Gonadotropin’sBioactivity Dr. Vincenzo Volpicelli Fertility Center Cardito Seconda Università...

Gonadotropin’s Gonadotropin’s

BioactivityBioactivity

Dr. Vincenzo VolpicelliDr. Vincenzo Volpicelli

Fertility Center Fertility Center CarditoCardito

Seconda Università degli Studi di Napoli Seconda Università degli Studi di Napoli

Dipartimento di Scienze della VitaDipartimento di Scienze della Vita

SUNfertSUNfert

Seconda Università degli Studi di Napoli Seconda Università degli Studi di Napoli

GonadotropinsGonadotropins

FSH, LH, HCGFSH, LH, HCGglycoproteinsglycoproteinsdimers dimers αα, , ββ (two peptide chain) (two peptide chain)

αα chain aspecific chain aspecificβ β chain specificchain specific ((provides specificity for receptor interaction)provides specificity for receptor interaction)

Glycoproteins are proteins that contain oligosaccharide chains covalently attached to their side-chains.

An oligosaccharide is a saccharide polymer containing a small number (typically three to ten) of

component sugars, also known as simple sugars.

FSHFSH

heterodimeric hormone: heterodimeric hormone:

• 92 amino acids 92 amino acids αα--chain chain

• 111 amino acids 111 amino acids ββ--chainchain

Ben-Rafael Z, Levy T, Schoemaker J. 1995 Pharmacokinetics of follicle-stimulating hormone: clinical significance. Fertil Steril. 63:689–700

Various types of FSH exist according to their sialic acid contentVarious types of FSH exist according to their sialic acid content

The half-life of FSH is 3-4 hours The half-life of FSH is 3-4 hours

LH

• The luteinizing hormone beta subunit gene is localized in the LHB/CGB gene cluster on chromosome 19q13.32

•The gene for the alpha subunit is located on chromosome 6q12.21.

LH/HCG bioactivityLH/HCG bioactivity LH & HCG: the same amino acids in sequence LH & HCG: the same amino acids in sequence LH & HCG both stimulate the same receptorLH & HCG both stimulate the same receptor the hCG the hCG ββ--subunit contains an additional 24 amino acids, subunit contains an additional 24 amino acids, both hormones differ in the composition of their sugar both hormones differ in the composition of their sugar

moieties. moieties. The different composition of these The different composition of these oligosaccharidesoligosaccharides

affects bioactivity and speed of degradation. affects bioactivity and speed of degradation. The biologic The biologic half-lifehalf-life::

LH: LH: 20 minutes20 minutes FSH: FSH: 3-4 hours3-4 hours hCG: hCG: 24 hours24 hours

FSH, LH, HCGFSH, LH, HCG

The protein dimer contains 2 polypeptide units, labeled alpha and beta subunits that are connected by two disulfide bridges

The alpha subunits of LH, FSH, TSH, and hCG are identical, and contain 92 amino acids

The The beta subunitsbeta subunits vary vary

Gn secretionGn secretion

estrogensestrogens

pituitary glandpituitary gland

hypothalamus hypothalamus (arcuate nucleus and preoptic area)(arcuate nucleus and preoptic area)

GnGn

(Gn-RH pulses)(Gn-RH pulses)

ovaryovary

feed-backfeed-back

Estradiol negative feed-backEstradiol negative feed-back

Pituitary gland embryology

Pituitary glandPituitary gland

by diencephalon

by diencephalon

(infundibulum)

(infundibulum)

by Rathke pouch

by Rathke pouch(mouth)(mouth)

Pituitary portal system

Pituitary gland histologyPituitary gland histology

GHGH

HPRLHPRL

FSHFSHLHLHTSHTSHACTHACTH

Gn mode actionGn mode action

• activate a PtdIns(phosphatidylinositol)-calcium second

messenger system

• membrane receptors

•Adenilcyclasi activation

Gn mode of actionGn mode of action

uterine blood uterine blood flow:flow:

increasesincreases the the uterine blood flow uterine blood flow during the early during the early luteal phase, a luteal phase, a periimplantation periimplantation stage stage

50

60

70

80

90

100

110

1° 5° 9° 14° 16° 19° 24°

(Index Resistance)

Gn mode of actionGn mode of action

increase in the number of receptor in preparation for ovulation

After ovulation, the luteinized ovary maintains LH-R-s that allow activation in case there is an implantation

receptors activationreceptors activation

~1% receptor sites activated

•binding LH to the external part of the membrane spanning receptor

•with LH attached, the receptor shifts conformation and thus•mechanically activates the G protein

•and activates the cAMP system

The seven transmembrane α-helix structure of a G protein-coupled receptor such as LHCGR

Gn-R expressionGn-R expression

•Its expression requires appropriate hormonal Its expression requires appropriate hormonal stimulation by FSH and estradiolstimulation by FSH and estradiol

present on:present on:• granulosa cells granulosa cells • theca cellstheca cells• luteal cells luteal cells • interstitial cellsinterstitial cells

Extragonadal Gn-RsExtragonadal Gn-Rs• Gn-Rs have been found in:Gn-Rs have been found in:

the uterus, the uterus, sperm,sperm, seminal vesicles, seminal vesicles, prostate, prostate, skin, skin, breast, breast, adrenals, adrenals, thyroid, thyroid, neural retina, neural retina, neuroendocrine cells, neuroendocrine cells, and (rat) brain.and (rat) brain.

• physiologic role largely unexploredphysiologic role largely unexplored.

Gn action in ovary Gn action in ovary

follicular maturation follicular maturation ovulationovulation luteal function luteal function

Gonadotropin’s avverse effectsGonadotropin’s avverse effects

OHSSOHSS Ovarian volume increasedOvarian volume increased Multiple pregnanciesMultiple pregnancies GynecomastiaGynecomastia

FSH in early follicular phaseFSH in early follicular phaseFSH thresholdFSH threshold: FSH serum concentrations needed to stimulate ovarian follicle : FSH serum concentrations needed to stimulate ovarian follicle growthgrowth (Brown 1978) (Brown 1978)

• At the onset of the menstrual cycle, a cohort of small (2–5 mm) antral follicles is present in each ovary

• This cohort will continue to grow in response to stimulation by FSH

• a process referred to as follicle recruitment • The follicle with the highest sensitivity will benefit most from

increasing FSH levels and will subsequently gain dominance (leader leader)

Brown JB. 1978 Pituitary control of ovarian function: concepts derived from gonadotropin Brown JB. 1978 Pituitary control of ovarian function: concepts derived from gonadotropin therapy. Aust NZ J Obstet Gynaecol. 18:47–54 therapy. Aust NZ J Obstet Gynaecol. 18:47–54

Scheele F, Schoemaker J. 1996 The role of follicle-stimulating hormone in the selection of Scheele F, Schoemaker J. 1996 The role of follicle-stimulating hormone in the selection of follicles in human ovaries: a survey of the literature and a proposed model. Gynecol follicles in human ovaries: a survey of the literature and a proposed model. Gynecol Endocrinol. 10:55–66. Endocrinol. 10:55–66.

FSH in early follicular phaseFSH in early follicular phase

• not increase much during a normal not increase much during a normal ovulatory cycle ovulatory cycle

• FSH concentrations only FSH concentrations only 10–30%10–30% above the above the threshold level is sufficient threshold level is sufficient to stimulate to stimulate normal follicle developmentnormal follicle development

**Brown JB. 1978 Pituitary control of ovarian function: concepts derived from Brown JB. 1978 Pituitary control of ovarian function: concepts derived from gonadotropin therapy. Aust NZ J Obstet Gynaecol. 18:47–54.gonadotropin therapy. Aust NZ J Obstet Gynaecol. 18:47–54.

****Messinis IE, Templeton AA. 1990 The importance of follicle-stimulating hormone Messinis IE, Templeton AA. 1990 The importance of follicle-stimulating hormone increase for folliculogenesis. Hum Reprod. 5:153–156.increase for folliculogenesis. Hum Reprod. 5:153–156.

FSH concentrations reach a maximum in the early follicular phase of the FSH concentrations reach a maximum in the early follicular phase of the normal menstrual cycle and decrease thereafternormal menstrual cycle and decrease thereafter

FSH in follicular phaseFSH in follicular phase

Stimulates:

1. follicular growth,

2. granulosa cell aromatase activity,

3. induction of LH receptors on the granulosa cell membrane,

4. estradiol secretion

•enzyme of the cytochrome P450 group

•mediate androgens aromatization:

producing estrogens producing estrogens sexual developmentsexual development

Aromatase

FSH in late follicular phaseFSH in late follicular phasedecreasedecrease due to increased ovarian secretion of:due to increased ovarian secretion of:

EE22

ββ--inhibin inhibin

Hotchkiss J, Knobil E. 1994 The menstrual cycle and its neuroendocrine control. In: Knobil Hotchkiss J, Knobil E. 1994 The menstrual cycle and its neuroendocrine control. In: Knobil E, Neill JD, eds. The physiology of reproduction. New York: Raven Press; 711–750. E, Neill JD, eds. The physiology of reproduction. New York: Raven Press; 711–750.

