1. The use of hCG in microdose to support ovarian folliculgenesis Michel Abou Abdallah, M.D.

2. FSH is currently considered the only stimulatory factor needed for ovulation induction which acts through specific receptors present on the granulosa cells of ovarian follicles

3. LH is another critical hormone involved in the control of the human menstrual cycle, its roles are traditionally believed to be limited to stimulating theca cells androgen production, triggering ovulation and supporting the corpus luteum

4. Nevertheless, granulosa cell LH receptors are expressed in more mature ovarian follicles (>10 mm in diameter) and that explains the efficacy of gonadotropin preparations containing LH

5. The addition of Rec-hLH to stimulation regimen of Rec-hFSH enhanced steroid and follicle development in patients with HH or patients undergoing stimulation with GnRH-A for ART. (Filicori et al Fertil Steril 1999 vol 72, No 6) (Sullivan et al, J Clin Endo Metab 1999, Vol.84, No.1) (Abou Abdallah et al, Fertil Steril 2000, Vol74 No 3S)

6. recruitmentmaturation FSHLH Menstrual cycle

7. E2 COH FSH LH E2 FSHLH recruitmentmaturation FSH ? E2 FSH

8. LH recruitmentmaturation FSH ? COH FSH LH E2 A 4, T E2 A 4, T FSH

9. Simon C. et al. Fertil Steril 1998;70:234-9 86 High responders previous failed IVF >3 good quality embryos 24 Step down 62 Regular protocol Detrimental effects of E2? 43221.5hCG Step down

10. The absolute concentration of LH present during the mid-through late follicular phase of the spontaneous cycle may play an important role in maintaining preovulatory folliculogenesis, and even in protecting the maturing follicle as FSH concentrations decline.

11. FSH operates in a threshold manner ( Brown J. 1978.Aust NZ J Obstet Gynecol.18:47-54) once adequate FSH concentrations in blood are achieved, follicles advance from antral stages until maturity, acquire LH receptors on granulosa cells and respond to either FSH or LH stimulation (Zeleznik and Hiller, 1984.Clin Obstet Gynecol.27:927-940)

12. Sullivan et al.1999 confirmed the hypothesis that a key mechanism by which the dominant follicle continues to develop in the face of decreasing concentration of FSH is by acquiring LH responsiveness.

13. 24 women down regulated with GnRH agonist received r-hFSH until a 14mm follicle appeared on ultrasound. They were randomized to 1 of 4 groups for a 2 day period: continued r-hFSH, saline only; r-hLH 150 IU bid, or r-hLH 375 IU bid.

14. Demographic characteristics of the treatment groups GroupAge (yr)BMI (kg/m2) Cycle length (days) Duration of infertility (yr) Saline (group A) 30.17 ± 3.225.52± 4.528.2 ± 3.33.5 ± 2.2 r-hFSH (group B) 31.00 ± 2.1 24.44 ± 2.4 28.5 ± 2.4 3.5 ± 2.4 r-hLH high (group C) 29.33 ± 1.222.20 ± 2.528.8 ± 0.84.0 ± 2.9 r-hLH low (group D) 31.33 ± 3.023.48 ± 4.428.5 ± 2.73.8 ± 1.7

15. Baseline parameters in the four treatment groups. Baseline Parameters Group AGroup BGroup CGroup DP Age (yr) 33.1 ± 0.933.1 ± 1.032.6 ± 1.130.4 ± 1.2NS Height (cm) 165 ± 1170 ± 2165 ± 2 NS Weight (kg) 58 ± 161 ± 157 ± 258 ± 2NS BMI (kg/m 2 ) 21.1 ± 0.321.2 ± 0.220.9 ± 0.221.3 ± 0.3NS Menstrual cycle duration (d) 27.9 ± 0.528.0 ± 0.527.9 ± 0.327.3 ± 0.5NS Mean ovarian volume (ml) 6.7 ± 0.464 ± 0.46.5 ± 0.36.4 ± 0.3NS LH (IU/liter) 4.7 ± 0.45.0 ± 0.64.4 ± 0.44.7 ± 0.5NS FSH (IU/liter) 6.5 ± 0.46.1 ± 0.65.8 ± 0.5 NS PRL (ng/ml) 16 ± 113 ± 214 ± 116 ± 3NS E2(pg/ml) 79 ± 761 ± 867 ± 874 ± 9NS P(ng/ml) 0.56 ± 0.040.49 ± 0.070.52 ± 0.040.58 ± 0.06NS T(ng/ml) 0.48 ± 0.050.42 ± 0.070.39 ± 0.040.40 ± 0.06NS

