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: 1.

2.

3.

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).

• 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 Primordial Primary Secondary Preantral Early antral (*) Antral (* +) Preovulatory (+) + IVF * IVM Follicle size (mm) 0.03

0.05

0.07

0.12

0.21

0.41

16.10

0.04

0.06

0.11

0.20

0.40

16.00

20.00

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

• Gonadotropin priming – None – hCG – FSH / FSH+hCG • Metformin • IVF / ICSI

Laboratory

• Culture conditions

HCG Priming

• Theoretically; – Promote invitro maturation – Improve pregnancy rates

IVM following hCG priming

• • • • • • • •

Cycles of IVM Age (yrs) Oocytes retrieved Maturation rate (%) 25 35.4



10.3



4.7

5.4

84 Fertilization rate (%) Cleavage rate (%) Embryos transferred 87 95 2.9



0.6

Clinical pregnancies - no (%) 10 (40) Chian et al New Engl J Med 1999; 341:1624-6

90 80 70 60 50 40 30 20 10 0 0 + HCG - HCG *p < 0.05

12 24 36 48 hours of culture Chian et al Hum Reprod 2000; 165-170

Response to LH in granulosa cells from follicles < 8 mm from ovulatory women (with normal ovaries or PCO compared to anovulatory women with PCO) Patients Ovulatory (normal and ovPCO) Anovulatory (anovPCO) Fold increase in steroid accumulation in response to LH above control Estradiol 1.0 (5.0 - 3.9); (n = 46) a 1.4 (0.7 - 25.4); (n = 17) b Progesterone 1.0 (0.3 - 2.5); (n = 42) c 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 No. of oocytes collected (mean + SEM) No. of MII-stage oocytes collected (%) No. of oocytes cultured No. of oocytes matured

in vitro

(%) Total no. of oocytes matured (%) No. of oocytes fertilized (%) No. of oocytes cleaved (%) No. of oocytes transferred (mean)

No. of pregnancies (%)

Group 1 (n=48) 922 (19.2 + 8.4) (14.6) 135 500 787 635 456 396 178

23

(63.5) (68.8) (71.8) (86.8) (3.7)

(47.9)

Group 2 (n=46) 854 (18.6 + 9.9) 0 854 535 535 419 377 173

13

P

NS (62.6) (62.6) (78.3) (90.0) (3.8)

(28.3)

NS NS NS NS NS NS

<0.05

Son WY et al. RBM Online. (2008), in press

Hormonal Priming

Regular cycling •

Beneficial

•

Wynn 1998

•

No difference

•

Trounson 1998

•

Suikkari 2000

•

Mikkelsen 2005

PCOS •

Beneficial

•

Mikkelsen 2001

•

No difference

•

Lin 2003

•

Chian 2000

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 oocyte Endometrial preparation is necessary – matured

Endometrial preparation

Endometrial thickness on day of oocyte retrieval <6 mm 10 - 12 mg estradiol 17ß (micronized) 6 - 8 mm >8 mm 8 - 10 mg estradiol 17ß (micronized) 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 Chian

et al

Cha

et al

(2000) Chian

et al

(1999) (2000) Mikkelsen and Lindenberg (2001) 25 94 11 13 12 24 PCOS PCOS PCOS PCOS Child Lin Chian (2004) Soderstrom-Anttila et

al

(2005) Cha Son

et al et al et al

Torre

et al et al

(2002) (2003) (2005) (2007) (2007) 107 35 33 254 PCO: 13 7 PCOS: 18 10 203 138 415 106 PCO/PCO S PCOS PCO/PCO S PCO: 13 7 PCOS: 18 10 PCOS PCOS PCO/PCO S No. of ET cycles at cleavage stage 25 85 11 13 9 21 107 35 33 NA 9 (IVF) 5 (ICSI) 17 (IVF) 9 (ICSI) 187 NA 415 106 (blastocyst) Gn Priming HCG None None HCG None FSH HCG FSH+HCG HCG HCG None None HCG HCG HCG Maturation Rate (%) 84 75.1