Groome NP, Illingworth PJ, O’Brien M, et al. 1996 Measurement of dimeric inhibin B Groome NP, Illingworth PJ, O’Brien M, et al. 1996 Measurement of dimeric inhibin B throughout the human menstrual cycle. J Clin Endocrinol Metab. 81:1401–1405.throughout the human menstrual cycle. J Clin Endocrinol Metab. 81:1401–1405.

negative feedback at the hypothalamic-pituitary levelnegative feedback at the hypothalamic-pituitary level

Thecal Cell Granulosa CellLHLH

cAMcAMPP

Protein Protein Kinase Kinase AA

cholesterol

pregnenolone

17-OH-P

DHEA

A

CYP11

CYP17

CYP17

3βHSD

A

E1

E2

FSHFSH

RR

RR

blood

Protein kinase

cAMP

P450

17βHSD

BBaasseemmeenntt

MMeemmbbrraannee

P4P4

AldostAldostCortisolCortisol

Steroidogenesis

FSH follicular decreasingFSH follicular decreasing

• strict relationship with dominant follicle strict relationship with dominant follicle developmentdevelopment

Schipper I, Hop J and Fauser B:Schipper I, Hop J and Fauser B: “ “The Follicle-Stimulating Hormone (FSH) Threshold/Window Concept Examined by The Follicle-Stimulating Hormone (FSH) Threshold/Window Concept Examined by

DifferentDifferent Interventions with Exogenous FSH during the Follicular Phase of the Normal Menstrual Cycle: Duration, Interventions with Exogenous FSH during the Follicular Phase of the Normal Menstrual Cycle: Duration, Rather Than Magnitude, of FSH Increase Affects Follicle Development”. The Journal of Clinical Endocrinology & Rather Than Magnitude, of FSH Increase Affects Follicle Development”. The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 4 1292-1298 Metabolism Vol. 83, No. 4 1292-1298

• As a consequence, other recruited As a consequence, other recruited follicles lackfollicles lack sufficient stimulation by sufficient stimulation by FSH and enter atresiaFSH and enter atresia

Zeleznik AJ, Hutchison JS, Schuler HM. 1985 Interference with the gonadotropin-Zeleznik AJ, Hutchison JS, Schuler HM. 1985 Interference with the gonadotropin-suppressing actions of estradiol in macaques overrides the selection of a single suppressing actions of estradiol in macaques overrides the selection of a single preovulatory follicle. Endocrinology. 117:991–999.preovulatory follicle. Endocrinology. 117:991–999.

FSH follicular decreasingFSH follicular decreasing

• Apparently, the maturing dominant follicle requires Apparently, the maturing dominant follicle requires less FSH to continue its growth. less FSH to continue its growth.

• It’s due to up-regulated FSH-sensitivity of leading It’s due to up-regulated FSH-sensitivity of leading follicle for: follicle for:

1.1. induction of locally various growth factors (IGF-I, induction of locally various growth factors (IGF-I, AMH, AMH, inibina B, leptina, ICAM-1, VCAM-1, VEGFinibina B, leptina, ICAM-1, VCAM-1, VEGF))

2.2. induction of LH receptors that enhance FSH induction of LH receptors that enhance FSH sensitivitysensitivity

•Erickson GF. 1996 The ovarian connection. In: Adashi EY, Rock JA, Rosenwaks Z, eds. Reproductive endocrinology, surgery, and technology. Philadephia: Lippincott-Raven; 1141–1160.

FSH in late luteal phaseFSH in late luteal phase

• At the end of the luteal phase, there is a At the end of the luteal phase, there is a slight rise in FSH that seems to be of slight rise in FSH that seems to be of importance to start the next ovulatory cycleimportance to start the next ovulatory cycle

• a cohort of small antral follicles is prevented a cohort of small antral follicles is prevented from undergoing atresia and is stimulated from undergoing atresia and is stimulated for further developmentfor further development

Hodgen GD. 1982 The dominant ovarian follicle. Fertil Steril. 38:281–300 Hodgen GD. 1982 The dominant ovarian follicle. Fertil Steril. 38:281–300

LH mode actionLH mode action With the rise in estrogens, LH receptors are also With the rise in estrogens, LH receptors are also

expressed on the maturing follicleexpressed on the maturing follicle estrogen rise leads via the hypothalamic interface to the estrogen rise leads via the hypothalamic interface to the

“positive LH feed-back” effect, a release of LH over a “positive LH feed-back” effect, a release of LH over a 24-48 hour period24-48 hour period

This 'LH surge' triggers ovulationThis 'LH surge' triggers ovulation LH is necessary to maintain luteal function (P4) for the LH is necessary to maintain luteal function (P4) for the

first two weeksfirst two weeks LH supports thecal cells in the ovary that provide LH supports thecal cells in the ovary that provide

androgens and hormonal precursors for estradiol androgens and hormonal precursors for estradiol productionproduction

In case of a pregnancy luteal function will be further In case of a pregnancy luteal function will be further maintained by the action of hCG (a hormone very maintained by the action of hCG (a hormone very similar to LH) from the newly established pregnancysimilar to LH) from the newly established pregnancy

FSH geneFSH gene

αα-chain gene-chain gene locate in arme 6p21.1-23 locate in arme 6p21.1-23

ββ-chain -chain gene: gene: locate in 11p13 only in gonadotrope cells of locate in 11p13 only in gonadotrope cells of

pituitary gland pituitary gland increased by Gn-RH and activine increased by Gn-RH and activine decreased by inhibinedecreased by inhibine

Deficient gonadotropin’s levelDeficient gonadotropin’s level

hypogonadism and amenorrhoeahypogonadism and amenorrhoea: Kallmann syndrome Kallmann syndrome Hypothalamic suppressionHypothalamic suppressionHypopituitarismHypopituitarismEating disorder (leptine)Eating disorder (leptine)HyperprolactinemiaHyperprolactinemiaGonadotropin deficiencyGonadotropin deficiencyGonadal suppression therapy Gonadal suppression therapy

• GnRH antagonistGnRH antagonist• GnRH agonist (downregulation)GnRH agonist (downregulation)

LH-R abnormalitiesLH-R abnormalities

• in females can lead to infertilityin females can lead to infertility

• masculinization masculinization

• In 46, XY pseudohermaphroditism, In 46, XY pseudohermaphroditism,

• hypospadiashypospadias

• micropenismicropenis

Antibodies to LH-R can interfere with LH-R activityAntibodies to LH-R can interfere with LH-R activity

High Gonadotropin levelsHigh Gonadotropin levels

Premature menopause Premature menopause Gonadal dysgenesis, Turner syndrome Gonadal dysgenesis, Turner syndrome CastrationCastration Swyer syndrome Swyer syndrome Polycystic Ovary Syndrome Polycystic Ovary Syndrome Certain forms of CAHCertain forms of CAH Testicular failureTesticular failure

Persistently high LH levels are indicative of situations where the normal restricting Persistently high LH levels are indicative of situations where the normal restricting feedback from the gonad is absent, leading to a pituitary production of both LH and FSH.feedback from the gonad is absent, leading to a pituitary production of both LH and FSH.

typical in the menopausetypical in the menopause

FSH in COHFSH in COH• multiple follicle development is induced by elevating

FSH concentrations far above the threshold • By starting with a lower dose of gonadotropins and

stepwise small increments, chances of inducing monofollicular growth should increase with a concomitant reduction of complications (step-up protocol)

• However, these stimulation protocols are characterized by FSH concentrations remaining above the threshold

Polson DW, Mason HD, Saldahna MBY, Franks S. 1987 Ovulation of a single dominant follicle during treatment with low-dose pusatile follicle stimulating hormone in women with polcystic ovary syndrome. Clin Endocrinol

(Oxf). 26:205–212. White DM, Polson DW, Kiddy D, et al. 1996 Induction of ovulation with low-dose gonadotropins in polycystic

ovary syndrome: an analysis of 109 pregnancies in 225 women. J Clin Endocrinol Metab. 81:3821–3824.

FSH gateFSH gate• the "FSH-gate" or "FSH-window" concept has been the "FSH-gate" or "FSH-window" concept has been

proposed, which adds the element of time to the FSH proposed, which adds the element of time to the FSH threshold theory and emphasizes the significance of a threshold theory and emphasizes the significance of a transient increase in FSH above the threshold level for transient increase in FSH above the threshold level for single dominant follicle development single dominant follicle development **

• Moreover, step-down dose regimen COH, has proven Moreover, step-down dose regimen COH, has proven successful in reducing the incidence of multiple follicle successful in reducing the incidence of multiple follicle developmentdevelopment ****

** ** van Santbrink EJP, Donderwinkel PFJ, van Dessel HJHM, Fauser BCJM. 1995 van Santbrink EJP, Donderwinkel PFJ, van Dessel HJHM, Fauser BCJM. 1995

Gonadotrophin induction of ovulation using a step-down dose regimen: single-centre clinical Gonadotrophin induction of ovulation using a step-down dose regimen: single-centre clinical experience in 82 patients. Hum Reprod. 10:1048–1053experience in 82 patients. Hum Reprod. 10:1048–1053

**Baird DT. 1987 A model for follicular selection and ovulation: lessons from superovulation. Baird DT. 1987 A model for follicular selection and ovulation: lessons from superovulation.

J Steroid Biochem. 27:15–23 J Steroid Biochem. 27:15–23

FSH windowFSH window

• the FSH window concept has been the FSH window concept has been proposed, proposed, stressingstressing the significance of the significance of the (limited) the (limited) duration of FSH elevationduration of FSH elevation above the threshold level above the threshold level

• rather than the height of the elevationrather than the height of the elevation of of FSH for single dominant follicle selectionFSH for single dominant follicle selection

Fauser BCJM, van Heusden AM. 1997 Manipulation of human ovarian function: physiological Fauser BCJM, van Heusden AM. 1997 Manipulation of human ovarian function: physiological concepts and clinical consequences. Endocr Rev. 18:71–106.concepts and clinical consequences. Endocr Rev. 18:71–106.

