Use of LH in IVF and IUI

Use of LH in IVF and IUI Differences between rec-hLH and LH

Activity in HMG Preparations

Middle East Lecture Tour, 2012

Sandro Esteves, MD, PhD Director, ANDROFERT

Center for Male Reproduction Campinas, BRAZIL

Esteves, 2

Role of LH in Reproductive Cycles LH window concept

What is in it for me?

Recent Advances in Injectable Gonadotropin Preparations Rec-LH Products Differences between rec-hLH and LH Activity in HMG Preparations

To Whom to Give LH Supplementation

Individualization of Patient Treatment Lecture Structure

Esteves, 3

Level Type of evidence 1a Obtained from meta-analysis of randomised trials 1b Obtained from at least one randomised trial 2a Obtained from one well-designed controlled study without

randomisation 2b Obtained from at least one other type of well-designed quasi-

experimental study 3 Obtained from well-designed non-experimental studies

(comparative and correlation studies, case series) 4 Obtained from expert committee reports or opinions or clinical

experience of respected authorities

Modified from Sackett et al. Oxford Centre for EBM Levels of Evidence (2009)

Level of evidence

Points I Consider Highly Relevant in Clinical Practice; Arguments Supported by Studies with High Level of Evidence.

Review this Lecture at: http://www.androfert.com.br/review

Esteves, 4

Use of LH in IVF and IUI Differences between rec-hLH and LH

Activity in HMG Preparations

Esteves, 5

Role of LH in Reproductive Cycles

2


3 Recent Advances in Injectable Gonadotropin Preparations Rec-LH Products Differences between rec-hLH and LH Activity in HMG Preparations


Presenter

Presentation Notes

These meta-analytic studies give us an overall idea but do not respond what we really want to know, that is,

Esteves, 6

• Mild Stimulation (low dose rec-hFSH + GnRH ant.):

• 5 oocytes retrieved;

• IR = 31%

• Conventional Stimulation :


• IR = 29%

Verberg et al. Hum Reprod Update 2009; 15: 5–12.

Promotion of Steroidogenesis (TCs) early FP

• Adequate estrogen production • Uterine/endometrial

changes

Stimulation of final Follicular Maturation (GCs) late FP

Esteves, 6 Alviggi et al. Reprod Biomed Online 2006;12:221.

Presenter

Presentation Notes

In the late stages of follicular development (when the follicle reaches the antral stage), FSH stimulates GCs to express LH receptors. GCs become receptive to LH stimulation. To summarize the role of LH in reproductive cycles, it can be said that: 1 - During the early follicular phase, LH plays a key role in promoting steroidogenesis. Androgens, which are produced in theca cells under LH stimulation, are transferred to GCs and transformed into estrogens via aromatization. Estrogens, in turn, induce uterine changes needed for embryo implantation. 2 - During the late follicular phase, LH promotes final follicular maturation through direct effects on the follicle.

Esteves, 7

Hypogonadotropic Hypogonadism Treated with FSH Alone

0

5

10

15

0 5 10 15 20 Days of Stimulation

Serum FSH

50 100

Follicles

Estradiol levels

r-hFSH

Estradiol (pg/ml)

Follicle Size (mm)

and FSH (IU/L)

0

9 Endometrial Thickness

(mm)

Presenter

Presentation Notes

In patients with hypogonadotropic hypogonadism, r-hFSH alone, even when administered continuously and in dose increments of 75UI every 7 days during follicular phase, estradiol production is inadequate despite being adequate to induce follicular growth (as shown by the white dots in the graph). Consequently, endometrium proliferation was also inadequate.

Esteves, 8

Evidence for LH threshold (1)

0 25

75

225

0

500

1000

1500

2000

2500

3000

Day 1 Day 5 Day 10 hCG

0 25 75 225

Day of Stimulation

Seru

m E

stra

diol

Lev

els

(p

mol

/L)

The European Recombinant Human LH Study Group, JCEM 1998; 83:1507

Rec-hLH suppementation (UI):

Presenter

Presentation Notes

It was shown that during the follicular phase, a clear relationship was observed between the dose of rLH and serum estradiol levels.

Esteves, 9

0 25

75 225

0

2

4

6

8

Day 1 Day 5 Day 10 hCG

0 25 75 225 rLH

Day of Stimulation

The European Recombinant Human LH Study Group, JCEM 1998; 83:1507

Endo

met

rial T

hick

enes

s (m

m)

Evidence for LH threshold (2)

Injected rLH dose (UI)

LH Cmax

75 UI 0.5 – 1.35 UI/L

Presenter

Presentation Notes

As a consequence, endometrial growth was obtained only if the two higher doses of LH were used. Interestingly, endometrial growth correlated to the estradiol levels up to 75 UI of rLH. No increment was seen by using 225UI of rLH, although serum estradiol levels were twice as high in the 225UI group compared to the 75UI one.

Esteves, 10

• Mild Stimulation (low dose rec-hFSH + GnRH ant.):


• IR = 31%

• Conventional Stimulation :


• IR = 29%

Verberg et al. Hum Reprod Update 2009; 15: 5–12. Esteves, 10 Balasch J, Fábreques F. Curr Opin Obstet Gynecol 2002, 14:265.

THRESHOLD

CEILING

• Suppression of GC proliferation • Follicular atresia (non-dominant follicles) • Premature luteinization • Oocyte development compromised H

igh

• Normal androgen and estrogen biosynthesis • Normal follicular growth and development • Normal oocyte maturation

Nor

mal

• Insufficient androgen (and estrogen) synthesis • Follicular growth and maturation impaired • Inadequate endometrial proliferation Lo

w

Presenter

Presentation Notes

According to this concept, in the absence of a minimum level of serum LH, E2 production will be insufficient for optimal follicular development and endometrial proliferation. On the other hand, exposure of the developing follicle to excessive LH (beyond a ceiling level) results in atresia and cessation of normal development. Evidence suggests that optimal follicular development occurs within an ‘LH window’, above a certain ‘LH threshold’ and below an ‘LH ceiling’.

Esteves, 11

Both FSH and LH are essential for normal estradiol biosynthesis.

75 UI recLH is sufficient to promote optimal follicular and endometrial growth as well as androgen production in most HH patients.

Evidence suggests that in reproductive cycles optimal follicular development occurs within an ‘LH window’, above a certain ‘LH threshold’ and below an ‘LH ceiling’ (1.2 to ? UI/L).

Esteves, 12





Presenter

Presentation Notes

These meta-analytic studies give us an overall idea but do not respond what we really want to know, that is,

High-quality oocyte yield

Cycle cancellation, OHSS, multiple

pregnancy

Central Paradigm

Minimize complications

and risks

Maximize beneficial effects

of treatment

Fauser BC et al: Predictors of ovarian response: progress towards individualized treatment in ovulation induction and ovarian stimulation. Hum Reprod Update 2008;14:1-14. Esteves, 13

Presenter

Presentation Notes

Before answering this question I believe you agree with me that: First: Ovarian stimulation with gonadotropins have a key role in ART treatments. In fact, OS has been applied to compensate for inefficiencies in the IVF procedure by increasing the number of oocytes retrieved thus enabling the selection of the best quality embryos for transfer. And… Second: that today, the central paradigm of all ovarian stimulation protocols is to maximize the beneficial effects of treatment (relating to high-quality oocyte yield) while minimizing the potential risks associated with OHSS and multiple pregnancy.

Factors Determining Response to Ovarian Stimulation

Demographics and

anthropometrics (Age, BMI, Race) Genetic profile Cause of Infertility Years of Infertility Health status Nutritional status

Esteves, 14

Presenter

Presentation Notes

Variability in the subfertile patient population excludes the possibility of a single approach to controlled ovarian stimulation (COS). In fact, the patient is the main variable in ovarian stimulation response. There are multiple factors interacting mutually, such as demographics and anthropometrics, genetic profile, cause and duration of infertility, health and nutritional status.

Esteves, 15

Negative Predictors

Positive Predictor

van Loendersloot et al. Hum Reprod Update 2010; 16: 577–589.

