r lh supplementation to rfsh in gnrh antagonist cycles

rLH SUPPLEMENTATION TO rFSH IN GnRH ANTAGONIST CYCLES:EFFECTS ON IMPLANTATION AND PREGNANCY RATE

A. Nazzaro, A Salerno

Two cell-two gonadotrophin

Physiology of follicular growth and differentiation

Pre-gonadotrophic folliculogenesis

KIT Ligand expression

It remains to be determined if activation can be influenced by gonadotrophins but it certainly seems to

proceed normally in their absence

Paracrine connectivity in ovarian follicles.

Hillier S G Mol. Hum. Reprod. 2009;15:843-850

© The Author 2009. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

The follicle responds to FSH and LH as a functional syncytium integrated by paracrine signalling

PrimarySe

cond

ary

• oocyte expansion

• granulosa cell proliferation

• LH responsive Techa cells

Secondary

FSH

TGF-β

Gonadotrophin dependence

1) enhance granulosa cell sensitivity and responsiveness to FSH

2) suppress LH-responsive thecal androgen synthesis

FSH

1) promote androgen synthesis2) Androgens in turn synergize with FSH to augment inhibin synthesis

The principle requirement for LH is to sustain androgen synthesis, without which there is no estrogen synthesis

Follicular antrum formation and antral expansion are absolutely dependent on FSH.

Early follicular phase

Late follicular phase

Huang Z , and Wells D Mol. Hum. Reprod. 2010;16:715-725

© The Author 2010. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Follicular activation

Meiotic resumption in late follicular phase

Zhang M et al. Mol. Hum. Reprod. 2009;15:399-409

LH stimulated paracrine signals are thereby able to impact oocytes even in follicles whose granulosa

cells do not express LH-R

• costly

• time consuming

• potentially unhealty

Gonadotrophic manipulation of paracrine signalling

Paracrine signalling explains how gonadotrophins are potentially able to benefit oocyte function

and by extension how inappropriate use of exogenous gonadotrophins might be deleterious

closely mimic physiology

controlled ovarian stimulation regimes might be reverse engineered from the natural follicular life cycle

Understanding the role of LH: myths and facts

Too much or too little LH activitycan have negative outcomes

LH window

Gynecol Endocrinol. 2012 Sep;28(9):674-7. doi: 10.3109/09513590.2011.652716. Epub 2012 Feb 8.Recombinant luteinizing hormone priming in multiple follicular stimulation for in-vitro fertilization in downregulated patients.Lisi F, et al.

follicular priming with LH

J Clin Endocrinol Metab. 1999 Aug;84(8):2951-6.Androgen and follicle-stimulating hormone interactions in primate ovarian follicle development.Weil S, et al.

Li M et al. Mol. Hum. Reprod. 2009;15:149-154

Granulosa cells express androgen receptor (AR) through which theca-derived testosterone amplifies FSH-stimulated PKA signalling

LH-driven thecal androgen production may be a component of the natural mechanism through which antral follicles initially acquire sensitivity to FSH.

LH priming

Durnerin C I et al. Hum. Reprod. 2008;23:421-426

© The Author 2007. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

revealed increased developmentof small antral follicles due to LH priming

a statistically significant increase in the number of normally fertilized embryos obtained after subsequent FSH treatment

no clinically important outcomes were significantly altered in the LH group versus controls

Evidence that LH priming can improve oocyte quality.

Hillier S G Mol. Hum. Reprod. 2009;15:843-850

© The Author 2009. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Evidence that follicular coasting with LH can promote mono-ovulation: a double-blind, placebo-controlled, multicentre pilot study in patients undergoing ovulation induction.

Hillier S G Mol. Hum. Reprod. 2009;15:843-850

© The Author 2009. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

OSF regulation of CC function and oocyte quality Model depicting oocyte–CC interactions and their impact on subsequent oocyte developmental potential.

Gilchrist R B et al. Hum. Reprod. Update 2008;14:159-177

© The Author 2008. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Oocyte quality is assured by LH-driven nutritional and informational signals emanating in cumulus cells

biphasic stimulation regimes using FSH followed by LH activity to target the gold standard

oocyte and its nearest neighbours (in developmental terms) could have potential clinical merit

Schematic overview of expected FSH and LH concentrations in various GnRH analogue regimens a and b are regimens without oral contraceptive pill (OCP) pretreatment: (a) long GnRH agonist protocol and (b) fixed day 6 GnRH antagonist protocol.