Gn dosage

• For assisted reproductive technology procedures, the usual initial dose is 150 IU to 225 IU daily for 5 days.

• The dose is then adjusted according to response and is usually continued for 6 to 12 days.

• When an adequate response is achieved, this medication is stopped and another medication, hCG, is given to induce ovulation.

FSH initial dosesFSH initial doses

patient’s agepatient’s age basal FSH basal FSH PCOSPCOS

HCG HCG

Gonasi fl i.m. 1000, 2000, 5.000 UIGonasi fl i.m. 1000, 2000, 5.000 UI

• HCGHCG• pregnant women urinepregnant women urine• made by the placenta made by the placenta • LH-activity likeLH-activity like• > half-life LH (4 h vs. 15 min)> half-life LH (4 h vs. 15 min)

hCG in normal pregnancy hCG in normal pregnancy

0

20000

40000

60000

80000

100000

120000

140000

0 7 8 9 10 11 12 13 14 16 19 20 39

HCG HCG

It is heterodimeric glycoprotein:It is heterodimeric glycoprotein:–α subunit identical to LH, FSH, TSH α subunit identical to LH, FSH, TSH –β subunit unique to hCGβ subunit unique to hCG–92 + 152 amino acids92 + 152 amino acids

HCG mode actionHCG mode action

interacts with the LHCG receptor interacts with the LHCG receptor

Follicle rupture inductionFollicle rupture induction

maintenance of the corpus luteum maintenance of the corpus luteum during the beginning of pregnancy, during the beginning of pregnancy,

causing it to secrete P4causing it to secrete P4

meiosis restarting meiosis restarting

HMGHMG

(Pergonal), (Metrodin), Menogon, Fostimon fl i.m. 75 UI

Menotropin (HMG) (1965s)Menotropin (HMG) (1965s)– Climateric women urine Climateric women urine

• FSH + LH (FSH + LH (~~ 50%)50%)– 5% Gn + 95% urinary proteins5% Gn + 95% urinary proteins

Urofollitropin (1983)Urofollitropin (1983)— Purified FSH (>95%)Purified FSH (>95%)

— purified by chromatographic techniquespurified by chromatographic techniques

Chinese hamster (Chinese hamster (Cricetulus griseusCricetulus griseus), white spotted type), white spotted type

r-FSHr-FSH

produced by inserting the genes produced by inserting the genes encoding for encoding for αα and and ββ subunits of subunits of FSH into expression vectors that FSH into expression vectors that are transfected into a Chinese are transfected into a Chinese hamster ovary cell linehamster ovary cell line

Gonal-F, Puregon fl s.c., penGonal-F, Puregon fl s.c., pen

Purification by immunochromatography using an antibody specifically binding FSH

r-FSHr-FSH

19951995

European Medicines European Medicines Evaluation Agency (EMEA)Evaluation Agency (EMEA)

Gonal-F, Puregon fl s.c., penGonal-F, Puregon fl s.c., pen

r-FSH propertiesr-FSH properties

knockdown degradation rateknockdown degradation ratereduced variability reduced variability interblocks firmnessinterblocks firmnesshigh degree of purenesshigh degree of purenessdiminished immunizationdiminished immunization

r-FSH r-FSH

r-LH (Luveris 75 IU fl s.c.)

β-follitropine (Puregon)

α-follitropine (Gonal F)

HMG vs. r-FSHHMG vs. r-FSH

• both products are probably equally both products are probably equally safe and similar in efficacy, based on safe and similar in efficacy, based on the available literature to datethe available literature to date.

Matorras R, Rodriguez-Escudero FG: “Debate. Bye-bye urinary gonadotropins?” . Hum Reprod . 2002;17:1675–1683

Suheil J. Muasher, Rony T. Abdallah, Ziad R. Hubayter: “Optimal stimulation protocols for in vitro fertilization”. Fertil Steril 2006; 86,2:267-273

FSH-HMG-HCG targetFSH-HMG-HCG target

targettarget FSH/FSH/HMGHMG LHLH HCGHCG

Follicles recruitm

ent+ + +

Oocytes maturati

on+ + + + + + +

Ovulation

trigger+ + + + +

E2 + + +P4 + + + +

Gonadotropin 75 UI 1.500 UI

Meropur € 12,28 € 245,6

Fostimon € 16,09 € 321,8

Puregon € 45,83 € 916,6

Gonal-F € 51,39 € 1027,8

Gn Gn availableavailable in the in the Italy marketItaly market

CCOOHH

PPRROOTTOOCCOOLLSS

CCCC

M. M. Quigley: M. M. Quigley: Annals of the New York Academy of Sciences, Vol 442, 1: 96-111Annals of the New York Academy of Sciences, Vol 442, 1: 96-111

•CC alone produces sufficientsufficient enhanced follicular recruitment

•Clomiphene alone had significantly fewer follicles

•necessary gonadotropin support to be continued to prevent atresia of some of the cohort of follicles

•supplemental hCG/P4 to corrected short luteal phases

CC + HMGCC + HMG

• CC 100 mg/day on day 3–7 of the menstrual cycle

• HMGHMG150 IU every other day starting on day 5starting on day 5

• HCG 10.000 on leading follicle >18 mm and at least two follicles >15 mm

• Pick-up or IUI 36-48 hours after

• HCG 5.000 UI 6 days after (staff conversion)HCG 5.000 UI 6 days after (staff conversion)• P4 50 mg/d i.m. on HCG day or E-T day

M. M. Quigley: M. M. Quigley: Annals of the New York Academy of Sciences, Vol 442, Issue 1 96-111Annals of the New York Academy of Sciences, Vol 442, Issue 1 96-111

CC + delayed HMGCC + delayed HMG

• CC 100 mg/day on day 1–5 day• HMG150 IU every other day starting on day 6 • HCG 10.000 on leading follicle >18 mm and at least

two follicles >15 mm• IUI or Pick-up 36-48 hours after• HCG 5.000 UI 6 days after (staff conversion)HCG 5.000 UI 6 days after (staff conversion)

• P4 50 mg/d i.m. on HCG day or E-T day

CC + HMGCC + HMG

1.1. best chance of COHbest chance of COH

2.2. minimize the disruption of the minimize the disruption of the subsequent luteal phasesubsequent luteal phase

3.3. increased pregnancy rateincreased pregnancy rate

M. M. Quigley: M. M. Quigley: Annals of the New York Academy of Sciences, Vol 442, Issue 1 96-111Annals of the New York Academy of Sciences, Vol 442, Issue 1 96-111

CC + HMG ovulation outcomeCC + HMG ovulation outcome

90% for cycle90% for cycle

CC/HMG Pregnancy OutcomeCC/HMG Pregnancy Outcome

USG pregnancy rate/cycle: 25% **

live-birth rates/cycle : 13-17% **

* * Published overallPublished overall

Miscarriage • integrins down regulation integrins down regulation

(markers of endometrial (markers of endometrial receptivity)receptivity)

• endometrial endometrial EE/P-r depletionEE/P-r depletion

• uterine artery flowuterine artery flow

impaired impaired endometrial endometrial developmentdevelopment

Low Pr in CC/Gn COH

premature LH surgepremature LH surge

immature oocytesimmature oocytes

desynchronized endometrial developmentdesynchronized endometrial development

CC/HMG Pr lower CC/HMG Pr lower

• over 38 years old

• low ovarian reserve

• poor quality sperm

• endometriosis

• tubal damage or pelvic scar tissue

• infertility >3 years

CC/HMG adverse effects CC/HMG adverse effects

3,5% twin 1/3 of admission in TIN Twin/mono mortality 10 +

PIH 5 – 10 + placenta previa Placenta detachment

CC + ECC + E22

• CC 100 mg/d on 3° cycle day

• EE 0.05 mg/d on days 8-12on days 8-12

• hCG 10,000 IU at least one follicle was >18 mm

• A single IUI/Pick-up 24–36 hours after

• progesterone 50 mg daily IM • on day of E-T

or• 3 days after IUI*• until β-hCG levels were evaluated

** Gerli: Intrauterine insemination. Fertil Steril 2000; 73,1:85-89 Gerli: Intrauterine insemination. Fertil Steril 2000; 73,1:85-89

CC + ECC + E22

endometrial thickness on the day of hCG administration.

= CC only = CC + ethinyl E2

0

5

10

15

20

25

30

>6 >9 >15

CC + ECC + E22

Characteristics and outcome of patients who received CC Characteristics and outcome of patients who received CC plus ethinyl E2 (group A) or CC alone (group B) in IUI cycleplus ethinyl E2 (group A) or CC alone (group B) in IUI cycle

CharacteristicCharacteristic Group AGroup A Group BGroup B P valueP value

No. of patientsNo. of patients 3232 3232 - -- -

Mean (±SD) age (y) Mean (±SD) age (y) 28.0 ± 5.628.0 ± 5.6 26.0 ± 4.226.0 ± 4.2 NSNS

Mean (±SD) duration Mean (±SD) duration of infertility (mo)of infertility (mo)

48.1 ± 18.548.1 ± 18.5 36.7 ± 9.636.7 ± 9.6 NSNS

Ongoing PregnancyOngoing Pregnancy 12 (37.5)12 (37.5) 2 (6.25)2 (6.25) <.05<.05

MiscarriedMiscarried 2 (6.25)2 (6.25) 6 (18.75)6 (18.75) <.05<.05

pulsatility index pulsatility index values values no differenceno difference no no

differencedifference - -- -

Traditional COHTraditional COH

HMG or r-FSH 300 IU on 2° day cycle

HCG 10.000 IU on leading follicle >17 mm and at least two follicles >15 mm

Pick-up after 33-36 h

P4 50 mg i.m. for luteal supplementation

Traditional Traditional COHCOH

• FSH remain elevatedFSH remain elevated

• recruitment and growth of ovarian follicles recruitment and growth of ovarian follicles continues throughout treatmentcontinues throughout treatment

* Filicori M: * Filicori M: Characterization of the physiological pattern of episodic Characterization of the physiological pattern of episodic gonadotropin secretion throughout the human menstrual cycle gonadotropin secretion throughout the human menstrual cycle . . J Clin J Clin Endocrinol Metab Endocrinol Metab . 1986;62:1136–1144. 1986;62:1136–1144

This FSH serum pattern profoundly diverges This FSH serum pattern profoundly diverges from the spontaneous menstrual cyclefrom the spontaneous menstrual cycle

Traditional COHTraditional COH

• heterogeneous size cohortsheterogeneous size cohorts of follicles of follicles are often found at hCG dayare often found at hCG day

• the optimal outcome of COH would be the optimal outcome of COH would be the selective attainment of numerous the selective attainment of numerous large mature homogeneous follicles.large mature homogeneous follicles.

