure. • Antimetabolites • methotrexate, bleomycin, 5-fluorouracil, actinomycin-D, mercaptopurine, vincristine) • impact the cells of the metabolically active ovarian follicles , are considered to be low risk for gonadal dysfunction • Platin - intermediate risk

15. chemotherapy • Women >40 years have a 90% chance of amenorrhea subsequent to multi-agent chemotherapy • younger patients 20% - 90%. • All patients exposed to chemotherapy might have a diminished ovarian reserve • significant predisposition for developing premature ovarian failure. Infertility > 40% @ 35 years. • The estimated probability of early menopause was at least 25% at age 30 years (Letourneau , Cancer 2011)

16. Radiotherapy • Direct damage to ovaries • Damage to hypothalamic-pituitary axis • Dose dependent; total dose and fractionation schedule • Age dependent; size of primordial follicle pool • Radiation field dependent • Could also affect uterine function (M/C, IUGR, Premature deliveries)

17. Radiotherapy • for a given dose of radiation, the younger patients are, the later the onset of premature menopause. • LD50 <2 Gy. • the effective sterilising dose falls with increasing age • effective sterilising doses are 20·3 Gy at birth, 18·4 Gy at 10 years, 16·5 Gy at 20 years, and 14·3 Gy at 30 years. • model to reliably predict the age of ovarian failure after treatment with a known dose of radiotherapy. (W Hamish B Wallace, Lancet 2005, International Journal of Radiation Oncology*Biology*Physics 2005)

18. Reproductive outcome • In animal studies, the risks of M/C and of malformed fetuses after chemotherapy are high. • In humans, no increases in fetal malformation or M/C after anticancer drug treatment. • No higher risk of genetic or chromosomal abnormalities in offspring from parents that had been cancer survivors (Winther JF 2004) • if conception occurs more than a year after cessation of the treatment • cancer treatment has mutagenic effects on the oocyte that are present in growing follicles and the risk of mutagenesis is related to the stage of oocyte development during exposure and the drug regime used.(Meirow 2005)

19. Fertility preservation

20. Fertility preservation Indications: • Cancer patients before gonadotoxic treatment • Other diseases before gonadotoxic treatment • Young patients with Turner syndrome, Fragile X premutation (FMR 1), Galactosemia • Women in mid-thirties without partner ?

21. Options for fertility preservation Should be tailored according to: • Patient’s age • Type of disease • Spread of the disease • Planned treatment • Time available • Whether she has a partner (Holzer Tan 2005)

22. Options for fertility preservation Ovarian protection: • Ovarian shielding • Ovarian transposition prior to local radiotherapy: • reduces dose to 5-15% • patients <40 • Laparoscopy • Location depends on the planned radiation. • Does not protect against chemotherapy. (Tulandi 2004)

23. Options for fertility preservation • GnRH agonist acts to protect gonads during chemotherapy by preferentially steering cells into less active cell cycle stage with decline in response to chemotherapeutic agents • Simulating pre-puberty. • Direct effect? • Decreased perfusion? • Young patients. (Blumenfeld 2007 ,Huser 2008)

24. Options for fertility preservation • 3 meta-analyses were published: • GnRHa are effective in preserving ovarian function and in reducing amenorrhoea (Clowse 2009, Ben Aharon 2010) • only prospective randomized studies: • odds ratio of 3.5 favouring the use of GnRHa. • Higher rates of resumption of menses and ovulation • No improvement of pregnancy rates (Bedaiwy 2010)

25. Options for fertility preservation • Some evidence that GnRHa has a protective effect. • the final conformation is still awaited. • GnRH antagnist – faster desensitization. • Flare up effect; combined treatment? (Mardesik 2004 von Wolff 2011) • The GnRHa could be used to induce ovulation in a stimulated cycle. • Reduced tumour cell sensitivity to chemo in E+ cases ?

26. Options for fertility preservation Apoptosis inhibition: • Ceramide – 2nd messenger signals apoptosis • Sphingosine 1 phosphate (S1P) ceramid antagonist • Interesting and promising; still far from clinical implementation (Morita, Paris, Perez, Tilly et al 1999,2000, 2002)

27. Transplantation of whole intact ovary by vascular anastomosis Wang et al Nature 2002; Imhof et al 2006; Bedaiwy et al 2007 Huge technical challenge, perfusion of the cryoprotectant In theory, risk of cancer cell transmission Cryopreservation for fertility preservation

28. Options for fertility preservation: Cryopreservation of ovarian tissue • Available for pre- and post-puberty patients • Hundreds to thousands of primordial follicles may be preserved. • No medical delay. • No ovarian stimulation. • Does not require a male partner • At least 2 surgical procedures (+ IVF) • Anesthesia- risks • Theoretic risk of neoplastic cells in transplanted tissue – recurrence?

29. Risk of presence of neoplastic cells in the transplanted tissue • Subclinical involvement of Hodgkin’s Lymphoma has not been identified in ovarian tissue(Seshadri Gook et al 2006) • detection of Hodgkin lymphoma within ovarian tissue taken at the time of harvest for cryopreservation. (Bittinger 2011) • 58 patients with hematological malignancies underwent ovarian tissue cryopreservation • after thawing, markers to detect minimal residual disease used • real-time RT-PCR positive in one patient with CML. • this alarming result avoided tissue transplantation(Meirow et al 2008) • Positive markers of CML and AML in cryopresrved harvested ovarian tissue (Dolmans 2010)

30. Risk of presence of neoplastic cells in the transplanted tissue • Other organ transplants: donor derived malignancy ( Kauffman 2002, Ison 2011 AM J Transplant) • Extreme caution is warranted before we assume that we understand tumour biology well enough to estimate the risk of transmission of malignant cells in autotransplanted ovarian cortex. • BRCA 1&2 carriers - potential of developing ovarian cancer (Colgan 2001) • Prophylactic BSO, transplant ovarian fragments?