69.1

84.3

44.0

59.0

76.0

76.5

71.9

78.8

60.6

49.2

54.3

53.2

NA 61.7

74.0

78.2

Fertilization Rate (%) 35.0

72.4

82.5

70.0

NA 62 80.1

80.5

87 67.9

83.9

90.7

69.0

70.0

78.0

75.8

69.5

69.2

Implantatio n Rate (%) Pregnancy Rate/ET (%) Miscarriage Rate (%) 32 6.9

24.8

16.6

0 21.6

9.5

9.5

11.3

11.1

40 27.1

27.3

38.5

0 33.3

26.2

31.4

36.4

24.0

20 26.1

0 40 57.1

26.1

13.0

13.3

0 34.5

12.5

5.5

10.9

9.7

26.8

22.2

0 52.9

22.2

21.9

24.5* 28.4

51.9

NA 0 33.3

50.0

36.8

42.3

NA 21.8

Outcome of IVM cycles from the literature in women with normal ovaries and regular cycles.

Authors (year) Child et al. (2001) No. of cycles 56 (normal) 53 (PCO) 68 (PCOS) No. of ET cycles at cleavage stage 50 52 67 Gn

Priming

Mean no. of oocytes retrieved Maturat ion Rate (%) HCG HCG HCG 5.1 10

± ±

3.7

5.1

11.3

±

9 79.5

75.9* Fertilization Rate (%) 67.7

71.6* Implanta tion Rate (%)

CPR/ET

(%) 1.5

8.9

9.6

4 23.1

29.9

Mikkelsen

et al. (2001)

132 83 None 3.9

60.1

72.9

NA 18 M/C Rate (%) 50 25 50 NA

Soderstrom

-Anttila et al. (2005) 92 (IVF) 100 (ICSI) 58 (IVF) 86 (ICSI) None 6.3

±

6.5

±

3.4

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

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

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

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 Mean age No. of mature oocytes retrieved No. of immature oocytes retrieved Mean oocyte maturation rate No. of oocytes vitrified and thawed No. of oocytes survived (mean % + SEM; range) No. of oocytes fertilized (mean % + SEM) No. of embryos transferred (median; range) No. of implantations (mean % + SEM) No. of pregnancies (%) No. of clinical pregnancies (%) No. of ongoing pregnancies (%) No. of live births (%) Mean birth weight (grams) 20 30.8 + 0.9

6 290 67.3 + 4.9

215 148 (67.5 + 5.8; range 23.5 -100.0) 96 (64.2 + 4.5) 64 (4; range 1 - 6) 4 (9.6 + 5.4) 4 (20.0) 4 (20.0) 0 (0) 4 (20.0) 3486 Chian et al, 2008, Fertil Steril, in press

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

Twin pregnancy rate Triplet pregnancy rate Mean birthweight (g) Mean gestational age (wks) Mean Apgar scores at 1 min Mean Apgar scores at 5 min Mean cord pH IVM 12.0% 4.0% 2,812 37 8 9 7.29

IVF 16.0% 2.0% 2,826 37 ICSI 14.0% 3.0% 2,801 36 8 9 7.30

8 9 7.30

Controls p-value 1.3% p<0.001

0 p<0.001

3,289 39 8 9 7.29

p<0.001

p<0.001

n/s n/s n/s

Congenital abnormalities following

Major malformations

IVM (n=55)

• ompalocele • small ventricuoloseptal defect 2 1 1 Minor malformations • patent ductus arteriosus • congenital hip dislocation 3 1 2

IVM IVF ICSI

Relative risk for any congenital abnormality compared with controls

RR 1.19

1.01

1.41

95% CI 0.35 – 3.25

0.52 – 1.90

0.72 – 2.68

Pregnancy outcomes per clinical pregnancy after IVM, IVF and ICSI

90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Live Birth (p<0.05) Miscarriage (p<0.005) IVM Ectopic IVF Late Pregnancy Loss ICSI Miscarriage in PCOS (NS) 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 Scandinavia Italy France Germany Rest of Europe Total Europe Deliveries and ongoing pregnancies 150 77 40 20 33 320

Deliveries and ongoing pregnancies (facts and educated guesses)

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

Deliveries and ongoing pregnancies (facts and educated guesses)

Countries Canada USA Australia Total Deliveries and ongoing pregnancies 120 5 5 130

Deliveries and ongoing pregnancies (facts and educated guesses)

Countries Asia Europe North America Australia Grand Total - one year ago !

Deliveries and ongoing pregnancies 679 320 125 5 1129

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

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

Acknolwedge

Dr. Ezgi Demirtas

Reproductive Centre McGill University