* * Arnot AM , Vandekerckhove P , DeBono MA , Rutherford AJ . Arnot AM , Vandekerckhove P , DeBono MA , Rutherford AJ . Follicular volume Follicular volume and number during in-vitro fertilization (association with oocyte developmental and number during in-vitro fertilization (association with oocyte developmental capacity and pregnancy rate) capacity and pregnancy rate) . . Hum Reprod Hum Reprod . 1995;10:256–261 . 1995;10:256–261

Traditional Traditional protocolprotocol

Long Long protocolprotocol

n° ampulesn° ampules 5.75.7 2525

Mature Mature oocytesoocytes 88 1616

Fertilization Fertilization raterate 83%83% 78%78%

PR/ETPR/ET 28%28% 31%31%

Cost-savingCost-saving + + ++ + + — —— —

MFMF — —— — + + ++ + +

StressStress — —— — + + ++ + +

Gn-RHGn-RH

• Gn-RH neurons are inside the Gn-RH neurons are inside the medium-basal hypothalamus (arcuate medium-basal hypothalamus (arcuate nucleus and median eminence)nucleus and median eminence)

• Lately scientists showed Gn-RH Lately scientists showed Gn-RH syntesis in pituitary gland toosyntesis in pituitary gland too

Gn-RH biochemistry (1977s)Gn-RH biochemistry (1977s)

a decapeptide (10 amino acids) in mammals. This chain is represented by: pyroGlu-His-Tyr-Ser-Gly-Leu-Arg-Pro-Gly-NH2

The identity of GN-RH1 was clarified by the 1977 Nobel Laureates Roger Guillemin and Andrew V. Schally

Pituitary gland histologyPituitary gland histology

Mel

anoc

yte-

stim

ulat

ing h

orm

one

(

Mel

anocy

te-s

timul

atin

g ho

rmon

e (M

SHM

SH))

is th

e pr

edom

inan

t hor

mon

e se

cret

ed

is th

e pr

edom

inan

t hor

mon

e se

cret

ed

by p

ars

inte

rmed

ia (

part

of aden

ohyphy

sis)

by p

ars

inte

rmed

ia (

part

of a

denoh

yphy

sis)

The pars nervosa stores stores • ADH ADH and • Oxytocin Oxytocin which were secreted by the hypothalamushypothalamus.

NEUROHYPOPHYSIS - PARS NERVOSANEUROHYPOPHYSIS - PARS NERVOSA

This region of the pituitary is non secretory. Its cells are neuroglial-like pituicytes.

Melatonin/steroidogenesisMelatonin/steroidogenesis

• The direct involvement of melatonin in modulation of ovarian steroidogenesis, the high levels of melatonin found in human follicular fluid, and the presence of melatonin binding sites in the ovary led us to hypothesize that melatonin acts as a modulator of ovarian function.

• the mechanism of melatonin action at the level of the ovary is still poorly understood

Melatonin/steroidogenesis +Melatonin/steroidogenesis +

HCGHCG

Melatonin-rMelatonin-r

Granulosa luteal cellsGranulosa luteal cells

PP44

Gn-RH secretion males/females

in males, in pulses at a constant frequencyin males, in pulses at a constant frequency • in females the frequency of the pulses varies in females the frequency of the pulses varies during the menstrual cycleduring the menstrual cycle

• there is a large surge of GN-RHthere is a large surge of GN-RH11 just before just before ovulationovulation

Gn-RH frequencyGn-RH frequency

•Low frequency Low frequency FSH releaseFSH release

•high frequency high frequency LH release LH release

The seven transmembrane α-helix The seven transmembrane α-helix structure of a G protein-coupled structure of a G protein-coupled

receptorreceptor

Gn-Rh analoguesGn-Rh analogues

• While Gn-RH1 has been synthesized and become available, its short half-life requires infusion pumps for its clinical use.

• Modifications of the decapeptide structure of Gn-RH1 have led to Gn-RH1 analog medications that either stimulate (Gn-RH1 agonists) or suppress (Gn-RH1 antagonists) the gonadotropins

Effects of Gn-RH analogues Effects of Gn-RH analogues

agonistagonist antagonistantagonist

Prevent premature luteinization + ++ + + + + + + +

Prevent premature ovulation + + + + + + + + + + To synchronize early follicular development + + ++ + + + +

Gn-RH agonistGn-RH agonist• is a synthetic peptide modeled after the hypothalamic

neurohormone Gn-RH that interacts with its receptor to elicit its biologic response, the release of the pituitary hormones FSH and LH

• Agonists do not quickly dissociate from the Gn-RH receptor

• As a result initially there is an increase in FSH and LH secretion (so-called flare-up effect)

• however after about ten days a profound hypogonadal effect is achieved through receptor down-regulation. Generally this induced and reversible hypogonadism is the therapeutic goal.

• Gn-RH agonists are synthetically modeled after the natural Gn-RH decapeptide with specific amino acid substitutions typically in position 6 and 10.

Gn-RH-aGn-RH-aAminoacid sequenceAminoacid sequence

namename actact 11 22 33 44 55 66 77 88 99 1010 forfor

Gn-RHGn-RH 11

PyroPyro-glu-glu

HiHiss

TrpTrp

sese

roro

toto

ninnin

TyrTyr

LeuLeu

LeLeuu

ArgArg PrProo

Gly-Gly-NHNH22

iviv

LeuprorelineLeuproreline** 1515 D-LeuD-LeuN-N-

EtNHEtNH22sc, imsc, im

Buserelin Buserelin * ** * 2020 D-SerD-SerN-N-

EtNHEtNH22sc, imsc, im

triptor triptor * ** * **D-D-

TripTrip sc, imsc, im

GoserelinGoserelin* * ** * *

**101000

D-SerD-SerAzGly-AzGly-

NHNH22depot scdepot sc

* * Enantone 3.75, 11.25 mg fl s.c. im; Enantone die 1 mg/die (0.2 ml) fl s.c.; Enantone 3.75, 11.25 mg fl s.c. im; Enantone die 1 mg/die (0.2 ml) fl s.c.;

* ** * Suprefact 5.5 ml fl s.c.; Suprefact spray nasale 10 gr (1 buff = 200 mg) Suprefact 5.5 ml fl s.c.; Suprefact spray nasale 10 gr (1 buff = 200 mg)

* * ** * * Decapeptyl 3.75, 11.25 mg fl s.c. im; Decapeptyl die 0.1 mg fl s.c. Decapeptyl 3.75, 11.25 mg fl s.c. im; Decapeptyl die 0.1 mg fl s.c.

* * * * * * * * ZoladexZoladex 3.6, 10.8 mg fl s.c. im3.6, 10.8 mg fl s.c. im

TriptorelinTriptorelin is an agonist with only a single substitution at position 6 is an agonist with only a single substitution at position 6

Gn-RH-a Gn-RH-a

brand namebrand name InjectionInjection mg/mlmg/ml

Leuproreline Leuproreline acetateacetate Enantone dieEnantone die

1 fl 1.6 ml 1 fl 1.6 ml

8 doses8 doses1 mg (0.2 ml) s.c1 mg (0.2 ml) s.c

TriptorelineTriptoreline

Decapeptyl Decapeptyl depotdepot 1 fl i.m.1 fl i.m. 3.75 mg3.75 mg

Decapeptyl dieDecapeptyl die14 fl 14 fl

pre-filledpre-filled

0.2 ml 0.2 ml

(0.1 mg) s.c. daily(0.1 mg) s.c. daily

BuserelinBuserelinSuprefact flSuprefact fl 5.5 ml5.5 ml 0.5 ml/d0.5 ml/d

sprayspray 1 flac1 flac 1 buff = 100 1 buff = 100 μμgg

triptorelin, buserelin and goserelin are equally effective triptorelin, buserelin and goserelin are equally effective

Gn-RH-a pharmacokinetics Gn-RH-a pharmacokinetics

two hours: peak serum. It rapidly binds to the LHRH receptor cells in

the pituitary gland thus leading to an initial increase in

production of LH (flare-upflare-up)

after 10 days: receptor desensitizationreceptor desensitization

and/or down-regulationdown-regulation

Gn-RH-aGn-RH-a

lysine replacement withlysine replacement with

ethylamide in 10 → half-time ethylamide in 10 → half-time (4 min (4 min vsvs 3 h) 3 h)

Gn-RH-aGn-RH-a

lysine replacement with D-lysine replacement with D-amynoacide in 6 amynoacide in 6 → → Increase Increase effectiveness (15-100 times)effectiveness (15-100 times)

D-aminoacid is hydrophobe chain D-aminoacid is hydrophobe chain carrier with enhancement receptor linkcarrier with enhancement receptor link

Gn-RH-a effectsGn-RH-a effects

Follicles synchronization ++++

Fewer small follicles on HCG day ++

Avoids premature luteinization ++++

Multiple pregnancies ≡ ≡ ≡

Decreases OHSS frequency ≡ ≡ ≡

Triptoreline depot serum levels

0

0,5

1

0 8 16 24 32 40

Lin

ee 3

D 1

DAYS

ng

/ m

l

TriptorelinTriptorelin[d-Trp6]GnRH[d-Trp6]GnRH

Goserelin*Goserelin*

* Zoladex 3.75 mg, 11.25 mg fl im (FDA, 1989)

D-Ser(But)66Azgly1010LHRH

Goserelin*Goserelin*

has a serum elimination half-life of two to four hours in patients with normal renal function.