Huirne J et al. Hum. Reprod. 2007;22:2805-2813

© The Author 2007. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

P. Platteau MD, MRCOGCentre for Reproductive Medicine

Brussels Free UniversityBelgium

Antagonist study

A.Nazzaro, MDA.Salerno MSc, PhD

AORN “G. Rummo Italy

Devroey et al., 2009

Ideal antagonist protocol

• Estroprogestin till Wednesday (last pill)• GnRh-antagonist started on Monday for 3 days• Blood test (E2 + P4 + LH)

• rFSH oh Thursday

• GnRh-antagonist added-back on the 6th day of COH• Blood test (E2 + P4 + LH) + US Tuesday and Thursday• OPU between Monday and wednesday

if P4 >1 ng/ml one more antagonist ampule (rFSH started one day later)

Ideal antagonist protocol

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

rFSH (Tailored)

GnRh-ant

HCG OPU ET

GnRh-ant

Menses

EP

rLH

rLH

Partially presented and awarded with first prize for scientific comunication at 2012 ASRM Annual Meeting – San Diego, Ca, 2012

Submitted for publication to Fertility and Sterility

Baseline characteristics of the two study groups.

rFSH (n=211) rFSH + rLH (n=211) P-value

Age (years) 36.7 ± 1.5 36.6 ± 1.6 NS

BMI (kg/m2) 22.6 ± 2.7 22.7 ± 2.8 NSInfertility duration (years) 4.4 ± 2.3 4.7 ± 2.4 NS

Basal plasma FSH (mIU/ml) 5.7 ± 2.4 5.1 ± 1.9 NS

Basal plasma LH (mIU/ml) 5.5 ± 2.3 4.0 ± 1.9 NS

Basal plasma oestradiol (pg/ml) 73.0 ± 29.1 66.9 ± 31.0 NS

r-FSH (n = 211) rFSH + rLH (n=211)

GnRH antagonist (days) 10.6 (1.8) 9.8 (1.3)

Duration of ovarian stimulation (days)

11.7 (0.9) 11.4 (1.1)

Cumulative r-HFSH dose (IU) 2562 (1187) 2453 (1884)

Plasma oestradiol (pg/ml) 2254 (989) 2325 (723)

follicle size (mm)

10 and <14 4.6 (2.8) 3.8 (1.6)

14 and <16 mm 3.4 (1.6) 4.6 (2.1)

16 and <18 mm 4.4 (1.2) 4.3 (1.1)

>18 mm 3 (1.2) 3 (1.1)

r-HFSH (n = 211) rFSH + rLH (n=211) P-value

Retrieved oocytes 8.9 + 4.9 11.8 + 4.3 0.036

Metaphase II oocytes 4.2 + 2.1 7.8 + 2.3 0.014

Fertilization rate 0.58 + 0,26 0.87 + 0.32 0.017

Embryos/cycle 5.4 + 2.1 9.6 + 1.2 0.092

Grade I-II embryos/cycle

4.3 + 0.8 7.6 + 1.3 0.086

Frozen embryo/cycle 1.0 + 0.4 4.3 + 1.2 0.008

Implantation rate (%) * 13 (105/829) 18 (148/826) 0.04

Clinical pregnancy rate (%) **

22 (84/380) 32 (98/305) 0.03

•GS/Embryo transferred• Pregnancy/Embryo Transfer

GnRH Antagonists vs. Agonists

LH, endometrium and embryo implantation

This scanning electron micrograph of a sample taken on day 7 after HCG administration from an oocyte donor in the ganirelix 2 mg group shows that the high-dose ganirelix treatment can produce the required epithelial response.

Simon C et al. Hum. Reprod. 2005;20:3318-3327

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Exogenous LH protects the endometrium from exposure to premature progesterone rises

Percentage of genes in common between stimulated and natural cycles during the receptive endometrium.

Haouzi D et al. Biol Reprod 2010;82:679-686

©2010 by Society for the Study of Reproduction

CONCLUSION

• The apparent beneficial effect of r-LH on implantation and pregnancy outcomes in our study could be explained by two different mechanisms:

1. Firstly, the embryo quality could be superior in the group supplemented with r-LH.

2. Secondly, there may be an effect of LH supplementation on the endometrium, which could promote implantation.

r-LH supplementation in GnRH-a cycles seems to improve implantation and pregnancy rates via an improvement in oocyte quality and/or uterine receptivity

Translational implications• The current emphasis on oocyte quality over quantity in ART procedures calls for a more intelligent approach to ovarian stimulation based on sound physiological principles

• Follicular responses in standard FSH/hMG alone regimes are inevitably asynchronous, yielding many oocytes that do not fertilize or are unable undergo normal embryonic development.

• We have highlighted paracrine pathways emanating in thecal cells and granulosa cells that potentially impact oocytes and which can be manipulated by sequential and/or combined use of FSH and LH

• We have to designe tactics to increase follicular responsiveness to FSH/LH, synchronize oocyte competence and maximize oocyte quality

There can be no one-size-fits-all approach to ovarian stimulation

Factors such as age, genotype and general health will always affect individual responses

However, treatment tailored to personal needs based on paracrine principles will always be more likely to achieve the desired outcome.