Female Age Duration of infertility Basal FSH Type of infertility Indication Fertilization method Number of oocytes retrieved Number of embryos transferred Embryo quality

All reflecting ovarian reserve

Level 1a

Presenter

Presentation Notes

When we consider the most important predictive factors for success in IVF, as done in this meta-analysis of 14 studies, we learn that advanced female age, long duration of infertility and elevated basal levels of FSH are negative predictors while No. oocytes retrieved (up to a limit) is a positive predictor. Interestingly, all these significant factors are age related and reflect ovarian reserve, thus indicating that ovarian reserve is a major determinant of success. Therefore, determination of a given patient ovarian reserve prior to OS has a key role in the understanding of her chances of pregnancy. The summary OR for pregnancy and female age was 0.95 (95% CI:0.94–0.96) indicating that increasing female age was associated with lower pregnancy chances in IVF. Negative predictors: pregnancy and female age [OR: 0.95, 95% confidence interval (CI): 0.94–0.96], duration of subfertility (OR: 0.99, 95% CI: 0.98–1.00) and basal FSH (OR: 0.94, 95% CI: 0.88–1.00). A positive association was found for the number of oocytes (OR 1.04, 95% CI: 1.02–1.07). Better embryo quality was associated with higher pregnancy chances. .

Esteves, 16

• ~80% normogonadotropic women undergoing Ovarian Stimulation1-3

Nor

mal

• 15-20% of NG women have less sensitive ovaries • Older patients (≥35 years)4

• Poor responders5

• Slow/Hypo-responders6

• Deeply suppressed endogenous LH levels (endometriosis)7

Low

1. Alviggi et al. Reprod Biomed Online 2006;12:221; 2. Tarlatzis et al. Hum Reprod 2006;21:90; 3. Esteves et al. Reprod Biol Endocrinol 2009;7:111; 4. Marrs et al. Reprod Biomed Online 2004;8:175;5. Mochtar MH, Cochrane Database, 2007; 6. Alviggi, et al.

RBMOnline 2009; 7. De Placido et al. Clin Endocrinol (Oxf) 2004;60:637;

Presenter

Presentation Notes

The general consensus regarding LH is that most women have sufficient levels of endogenous LH and do not require supplementation. However, certain subgroups with low levels of LH may benefit from additional LH. Early studies identified a subgroup of normogonadotrophic patients who have normal estimated ovarian reserves but suboptimal responses to FSH stimulation (De Placido et al., 2001, 2004, 2005; Mochtar et al., 2007). Such women express ovarian resistance to FSH but seem to be distinct from classical poor responders because some investigators suggest that luteinizing hormone (LH) supplementation improves their ART treatment outcomes (Alviggi et al., 2006). The subgroups which may benefit from LH supplementation include women over the age of 35 years, those with a poor response to ovarian stimulation or women with highly suppressed levels of endogenous LH. The general consensus regarding LH is that most women have sufficient levels of endogenous LH and do not require supplementation, although certain subgroups with low levels of LH may benefit from additional LH (Alviggi et al. Reprod Biomed Online 2006;12:221–233; Tarlatzis et al. Hum Reprod 2006;21:90–94; Esteves et al. Reprod Biol Endocrinol 2009;7:111; Marrs et al. Reprod Biomed Online 2004;8:175–182). The subgroups which may benefit from LH supplementation include women over the age of 35 years, those with a poor response to ovarian stimulation or women with highly suppressed levels of endogenous. It has been suggested that a subgroup of women with adequate AFC and FSH levels with hypo-response to ovarian stimulation may also benefit from LH supplementation (Alviggi, et al. RBMOnline 2009). These patients harbor single nucleotide polymorphisms of FSH and LH receptors. After pituitary suppression, residual circulating levels of endogenous LH are usually adequate to support multiple follicular growth and oocyte development, also even when drugs containing FSH with low or absent LH activity are administered (Loumaye et al., 1997; Sills et al., 1999). It has been demonstrated that only 1% of LH receptors need to be occupied to drive adequate ovarian steroidogenesis for reproduction. Nevertheless, in a subset of normogonadotrophic patients, the ovarian response to this association is suboptimal. It has been suggested that this may be due to a profound suppression of endogenous LH in some women whose activity may fall below an hypothetical threshold value. Thus, it could be hypothesized that those subjects may benefit from the use of LH-containing gonadotrophin preparations (Laml et al., 1999; Fleming et al., 2000; Levy et al., 2000; Westergaard et al., 2000; De Placido et al., 2001). Nagawa, 2008: pts with very low LH levels after down-regulation had lower pregnancy and implantaion rates. A possible mechanism behind the beneficial effect of exogenous LH supplementation in older women may relate to decreasing numbers of functional LH receptors with increasing age (Vihko et al. 1996). It suggests that younger women, due to a higher number of LH receptors, do not require exogenous LH, while supplementation with exogenous LH in the older woman secures a sufficient LH-induced response. In addition, Piltonen et al. (2003) found that ovarian androgen secretion, i.e. oestrogen precursor secretion capacity, starts to decline as early as before the age of 30 years, again suggesting a diminished capacity of the ovary to respond to LH stimulus with age. Another subgroup of women that seems to benefit from exogenous LH supplementation is the group of women with high endogenous LH concentrations on day 8 of stimulation (i.e. >1.99 IU/l). r-hLH supplementation in this subgroup resulted in a significantly higher implantation rate and a marginally higher pregnancy rate (P = 0.07) as compared with the non-supplemented subgroup. This is an interesting and surprising finding. A possible explanation for this phenomenon could be a desensitization of the ovarian LH receptor due to high concentrations of circulating endogenous LH, leading to receptor down-regulation (Zor et al., 1976; Amsterdam et al.. 2002). LH receptor polymorphism also seems to play a role. In summary: less LH receptors, less sensitive LH receptors, Low LH levels.

Esteves, 17

• Older patients (≥35 years)

• Poor responders

• Slow/Hypo-responders

• Deeply suppressed endogenous LH (endometriosis)

Less

Sen

sitiv

e O

varie

s

Marrs et al. Reprod Biomed Online 2004;8:175 De Placido et al. Clin Endocrinol (Oxf) 2004;60:637; Ferraretti et al. Fertil Steril. 2004; 82:1521-6;

Mochtar MH, Cochrane Database, 2007; Alviggi, et al. RBMOnline 2012

Poor Responders* At least 2 of the following: Advanced maternal age (≥40 years) Previous POR (≤3 oocytes with a conventional stimulation protocol) Abnormal ovarian reserve test (AFC<5; AMH <1.1) Or: 2 episodes of POR after maximal stimulation

Hypo/Slow Responders Normal markers of ovarian reserve Hypo-responders: d1-d7: normal initial follicullar recruitment using fixed starting dose of FSH; d7- d10: plateau on follicullar growth despite continuing same FSH dosage Slow responders: High doses of FSH (>3,000UI) to promote follicular growth; May indicate genetic polymorphisms of LH and/or FSH receptor

Up to 45% Infertility Patients

aged 35 or above

Presenter

Presentation Notes

In the ovary, granulosa cells are the only target cells of FSH action, whereas both theca and late-stage (In the ovary, granulosa cells are the only target cells of FSH action, whereas both theca and late-stage (luteinizing) granulosa cells contain LH receptors. Increased FSH drive has been shown to be of only limited value in the less gonadotrophin-sensitive ovary, and there is a potential need for LH activity to be part of an individualized treatment regimen tailored for the biologically older ovary. LH half-life: 20 minutes; hCG: 24 hours

Increase in FSH drive FSH

Theca cells

Granulosa cells

LH

LH

Increase in LH drive

Esteves, 18

Pregnancy rates

% Cycle cancellation

Number oocytes retrieved

Increasing the Stimulation Dose of

FSH… …is not associated with better

IVF outcome Manzi et al, 1994

Klinkert et al, 2004 Berkkanoglu & Ozgur,

2010

Level 1b

Presenter

Presentation Notes

In the ovary, granulosa cells are the only target cells of FSH action, whereas both theca and late-stage (In the ovary, granulosa cells are the only target cells of FSH action, whereas both theca and late-stage (luteinizing) granulosa cells contain LH receptors. Increased FSH drive has been shown to be of only limited value in the less gonadotrophin-sensitive ovary, and there is a potential need for LH activity to be part of an individualized treatment regimen tailored for the biologically older ovary. LH half-life: 20 minutes; hCG: 24 hours