After administration, peak serum concentrations are reached in about two hours

after a period of about 14-21 days, production of LH is greatly reduced due to receptor downregulation

Gn-RH-a protocolsGn-RH-a protocols

long protocollong protocol short (“flare-up”) protocol short (“flare-up”) protocol ultrashort protocolultrashort protocolmicrodose flare protocolmicrodose flare protocol

Long protocol:Long protocol:

1. Avoid pre-menses FSH surge2. Follicles timing3. Avoid premature LH surge4. Higher follicular recruitment

(synchronization)5. Improvement immune attitude6. Expensive cost

High respondersHigh respondersPCOSPCOS

short protocolsshort protocols

1. follicles timing 2. avoid premature LH surge3. lower follicular recruitment4. make procedures easier

Poor respondersPoor responders

Serum levelsSerum levels Short protocolShort protocol Long protocolLong protocol

E2 idem Idem

D4 ++++++ ++depression ++++++ ++ Pregnancy rate/cicle 9.2%9.2% 16. 5%16. 5%

PR/transfer 9..9%9..9% 23. 5%23. 5%

patients

«poor responders» «High responders»

PCOS

> 40 years hyrsutism

HMG ampules + + - -+ + - - + + + ++ + + +

Cancelled cycles + + - -+ + - - + + + ++ + + +

Short/long protocol (Volpicelli V. 2003)

PR/transfer in Gn-RH-aPR/transfer in Gn-RH-a

FIV nel periodo 92-96 (da FIV-NAT ’97) sec. FIV nel periodo 92-96 (da FIV-NAT ’97) sec. Barrière et al. 1999Barrière et al. 1999

Flare-up Flare-up protocolprotocol 19.2%19.2%

Long protocol Long protocol 25.7%25.7%

MediaMedia 24.8%24.8%

without without analoguesanalogues 23.2%23.2%

Gn-RH-a Long protocolGn-RH-a Long protocol

• Gn-Rh-a depot 3.75 mg in one dose on 21Gn-Rh-a depot 3.75 mg in one dose on 21stst day only of day only of previous cycle previous cycle

• Gn-Rh-a low-dose daily on the 21Gn-Rh-a low-dose daily on the 21stst day of previous cicle to day of previous cicle to HCG day:HCG day:

• Buserelin (Suprefact fl 5.5 ml) 0.3 ml fl s.c. Buserelin (Suprefact fl 5.5 ml) 0.3 ml fl s.c. • Buserelin nasally 1 buff x 3/d (300 Buserelin nasally 1 buff x 3/d (300 μμg)g)• Leuproreline (Enantone die fl s.c.) 0.2 ml/dayLeuproreline (Enantone die fl s.c.) 0.2 ml/day• Triptoreline (Decapeptyl die fl s.c.) 0.2 mlTriptoreline (Decapeptyl die fl s.c.) 0.2 ml

oror• on any day when:on any day when:

» LH <0.5 LH <0.5 » EE22 <30 <30» No ovarian cyst >10 mmNo ovarian cyst >10 mm

88

Gn-RH-a long protocolGn-RH-a long protocol

•r-FSH/HMG 300-650 IU/day on 2r-FSH/HMG 300-650 IU/day on 2ndnd cycle day to HCG day cycle day to HCG day

•HCG 10.000 IU on the least two follicles >18 mmHCG 10.000 IU on the least two follicles >18 mm

•Pick-up after 33-36 hoursPick-up after 33-36 hours

•P4 supplementationP4 supplementation

•HCG 5.000 IU six days after E-THCG 5.000 IU six days after E-T

88

Short (flare-up) protocolShort (flare-up) protocol

• Gn-RH-a 3.75 mg depot ½ fl i.m. on 2° cycle Gn-RH-a 3.75 mg depot ½ fl i.m. on 2° cycle day onlyday only

• r-FSH 225-600 IU/d on 3th day (step-down regimen)

• HCG 10.000 IU (18 mm + 15-16)• Pick-up after 33-36 h• HCGHCG (+ P4)

poor responder

99

Gn-RH-a flare low dose protocol

• on 1on 1stst cycle day at HCG day: cycle day at HCG day:» Triptoreline (decapeptyl die) 0.2 ml (0.1 mg) s.c. dailyTriptoreline (decapeptyl die) 0.2 ml (0.1 mg) s.c. daily» Leuproreline acetate (enantone die) 0.2 ml (1 mg) s.c. dailyLeuproreline acetate (enantone die) 0.2 ml (1 mg) s.c. daily» Buserelin (Suprefact flac 5.5 ml) 0.3 ml s.c.Buserelin (Suprefact flac 5.5 ml) 0.3 ml s.c.» Buserelin nasally 3 buff/day (300 Buserelin nasally 3 buff/day (300 μμg)g)



• r-FSH/HMG 300-650 UI/d on 3r-FSH/HMG 300-650 UI/d on 3rdrd cycle day cycle day

After administration s.c. enantone die reachs a serum peak of 32.3 mg/ml in 0.6 hAfter administration s.c. enantone die reachs a serum peak of 32.3 mg/ml in 0.6 h

•EE-P for 1-2 cyclesEE-P for 1-2 cycles

Gn-RH-a ultrashort protocolGn-RH-a ultrashort protocol

• on 2on 2nd nd cycle day for three days:cycle day for three days:» Triptoreline 0.2 ml s.cTriptoreline 0.2 ml s.c» Leuproreline 0.2 ml s.c.Leuproreline 0.2 ml s.c.» Buserelin 0.5 ml s.c.Buserelin 0.5 ml s.c.» Buserelin nasally 3 buff/day Buserelin nasally 3 buff/day



• r-FSH/HMG on the 2r-FSH/HMG on the 2ndnd cycle day cycle day

1111

1111

UltrashortUltrashort LongLongHMG ampoules + + ++ + +cancelled cycles ~ ~ ~~ ~ ~ ~ ~ ~~ ~ ~n. oocytes ~ ~ ~~ ~ ~ ~ ~ ~~ ~ ~fertilization rate ~ ~ ~~ ~ ~ ~ ~ ~~ ~ ~embryo cleavage rate ~ ~ ~~ ~ ~ ~ ~ ~~ ~ ~supernumerary embryos

+ + ++ + +

Samuel F. Marcus: “ “Comparative trial between an ultra-short and long protocol of luteinizing hormone-releasing hormone agonist for ovarian stimulation in in-vitro fertilization”. Human Reproduction, 1993; Vol. 8, No. 2, pp. 238-243

HCG low-dose long protocol HCG low-dose long protocol • Granulosa cells in ovarian follicles of largerin ovarian follicles of larger size (>10–

12 mm) normally express the LH/hCG receptor and become sensitive to LH activity stimulation (1).

• For a long time it was thought that this physiologic phenomenon was finalized to make mature follicles susceptible to the midcycle LH surge and thus ovulate.

• Nevertheless, GCs LH/hCG receptors may also be highly relevant to permit continued dominant follicle growth in the spontaneous mid-late follicular phase, at a time when the physiologic serum FSH decline may curtail adequate GC support and growth.

• At this time LH appears capable of exerting virtually all LH appears capable of exerting virtually all the physiologic actions of FSHthe physiologic actions of FSH on GCs (2).

1212

1. Zeleznik AJ , Hillier SG . The role of gonadotropins in the selection of the preovulatory follicle . Clin Obstet Gynecol . 1984;27:927–940 . 2. Campbell BK , Dobson H , Baird DT , Scaramuzzi RJ . Examination of the relative role of FSH and LH in the mechanism of ovulatory follicle selection in sheep . J Reprod Fertil . 1999;117:355–367

HCG low-dose in a-long protocol HCG low-dose in a-long protocol • Based on this information we postulated

that LH activity could substitute FSH administration in the late stages of COH to allow larger follicles growth and maturation.

1212

1. Filicori M , Cognigni GE , Taraborrelli S , Parmegiani L , Bernardi S , Ciampaglia W . Intracytoplasmic sperm injection pregnancy after low-dose human chorionic gonadotropin alone to support ovarian folliculogenesis . Fertil Steril . 2002;78:414–416

HCG low-dose long protocol HCG low-dose long protocol • The longer half-life and greater affinity for the

LH/hCG receptor of hCG account for a potency ratio estimate of hCG-to-LH of around 1:6 (1,2).

• hCG alone (200hCG alone (200 IU/d), corresponding to IU/d), corresponding to roughly 1,200 IU/d of LHroughly 1,200 IU/d of LH

• The hCG is also drastically less expensive than recombinant FSH or hMG .

