The document discusses the evolution and advantages of recombinant gonadotropins (rFSH) compared to urinary-derived gonadotropins (uFSH) in fertility treatments, emphasizing better consistency, fewer side effects, and lower contamination risks associated with rFSH. It outlines various studies that demonstrate the superior quality and efficacy of rFSH, including higher oocyte retrieval rates and consistent hormonal responses, ultimately leading to improved clinical outcomes. The potential risks linked to urinary-derived products, including immunogenicity and contamination, are also addressed, highlighting the shift towards recombinant options in reproductive medicine.

1. The epic war of
Gonadotropins
Dr .Raju Nair
Mitera Hopital
Kottayam
Kerala ,India

2. 1995
1930’s
Horse
PMSG
Pig
FSH
Pituitary
FSH
u-hMG
u-FSH
r-hFSH
1950 1980
1960
Antibodies Local, systemic reactions
Local
rections &
potentiel
side effects
CJD
Lunenfeld. RBM Online 2002;4(suppl 1):11
Discovery Timelines: Gonadotropins
1946

3. The Move From Urinary FSH to rFSH
Better ovarian response.
Lower FSH doses.
Fewer dosage adjustments.
Shorter treatment time.
No risk of contamination / infection.

4. Gonadotropin
 Advantages of recombinant FSH
 High purity
 High specific FSH activity (about 10,000
IU/mg protein)
 Identical amino-acid sequence to natural
FSH
 No contamination with urinary proteins
of undetermined origin
 No LH activity
 Good source control, providing good
batch-to-batch consistency
 Disadvantages of urinary FSH
 Variable purity
 Variable specific FSH activity
 Amino-acid sequence of more acid
profile as in postmenopausal women
 May contain >95% proteins
of urinary
origin
 There is always some LH contamination
 No absolute source control, resulting
in batch-to-batch inconsistency.
 Collection of urine is cumbersome

5. Is it true or….
 Favouring medical modernity over older-line products
 Marketing strategy of recombinant gonadotrophins.

6. Concerns About Batch to Batch
consistency

7. Comparison – Batch to Batch Consistency
Urinary gonadotropins (-20%,+25%) Follitropin alfa (2%)
1. Bassett et al. Reprod Biomed Online 2005;10:169–177; 2. Driebergen et al. Curr Med Res Opin 2003;19:41–46

8. Steelman-Pohley (1953)
Rat Ovarian Weight Gain Bioassay for hFSH
Kill on day 4
to collect ovaries
Ovaries weighed
and data processed
FSH injected sc
1 x 3 days
21-22 day old female rats
Randomized
+ hCG primed

9. 1. Bassett et al. Reprod Biomed Online 2005;10:169–177
2. Driebergen et al. Curr Med Res Opin 2003;19:41–46
Conventional
Bioassay
High
variability
(~20%)
in vivo (rat)
Novel analytical
method
Physiochemical
technique
Minimal batch-to-
batch variability
(1.6%)1,2
Gonadotropins: an overview Product Quality: Filled
by Mass (FbM)

10. Rat Bioassay vs.
SE-HPLC (Filled-by-Mass)
( Size exclusion high performance liquid chromatography )
Filled-by-Rat =
ovary weight gain
Variability: + 25/- 20%
Filled-by-Mass =
SE HPLC
Variability: +/- 2%

11. Clinical Relevance of r- hFSH
Filled-by-Mass
 Consistent r-hFSH isoforms.
 Consistent and accurate r-hFSH dosing.
 More consistent ovarian response.
Lower risk of cancellation?
Less fluctuation in pregnancy rate

12. Concept of Dose Precision: Clinical implications
Batch variability
+20%, -25%
225
270
170
IU
Bioassay
Urinary
16.5 mcg
(225 IU)
Filled by Mass
R-FSH
Batch variability
 2%
Risk of OHSS
Poor response

13. Concerns About Purity

14. Comparison - Purity
Product
Purity
(FSH content)
Mean specific
FSH activity
(IU/mg
Injected
/75IU
(mcg)
U HMG <5 % ˜ 100 ˜ 750
HMG - HP <70% 2000 - 2500 ˜ 33
Rec FSH
Follitropin β 7000 - 8000 8.1
Rec FSH
Follitropin α
>99% 13,645 6.1
Bassett et al. Reprod Biomed Online 2005;10:169–177

15. Gonadotrophins
LH Protein Activity
 hMG 75 98% 40IU
 hMG P 1 95% 150IU
 hMG HP 0.1 5% 9000IU
 rFSH Nil None 10,000IU

16. Urinary Vs Recombinant GN - sources
HMG – Menopausal urine
 One 75 IU ampoule requires 2.5 liters
of urine
 250,000,000 liters of urine/year
would be required to meet current
demands
 Impossible to trace donor source
Recombinant human FSH
(r-hFSH)
 Chinese hamster –
recombinant technology
 Provides the ability to
make a virtually unlimited
quantity
Concerns about source, purity and
batch-to-batch consistency of
urinary products
No Such Concerns
Adapted from Ludwig, et al. RBM Online 2002; 5 (Suppl 1): 73