Reduced oocyte quality

Reduced Fertilization Rate

Reduced Embryo Quality

Increase Miscarriage Rates

Reduced ovarian

paracrine activity

Hurwitz & Santoro 2004

LH receptor poly-

morphisms

Alviggi et al., 2006

Androgen secretory capacity reduced

• Piltonen et al., 2003

Decreased numbers of functional

LH receptors

• Vihko et al. 1996

Reduced LH

bioactivity while

imnuno-reactivity

unchanged

• Mitchell et al. 1995; Marama et al 1984

Esteves, 19

Less

Sen

sitiv

e O

varie

s

Westergaard et al., 2000; Esposito et al., 2001; Humaidan et al., 2002

Presenter

Presentation Notes

It has been shown, in retrospective studies, that low concentrations of circulating follicular phase LH in women undergoing GnRH-agonist long-protocol cycles might be associated with impaired oestradiol synthesis and/or a low oocyte yield as well as low fertilization rates, low pregnancy rates and high miscarriage rates (Westergaard et al., 2000; Esposito et al., 2001; Humaidan et al., 2002). The potential benefit of LH administration for older patients could be explained by two different mechanisms. On the one hand, the endocrine changes occurring with ovarian aging include an increase of the serum FSH levels in the early follicular phase, which are not accompanied by an LH increase but by a progressive decrease of the basal androgen levels. Moreover, the follicular capability for inducing androstenedione synthesis after rFSH administration is significantly impaired in older patients compared with younger reproductive-aged patients, whereas E2 secretion is preserved by increased aromatase function. Action of LH at the follicular level that increases androgen production for its later aromatization to estrogens in a dose dependent manner may restore the follicular milieu in these patients to recover oocyte quality and, therefore, embryo quality and implantation rates. Follicle-stimulating hormone acts on granulosa cells to promote the conversion of cholesterol into P, which is passed to the thecal cells to be converted into androgens under the influence of LH, therefore reducing circulating P. All in all, this could explain why stimulation with rFSH alone can produce poorer outcomes in older and low-responding patients because they usually receive higher FSH doses for COS, they show higher P levels at the end of stimulation, and, subsequently, their endometrium receptivity diminishes.

LH Supplementation in Poor Responders…

Regimen Outcome Effect on Pregnancy

Mochtar et al, 2007 3 RCT (N=310) Poor responders

r-hFSH+rLH vs. r-hFSH alone* OPR OR 1.85

(95% CI: 1.10; 3.11)

Bosdou et al, 2012 7 RCT (N= 603) Poor responders

r-hFSH+rLH vs. r-hFSH alone*

CPR

LBR (only 1 RCT)

RD: +6%, (95% CI: -0.3; +13.0)

RD: +19%

(95% CI: +1.0; +36.0%)

Hill et al, 2012 7 RCT (N=902) Women advanced age ≥35 yrs.

r-hFSH+rLH vs. r-hFSH alone

CPR

OR 1.37

(95% CI: 1.03; 1.83)

*long GnRH-a protocol; OR=odds-ratio; RD=risk difference

Mochtar MH et al. Cochrane Database Syst Rev. 2007;2:CD005070; Bosdou JK et al, Hum Reprod Update 2012; 8(2):127-45. Hill MJ et al. Fertil Steril 2012; 97:1108-4. Esteves, 20

Level 1a

Presenter

Presentation Notes

It has been shown, in retrospective studies, that low concentrations of circulating follicular phase LH in women undergoing GnRH-agonist long-protocol cycles might be associated with impaired oestradiol synthesis and/or a low oocyte yield as well as low fertilization rates, low pregnancy rates and high miscarriage rates (Westergaard et al., 2000; Esposito et al., 2001; Humaidan et al., 2002). The beneficial effect of LH supplementation in down-regulated poor responders undergoing COH with rFSH has been demonstrated in this meta-analysis. The potential benefit of LH administration for older patients could be explained by two different mechanisms. On the one hand, the endocrine changes occurring with ovarian aging include an increase of the serum FSH levels in the early follicular phase, which are not accompanied by an LH increase but by a progressive decrease of the basal androgen levels. Moreover, the follicular capability for inducing androstenedione synthesis after rFSH administration is significantly impaired in older patients compared with younger reproductive-aged patients, whereas E2 secretion is preserved by increased aromatase function. Action of LH at the follicular level that increases androgen production for its later aromatization to estrogens in a dose dependent manner may restore the follicular milieu in these patients to recover oocyte quality and, therefore, embryo quality and implantation rates. Follicle-stimulating hormone acts on granulosa cells to promote the conversion of cholesterol into P, which is passed to the thecal cells to be converted into androgens under the influence of LH, therefore reducing circulating P. All in all, this could explain why stimulation with rFSH alone can produce poorer outcomes in older and low-responding patients because they usually receive higher FSH doses for COS, they show higher P levels at the end of stimulation, and, subsequently, their endometrium receptivity diminishes.

Jamnongjit M et al. PNAS 2005;102:16257-16262

Action of LH at the follicular level that increases androgen production for its later aromatization to estrogens in a dose dependent manner may restore the follicular milieu in these patients to recover oocyte quality and, therefore, embryo quality and implantation rates.

Presenter

Presentation Notes

Model for gonadotropin-induced oocyte maturation. LH binds to its receptors on theca cells. Activation of the LH receptor results in MMP-mediated cleavage of membrane-bound EGF molecules. Upon release, these EGFs then bind to EGFRs on cumulus cells (and likely others) to enhance the production of steroids and other factor(s) that promote oocyte maturation.

Esteves, 22

6 9 11 10 14

18 22

32 40

FSH step-up (+150 UI) LH supplementation(+150 UI)

Normal Responders

Mean No. oocytes retrieved IR (%) OPR (%)

De Placido et al. Hum Reprod. 2004; 20: 390-6.

• RCT 260 pts with “steady” response on COS D8 (E2 <180pg/mL; >6 follicles <10mm) • 3 groups:

LH Supplementation in Hypo/Slow Responders (1)

Level 1b

Presenter

Presentation Notes

De Placido et al. studied the effects of LH supplementation in women showing a poor response to ovarian stimulation. ‘Poor responders’ were defined as those who had serum oestradiol levels below 180 pg/ml and no follicles over 10 mm on day 8. These women were randomized to a daily dose of either 75 IU or 150 IU r-hLH. Women classified as ‘good responders’ received no additional r-hLH (control group). They found that the high dose of r-hLH was significantly more effective in terms of oocyte development and maturation than the increasing FSH drive. Furthermore, there was no significant difference between the high-dose group and controls in terms of these outcomes.

Esteves, 23

8 11 11 10 22

41

18

37

increase in r-hFSH dose

(max. 450UI)

increase in r-hFSH dose + r-hLH (75-150UI)supplementation

increase in r-hFSH dose + LHsupplementation

with HMG

controls

Mean No. oocytes retrieved LBR (%)

Ferraretti et al. Fertil Steril. 2004; 82: 1521-6.

• RCT • 126 pts. follicular stagnation during d7-d10 COS • 4 groups:

LH Supplementation in Hypo/Slow Responders (2)

Level 1b

Presenter

Presentation Notes

It is believed that androgens, which are endogenously produced in response to LH stimulation, are involved in sensitizing small antral follicles to FSH (Durnerin et al., 2008). Profound suppression of endogenous LH by GnRH agonists combined with the use of rhFSH may cause LH activity to fall below a hypothetical threshold value in some women. An observational trial by Alviggi et al. (2006) showed that a common polymorphism of LH (v-LH) was present in a higher proportion of patients who required .3500 IU compared with ,3500 IU FSH for follicular maturation (35% versus 2.6%). v-LH has a short half-life and may be ineffective in supporting FSH-stimulated multifollicular growth.