1212

1. Stokman PG , de Leeuw R , van den Wijngaard HA , Kloosterboer HJ , Vemer HM , Sanders AL . Human chorionic gonadotropin in commercial human menopausal gonadotropin preparations . Fertil Steril . 1993;60:175–178

2. Sullivan MW , Stewart-Akers A , Krasnow JS , Berga SL , Zeleznik AJ . Ovarian responses in women to recombinant follicle-stimulating hormone and luteinizing hormone (LH) (a role for LH in the final

stages of follicular maturation) . J Clin Endocrinol Metab . 1999;84:228–232

HCG low-dose in long protocol HCG low-dose in long protocol • Gn-RH-a long protocol

• r-FSH/hMG (1:1/2) 225-300 IU on 2° day at least six follicles >12 mm and E2 >300 pg/ml

• hCG 250 IU/day alonehCG 250 IU/day alone until the end of COH

Filicori M: Fertil Steril 2005: 84, 2:394-401

•reduced r-FSH/hMG consumptionreduced r-FSH/hMG consumption•outcome comparable to traditional COH regimens; outcome comparable to traditional COH regimens; •reduced number of small preovulatory follicles;reduced number of small preovulatory follicles;•did not cause premature luteinization; did not cause premature luteinization; •more estrogenic intrafollicular environmentmore estrogenic intrafollicular environment 1212

or

variable amounts of r-FSH and low-dose (10-50) IU hCGvariable amounts of r-FSH and low-dose (10-50) IU hCG

gonadotropin and steroid at HCG daygonadotropin and steroid at HCG day

Group A Group B P value

(no hCG) (hCG)

• LH (IU/L) 0.6±0.1 0.7±0.3 NS

• FSH (IU/L) 11.3±1.211.3±1.2 4.3±0.64.3±0.6 <.001

• hCG (IU/L) 0.4±0.2 8.1±0.5 <.001

• E2 (pg/mL) 2.358±2342.358±234 3.235±3173.235±317 <.05

• P (ng/mL) 1.1±0.1 1.1±0.1 NS

• T (ng/mL) 0.9±0.1 1.1±0.1 <.05


12

Clinical outcomeClinical outcomeno-HCGno-HCG HCGHCG

COH daysCOH days 11.6±0.211.6±0.2 11.9±0.111.9±0.1 NSNS

r-FSH/hMG daysr-FSH/hMG days 11.6±0.211.6±0.2 8.6±0.18.6±0.1 <.001<.001

Daily hCG duration (days)Daily hCG duration (days) — — 3.3±0.13.3±0.1 ——

r-FSH/hMG dose (IU)r-FSH/hMG dose (IU) 2,779±1602,779±160 1,960±991,960±99 <.001<.001

Immature oocytes (n)Immature oocytes (n) 1.4±0.21.4±0.2 1.6±0.31.6±0.3 NS NS

Mature oocytes (n)Mature oocytes (n) 8.0±0.78.0±0.7 8.2±0.68.2±0.6 NSNS

Fertilization rate (%)Fertilization rate (%) 48±4% (0–100)48±4% (0–100) 74±3% (36–100)74±3% (36–100) <.001<.001

Good quality embryos (%)Good quality embryos (%) 86±6%86±6% 84±5%84±5% NSNS

Embryos transferred (n)Embryos transferred (n) 2.3±0.22.3±0.2 2.5±0.12.5±0.1 NSNS

Implantation rates (%)Implantation rates (%) 11%11% 12%12% NSNS

Pregnancy rates (%)Pregnancy rates (%) 21%21% 25%25% NSNS


FSH/HMG long protocol FSH/HMG long protocol

• Gn-RH-a depot on 21° day of previous cycle only• or Gn-RH-a low dose on 21° day up HCG day

• r-FSH 225-450 UI, step-down regimen, on 2nd at 8th cycle day

•HMGHMG on 9th until HCG day (if LH < 1 mUI/ml)until HCG day (if LH < 1 mUI/ml)

•r-FSH continuedr-FSH continued until HCG day (if LH ≥5 mUI/ml)until HCG day (if LH ≥5 mUI/ml)

oror

Ye H: Fertil Steril 2006;86,3S:S420-S421Ye H: Fertil Steril 2006;86,3S:S420-S421

8

1313

r-FSH/HMG Long protocol

11 22 33 44 55 66 77 88 991100

1111

1122 . . . .. . . .

LH >5 LH >5 mIU/mlmIU/ml

LH <1 LH <1 mIU/mlmIU/ml

r-FSHr-FSH HMGHMG Gn-RH-a low dose

21

° G

n-R

H-a

de

po

t o

r l

ow

do

se

lo

ng

pro

toc

ol

21

° G

n-R

H-a

de

po

t o

r l

ow

do

se

lo

ng

pro

toc

ol

1313

r-FSH/HMG Long protocol r-FSH/HMG Long protocol

Normal LHNormal LH Low LHLow LH

r-FSH r-FSH r- & HMG r- & HMG r-FSH r-FSH r- & HMG r- & HMG

Oocytes MII 1414 1212 1313 1010

Oocyt fert 10.510.5 8.88.8 9.99.9 7.27.2

Embryos 2.42.4 1.51.5 1.91.9 1.31.3

Implant % 35.8%35.8% 31.4%31.4% 40.7%40.7% 32.3%32.3%

Pregn rate 55.2%55.2% 43.8%43.8% 61.7%61.7% 54.1%54.1%

miscarriage 00 7.1%7.1% 18.9%18.9% 3.0%3.0%

1313

Antagonists (1990s)Antagonists (1990s)

**Orgalutran, Cetrotide 0.25 mg flOrgalutran, Cetrotide 0.25 mg fl s.c s.c

•They bind immediately to the receptor

•this leads to immediate pituitary down-regulation

•and do not activate classic postreceptor events;

•Receptor target

•no “flare-up”

Gn-RH Antagonists

Lubecca Method, delayed somministration

0.25 mg s.c. on 6° COH day or leading follicle >14 mm until HCG day

California methodearly administration (very high-responders)

On 1° COH day until leading follicle ≥18 mm and at least two follicles ≥ 15 mmOvulation triggering with Gn-RH-a long-acting

on 1° days Gn stimulation on 1° days Gn stimulation

on 5°-6° dayson 5°-6° days

on leading follicle on leading follicle ≥14 mm≥14 mm

• HMG or r-FSH + HMG or r-FSH + LH addedLH added

Antagonists protocolAntagonists protocol

Fixed and early start of the antagonist is probably more Fixed and early start of the antagonist is probably more effective than an individualized and late start. effective than an individualized and late start.

Gn-RH AntagonistGn-RH Antagonist

advantages:• Prevention surge LHPrevention surge LH• larger cohort of larger cohort of

follicles follicles • Avoidance of adverse Avoidance of adverse

effects of agonistseffects of agonists• More friendly More friendly

stimulation protocolstimulation protocol OHSSOHSS

disavantages • LDP peak E2 on HCG

day mature follicles oocytes embryos PR

LH addedLH added

• The early follicular phase is characterized by the presence of LH receptors on theca cells and the presence of FSH receptors on granulosa cells, with a prevalence of FSH activity.

• The middle-late follicular phase is characterized by the presence of LH receptors on both theca and granulosa cells, with a prevalence of LH activity and declining FSH levels.

• This leads to a selection of the dominant follicle and monofollicular ovulation.

. Filicori M. Use of luteinizing hormone in the treatment of infertility: time for reassessment? . Filicori M. Use of luteinizing hormone in the treatment of infertility: time for reassessment? Fertil Steril 20003;79:253–5. Fertil Steril 20003;79:253–5.

LH addedLH added• Granulosa cells in ovarian follicles of larger size (>10–Granulosa cells in ovarian follicles of larger size (>10–

12 mm) normally express the LH/hCG receptor and 12 mm) normally express the LH/hCG receptor and become sensitive to LH activity stimulation become sensitive to LH activity stimulation **

• Campbell et al. showed that pulsatile LH Campbell et al. showed that pulsatile LH administration in sheep maintained elevated ovulatory administration in sheep maintained elevated ovulatory rates despite FSH withdrawal rates despite FSH withdrawal ****

• LH/hCG receptors may also be highly relevant to LH/hCG receptors may also be highly relevant to permit continued dominant follicle growth in the permit continued dominant follicle growth in the spontaneous mid-late follicular phase, at a time when spontaneous mid-late follicular phase, at a time when the physiologic serum FSH decline the physiologic serum FSH decline * ** *

* Zeleznik AJ , Hillier SG .: * Zeleznik AJ , Hillier SG .: Clin Obstet Gynecol Clin Obstet Gynecol . 1984;27:927–940 . 1984;27:927–940 * * Campbell BK , Dobson H , Baird DT , Scaramuzzi RJ .: J Reprod Fertil . * * Campbell BK , Dobson H , Baird DT , Scaramuzzi RJ .: J Reprod Fertil . 1999;117:355–367 1999;117:355–367

− LH <1 UI/ml at the start of Gn LH <1 UI/ml at the start of Gn stimulationstimulation

− Gn-RH-a flare protocol (LH Gn-RH-a flare protocol (LH suppression)suppression)

− Gn-RH antagonist during stimulationGn-RH antagonist during stimulation− >35 years>35 years− Poor respondersPoor responders− High responders (LH prevalence activity High responders (LH prevalence activity

decrease n. small follicles and OHSS decrease n. small follicles and OHSS risk)risk)

LH ADDED target

LH added targetLH added target

0

20

40

60

80

100

120

0 5 10 15 20

T-LH-r

G-FSH-r

G-LH-r

LH added targetLH added target• the early follicular phase is characterized by the

presence of LH receptors on theca cells and FSH receptors on granulosa cells, with a prevalence of FSH activity.

