Hong Kong Sanatorium & Hospital, China WORLD IVM EXPERIENCE Milton K. H. Leong, M.D. IVF Centre Hong Kong Sanatorium & Hospital, China

LEARNING OBJECTIVES At the conclusion of this presentation, participants should be able to: Describe the indications IVM Outline the various IVM approaches undertaken currently. Evaluate the IVM outcomes with regard to the treatment success rates and the babies born as a result of IVM treatment.

DISCLOSURE Milton K. H. Leong, MD None

Development of IVM It was first demonstrated in 1935 that the immature oocytes have the ability to resume meiosis spontaneously when removed from the follicle. Pincus G, Enzmann EV. J. Exp. Med. 62, 665-675 (1935) Edwards showed that in-vitro matured human oocytes could be fertilized. Edwards RG, Bavister BD, Steptoe PC. Nature. 221(5181), 632-5 (1969).

1994-first IVM pregnancy with a patient’s own oocytes. the immature human oocytes retrieved during gynecologic surgery in an oocyte donation program resulted in the first IVM pregnancy in 1991. Cha et al., Fertil Steril 55; 109-13 (1991). 1994-first IVM pregnancy with a patient’s own oocytes. Trounson A, Wood C, Kausche A. Fertil Steril 62; 353-62 (1994)

Development of the follicle Stage Follicle size (mm) Primordial 0.03 - 0.04 Primary 0.05 - 0.06 Secondary 0.07 - 0.11 Preantral 0.12 - 0.20 Early antral (*) 0.21 - 0.40 Antral (* +) 0.41 - 16.00 Preovulatory (+) 16.10 - 20.00 + IVF * IVM Gougeon, Hum Reprod 1986;1:81-7

Target patient group Women with high AFC; PCOS PCO with regular cycles The most significant factor which determines the success of IVM treatment is the AFC of the woman (Tan, 2002. Am. J. Obstet. Gynecol. 186; 684-9)

Patient selection for IVM Suikkari 2007; Best Practice & Res Clin Obstet Gynecol 21; 145-155

promising outcomes are also reported in “regular cycling” women Better prognosis if AF basale count > 7 Suikkari 2007; Best Practice & Res Clin Obstet Gynecol 21; 145-155

Common Indications for IVM failure after > 6 cycles of ovulation induction women having IVF with high AFC repeated poor embryo quality in previous IVF cycles for no obvious reason repeated poor responders to ovarian stimulation

however low implantation rates when compared to conventional stimulated cycles. asynchrony in the cytoplasmic and nuclear maturation of the oocyte asynchrony in the endometrium culture conditions

Various approaches to improve implantation rates in IVM Clinical Laboratory Gonadotropin priming None hCG FSH / FSH+hCG Metformin IVF / ICSI Culture conditions

HCG Priming Theoretically; Promote invitro maturation Improve pregnancy rates

IVM following hCG priming Cycles of IVM 25 Age (yrs) 35.4  4.7 Oocytes retrieved 10.3  5.4 Maturation rate (%) 84 Fertilization rate (%) 87 Cleavage rate (%) 95 Embryos transferred 2.9  0.6 Clinical pregnancies - no (%) 10 (40) Chian et al New Engl J Med 1999; 341:1624-6

% of metaphase II *p < 0.05 hours of culture Chian et al Hum Reprod 2000; 165-170

Fold increase in steroid accumulation in response to LH above control Response to LH in granulosa cells from follicles < 8 mm from ovulatory women (with normal ovaries or PCO compared to anovulatory women with PCO) Fold increase in steroid accumulation in response to LH above control Patients Estradiol Progesterone Ovulatory (normal and ovPCO) 1.0 (5.0 - 3.9); (n = 46)a 1.0 (0.3 - 2.5); (n = 42)c Anovulatory (anovPCO) 1.4 (0.7 - 25.4); (n = 17)b 1.3 (0.3 - 7.0); (n = 20)d a vs b, P<0.0003 c vs d, P<0.03 Willis et al., Journal of Clinical Endocrinology and Metabolism 1998; 83:3984-91

Duration between HCG administration and oocyte retrieval When the durations of 35 hours vs. 38 hours between hCG administration and the oocyte retrieval were compared, the 38 h group yielded significantly higher number of mature oocytes. In-vitro maturation rate after 24 h in the culture was significantly higher, and the clinical pregnancy rate in the 38 h group was higher compared to the 35 h group in the unstimulated cycles, 40.9% vs. 25%. Son et al. Fertil Steril 88(Suppl. 1), S24-S25 (2007).