16. Serum FSH concentrations Results show that the mean serum FSH concentration (International units per L) ± SEM of the four treatment groups (A-D) on the day of randomization (day 0; black bars) and the day hCG administration (day 2; gray bars). The mean concentration of FSH was maintained in the group receiving r-rFSH (group B) as the mean concentration of FSH fell in groups A,C and D (p<0.05)

17. Serum LH concentrations Results show that the mean serum LH concentration (international units per L) ± SEM of the four treatment groups (A-D) on the day of randomization (day 0; black bars) and the day hCG administration (day 2; gray bars). Note that the mean LH concentration of the groups receiving r-hLH (group C and D) were greater than those of the groups not receiving r-hLH ( groups A and B; P<0.05)

18. Serum E2 concentrations Results show that the mean serum E2 concentration (international units per L) ± SEM of the four treatment groups (A-D) through the study period (day 0,1, and 2). Note that the serum E2 concentrations increased throughout the study period in the groups receiving gonadotropin (groups B, C and D), whereas serum E2 concentrations decreased toward the baseline in the baseline in the saline-treated group ( group A).

19. HCG is usually used as a surrogate to LH to stimulate ovulation

20. The effect of supplementing FSH treatment with LH activity in the form of low dose hCG therapy was reported by Filicori et al in 1999 (Fertility and Sterility vol. 72,No 6, Dec 1999) the time where Rec-hLH was not clinically available

21. The dosage of menotropins and levels of estrogen during treatment in a hypogonadal patient. Filicori. Human chronic gonadotropin. Fertil Steril 1999. Amps=ampules; HP= highly purified. (B), supplementation of highly purified FSH with low dose hCG therapy (50 IU/d). (A) dministration of highly purifies FSH alone.

22. The low dose hCG (50 IU/d) was highly effective at enhancing ovarian stimulation with highly purified FSH in GnRH agonist- suppresed women undergoing COH for ART, without causing follicle luteinization or excessive theca cell stimulation ( Filicori et al, J Clin Endocrinol Metab 1999;84:2659-63.)

23. Several days of low-dose hCG (200 IU/d) alone can be used to stimulate folliculogenesis, complete FSH initiated follicle/oocyte maturation, and achieve pregnancy in assisted reproduction technology. (Filicori et al. Fertil Steril2002; 78:414-6)

24. Ovarian follicles detected at transvaginal pelvic ultrasound and daily hormone serum levels during gonadotropin administration for ovulation induction in patient MC. The vertical arrow in the upper right corner of the figure indicates high dose (10,000 IU) hCG administration to trigger final follicle and oocyte maturation. Filicori. ICSI pregnancy after low dose hCG. Fertliti Steril 2002 No. of days of treatment hMG 225 IU/dayhCG 200 IU/day Ovarian follicles (n) T (ng/ml) P (ng/ml) E 2 (pg/ml) hCG (IU/L) FSH (IU/L) LH (IU/L) <10mm 10-14mm >14mm

25. In the same year 2002, Filicori et al, tested on 40 studied women the hypothesis that in the late stages of ovulation induction, LH activity can stimulate and selectively modulate ovarian follicle function and growth, independently of FSH administration. (Filicori et al. J Clin Endocrinol Metab 87:1156-1161, 2002)

26. Filicori M et al. JCEM 2002;87:1156-61. Stimulation and growth of antral ovarian follicles by selective LH activity administration in women. mid-luteal DT 3.75 mg 2 weeks rFSH (150IU) hCG 10’000IU IUI 2 weeks Luteal sup. Vag P Experimental protocol 40 women suffering from unexplained or male related infertility w/ reg cycles. Study population No hormone therapy for 3 mo before study. > 8 ds B A C D hFSH 150 IU hFSH 50 IU hCG 100 IU hFSH 25 IU hCG 50 IU hCG 200 IU Foll. >14mm E2 800-1’500 rFSH: Puregon