• The middle-late follicular phase is characterized by the presence of LH receptors on both teca and granulosa cells, with a prevalence of LH activity and declining FSH levels*

* Filicori 2003


Cycle’s

phaseFSH rec LH rec

Prevalence activity of

early follicolar

+ + + (G) + (T) FSH

late follicolar

+ + (G)+ + + (T&G)

LH

luteal - - + + + (CL) LH


* Filicori 2003

• FSH: earlier cycle follicular phase:» follicles recruitment

» Follicles growth

• LH: late cycle follicular phase:» mature oocytes» Ovulation

• LH: Luteal cycle phase: »corpus luteum, LDP

Rationale for LH added (Sullivan 1999)

• The rationale for this hypothesis is that the FSH-stimulated induction of LH receptors on granulosa cells could enable the maturing follicle to respond to LH and thereby continue to mature in the presence of continuously declining FSH concentrations

Rationale for LH added (Sullivan 1999)• It is generally accepted that E2 production by the maturing follicle

occurs by way of the two-cell, two-gonadotropin model. • In this model, theca cells produce androstenedione and theca cells produce androstenedione and

testosterone under LH stimulationtestosterone under LH stimulation, and FSH induces granulosa FSH induces granulosa cell aromatasecell aromatase, thus enabling the thecally derived androgens to be metabolized to E2.

• Assuming the validity of this model in humans, our results indicate that thecal androgen production is exquisitely sensitive to LH, as a plasma LH concentration of 1.5 IU/L was sufficient to maintain E2 production as well as plasma androstenedione concentrations.

• Our observation of E2 production despite very low serum LH concentrations is in agreement with other published data showing that women treated with GnRH agonists to suppress gonadotropin secretion maintain E2 production in the presence of very low levels of serum LH (<0.5 IU/L).

Our current study also indicates that although LH concentrations of approximately 1.5 IU/L are able to sustain thecal androgen production, these levels of LH are unable to maintain granulosa cell aromatase activity when FSH concentrations decline. (vedi (vedi iperandrogenismo in PCOS)iperandrogenismo in PCOS)

LH Added protocolLH Added protocol 1515

r-LH 375 IU twice a day (7.30 and 19.30 h) for the last 2 days of COH r-LH 375 IU twice a day (7.30 and 19.30 h) for the last 2 days of COH

ororLeading follicle ≥14 mmLeading follicle ≥14 mm

*Sullivan MW et al: “Ovarian Responses in Women to Recombinant Follicle-Stimulating Hormone and *Sullivan MW et al: “Ovarian Responses in Women to Recombinant Follicle-Stimulating Hormone and Luteinizing Hormone (LH): A Role for LH in the Final Stages of Follicular Maturation”Luteinizing Hormone (LH): A Role for LH in the Final Stages of Follicular Maturation” J J Clin Endocrinol Metab Clin Endocrinol Metab . 1999;84:228–232 .. 1999;84:228–232 .

leuprolide acetate 1 mg daily, sc, from menstrual day 21 for 14 days (+ 7 days) leuprolide acetate 1 mg daily, sc, from menstrual day 21 for 14 days (+ 7 days)

LH <2.5 IU/L and E2 <20 pg/mL

r-FSH starting at 150 IU sc daily at 07.30 h. for 4 days

excluded from further treatment if E2 >20 pg/ml and/or LH >2,5 IU after 21 days leuprolide

On 5° day •If serum E2 levels were less than 100 pg/mL, the r-FSH dose was increased to 225 IU •If serum E2 levels were greather than 100pg/mL, the r-FSH was If serum E2 levels were greather than 100pg/mL, the r-FSH was maintained at 150 maintained at 150 IU/day IU/day

LH added vs. HMG LH added vs. HMG in over 38 in over 38 **

** Gomez-Palomares J. L. ; Acevedo-Martin B. ; Andres L. ; Ricciarelli E. ; Hernandez E. R.; Reproductive biomedicine online ISSN 1472-6483; 2008

r-FSH + HMG 75 UI (group I) and r-FSH + r-LH 75 UI (group II)

HMG group HMG group LH groupLH group

n. follicles on 6 day 6.72 2.22 5.87 1.29

COH days 10.5 1.7 12 1.8

M II oocytes 75.3% 93.1%

Pregnancy rate 26% 47%47%

Luteal supplementation in Luteal supplementation in agonists/antagonists protocolsagonists/antagonists protocols

• Pituitary depletion

• Pituitary desensitization

• Negative estrogen feed-back

• Compulsory supplementation E/P

HCG supplementation absolutely necessary !!!HCG supplementation absolutely necessary !!!

PP4 4 secretionsecretion

Follicular Follicular phasephase

Luteal phase Luteal phase **

OvaryOvary 48%48% 95%95%

Adrenal glandAdrenal gland 48%48% 4%4%

from from pregnenolonepregnenolone 4%4% 1%1%

*P*P44 serum level: serum level: 4 ng/ml is low level; 40 ng/ml is high4 ng/ml is low level; 40 ng/ml is high

luteal Pluteal P4 4 supplementationsupplementation

☻Few studies in the last 20 yearsFew studies in the last 20 years☻Currently, no reliable method for specific diagnosis Currently, no reliable method for specific diagnosis of Pof P44 deficiency in luteal phase deficiency in luteal phase☻Regimens often determined by clinical experienceRegimens often determined by clinical experience

The rationale for PThe rationale for P44 supplementation: supplementation:

1.1. Aspiration of the granulosa cellsAspiration of the granulosa cells2.2. Presence of high levels of EPresence of high levels of E22

3.3. Analogues Analogues poor luteal function poor luteal function (due to (due to

residual suppression of pituitary LH secretion)residual suppression of pituitary LH secretion)ASRM Practice Committee: “Exogenous progesterone supplementation” Fertil Steril ASRM Practice Committee: “Exogenous progesterone supplementation” Fertil Steril

2008;89,4:789-792.2008;89,4:789-792.

luteal Pluteal P44 supplementation supplementation

PP44 50 mg/d i.m. Or 50 mg/d i.m. Or200-600 mg/day vaginally200-600 mg/day vaginally

Starting: Starting: 3 days after IUI3 days after IUI

ororat E-T dayat E-T day

Prontogest fl im 100 mg Prontogest fl im 100 mg

luteal Pluteal P44 supplementation supplementation

Higher pregnancy rate Higher pregnancy rate **

Lack of evidenceLack of evidence in literature in literature * ** *Increased of hypospadias Increased of hypospadias (progestins derived from (progestins derived from

androgens and that bind to androgen receptors) androgens and that bind to androgen receptors) * * ** * ** * Yovich JL et al: “Early luteal serum progestyerone concentration are higher in pregnancy cycles”. Fertil Steril Yovich JL et al: “Early luteal serum progestyerone concentration are higher in pregnancy cycles”. Fertil Steril 1985;44:185-189. 1985;44:185-189.

**** Ziad R. Hubayter: “luteal supplementation in in vitro fertilization: Ziad R. Hubayter: “luteal supplementation in in vitro fertilization: more question than answersmore question than answers”. Fertil ”. Fertil

Steril 2008; 89,4:749758.Steril 2008; 89,4:749758.

******ASRM Practice Committee: “Exogenous progesterone supplementation” Fertil Steril 2008;89,4:789-792.ASRM Practice Committee: “Exogenous progesterone supplementation” Fertil Steril 2008;89,4:789-792.Carmichael SL et al: “Maternal progestin intake and risk of hypospadias”. Arch Pediatr Adolesc Med 2005;159:957Carmichael SL et al: “Maternal progestin intake and risk of hypospadias”. Arch Pediatr Adolesc Med 2005;159:957

PP44

**Posaci C, Smitz J, Camus M, Osmanagaoglu K, Devroey P: “Progesterone for the luteal Posaci C, Smitz J, Camus M, Osmanagaoglu K, Devroey P: “Progesterone for the luteal support of ART: clinical options”. Human Reprod 2000; 15,S1:129-148.support of ART: clinical options”. Human Reprod 2000; 15,S1:129-148.

Orally:Orally: • bioavailabilty diminished by the liver first passbioavailabilty diminished by the liver first pass• the serum level typically returns to baseline level by the serum level typically returns to baseline level by 6 hours6 hours•dizziness and somnolencedizziness and somnolence•fatigue, headache, urinary frequencyfatigue, headache, urinary frequency

vaginal route:vaginal route: •the level remains elevated for up the level remains elevated for up 48 hours48 hours•Crinone gel 8% once a day and contains 90 mg of PCrinone gel 8% once a day and contains 90 mg of P44

•Progeffik gel 200 mg 1-3/dayProgeffik gel 200 mg 1-3/day•Uterine tissue higher level PUterine tissue higher level P44 despite a lower serum P despite a lower serum P44

•Vaginal irritationVaginal irritation

Intramuscular: Intramuscular: •P4 in oil result in higher plasma concentration P4 in oil result in higher plasma concentration •and and longer durationlonger duration ** •Severe allergic reaction Severe allergic reaction •Adult respiratory distress syndromeAdult respiratory distress syndrome•Eosinophilic pneumonitisEosinophilic pneumonitis

luteal supplementation in luteal supplementation in agonist/antagonist protocolsagonist/antagonist protocols

Pituitary desensitization for 2-3 w after Pituitary desensitization for 2-3 w after last administrationlast administration

* * Belaisch-Allart J et al: “ JL et al: “The effect of HCG supplementation after combined Belaisch-Allart J et al: “ JL et al: “The effect of HCG supplementation after combined Gn-RH agonist/HMG treatment in an IVF programme”. Human Reprod 1990;5:163-166.Gn-RH agonist/HMG treatment in an IVF programme”. Human Reprod 1990;5:163-166.