20. Prion proteins can be detected in hMG preparations by MS, suggesting that there is potential
risk for developing prion disease in using hMG preparations.
Kuwabara Y et al, J Reprod Med 2009;54 (8):459–4
Prion Protein identified
Presence of Prion’s in urinary

22. Other Contaminants
Contaminating Active Proteins
 Epidermal growth factor (EGF)
 TNF-binding protein 1
 Tamm-Horsfall glycoprotein
 Urokinase
 Leukocyte elastase inhibitor
 Protein C inhibitor
 Human zinc-2-glycoprotein
 Inhibitor Alpha-2-antiplasmin precursor
 IGFBP7 precursor
 FLJ13710 - Tromospondin type 1
Non-gonadotropin protein
These are not exactly
needed by FSH or
LH to induce
follicle development
33 contaminants

23. Human Reproduction, Vol. 19, No. 5, 1236-1237, May 2004
© 2004 European Society of Human Reproduction and Embryology
Creutzfeldt–Jakob disease and urinary gonadotrophins
 To date, there is no strong evidence to support the suggestion that vCJD (or
in fact sporadic CJD) has been acquired through receiving urinary
gonadotrophins.
 Of 143 cases of vCJD to date in the UK, 63 were females and one of these cases had a
history of treatment for infertility from 1998 to 1999, with a latency of 20 months from the
start of treatment to the onset of clinical symptoms

24. Balen, A. (2002) Bye-bye urinary gonadotrophins?: Is there a risk of prion disease after
administration of urinary-derived gonadotrophins? Hum. Reprod., 17, 1676–1680
 In over 30 years of clinical use of urinary gonadotrophins, not a single case of
infectious contamination has been reported. Even cases of slow viruses should, in
such a time span, have clinically become apparent

25. Gleicher, N. (2002) Some thoughts on the autoimmune Reproductive Failure Syndrome
(RAFS) and Th-1 versus Th-2 immune responses. Am. J. Reprod. Immunol., 48, 252–255
 Urinary Gonadotropins
 Allergic reactions are of concern,
 activate immune processes
 hostile to implantation and increase miscarriage risks.
 allergic reactions can be associated with significant shifts in Th1/Th2 activities, which can
include APA-responses,
 closely linked to an increased risk of infertility and pregnancy wastage

26. Human Reproduction, Vol. 18, No. 3, 476-482, March 2003
Bye-bye urinary gonadotrophins?
Recombinant FSH: A real progress in ovulation induction and IVF?*
 The conclusion has, therefore, to be reached that recombinant medications, indeed,
are less immunogenetic than the older urinary-derived medication and, at least
from this point of view, are preferable.

27. LH activity :
hcg driven ?
FSH and LH are major components in the process of follicle development
hCG is essential for the maintenance of the corpus luteum and implantation
process, but potentially too strong signal for the production of healthy oocytes

28. Results: LH activity in HMG
 Approximately 95% of LH bioactivity in filtered
hMG is due to presence of hCG
 1 unit of hCG bioactivity is ~ 6-8 IU LH
bioactivity
Van de Weijer, et al. RBM Online 2003;7:547
0
2
4
6
8
10
LH hCG
3
10
IU/vial
If human Menopausal Gonadotropin normally has 75 IU of FSH + 75 IU of LH,
why contains hCG?
The more you filter , the more LH you loose, so to reestablish the 75:75 ratio you
need to spike with hCG
Why not spike with natural LH or rLH?
Much more expensive and not as easy to get as hCG

29. hCG in urinaries is different from endogenous LH
LH and hCG are different molecules, but they act through the same
receptor
▪ Different size and glycosylation patterns
▪ Different source: anterior pituitary gland (LH) vs. throphoblastic embryonic cells
(hCG)
▪ Different half-life: hCG ~3-4x longer than LH (1,2)
 92aa, b 121aa,
3 glycos. sites
28kDa
 92aa, b 145aa,
8 glycos. sites
37kDa
LHCGR
1. Le Cotonnec JY, et al. Fertil Steril. 1998;69(2):195-200.
2. Trinchard-Lugan I, et al. Reprod BioMed Online. 2002;4(2):106-115.