27. Clinical & hormonal results of gonadotropin stimulation in the four treatment groups Group AGroup BGroup CGroup Dp Daily r-hFSH dose (IU), d 8 onward 15050250 Daily hCG dose (IU), d 8 onward 050100200 Results of gonadotropin administration Days of gonadotropin administration 13.7 ±1.0(11-19)13.4 ± 0.7(10-16)13.1 ± 0.6(11-17)12.7 ± 0.6(10-15)NS Total r-hFSH dose received(IU) 1,920 ± 146 a 1,325 ± 40 a 1,180 ± 15 a 1,050 ± 0 a < 0.001 Total hCG dose received (IU)0275 ± 40 b 520 ± 59 b 940 ± 112 b < 0.001 Preovulatory E2 (pg/ml)1,034 ± 511,274 ± 1131,223 ± 1061,271 ± 105NS Follicular phase hormone levels LH ( IU/liter-d)12.0 ± 3.312.3 ± 1.313.8 ± 1.012.4 ± 1.7NS FSH ( IU/liter-d)96.6 ± 12.391.2 ± 3.479.8 ± 7.080.7 ± 5.5NS hCG ( IU/liter-d)ND10.2 ± 3.0 b 11.8 ± 2.8 b 38.5 ± 8.6 b < 0.005 E2( pg/ml-d)3,651 ± 4663,695 ± 6623,929 ± 7983,902 ± 677NS p (ng/ml-d)7.4 ± 1.0 c 10.7 ± 0.8 c 8.1 ± 0.7< 0.01 T (ng/ml-d)4.2 ± 0.6 d 6.8 ± 0.6 d 4.9 ± 0.44.9 ± 0.7< 0.05 a P<0.05 group A vs. groups C & D, group B vs. group D. b P< 0.05 group D vs. group B & C. c P< 0.05 group A vs. group B & C. d P< 0.05 group A vs. group B.

28. Filicori et al. Follicular Support by LH. Gonadotropin concentrations. Daily gonadotropin serum levels mean ±SEM) in the 4 groups of patients participating in the study. Days of treatment hCG (IU/L) FSH (IU/L) LH (IU/L)

29. J Clin Endocrinol Metab, March 2002, 87 (3):1156-1161 Steroid concentrations. Daily gonadal setroid serum levels (mean ±SEM) in the 4 groups of patients participating in the study. Days of treatment T (ng/ml) P (ng/ml E 2 (pg/ml)

30. Filicori M et al. JCEM 2002;87:1156-61. Stimulation and growth of antral ovarian follicles by selective LH activity administration in women. D hCG 200 IU CB A >14 >10-14 <10 day of hCG Days of treatment <10 >10-14 >14

31. Human Chorionic gonadotropin is approximately 6 times more potent than LH ( Stokman et al 1993. Fertil Steril 60:175-178), has a longer half-life than does LH and offers more prolonged and stable occupancy of LH receptors between hormone administration (Filicori et al; Fertil Steril 2002; 76(suppl):s104-5) (Damewood et al. Fertil Steril 52:398-400)

32. hCG can promote angiogenesis, a crucial process involved in embryo implantation. (Mannaerts et al; Hum Repod update 1996;2:153- 61)

33. Low dose hCG can hasten small follicle development and reduce the dose and duration of treatment with highly purified FSH without causing follicle Luteinization or excessive theca cell stimulation (Filicori et al. 1999. J.Clin Endocrinol Metab 84:2659–2663 ) (Filicori et al 1999. Fertil Steril 72:1118–1120)

34. COH FSH LH E2 A 4, T mini hCG 75 IU, when foll. >13mm FSHLH recruitmentmaturation FSH ? mini hCG E2 A 4, T FSH

35. In addition to its lower price, low dose hCG therapy can: a.reduce the overall cost of ovulation induction by dramatically reducing the dose of exogenous FSH required to achieve adequate folliculogenesis and by reducing the duration of treatment and the need for monitoring procedures. b.Selectively reduce the occurrence of small preovulatory ovarian follicles and, potentially of OHSS. ( Navot D et al 1988. Am J Obstet Gynecol 159:210–215) Thus improving the safety of ART procedures.

36. Although, it cannot be excluded that low levels of FSH activity may still be needed to optimize the late folliculogenesis stages, novel and unconventional protocols could be envisioned, consisting of initial higher dose FSH administration to boost follicle recruitment, followed by FSH curtailment or discontinuation along with LH activity administration until ovulation, leading to selective promotion of larger follicle developments. (Filicori M, et al. 2001. J Clin Endocrinol Metab 86:1437– 1441)

37. Such an approach may profoundly modify the current management of anovulation and ART. Additional investigations will be needed to further assess the specific effects of r-hLH and low dose hCG administration in different clinical conditions and regimens.

38. MEFS/STGO 2008 October 15-18, 2008 The Royal hotel Hammamet, Tunisia See you in Tunis