Clinical outcome in hCG-primed IVM cycles with (Group 1) and without (Group 2) MII-stage oocytes on the day of retrieval Groups Group 1 (n=48) Group 2 (n=46) P No. of oocytes collected (mean + SEM) 922 (19.2 + 8.4) 854 (18.6 + 9.9) NS No. of MII-stage oocytes collected (%) 135 (14.6) No. of oocytes cultured 787 854 No. of oocytes matured in vitro (%) 500 (63.5) 535 (62.6) Total no. of oocytes matured (%) 635 (68.8) No. of oocytes fertilized (%) 456 (71.8) 419 (78.3) No. of oocytes cleaved (%) 396 (86.8) 377 (90.0) No. of oocytes transferred (mean) 178 (3.7) 173 (3.8) No. of pregnancies (%) 23 (47.9) 13 (28.3) <0.05 Son WY et al. RBM Online. (2008), in press

Hormonal Priming Regular cycling PCOS Beneficial No difference Wynn 1998 No difference Trounson 1998 Suikkari 2000 Mikkelsen 2005 Beneficial Mikkelsen 2001 No difference Lin 2003 Chian 2000

May improve endometrium FSH Priming Results are conflicting Potential benefits: Larger ovarian size Easier retrieval Higher E2 levels More maturational competence May improve endometrium

Overview of IVM treatment cycle Withdrawal bleed U/S scan day 2-4 to identify if PCO and measure AFC Repeat u/s scan on day of hCG to measure endometrial thickness s/c hCG 10,000 IU when ET 6-8 mm, largest follicle 10-12 mm and oocyte retrieval 38 hours later

Transvaginal U/S-guided oocyte retrieval vaginal vault cleansed with sterile water i.v. sedation sedation with fentanyl and L.A. 19 G single single-lumen needle reduced aspiration pressure (7.5 kPa) multiple punctures 10 ml culture tubes with 2ml warm 0.9% saline with 2 IU heparin

In-vitro maturation of oocytes GV oocytes cultured in IVM medium supplemented with 75mIU/ml FSH + LH for 24 - 48 hrs, checked every 12 hours all MII oocytes undergo ICSI ET day 2 or 3 following ICSI Patients receive estradiol-17ß (micronized) immediately following OR and progesteron following ICSI

Endometrial Priming Endometrium is exposed to lower E2 levels Dyssynchrony between phase of endometrium – matured oocyte Endometrial preparation is necessary

Endometrial preparation Endometrial thickness on day of oocyte retrieval <6 mm 10 - 12 mg estradiol-17ß (micronized) 6 - 8 mm 8 - 10 mg estradiol-17ß (micronized) >8 mm 6 mg estradiol-17ß (micronized) Progesterone support (50 mg I/M or 200mg tid, pv) started following ICSI

Timing of Oocyte Retrieval Dominant follicle Early atretic follicles Still competent to Embryonic development Can be used in IVM But; TIMING ?

Timing of Oocyte Collection Russell et al. (1999) When the leading follicle > 13 mm Less oocytes Less fertilization Fewer embryos

Timing of Oocyte Collection Cobo et al. (1999) When the leading follicle < 10 mm Higher blastocyst formation

Metformin in IVM 56 women, 70 cycles Metformin, 500 mg bid for 12 weeks before the IVM treatment HMG for 5 days and hCG 10,000 IU, 36 h prior to OPU number of immature oocytes, oocyte maturation, fertilization and cleavage rates in were comparable to the control group significantly higher implantation and clinical pregnancy rates were obtained in the metformin-treated group (15.3% and 38.2% respectively) compared to the controls (6.2% and 16.7%) Wei Z et al. Fertil Steril 2007 Nov 15

IVM outcomes

Outcome of IVM cycles from literature in women with PCO/PCOS. Authors (year) No. of cycles Indication No. of ET cycles at cleavage stage Gn Priming Maturation Rate (%) Fertilization Implantation Pregnancy Rate/ET (%) Miscarriage Chian et al (1999) 25 PCOS HCG 84 87 32 40 20 Cha et al (2000) 94 85 None 75.1 67.9 6.9 27.1 26.1 Chian et al (2000) 11 13 69.1 84.3 83.9 90.7 24.8 16.6 27.3 38.5 Mikkelsen and Lindenberg (2001) 12 24 9 21 FSH 44.0 59.0 69.0 70.0 21.6 33.3 57.1 Child et al (2002) 107 PCO/PCOS 76.0 78.0 9.5 26.2 Lin et al (2003) 35 33 FSH+HCG 76.5 71.9 75.8 69.5 11.3 31.4 36.4 13.0 Chian (2004) 254 NA 78.8 69.2 11.1 24.0 Soderstrom-Anttila et al (2005) PCO: 13 7 PCOS: 18 10 9 (IVF) 5 (ICSI) 17 (IVF) 9 (ICSI) 60.6 49.2 54.3 53.2 35.0 72.4 82.5 13.3 34.5 12.5 22.2 52.9 - 50.0 Cha et al (2005) 203 187 5.5 21.9 36.8 Torre et al (2007) 138 61.7 62 10.9 24.5* 42.3 Son et al (2007) 415 106 106 (blastocyst) 74.0 78.2 80.1 80.5 9.7 26.8 28.4 51.9 21.8