30. When is LH activity needed?
There is an upper and a lower limit for LH levels to ensure
optimal follicular development
▪ LH receptors down-regulation
▪ Suppression of granulosa cell proliferation
▪ Follicular atresia (nondominant follicles)
▪ Premature luteinization (preovulatory follicle)
LH
▪ Follicular growth impaired
▪ Inadequate androgen (and estrogen) synthesis
▪ No full oocyte maturation
Optimal range: 1.2 IU/L – 5 IU/L
O’Dea et al. Curr Med Res Opin. 2008 Oct;24(10):2785-93.
Balasch J, Fábregues F. Curr Opin Obstet Gynecol. 2002;14:265-274.
Implantation rate
Marrs R, et al. Reprod
BioMed Online.
2003;8(2):175-182.
35-39 yr
27.8
28.6
18.9
26.7
<35 yr
FSH + LH
FSH
Pregnancy rate
49.3%
55.3%
57.0%
41.5%
35-39 yr
<35 yr
35-39 yr
<35 yr
37.3%
33.5%
37.3%
25.3%
Ongoing pregnancy
rate (ITT analysis)
Clinical pregnancy rate per cycle
≥35 yr
<35 yr
45.8%
39.8%
35.4%
28.3%
33.3%
22.2%
Bosch E, et al. Fertil Steril. 2008;90(suppl):S41.
Humaidan P, et al. Reprod
BioMed Online.
2004;8(6):635-643.
LH appears to helpful in patients with advanced maternal age

31. Comparing Efficacy of
Recombinants
VS
Urinary

32. rFSH is more potent than urinary hMG/FSH preparations
14
2 12
0 8 10
4 6
34%
HP-hMG 11.2
9%
12.1
8.4
10.4
u-hFSH-HP
rFSH
rFSH
u-hFSH-HP
rFSH 12.3
12.7
14%
Number of follicles
≥12mm on hCG day1
14
12
8
4
0 2 6 10
10.0
8.8
11.0
11.8
38%
7.6
12.2
15%
20%
Number of oocytes
retrieved
6 8
0 2 4 12 14
10
6.3
8.1
7.4
3.5
4.7
5.0
30%
15%
42%
Number of embryos
generated
Bergh C,
et al. (1997)
Frydman R,
et al. (2000)
Andersen AN,
et al. (2006)
Bergh C, et al. Hum. Reprod. 1997; 12(10): 2133-9
Frydman R, et al. Hum. Reprod. 2000; 15(3):520-5
Andersen AN, et al. Hum. Reprod. 2006; 21(12): 3217-27
p<0.002
p=0.005
p=0.003
p<0.0001
p=0.002
p<0.001
p<0.0001
p=0.0001
p=0.002

33. rFSH produces significantly more oocytes than urinaries with lower doses and
shorter treatment time
Mean total
dose per
cycle (IU)
rFSH hMG-HP
2072 2540
p-value
<0.01 (1)
2385 2508 0.006 (2)
(3)
1353 1433 0.009
rFSH uFSH p-value
11.7 14.5 <0.05
27.6 40.7 <0.05 (4)
# of 75 IU
ampoules
Days of FSH
stimulation
1. Trew GH, et al. Reprod Biol Endocrinol. 2010;8:137.
2. Nyboe Andersen A, et al. Hum Reprod.
2006;21(12):3217-3227.
3. Devroey P, et al. Fertil Steril. 2012;97(3):561-571.
4. Frydman R, et al. Hum Reprod. 2000;15(3):520-525.
Ref.
(4)
Ref.

34. More oocytes allow for more frozen embryo transfers, thereby
increasing cumulative pregnancy and live birth rates
Bosch E and Ezcurra D Reprod Biol
Endocrinol. 2011; 9:82
More oocytes yield in higher
pregnancy rate
More oocytes yield in higher live birth rate
Predicted live birth probability given egg number and age
Sunkara SK, et al. Hum Reprod. 2011;26(7):1768-74

35. Major studies …

36. R- Lh will help or not ?

37.  Although live birth is the most clinically meaningful goal of ART, the number of
oocytes retrieved following OS is frequently used as a surrogate measure of clinical
success
R-FSH HMG -HP P VALUE
bOSCH 14.4 11.3 0.0001
MERIT 11.8 5.4 0.001
MEGASET 9.7 7.8 0.004
MEGSET HR 22 15
PERSIST 10.9 9.7 (r-FSH +r-LH) NS
ESPART 3.3 3.6 (r-FSH +r-LH) NS

38. MERiT trial
 The MERiT trial (a prospective, randomised, controlled, multicentre study), primarily
investigated the clinical outcome of 731 IVF patients treated with HP-hMG (Menopur®)
versus rFSH alfa
 Progesterone levels were significantly higher at the end of stimulation in the rFSH alfa
group versus HP-hMG treatment (23% higher on the last day of stimulation,
3.4 ± 1.7 nmol/L vs 2.6 ± 1.3 nmol/L, P < 0.001)
 31% higher at oocyte retrieval, 36.3 ± 25 vs 24.5 ± 15.6, P < 0.001)
 higher number of patients developed progesterone levels of > 4 nmol/L at the end of
stimulation in the rFSH alfa group compared with the HP-hMG group (23% vs 11%,
respectively), which was linked with reduced pregnancy rates

39. MERiT trial
 ongoing pregnancy rate (OPR) and live birth rate (LBR) from the primary study
 OPR: 27% with HP-hMG group vs 22% with rFSH alfa, P = 0.204;
 LBR: 26% HPhMG vs 22% rFSH alfa, P = 0.236
 the data suggest that the differences in outcome observed between the HPhMG
and rFSH alfa groups may have been influenced by the different endocrine profiles
 The pregnancy rate is not affected in FET cycles with those with high p4 level on
trigger day .