LH Supplementation in OI and IUI

LH levels 1.2 UI/L (WHO group I) Higher follicular development pts. receiving LH (67% vs 20%; p=0.02): Shoham et al., 2008.

Similar follicular development HMG vs FSH+rLH; higher cumulative PR after 3 cycles in FSH+LH (56% vs 23%; p=0.01): Carone et al., 2012.

Level 1b

Esteves, 24

WHO group II Clomiphene-resistant: fewer intermediate-sized follicles and OHSS in

LH-supl. vs FSH group; similar ovulation rate (Plateau, 2006); Previous over-response: higher monofollicular development in LH group

(32% vs 13%; p=0.04): Hughes et al., 2005; IUI: higher monofollicular development in LH group without

intermediate-size (42% vs 11%; p=0.03); lower cycle cancellation due to risk OHSS (-7% difference): Segnella et al., 2011.

Presenter

Presentation Notes

Hypogonadotrophic hypogonadism Management options include exogenous replacement of gonadotropins and pulsatile GnRH agonist administration. In women with intact pituitary function, pulsatile gonadotropin releasing hormone (GnRH) therapy can be used. Exogenous gonadotropins administration is the alternative therapeutic option in hypothalamic dysfunction and the first line treatment if the defect is primary pituitary failure. Currently available evidence indicates that rFSH alone may not be sufficient to promote optimum follicular growth in severely gonadotropin deficient women. It has been suggested that a minimum threshold of serum LH is required to re-establish meiosis and final stages of growth of antral follicles. The European Recombinant Human LH Study Group, 1998: A dose finding trial included 38 WHO type I anovulatory patients, who were randomly assigned to receive either 0, 25, 75, or 225 IU rLH once daily in addition to 150 IU follitropin alpha once daily. None of the 8 patients who received follitropin alpha alone ovulated in the absence of rLH. Fourteen percent of patients who received follitropin alpha and 25 IU/L rLH ovulated compared to 66% and 80% of those who received 75 IU/L and 225 IU/L, respectively. Significant dose dependent increases in the rate of optimal follicular growth were observed in women receiving follitropin alpha with different doses of rLH varying from 0 to 225 IU/day. Shoham et al, 2008: Double-blind, randomized, placebo-controlled trial conducted in 25 medical centres in four countries. Thirty-nine patients with LH < 1.2 IU/l and FSH < 5.0 IU/l were treated with concomitant 75 IU lutropin alfa and 150 IU follitropin alfa or concomitant placebo and 150 IU follitropin alfa. With LHr: 66.7% achieved follicular development compared with 20.0% of patients receiving placebo (P = 0.023). Burgues et al, 2001: A case series from Spain included 38 hypogonadotrophic anovulatory (WHO group I) women undergoing 84 ovulation induction cycles where patients received 150 IU/day rFSH and 75 IU/day rLH. Sufficient follicular growth was observed in 79 (94%) out of 84 initiated cycles. The cumulative pregnancy rate following three cycles of stimulation with follitropin alpha and lutropin alpha was 39.5%. Kaufman et al, 2007: An open-label, noncomparative study with 31 hypogonadotrophic hypogonadal women. They receive Lutropin alfa 75 IU and follitropin alfa (75-225 IU). In a total of 54 cycles, 27 of 31 (87.1%) achieved follicular development within three cycles. Twenty of 27 patients (74.1%) who achieved follicular development and received hCG became pregnant; 16 (59.3%) continued to clinical pregnancy. Carone et al, 2012: Two-arm randomized open-label study, 35 women with hypogonadotropic hypogonadism, comparing: r-hFSH + r-hLH (2:1) X hMG-HP in women with. Following a total of 70 cycles, 70% of r-hFSH/r-hLH treated patients ovulated vs. 88% in hMG-HP group (p=0.11). However, PR in r-hFSH/rhLH group was 55.6% compared to 23.3% in hMG-HP group (p=0.01). OI in WHO group II anovulation The large majority of women with PCOS would have excess elevated LH concentrations when measured at the appropriate time. This may justify the potential advantages in preparations devoid of LH activity as follitropin alpha. Currently, there is no role of lutropin alpha in the management of women with PCOS. (conclusao de uma revisao de gibreel et al, 2009). Mas ha 2 estudos com inducao de ovulacao neste tipo de pacientes: Plateau et al, 2006: A randomized, open-label, assessor-blind, multinational study. Women with anovulatory infertility WHO Group II and resistant to clomiphene citrate were randomized to stimulation with HP-HMG (n=91) or rFSH (n=93) using a low-dose step-up protocol. The ovulation rate was 85.7% with HP-HMG and 85.5% with rFSH (per-protocol population), and non-inferiority was demonstrated. Significantly fewer intermediate-sized follicles were observed in the HP-HMG group (P<0.05). The singleton live birth rate was comparable between the two groups. The frequency of ovarian hyperstimulation syndrome and/or cancellation due to excessive response was 2.2% with HP-HMG and 9.8% with rFSH (P=0.058). Hugues et al, 2005: 153 wHO group ii anovulation women that during ovulation induction had over-response (at least 3 follicles 11-15mm and no one >15) were randomized to receive, in addition to 37.5IU recombinant human FSH (rFSH), either placebo or different doses of rLH (6.8, 13.6, 30 or 60mg) daily for a maximum of 7 days. The proportion of patients with exactly one follicle ≥ 16mm ranged from 13.3% in the placebo group to 32.1% in the 30 mg rLH group (P =0.048). The pregnancy rate ranged from 10.3% in the 60 mg group to 28.6% in the 30 mg rLH group. Adverse events were similar between groups. CONCLUSIONS: In patients over-responding to FSH during ovulation induction, doses of up to 30 mg rLH/day appear to increase the proportion of patients developing a single dominant follicle (≥ 16mm). Our data support the ‘LH ceiling’ concept whereby addition of rLH is able to control development of the follicular cohort. IIU 1 RCT comparing HP-hmg com FSHr Segnella et al, 2011: 523 patients with unexplained infertility or mild male infertility undergoing controlled ovarian hyperstimulation for IUI. Patients were randomized for treatment with rFSH (262 patients) or HP-hMG (261 patients). Insemination was performed 34-36 hours after hCG injection. The primary outcome was clinical pregnancy rate (PR). The secondary outcome was the number of interrupted cycles for high risk of ovarian hyperstimulation syndrome (OHSS) and multiple pregnancy. -Regarding follicular development, there was a significantly lower average number of intermediate-size follicles (14–16 mm) at the end of stimulation in the HP-hMG group (0.73 ± 1.00 in HP-hMG and 1.96 ± 1.54 in rFSH; P=0.001); - furthermore, the number of follicles ≥17 mm was significantly higher in rFSH cycles (1.27 ± 0.45 in HP-hMG and 1.69 ± 0.84 in rFSH; P1⁄=0.03; -Development of one dominant follicle (≥17 mm) without intermediate-size follicles was achieved for 42.3% in the HP-hMG cycles versus 11.5% in the rFSH cycles (P1⁄=0.03). -On the hCG day, E2 levels were significantly higher in the rFSH group compared with HP-hMG (833.19 ± 385.80 pg/mL and 551.75 ± 240.06 pg/mL, respectively; P1⁄=0.004). -No significant difference in endometrial thickness were observed. -Higher P levels were observed in the rFSH cycles (37.77 ± 26.22 ng/mL in rFSH and 23.52 ± 13.39 ng/mL in HP-hMG; P=0.02) The clinical PR was 19.7% (95% confidence interval [CI] 15.3%-25.1%) in the HP-hMG group and 21.4% (95% CI 16.9%-26.8%) in the rFSH group [absolute difference -1.7% (95% CI -8.6% to 5.2%)]; The number of interrupted cycles for OHSS risk and multiple pregnancy was significantly higher in the rFSH group, 8.4% (95% CI 5.6%-12.4%) than in the HP-hMG group 1.2% (95% CI 0.4%-3.3%) [absolute difference -7.27% (95% CI -11.3 to -3.7)]. CONCLUSION(S): HP-hMG is not inferior compared with rFSH regarding clinical PR. Comments: Bi/multifollicular development was significantly higher in the rFSH cycles, contributing to the significantly higher estradiol concentration compared with HP-hMG cycles. In contrast, the development of a single dominant follicle without intermediate-size follicles was significantly higher in the HP-hMG cycles. The lower number of follicles obtained in HP-hMG cycles could reflect an LH effect during the follicular phase; the gonadotropin might induce follicular atresia. In the present study, despite significant differences between the two treatment groups regarding number of dominant follicles and hormonal environment, the endometrial thickness and the clinical pregnancy rate were similar between the two groups. We could hypothesize that exogenous LH/hCG activity might positively influence oocyte quality and development. This observation could explain the benefit regarding PR for those normo-ovulatory women with unexplained infertility who achieved pregnancy even though they developed a monofollicular response. Interesting data in the present study concern the different mean age of pregnant patients. Mean age of pregnant patients was higher in the HP-hMG group compared with the rFSH group. This observation led us to hypothesize that, in older women, the addition of LH/hCG activity could positively affect oocyte quality and steroidogenesis. This hy-pothesis is supported by data demonstrating that in poor responders, superimposing rLH on rFSH improves outcome data. Finally, because one of the main challenges for clinicians involved in ovulation induction is controlled ovarian hyperstimulation cycle cancellation, another end point of our work was to evaluate the frequency of interrupted cycles for high risk of OHSS and multiple pregnancy. We observed that the number of interrupted cycles was higher in the rFSH group; therefore, HP-hMG treatment might besafer in women who tend to a multifollicular response. References: The European Recombinant Human LH Study Group. Recombinant human luteinizing hormone (LH) to support recombinant human follicle-stimulating hormone (FSH)-induced follicular development in LH- and FSH-deficient anovulatory women: a dose-finding study. J Clin Endocrinol Metab. 1998 May;83(5):1507-14. ShohamZ,SmithH,YekoT,O’BrienF,HemseyG,O’DeaL.Recombinant LH (lutropin alfa) for the treatment of hypogonadotrophic women with profound LH deficiency: a randomized, double-blind, placebo-controlled, proof-of-efficacy study. Clin Endocrinol (Oxf). 2008;69(3):471–478. Burgues S. Spanish Collaborative Group on Female Hypogonadotrophic Hypogonadism. The effectiveness and safety of recombinant human LH to support follicular development induced by recombinant human FSH in WHO group I anovulation: evidence from a multicentre study in Spain. Hum Reprod. 2001;16(12):2525–2532. Kaufmann R, Dunn R, Vaughn T, et al. Recombinant human luteinizing hormone, lutropin alfa, for the induction of follicular development and pregnancy in profoundly gonadotrophin-deficient women. Clin Endocrinol (Oxf). 2007;67(4):563–569. Carone D, Caropreso C, Vitti A, Chiappetta R. Efficacy of different gonadotropin combinations to support ovulation induction in WHO type I anovulation infertility: clinical evidences of human recombinant FSH/human recombinant LH in a 2:1 ratio and highly purified human menopausal gonadotrophin stimulation protocols. J Endocrinol Invest. 2012 Oct 22. Platteau P, Andersen AN, Balen A, et al. Ovulation Induction (MOI) Study Group. Similar ovulation rates, but different follicular development with highly purified menotrophin compared with recombinant FSH in WHO Group II anovulatory infertility: a randomized controlled study. Hum Reprod. 2006;21(7):1798–1804. Recombinant LH Study Group. Does the addition of recombinant LH in WHO group II anovulatory women over-responding to FSH treatment reduce the number of developing follicles? A dose-finding study. Hum Reprod. 2005;20(3):629–635. Sagnella F, Moro F, Lanzone A, Tropea A, Martinez D, Capalbo A, Gangale MF, Spadoni V, Morciano A, Apa R. A prospective randomized noninferiority study comparing recombinant FSH and highly purified menotropin in intrauterine insemination cycles in couples with unexplained infertility and/or mild-moderate male factor. Fertil Steril. 2011 Feb;95(2):689-94. Epub 2010 Sep 25.