Mean no. of oocytes retrieved Outcome of IVM cycles from the literature in women with normal ovaries and regular cycles. Authors (year) No. of cycles No. of ET cycles at cleavage stage Gn Priming Mean no. of oocytes retrieved Maturation Rate (%) Fertilization Implantation CPR/ET (%) M/C Child et al. (2001) 56 (normal) 53 (PCO) 68 (PCOS) 50 52 67 HCG 5.1 ± 3.7 10 ± 5.1 11.3 ± 9 79.5 75.9* 67.7 71.6* 1.5 8.9 9.6 4 23.1 29.9 25 Mikkelsen et al. (2001) 132 83 None 3.9 60.1 72.9 NA 18 Soderstrom-Anttila et al. (2005) 92 (IVF) 100 (ICSI) 58 (IVF) 86 (ICSI) 6.3 ± 3.4 6.5 ± 3.6 66.9 54.5 35.9 67.1 22.6 15 31 21 33.3 16.7 * PCO and PCOS groups pooled together.

IVM for other indications

IVM oocyte donation 12 oocyte donors (29.7 yrs; AFC 29.7) oocyte retrieval days 9-18 of unstimulated cycle mean of 12.8 GV oocytes retrieved 8.67 mature oocytes and 5.9 fertilized oocytes 3.9 embryos transferred implantation rate 19.1%; 6/12 clinical pregnancy – 4 delivered Holzer et al Fertil Steril 2007; 88: 62-67

IVM +/- natural cycle IVF and PGD 35 yr old with RM failed 2 IUI and 2 IVF IVM offered because of PCO; 1 M II and 14 GV oocytes; ICSI performed 8 embryos, 6 biopsied, 1 embryo from MII oocyte and 1 from GV oocyte chromosomally normal for 6 autosomes and X and Y chromosome 2 ET – one blastocyst from MII oocyte and one morula from GV oocyte ß-hCG 399 IU 14 days after ET and livebirth in May 2005 Ao et al Fertil Steril 2006;85:1510-12

these oocytes can be matured in-vitro IVM as a Rescue Some cycles are cancelled due to Risk of OHSS Poor pesponse Can IVM be a rescue ? these oocytes can be matured in-vitro

IVM as a rescue + Risk of OHSS Immature oocyte retriaval IVM 10,000 IU HCG Leading follicle = 12-14 mm Immature oocyte retriaval + IVM 47 % CLINICAL PREGNANCY No OHSS Lim et al. Fertil Steril 2002

Immature oocyte retrieval + IVM IVM as a rescue In POOR RESPONSE = E2 < 1000 pg/ml < 4 mature oocytes Poor responders no HCG Immature oocyte retrieval + IVM 37,5 % Pregnancy rate Liu et al. Fertil Steril 2003

IVM for Fertility Preservation

Fertility preservation for young women Best option; embryo cryopreservation, after ovarian stimulation followed by oocyte retrieval and fertilization of oocytes by sperm; IVF or ICSI Probably second best; oocyte cryopreservation after ovarian stimulation followed by oocyte retrieval

Ovarian stimulation is not suitable for certain cancer patients; no sufficient time and/or ovarian stimulation contraindicated Solution ? Trial: Retrieval of immature oocytes from unstimulated ovaries, and maturation in-vitro followed by cryopreservation of oocytes by vitrification

Viability and pregnancy outcome of vitrified IVM oocytes No. of patients 20 Mean age 30.8 + 0.9 No. of mature oocytes retrieved 6 No. of immature oocytes retrieved 290 Mean oocyte maturation rate 67.3 + 4.9 No. of oocytes vitrified and thawed 215 No. of oocytes survived (mean % + SEM; range) 148 (67.5 + 5.8; range 23.5 -100.0) No. of oocytes fertilized (mean % + SEM) 96 (64.2 + 4.5) No. of embryos transferred (median; range) 64 (4; range 1 - 6) No. of implantations (mean % + SEM) 4 (9.6 + 5.4) No. of pregnancies (%) 4 (20.0) No. of clinical pregnancies (%) No. of ongoing pregnancies (%) 0 (0) No. of live births (%) Mean birth weight (grams) 3486 Chian et al, 2008, Fertil Steril, in press 42