40. Why s.p4 is low with HMG
 Treatment supplementation with LH or hCG during the stimulation is thought to
instead drive the conversion of pregnenolone to androgens (via the Delta 5
pathway), which are further converted by FSH into oestrogens, thereby limiting the
conversion of pregnenolone to progesterone.
 As a result, less progesterone is available to enter the bloodstream
 So Hcg driven LH activity is not a bad choice

41. Number of oocytes retrieved and proportion of top-quality embryos
achieved with different gonadotrophin preparations
comparing a 2:1 formulation of rFSH alfa plus rLH (Pergoveris®) administered from Day 1
versus rFSH alfa (Gonal-F®) administered during Days 1–5 and supplemented with rLH from
Day 6 of the stimulation cycle did not report a significant difference in terms of oocytes
retrieved (9.7 vs 10.9, 95% confidence interval [CI]: − 3.15 to 0.59), therefore failing to meet its
primary endpoint and yielding inconclusive evidence on the influence of rLH on oocyte yield
Persist Trial :

42. The impact of improved embryo quality on OPR
and LBR – NORMO RESPONDER
 MERiT study in normal responders indicated that top-quality embryos obtained
from HP-hMG-treated patients are associated with a numerically higher OPR and
LBR compared with top quality embryos from rFSH alfa-treated patients
 OPR: 27% HP-hMG vs 22% rFSH alfa, [OR]: 1.25, 95% CI: 0.89 to 1.75, P = 0.204
 LBR: 26% HP-hMG vs 22% rFSH alfa, P = 0.236

43. MEGASET Study
 MEGASET study ;despite a significantly reduced number of oocytes retrieved in the
HP-hMG group
 a similar number of top-quality embryos between groups
 (31 ± 30% in the HP-hMG group vs 31 ± 28% in the rFSH beta group, P = 0.546),
 non-significant trend towards an increased OPR with HP-hMG relative to rFSH beta
(30% vs 27)

44. The impact of improved embryo quality on
OPR and LBR Poor responders
 ESPART trial a large RCT undertaken to determine if there is a difference in the
efficacy and safety of rFSH alfa/rLH (Pergoveris®) versus rFSH alfa monotherapy
(Gonal-F®) when administered for OS in poor responders
 the study failed to show superiority of rFSH alfa/rLH versus rFSH alfa monotherapy
in regard to the primary endpoint of number of oocytes retrieved
 (3.3 rFSH alfa/rLH vs 3.6 Rfsh alfa; adjusted P-value of 0.182).
 Considering these data alongside the trials comparing HP-hMG with CFA in poor
responders, the results suggest that the positive outcomes in terms of oocyte yield
associated with HP-hMG-only protocols may be attributable to the hCG content
(not LH)

45. MEGASET-HR
 The MEGASET-HR trial comparing HP-hMG and rFSH alfa in predicted high responders.
 HP-hMG was associated with a trend towards increased OPR/cycle start relative to rFSH
alfa
 Oocyte yield : HMG -15.1 VS r-FSH -22
 OPR : HMG 35.5% vs r-FSH 30.7, 95% CI: − 2.7 to 12.1), NS
 Less OHSS with HMG
 Cumulative live birth rate per cycle start was comparable between
HP-hMG and rFSH alfa (HP-hMG 50.6% vs rFSH alfa 51.5%),

46. META –ANALYSIS ( comparing rfsh vs hmg)
 Coomarasamy -2008
 Al inany – 2008
 Van wely -2011
 A separate, large meta-analysis investigated pregnancy outcomes with rFSH alfa/beta
treatment versus urinary derived gonadotrophins (hMG, purified FSH or HPFSH),
 42 trials and more than 9000 couples
 The investigators concluded that there were no significant differences in the LBR or OHSS
rates with rFSH alfa/beta versus all other gonadotrophin treatments combined.

47. Conclusions
 Better ovarian response.
 Lower FSH doses.
 Fewer dosage adjustments.
 Shorter treatment time.
 No risk of contamination / infection
 More oocytes
 Better quality embryo with HMG ?
 More or less equal pregnancy rates