Esteves, 25

What is the optimal LH supplementation protocol?

Existing studies give us some clues but the optimal LH protocol has yet to be established How much LH should be used? Should the dose be fixed or flexible? At what stage of the cycle should LH be

administered? Is LH needed in a GnRH antagonist Protocol?

FSH

LH

2:1? 1:1? Fixed? Mimic of natural LH levels?

Presenter

Presentation Notes

Even if we are able to identify those women who may require LH supplementation, there is currently no consensus on the most suitable LH protocol. Indeed, the protocol used may even depend on the subgroup being treated. Some of the treatment parameters that still need to be established are below. How much LH should be used? Should the dose be fixed or flexible? At what stage of the cycle should LH be administered?

Is LH needed in a GnRH antagonist Protocol?

Mochtar et al. 3 RCT (N=216)

Kolibianakis et al. 2 RCT (N=176)

Baruffi et al. 5 RCT (N= 434)

Estradiol on hCG day (pg/ml)

WMD 571 (95% CI 259; 882)

- WMD 514 (95% CI 368; 660)

No. retrieved oocytes

WMD 0.50 (95% CI -0.68; 1.68) - WMD 0.41

(95% CI -0.44; 1.3)

CPR†/LBR* †OR 0.79

(95% CI: 0.26; 2.43) *OR 0.86

(95% CI: 0.04; 1.85) †OR 0.89

(95% CI: 0.57; 1.39)

Unselected women undergoing COS; r-hFSH+r-hLH vs. r-hFSH alone in antagonist cycles

Mochtar MH et al. Cochrane Database Syst Rev. 2007;2:CD005070; Kolibianakis et al, Hum Reprod Update. 2007;13:445-52; Baruffi RL et al, Reprod Biomed Online. 2007;14:14-25. Esteves, 26

Level 1a

WMD weight mean difference

Presenter

Presentation Notes

WMD weight mean difference *No difference in Gonadotrophin total dose, OPR, OHSS or miscarriage rate **No difference in Gonadotrophin total dose, days of stimulation, implantation rate or miscarriage rate Há essas tres meta-anlises que avaliaram LH x sem LH em antagonista. A do Baruffi viu só antagonista mesmo enquanto a do Mochtar e é a da cochrane, que viu em geral e depois separou agonista de antagonista, assim como kobilianakis. só tem mais estradiol no dia do hcg em ambas. Na do baruffi tem mais oocito maduro. Baruffi: "a good effect of rLH supplementation in ovarian stimulation was observed in serum oestradiol concentrations on the day of HCG administration. Significantly higher serum oestradiol concentrations were found in 221 women using r-LH compared with 205 women not using it (P < 0.0001; WMD = 514,95% CI 368,660). This result demonstrates that the association of rLH with rFSH may prevent a decrease in oestradiol after antagonist administration. In addition, significantly higher numbers of mature oocytes were retrieved in 214 women using r-LH compared with 193 women not using it (P = 0.0098; WMD = 0.88, 95% CI -0.21, 1.54).

Yes, for women aged >35 yo

61%

25% 19%

68%

33% 27%

%2PN Ongoing PR Implantation

rFSH rFSH + rLH

Esteves, 27

RCT; 292 NG women aged 36-39; Fixed (D6) antagonist COH protocol

Bosch et al. Fertil Steril. 2011; 95:1031-6.

Is LH needed in a GnRH antagonist Protocol?