31. Options for fertility preservation: Cryopreservation of ovarian tissue • Loss of follicles during transplantation and initial ischemia. Absence of inhibitory mechanism? Longevity of the graft? • 14 pregnancies and live births reported : • Donnez, Meirow* • Rosendahl* (6 pregnancies,3 live births, 2 women)/12 cases. • Demeestere (OTx2) *IVF • Bias due to selective reporting?

32. Options for fertility preservation: Cryopreservation of ovarian tissue • Culture and IVM of primordial follicles: 2 step culture system: culture of tissue followed by isolation of follicles and culture. Or using 3D supportive matrix. Culturing to MII is the next chalange (Abir et al. Histol. Histopatho 2006 Picton et al. Reproduction 2008, Tefler et al. Hum Rep. 2008, Woodruff 2009) • Suspension of isolated primordial follicles (Dolmans et al 2008) • Xenotransplantation: human ovarian tissue to SCID mice. Aberrant microtubule and chromatin patern. Transmission of pathogens, short life span ethical issues, and …( Lucifero 2002, Kim 2001)

33. Embryo cryopreservation • Integral part of IVF programs >25 years • Success rates 30-50% per embryo transfer, depending on the age at the time of oocytes retrieval. • Only well-established option of fertility preservation • Post pubertal patients. • Partner required • Donor sperm?

34. 1st live birth 1986 (Chen et al) But: oocytes vulnerable to freezing process intracellular ice formation membrane rupture, abnormal cortical granule reaction, zona hardening, meiotic spindle and cytoskeleton damage 1986-1997: 5 live births No male partner: Oocyte cryopreservation

35. No male partner: Oocyte cryopreservation • Using PROH/sucrose slow freezing protocol and introduction of ICSI improved pregnancy rates (Porcu et al 1997) • Reported survival rates for mature oocytes (collected from stimulated ovaries) are 50-70%

36. Oocyte vitrification Cryoprotectants in high concentration used to induce glasslike state, cell then rapidly frozen avoiding formation of intracellular ice Kuleshova et al, 1999:survival rate 65%, PR/ET 21% Yoon et al, 2003:survival rate 69%, implantation rate 6.4%, PR/ET 21.4% Katayama et al, 2003:survival rate 94%, PR/ET 33%

37. Oocyte vitrification 165 live births (Chian et al ASRM 2007: McGill Reproductive Center in Canada, Instituto Mexicano de Infertilidad in Mexico and CECOLFES in Colombia) A few thousand babies Vitrification is emerging to be a better technique.

38. Embryo or oocyte cryopreservation after ovarian stimulation • Pregnancies reported are result of fertilization of frozen/thawed oocytes collected after ovarian stimulation • However: • time interval needed for ovarian stimulation 2-6 weeks. • Starting during menstruation • Time may not be available for cancer patients • Recent studies: stimulation luteal phase. (Von Wolff 2009, Sonmezer 2011) • ovarian stimulation associated with high estrogen levels which may not be safe in cases of hormone sensitive tumors such as breast cancer • Estrogens may have an indirect mitogenic effect on receptor negative cancers. (Gupta PB 2006)

39. Embryo or oocyte cryopreservation after ovarian stimulation Aromatase inhibitor + FSH: letrozole started 2 days before FSH administration, then given concomitantly, no increased risk of relapse (Oktay 2005, 2007, Azim 2008) Lower estradiol levels does not totally avoid stimulation Early follicular start of treatment. More studies are needed

40. Collection of immature oocytes from unstimulated ovaries • Pincus, J Exp Med1935; Edwards, Nature, 1965,1969. • Cha et al,1991: Immature oocyte laparotomy for oocyte donation • Trounson et al, 1994: IVM: vaginal collection of immature oocytes, for PCO related infertility

43. Mature oocytes at oocyte collection in IVM cycles 8mm 12mm 14mm

44. Vitrification of IVM oocytes? • Could oocytes collected from unstimulated ovaries, matured in vitro, then vitrified survive thawing and be fertilized? • Could transfer of these embryos result in a viable pregnancy ?

45. Vitrification of IVM oocytes Holzer et al ESHRE 2007

46. Conclusions Vitrification of in-vitro matured oocytes collected from unstimulated ovaries followed by later thawing and fertilization can result in successful pregnancies and live births

47. Conclusions • Preliminary results • 20 patients, all with PCO • 20% pregnancy rate (vs. 35% in “Fresh IVM” ) • Lower implantation rate (10.8% vs. 14.4%) • Learning curve?

48. Conclusions • Collection of immature oocytes from unstimulated ovaries followed by IVM and vitrification of mature oocytes could be offered to patients with hormone-sensitive disease and/or when there is not enough time to stimulate ovaries • no risk of aggravating disease • no theoretic risk of re-instituting metastatic malignant disease • does not require same amount of time as that needed for ovarian stimulation, no need to wait for next cycle

49. When ovarian tissue is being harvested • Additional strategy of fertility preservation combines ovarian tissue cryobanking with retrieval of immature oocytes from excised ovarian tissue, followed by in vitro maturation (IVM) and vitrification (Huang, Tulandi, Holzer, Tan, Chian Fertil Steril 2007) • When surgery performed to remove ovarian tumor or for other therapeutic indications

50. Fertility preservation in prepubertal children • Childhood and adolescence period of emotional and psychological instability; issues of sexuality, including fertility, are of particular importance • Depletion of primordial oocytes after gonadotoxic treatment proportional to size of oocyte pool • Younger patients have more oocytes; thus gonadal damage could seem to be less severe than in older patients