Fertility preservation strategies offered for women at MRC with cancer Chemotherapy cannot be delayed and/or hormonal stimulation contraindicated Chemotherapy can be delayed and hormonal stimulation not contraindicated Ovarian wedge resection or oophorectomy Immature oocyte retrieval Ovarian stimulation mature oocyte retrieval Immature oocyte retrieval from ovarian tissue IVM Male partner available (ICSI) Male partner available No male partner available No male partner Ovarian tissue cryopreservation Embryo cryopreservation Embryo cryopreservation Ooycte vitrification Ooycte vitrification

Obstetric and perinatal outcomes of the IVM pregnancies

Outcome of IVM, IVF, ICSI and normal pregnancies obstetrical and perinatal outcome of 432 babies (55 IVM, 217 IVF, 160 ICSI) compared with 1,296 age-matched spontaneous pregnancies (controls) delivered at a single hospital (MUHC) Buckett et al. Obstet Gynecol 2007; 110:885-91

Perinatal outcome IVM IVF ICSI Controls p-value Twin pregnancy rate 12.0% 16.0% 14.0% 1.3% p<0.001 Triplet pregnancy rate 4.0% 2.0% 3.0% Mean birthweight (g) 2,812 2,826 2,801 3,289 Mean gestational age (wks) 37 36 39 Mean Apgar scores at 1 min 8 n/s Mean Apgar scores at 5 min 9 Mean cord pH 7.29 7.30

Congenital abnormalities following IVM (n=55) Major malformations 2 ompalocele small ventricuoloseptal defect 1 Minor malformations 3 patent ductus arteriosus congenital hip dislocation

Relative risk for any congenital abnormality compared with controls RR 95% CI IVM 1.19 0.35 – 3.25 IVF 1.01 0.52 – 1.90 ICSI 1.41 0.72 – 2.68

Pregnancy outcomes per clinical pregnancy after IVM, IVF and ICSI Buckett et al Fertil Steril 2007

Pregnancy Outcome in IVM Mikkelsen et al. (2005) ----- 47 IVM babies 2 twins 1 NT Normal karyotype 2 preterm deliveries 1 stillbirth (42 weeks) 1 chromozomal abnormality

Pregnancy Outcome in IVM Malformation: Cha, Fertil. Steril. 2005 5,3% major malformation rate Later neuromotor development: Soderstrom-Anttila, Hum. Reprod. 2006 ))) Minor developmental delay at first year ))) No Difference in the second year

Deliveries and ongoing pregnancies (facts and educated guesses) Countries Deliveries and ongoing pregnancies Scandinavia 150 Italy 77 France 40 Germany 20 Rest of Europe 33 Total Europe 320

Deliveries and ongoing pregnancies (facts and educated guesses) Countries Deliveries and ongoing pregnancies Middle East 21 Japan 100 Vietnam 26 China (incl. HK) 60 Korea (Cha Hosp.) 57 Korea (Maria Cl.) ≈ 400 Rest of Asia 15 Total Asia 679

Deliveries and ongoing pregnancies (facts and educated guesses) Countries Deliveries and ongoing pregnancies Canada 120 USA 5 Australia Total 130

Deliveries and ongoing pregnancies (facts and educated guesses) Countries Deliveries and ongoing pregnancies Asia 679 Europe 320 North America 125 Australia 5 Grand Total 1129 - one year ago !

Korea 455 Taiwan 20 Colombia 7 Canada 131 Finland 52 Turkey 8 China 58 Japan 51 Vietnam 42 Hong Kong 18 Denmark 34 Italy 56 UK Total 930

Conclusions IVM simplifies treatment, reduces costs and eliminates OHSS IVM successful in women with high AFC hCG increases final number of MII oocytes and rate of maturation IVM may be helpful in women with repeated poor embryo quality in previous IVF cycles for no obvious reason, or repeated poor responders to ovarian stimulation

Conclusions IVM produces CPR/C of 35%, and up to 48% in selected cases, in women up to 35 . obstetric and perinatal outcomes of IVM pregnancies comparable with IVF and ICSI IVM may be useful for oocyte donation or PGD IVM may offer a chance for fertility preservation to young women with cancer and undergoing cytotoxic treatment. IVM may not replace standard IVF but appears to play increasingly important role in ART

Dr. Ezgi Demirtas Reproductive Centre McGill University Acknolwedge Dr. Ezgi Demirtas Reproductive Centre McGill University