OR=1.49 95% CI 0.93-2.38

OR=1.56 95% CI 1.04-2.33

Level 1b

P= 0.027

Presenter

Presentation Notes

Randomized, open-label, controlled trial; Initial gonadotropin dose: 300 IU/d for both study groups: 300UI of rFSH-alone or 225UI of rFSH + 75UI rLH, starting day 1 of COS. FSHr dose adjusted on day 6 but no changes in dose of rLH. No difference in outcomes for women <36. The potential benefit of LH administration for older patients could be explained by two different mechanisms. On the one hand, the endocrine changes occurring with ovarian aging include an increase of the serum FSH levels in the early follicular phase, which are not accompanied by an LH increase but by a progressive decrease of the basal androgen levels. Moreover, the follicular capability for inducing androstenedione synthesis after rFSH administration is significantly impaired in older patients compared with younger reproductive-aged patients, whereas E2 secretion is preserved by increased aromatase function. Action of LH at the follicular level that increases androgen production for its later aromatization to estrogens in a dose dependent manner may restore the follicular milieu in these patients to recover oocyte quality and, therefore, embryo quality and implantation rates. Follicle-stimulating hormone acts on granulosa cells to promote the conversion of cholesterol into P, which is passed to the thecal cells to be converted into androgens under the influence of LH, therefore reducing circulating P. All in all, this could explain why stimulation with rFSH alone can produce poorer outcomes in older and low-responding patients because they usually receive higher FSH doses for COS, they show higher P levels at the end of stimulation, and, subsequently, their endometrium receptivity diminishes.

Esteves, 28

Women with less sensitive ovaries (ovarian aging) have poorer IVF outcomes.

Androgen secretory capacity decreases with ovarian ageing. Mechanisms include decreased number of functional LH receptors and ovarian paracrine activity resulting in reduced LH bioactivity. LH-r polymorphisms possibly involved in hypo-responders.

LH supplementation to COS is an evidence-based strategy to maximize pregnancy results.

4 subgroups benefit of LH supplementation in COS: Poor responders Slow/hypo-responders Age >35 years Deeply suppressed endogenous LH levels

Esteves, 29

3 subgroups clearly benefit of LH supplementation in OI and IUI: WHO group I anovulation WHO group II clomiphene resistant WHO group II with previous over-response to OS

Other potential indications include: Poor responders Slow/hypo-responders Age >35 years Deeply suppressed endogenous LH levels

Esteves, 30





1930s 1950 1980 1995 2003

Pituitary FSH u-hMG

u-FSH

u-FSH HP r-hFSH

r-hFSH FbM

Intramuscular administration sc Injector pens

sc, subcutaneous; FbM, filled by Mass; HP, highly-purified

Safety, Quality, Consistency and Patient

Convenience

Esteves, 31

Long-acting

r-hFSH;

2010

Puriity and

SpecificActivity

Adapted from Lunenfeld. Hum Reprod Update 2004;10:453–67

r-hFSH +r-hLH

FbM

2007

Horse PMSG

1962 1993

r-hLH

2000

Presenter

Presentation Notes

Therapeutic armamentarium for ovarian stimulation has increased over the last years and clinicians face the problem of which drug regimen to use.

Esteves, 32

*

*Pergoveris™: rec-hFSH (150 UI) + rec-hLH (75 UI); Bosch et al. Expert Opin Biol Ther 2010;10:1001-9.

Launched in Germany, 2007

Presenter

Presentation Notes

• Same injection device

design for all gonadotropins;

• Color-coded for differentiation;

• Pre-filled, ready-to-use family of pens for fertility treatment.

Esteves, 33

Presenter

Presentation Notes

GF family of pens in india will be launched in June, for Ovitrelle & Luveris, it will be early part of next year.

Bassett et al. Reprod Biomed Online 2005;10:169–177; Driebergen et al. Curr Med Res Opin 2003;19:41–46.

Conventional Bioassay

High

variability

Rat ovary weight gain

FbM: Novel analitycal method

Protein content by mass

Minimal batch-to-batch variability (1.6%)

Urinary gonadotropins Follitropin beta Follitropin alfa and rec-hLH

Esteves, 34

Presenter

Presentation Notes

Vitual elimination of batch-to-batch variation. The enhanced purity and increased specific activity allow SC delivery in very small volumes. A further improvement towards quality and patient convenience came with the development of an innovative method for measuring the gonadotropin content that comes in each vial. The conventional method to quantify the activity of gonadotropin products is the Steelman–Pohley assay, which is an in vivo rat bioassay. As well as being costly and subject to ethical concerns related to the use of animals, this technique has an inherent variability of up to 20%. In 2003, a novel physiochemical method was developed for measuring FSH content (by protein mass), that improved batch-to-batch consistency of r-hFSH (follitropin alfa) in terms of specific activity, isoform pattern and sialylation profile. Following this, These advances led to the development of GONAL-f® FbM, which is formulated based on r-hFSH protein content. Driebergen et al. Curr Med Res Opin 2003;19:41–46 Bassett et al. Reprod Biomed Online 2005;10:169–177

*derives primarily from the hCG component, which preferentially is concentrated during the purification process and sometimes was added

to achieve the desired amount of LH-like biological activity.

Alfa Unit Beta unit (biological action

and receptor affinity)

Carboxyl terminal segment

(determines half-life)

LH 92 AA; 121 AA Absent; half life of 20’ hCG Identical to LH 144 AA

Higher receptor affinity Present; half-life of

24h

Purity (LH content)

FSH activity (IU/vial)

LH activity (IU/vial)

Rec-hLH >99% 0 75

Rec-hLH + rec-hFSH >99% 150 75

hMG-HP Unknown* 75 75*

ASRM Practice Committee. Fertil Steril. 2008; 90:S13-20. Esteves, 35

Presenter

Presentation Notes

Human chorionic gonadotropin (hCG) is similar in structural attributes compared with follitropin. A notable exception is the presence of a long carboxy terminal segment that is O-glycosylated (O-linked CHO). This segment is not visible in the hCG crystal structure (PDB files 1XUN and 1HCN), but it is shown here for illustrative purposes. Of importance is that this extension confers a long half-life to hCG; when grafted onto the hFSH beta subunit, this extension likewise confers a longer circulatory half-life to hFSH. The C-terminal extended hFSH is currently in clinical trials. Although the alpha subunits of LH and hCG are identical to that of FSH, the beta subunits are different. Luteinizing hormone has a beta subunit containing 121 amino acids that confers its specific biologic action and is responsible for its interaction with the LH receptor. This beta subunit of LH contains the same amino acids in sequence as the beta subunit of hCG, but the hCG beta subunit contains an additional 23 amino acids. The two hormones differ in the composition of their carbohydrate moieties which, in turn, affects bioactivity and half-life. LH activity in serum is 30 times higher when hCG is used. The half-life of LH is 20 minutes, and that for hCG is 24 hours.

Esteves, 36 Buhler KF, Fisher R. Gynecol Endocrinol 2011; 1-6.

• Matched case-control study; • N=4,719 pts.; long GnRH-a protocol • 3 groups

31 26 25

0

5

10

15

20

25

30

35

2:1 r-hFSH+r-hLH

HMG rec-hFSH +HMG

Duration ofStimulation (days)

Mean No. oocytesretrieved

IR (%)

CPR per transfer(%)

P=0.02

Level 2a

Presenter

Presentation Notes

New fixed combination of rec-hFSH and rec-hLH filled by Mass at a 2:1 ratio; Pergoveris™: rec-hFSH (150 UI) + rec-hLH (75 UI); Bosch et al. Expert Opin Biol Ther 2010; 10: 1001-9.

Lower expression of LH/hCG receptor gene as well as genes involved in in biosynthesis of cholesterol and steroids in granulosa cells in pts. treated with HMG preparations May reflect down-regulation of LH receptors, as shown in animals: Caused by a constant ligand exposure during the follicular phase due to longer half life and higher binding affinity of hCG to LHr May explain the observed lower progesterone levels: Caused by lower LH-induced cholesterol uptake, a decrease in the novo cholesterol synthesis and a decrease in steroid synthesis.

Trinchard-Lugan I et al. Reprod Biomed Online 2002; 4:106-115; Menon KM et al. Biol Reprod 2004; 70:861-866; Grondal ML et al. Fertil Steril 2009; 91: 1820-1830. Esteves, 37

Level 1a

Presenter

Presentation Notes

Possible reasons: 1 - longer half life of hCG and higher binding afinity to LH receptors: Trinchard-Lugan I et al: Pharmacokinetics and pharmacodynamics of recombinant human chorionic gonadotrophin in healthy male and female volunteers. Reprod Biomed Online 2002; 4:106-115. 2 - Down-regulation of LH receptors for up to 48h as shown in animal models: Menon KM et al: Regulation of luteinizing hormone/human chorionic gonadotropin receptor expression: A perspective. Biol Reprod 2004; 70:861-866. 3 - Differences in gene expression profiles in granulosa cells after COS with r-hFSH and u-HMG: consistent lower expression of LH/hCG receptor gene in granulosa cells of in the u-HMG group. The lower gene expression may reflect down-regulation caused by a constant ligand exposure during the follicular phase. This would also explain the lower progesterone levels reported in these patients. The expression level of hCG/LH receptors genes and genes involved in in biosynthesis of cholesterol and steroids were expressed at lower levels in the HMG treated group; These findings indicate that granulosa cells of u-HMG treated pts have lower LH-induced cholesterol uptake, a decrease in the novo cholesterol synthesis and a decrease in steroid synthesis. Contrary, r-hFSH induced up-regulation of LH/hCG receptors that may increase cholesterol and steriod synthesis in pts treated with recFSH: Grondal ML et al. Differences in gene expression of granulosa cells from women undergoing controlled ovarian hyperstimulation with either recombinant follicle-stimulating hormone or highly-purified human menopausal gonadotropin. Fertil Steril 2009; 91: 1820-1830. As hCG has a relatively long half-life of 48 hours and accumulates significantly over the first week of administration before reaching a plateau, there is a possibility that LHR down-regulation and cell desensitisation can occur due to constant ligand exposure. The expression of LHR is linked to the synthesis of progesterone during COS, and the levels of LHR and progesterone synthesis vary depending on the hormones used during the stimulation protocol. Administration of standard FSH leads to higher FSH bioactivity than stimulation with hMG. Treatment with FSH leads to the development of more follicles and granulosa cells, higher numbers of oocyte retrieved, higher numbers of embryos produced, lower total amounts of gonadotrophins utilised and fewer days of stimulation than treatment with hMG. FSH also leads to the up-regulation of LHR, whereas LHR expression is down-regulated following administration of hMG (through a specific effect of hCG; see Figure). Subsequently, endogenous LH results in higher levels of progesterone synthesis following treatment with FSH than hMG. This is due to the combination of higher levels of LHR expressed on granulosa cells and the increased number of granulosa cells following FSH treatment. This indicates that it may be possible to determine LH activity during COS through quantitation of serum levels of progesterone.

Recombinants vs Urinary products

Esteves, 38

Recombinant LH preparations have 3 major differences compared to urinary products: Higher purity and specific activity (SC delivery in

very small volumes)) Higher dose precision (FbM) LH activity in u-HMG is hCG dependent:

hCG is concentrated during purification or added to achieve the desired amount of LH-like biological activity; hCG has higher half-life and biological activity compared to rec-hLH.

Lower expression of LH receptor gene in pts. treated with HMG (LH-r down-regulation).

Preparations used for COS are important for granulosa cell function and may influence the developmental competence of the oocyte and the function of corpus luteum.

Esteves, 39

Differences between rec-hLH and LH Activity in HMG Preparations

Esteves, 41

Use of LH in IVF and IUI Progesterone Issues

Supplementary Material

Steroidogenesis During Normal Follicular Phase and Following COS

The expression of LH-R (GCs) is linked to the synthesis of progesterone during COS.

Levels of LH-R and progesterone synthesis vary depending on the hormones used during the stimulation protocol.

Steroidogenesis Consensus Meeting III, Copenhagen, Denmark, May 2011. Esteves, 42

Presenter

Presentation Notes

there are two sources of progesterone in the follicle (i: produced by the granulosa cells under FSH drive and ii: theca cells), but only one step of further metabolism (catabolism of progesterone to androgens) which is driven by LH activity in theca cells. A lack of LH drive to the thecal cells is therefore likely to leave more progesterone to find its way into the general circulation. Follicle-stimulating hormone acts on granulosa cells to promote conversion of cholesterol to progesterone, which is passed to the thecal cells to be converted to androgens under the inﬂuence of LH. The androgens are then passed to the granulosa cells to be converted to estradiol; the classical ‘2-cell, two gonadotrophin’ hypothesis (Moon et al., 1978) It can be predicted that an ovary with a large number of growing follicles, stimulated by maintained high FSH concentrations by daily FSH injections, will produce and secrete more progesterone into the ovarian vein than a single follicle in the normal mid-follicular phase. In the event of ovarian stimulation-induced multiple-follicle growth, the progesterone output to the periphery will be magnified in accord with the number of follicles and the FSH drive. This is likely to impact upon progesterone concentrations in the periphery and may influence endometrial development. Thus, the three major components to the degree of progesterone secretion from the ovaries will be: (i) the number of follicles (or granulosa cells); (ii) the degree of trophic stimulus (FSH drive to granulosa cells); and (iii) the degree of LH drive to thecal cells, which will encourage conversion of progesterone to androgens and oestrogen.

Endogenous LH then results in higher levels of progesterone synthesis following treatment with FSH than hMG: higher levels of LHR expressed on granulosa cells and increased number of granulosa cells.

Steroidogenesis in COS

Steroidogenesis Consensus Meeting III, Copenhagen, Denmark, May 2011. Esteves, 43

Presenter

Presentation Notes

The optimal dose and timing of LH/hCG administration during COS is a topical issue. Serum progesterone increases mainly occur during the final stages of COS, and in normal responders this is driven by the degree of ovarian stimulation. A retrospective analysis of 708 cycles suggests that there is a higher progesterone rise in gonadotrophin-releasing hormone (GnRH) agonist cycles than antagonist cycles, which is related to stronger ovarian stimulation and an increase in serum LH. These data suggest that there is a positive correlation between serum LH and progesterone levels, at least in GnRH agonist cycles (Hugues JN, Massé-Laroche E, Reboul-Marty J, Boîko O, Meynant C, Cédrin-Durnerin I. Impact of endogenous luteinizing hormone serum levels on progesterone elevation on the day of human chorionic gonadotropin administration. Fertil Steril 2011;96:600–4). Following treatment with FSH, endogenous LH induces the synthesis of progesterone. It is likely that LH stimulates progesterone secretion in the late follicular phase. The effect of high progesterone levels at the time of hCG administration on pregnancy outcome is still controversial. Further detailed analyses are required to understand why, when and how much progesterone is detrimental for implantation rates.

Progesterone on the Day of hCG and Probability of Pregnancy in IVF

Venetis et al, 2007 Kolibianakis et al, 2012 GnRH

analogue Agonists n = 2,624

Antagonists n = 109

Antagonists n = 109

CPR OR: 0.86 (95% CI: 0.59; 1.25)

OR: 0.57 (95% CI: 0.09; 3.56)

WMD: -9% (95% CI: -17; -2)

E2 levels on the day of

hCG (pg/mL)

WMD: 413.06 (95% CI: 240.14; 585.99)

WMD: 956 (95% CI 248; 1664)

Number of retrieved oocytes

WMD: +2.96 (95% CI: +1.74; +4.18)

WMD: 0.00 (95% CI: -2.98; +2.99)

WMD: +2.9 (95% CI: +1.5; +4.4)

Venetis et al, Hum Reprod Update. 2007;13:343-55; Kolibianakis et al, Curr Pharm Biotechnol. 2012;13:464-70.

Progesterone Elevation x No Progesterone Elevation

heterogeneity of the studies included; arbitrary serum progesterone threshold values

Level 1a

Esteves, 44

Presenter

Presentation Notes

Venetis et al, 2007: 2733 patients were reviewed (GnRH antagonists: n = 109, GnRH agonists: n = 2624). Limitacao: heterogeneidade e estudos consideraram alta se acima de 1,2 em alguns estudos e 0,9 em outros. No difference in CPR, FSH requirement , Duration of FSH stimulation and fertilization rate In the group of agonist studies significantly more oocytes were retrieved in the progesterone elevation group compared with the group without progesterone elevation This difference was not present when the two antagonist studies were pooled. higher serum estradiol levels on the day of hCG administration Kolibianakis et al, 2012 5 estudos com antagonist, 585 no difference duration of stimulation and total dose of gonadotrophins higher serum estradiol levels on the day of hCG administration and more COCs retrieved and less pregnancy per cycle

Progesterone on the Day of hCG and Probability of Pregnancy in IVF Bosch et al. 2010 (N=4,032)

OPR: inversely associated with serum P levels on the day of hCG irrespective of the GnRH analogue: CUT-OFF: 1.5 ng/mL Serum P levels ≤1.5 ng/ml: ↑OPR 31% versus 19.1%; P = 0.00006; OR: 0.53 (95% CI, 0.38 – 0.72)

FSH dose

No. oocytes

Estradiol (day of hCG)

Serum P levels: agonists: 0.84 ± 0.67 vs antagonists: 0.75 ± 0.66 (P = 0.0003)

positively associated

with P levels (P < 0.0001 for

all).

Bosch et al. Hum Reprod. 2010; 25(8):2092-100.

Level 2b

Esteves, 45

Presenter

Presentation Notes

non-interventional, retrospective, observational, single-centre cohort study Este estudo do bosh mostra que o melhor cut-off seria 1.5. Acima deste valor, cai a taxa de gravidez. Isso vale para agonista e antagonista. Este e o proximo criticam a meta-analise e estudos previos pois eles definaram um cut-off de 0,9 ou 1,2 que, alem de baixo, foi arbitrario. Nesses dois estudos eles calcularam de acordo com a curva de resposta, qual seria o corte ideal.

Progesterone on the Day of hCG and Probability of Pregnancy in IVF Xu et al, 2012 (N=11,055 long agonist protocol)

For fresh cycles, OPR inversely associated with serum P levels on hCG day

FSH dose No. oocytes Estradiol (day of hCG)

Positively associated

with P levels

■ Fresh ■ FET

Ovarian response

Number of oocytes

Serum P threshold (ng/mL)

Poor ≤4 1.5 Intermediate 5-19 1.75

High ≥20 2.25

Xu et al. Fertil Steril 2012;97:1321–7.

Level 2b

Esteves, 46

Presenter

Presentation Notes

noninterventional, retrospective Nesse estudo do xu, ele mostra que o aumento da progesterona nao qualidade embrionaria (pr em fet, fertiliztion rate and cleavage rate without relation to level of progesterone. Ele mostra que a curva que o bosch fez no estudo anterior varia se baixa, normo ou hiporrespondedora, calculando o melhor threshold este da tabela. Para cada nivel de resposta. Na conclusao ele propoe o congelamento de acoro com esse nivel.


The rise in progesterone levels seen during COS for IVF/ICSI cycles cannot be explained by luteinization of

granulosa cells

FSH activity Conversion

of cholesterol to progesterone

LH bioactivity

Conversion of progesterone

to androgens

Granulosa cell

Teca cell

FSH dose, number of follicles and rec-hFSH (x HMG): correlation to P increase on hCG day

Bosch et al. Hum Reprod. 2010;25:2092-100; Xu et al. Fertil Steril 2012;97:1321–7; Smitz J et al. Hum Reprod 2007;22:676–87. Esteves, 47

Presenter

Presentation Notes

The rise in progesterone levels seen during COS for IVF/ICSI cycles cannot be explained by luteinization of granulosa cells, since this occurs in the presence of GnRH analogues and, hence, low LH levels. FSH stimulation, leading to increased granulosa cell steroidogenic activity Follicle-stimulating hormone acts on granulosa cells to promote conversion of cholesterol to progesterone Therefore, in an ovary with multiple follicles stimulated by high FSH concentrations, greater progesterone production could be anticipated than with a single follicle in the normal mid-follicular phase,


Hofmann et al, Fertil Steril. 1993;60:675-9; Xu et al. Fertil Steril 2012;97:1321–7; Huang et al. Fertil Steril 2012; 98:664–70; Melo et al. Hum Reprod 2006; 21:1503–1507.

Levels 2b, 3

Esteves, 48

Presenter

Presentation Notes

Studies with GnRH agonists suggest that elevated progesterone levels may act at the level of the endometrium, since adverse effects on the oocyte and embryo quality have not been observed (Hofmann et al., 1993; Fanchin et al., 1997a; Ubaldi et al., 1997; Fanchin et al., 1999; Andersen et al., 2006). Moreover, a study performed in an oocyte donation programme suggested that pregnancy rates of recipients were not influenced by progesterone levels of the donors at the end of stimulation (Melo et al., 2006).. The increase in progesterone has the potential to advance the endometrium, without influencing the embryo. This can, in turn, lead to a state of asynchrony between embryo and endometrial dating, which may result in reduced implantation (Bourgain and Devroey, 2003) and, consequently, a reduced pregnancy rate.

Most circulating P4 (95%) is produced in the intrafollicular compartment by the granulosa cells;

Intrafollicular P4 and Hydroxi-progesterone are terminal products and cannot be converted to estradiol by GCs under the effect of LH/hCG activity contained in hMG, due to lack of expression of an hydrogenase and P450-17α needed for this pathway;

Higher serum Progesterone increments are related with more follicles developed and more oocytes retrieved and it’s effect in pregnancy still controversial. Increments up to >7nmol/L seems not to affect clinical pregnancy rates.

Serum Progesterone and IVF Outcome

Treatment with FSH results in higher levels of progesterone than treatment with hMG.

A large number of developing follicles leads to increased levels of progesterone.

The higher the level of LH present, the higher the level of progesterone.

The effect of high progesterone levels at the time of hCG administration on pregnancy outcome is still controversial. Further detailed analyses are required to understand why, when and how much progesterone is detrimental for implantation rates.

Esteves, 50

Serum Progesterone and IVF Outcome

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Presentation Notes

What we don’t know: Whether AMH levels are directly regulated by LH. The identity of the signal that determines if pregnenolone follows the D4 or D5 pathway. If LHRs are functional in granulosa cells in follicles 3–9 mm in size. The LH/hCG response in granulosa cells that have not been exposed to hCG. The nature of the molecular changes that occur during luteinisation, how long luteinisation takes and how much LH is required for the process. At what point expression of progesterone constitutes premature elevation, and what levels of premature progesterone are needed to cause changes in the endometrium.

Presenter

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LH or hCG cannot convert free P4 in E2 by the action of P450-17α because this enzyme does not exist in the follicular compartment. Importantly, CYP17 is located exclusively in thecal/interstitial cells (i.e. the extrafollicular compartment of the ovary), whereas CYP19 (aromatase), which converts androgens to oestrogens, is expressed only in the granulosa cells (the intrafollicular component). The theca cells therefore provide the androgens required by the developing follicles for conversion into oestrogens by the granulosa cells. Increases in progesterone may instead reﬂect the mature granulosa cell response to high FSH exposure: reflect increased granulosa cell steroidogenic activity (Bosch et al., 2003). T herefore, in an ovary with multiple follicles stimulated by high FSH concentrations, greater progesterone production could be anticipated than with a single follicle in the normal mid-follicular phase, particularly if the FSH action is not balanced by LH activity. Thus, 17-hydroxyprogesterone is the end product of the D4 pathway in humans and the only route for oestradiol synthesis in the human ovary is via the D5 pathway. Progesterone: conversion factor 3.18: ng/mL to nmol/L. The Nordic LH study proved that high P4 level up to >7 nmol/l (2.2 ng/ml) at the time of LH peak “Is not related with Reduced Pregnancy rates” and the higher the LH the higher the number of follicles, oocytes and progesterone!! Conclusions: Progesterone (or 17OH-progesterone) produced by the human ovary is a terminal product that cannot be further converted through normal ovarian steroidogenesis. Human CYP17 cannot convert 17OH-progesterone to androstenedione; 3β-HSD acts essentially as a one-way enzyme from the D5 to the D4 pathway. Progesterone is naturally produced by human follicles and its concentration increases towards ovulation. Oestradiol produced by human follicles is almost exclusively derived through the D5 pathway. LH appears to play a central role in regulating follicular phase progesterone levels.

Use of LH in IVF and IUI

Health & Medicine

Transcript of Use of LH in IVF and IUI