Worldwide standard practiceWorldwide standard practice **

luteal suppl agonist/antagonist protocolsluteal suppl agonist/antagonist protocols

PP44

**Martinez F: “ Human Corionic Gonadotropin and intravaginal natural progesterone are Martinez F: “ Human Corionic Gonadotropin and intravaginal natural progesterone are equally effective for luteal phase support in IVF”. Gynecol Endocrinol 2000; 14:316-320.equally effective for luteal phase support in IVF”. Gynecol Endocrinol 2000; 14:316-320.

HCGHCG : : •more effectivemore effective•Increased production of EIncreased production of E22 and P and P44

•Better endocrine profileBetter endocrine profile•No differences in pregnancy outcomeNo differences in pregnancy outcome•OHSS risk (EOHSS risk (E22 peak at HCG day) peak at HCG day)

or/andor/and

Luteal ELuteal E2 2 supplementationsupplementation

• E2 orally 2-6 mg/d (Progynova cpr 2 mg) *• Start on:

» E-T day E-T day or or » 7 days after E-T7 days after E-T

• Increases implantation rate• Increases pregnancy rate

In IVF cycles, the levels of EIn IVF cycles, the levels of E2 2 and Pand P44 drop in the mid-late luteal phase drop in the mid-late luteal phase

Lower ELower E22 at 11 days after pick-up is associated with lower pregnancy rate at 11 days after pick-up is associated with lower pregnancy rate

* Lukaszuk K: Fertil Steril 2005;83:1372-1376* Lukaszuk K: Fertil Steril 2005;83:1372-1376

PROTOCOLS:PROTOCOLS:

• 66

• 99

• 1010

• 1111

• 16-2716-27

Poor responsePoor response— the devil is in the — the devil is in the

definitiondefinition • The original definition of low response to

ovarian response by Garcia et Acosta was based on low peak E2 concentrations alone

• They stimulated patients with hMG (150 IU IM daily) and defined low responders as patients with a peak E2 concentration of <300 pg/mL

Poor respondersPoor responders • diminished ovarian reserve• A lower expression of FSH receptor in the granulosa

cells • Advanced maternal age• E2 < 500 pg/mL on day of hCG • <4 de Graaf follicles on HCG day• lower fertilization rates • lower cleavage rates • lower resulting embryos• Lower implantation rate• lower pregnancy rates

10–25% of the ART population*10–25% of the ART population*

* * Keay Keay et alet al., 1997 ; Karande and Gleicher, 1999 ; Fasouliotis ., 1997 ; Karande and Gleicher, 1999 ; Fasouliotis et alet al., 2000 ; Tarlatzis ., 2000 ; Tarlatzis et alet al., 2003., 2003

““occult ovarian failure”

occult ovarian failure”

increase Gn dose increase Gn dose

• first and simplest approach

• limited benefit to 450 IU per day

• 300 IU r-FSH + hMG 150 IU

• beyond this amount little or no improvement

1616

Murat Arslan: Fertil Steril 2005; 84,3:555-569

the stop Gn-RH-a protocolthe stop Gn-RH-a protocol

• Gn-RH-a low dose on 21° day until the beginning of menstruation.

• Stop analogues

• gonadotropins from day 2 of the cycle until HCG day

Target of this protocol:Target of this protocol:Stop to pre-menstrual FSH Stop to pre-menstrual FSH and, subsequently, and, subsequently, stop to size discrepancy in the developing folliclesstop to size discrepancy in the developing follicles

1717

CC + HMG + AntagonistCC + HMG + Antagonist

• CC 150 mg/d on 1°-5° days • HMG (r-FSH) large dosage (450-600 IU/d) on

2-3° cycle day

Antagonist delayed administration: Antagonist delayed administration: •on 6°-8° stimulation dayson 6°-8° stimulation days•or leader follicle ≥ 14 mm if very few folliclesor leader follicle ≥ 14 mm if very few follicles

• HCG 10.000 UI on day dominant follicle ≥18 mm

1818

•Luteal supplementation: HCG 2.000 IU/d (+ PLuteal supplementation: HCG 2.000 IU/d (+ P44) )

E-P Antagonist protocolE-P Antagonist protocol Farmakon Dosage Time length

E-P pillE-P pillprevious

cycle14-21 days

FSH/HMG300 + 150

2° cycle d up HCG d

CC 150 mg/d 1° cycle d 5-7 days

Antagon0.25 mg/d

7-8° d up HCG d

HCG 10.000 IU >18 mm

1919

Age >40Age >40 Long protocolCC + HMG + Antagonist

n° ampules 50 83

follicles >14 3.7 5.8

E2 on day 5 36 74

E2 on day 9 169 945 (400-1480)

E2 on HCG day 744 833 (410-2160)

Cancellation rate 34% 4.8%

total oocytes 3.3 5.5

Mature oocytes 2.6 4.29

n° embryo 1.4 1.6

PR 15.3% 22.2%

Implant rate 7.6% 13.5%

Weghofer, 2004Weghofer, 2004

Luteal estradiol protocolLuteal estradiol protocol

• Oral micronized E2 2 mg twice a day» On luteal day 21» At 3 days of COH

• r-FSH 375-450 IU/d down regimen on 2° day

microdose flare Gn-RH-a on 3° COH daymicrodose flare Gn-RH-a on 3° COH day

orordelayed Gn-RH antagonist delayed Gn-RH antagonist

Dragisic KG Fertil Steril 2005;84:1023-1026

2020

HCG low-dose (10-50 IU/d) on 8° dayHCG low-dose (10-50 IU/d) on 8° day

lowering FSH levels with estrogen, the ovary will respond when high doses of FSH are lowering FSH levels with estrogen, the ovary will respond when high doses of FSH are added in COH protocoladded in COH protocol

Luteal estradiol protocol Luteal estradiol protocol **

outcomeoutcome All cyclesLuteal

EstradiolStandard protocol

Clinical Pr 38,3% 40,9% 31,3%

Miscarriage rate

43,5% 38,9% 60,0%

Delivery rate

20.0% 25.0%25.0% 12.5%

* Frattarelli J, et al: “A luteal estradiol protocol for expected poor-responders improves * Frattarelli J, et al: “A luteal estradiol protocol for expected poor-responders improves embryo number and quality” Fertil Steril 2008;89,5:1118-22embryo number and quality” Fertil Steril 2008;89,5:1118-22

AACEP ProtocolAACEP Protocol• E-P pills for 1 to 3 weeksE-P pills for 1 to 3 weeks

• Gn-RH-a low-dose in a standard long protocol overlapping the last 5 to 7 Gn-RH-a low-dose in a standard long protocol overlapping the last 5 to 7 days of E-P pills until onset of menses days of E-P pills until onset of menses

• Gn-Rh antagonist low-dose (0.125 mg/day) on cycle day 2Gn-Rh antagonist low-dose (0.125 mg/day) on cycle day 2

• Estradiol valerateEstradiol valerate** 2 mg/d on 1° to 10° cycle day 2 mg/d on 1° to 10° cycle day• Estrogen suppositoriesEstrogen suppositories**** were used to maintain the endometrium until at were used to maintain the endometrium until at

last one follicle measured 15 mmlast one follicle measured 15 mm

• r-FSH in initial doses of 600 or 750 IU/day, decreasing to 225 IU/day of r-r-FSH in initial doses of 600 or 750 IU/day, decreasing to 225 IU/day of r-FSH. FSH.

Fisch JD, Keskintepe L and Sher G: “Gonadotropin-releasing hormone agonist/antagonist conversion with estrogen priming in low responders with prior in vitro fertilization failure”. Fertil Steril 2008;89,2:342-347

* Progynova cpr 2 mg * Progynova cpr 2 mg ** ** Vagifem cpr vaginali 0.025 mgVagifem cpr vaginali 0.025 mg

AACEP ProtocolAACEP Protocol

EE--P P

pp

II

ll

ll

ss

**aa

gg

oo

nn

ii

ss

tt

1 2 3 4 5 6 7 8 9 10 12 13 14 15 16 17 …

E T

PP44PP

44PP44

AA AA AA AA AA AA AA AA AA AA AA PP44PP

44PP44

EE EE EE EE EE EE EE EE EE EE

HHCCGG

r-FSH 150 IUr-FSH 150 IU A antagonistA antagonist

AndrogensAndrogens• androgens may influence the responsiveness of

ovaries to gonadotrophins • positive regulators of follicular development • augments follicular FSH-receptor expression in

granulosa cells • IGF-I oocyte expression• promotes initiation of primordial follicle growth• increases the number of growing preantral and

small antral follicles

Vendola K, Zhou J, Wang J, Famuyiwa OA, Bievre M, Bondy CA. Androgens promote Vendola K, Zhou J, Wang J, Famuyiwa OA, Bievre M, Bondy CA. Androgens promote oocyte insulin-like growth factor I expression and initiation of follicle development in the oocyte insulin-like growth factor I expression and initiation of follicle development in the primate ovary. primate ovary. Biol ReprodBiol Reprod 1999; 61:353–357. 1999; 61:353–357.

2222

AndrogensAndrogens

TTDHTDHTDHEADHEALetrolozole Letrolozole

DHEADHEA

For women under 50, DHEA levels of less For women under 50, DHEA levels of less than 150 ng/dL are considered lowthan 150 ng/dL are considered low

DHEA is the cornerstone to all sex hormonesDHEA is the cornerstone to all sex hormones

Casson PR: Casson PR: “ “Dehydroepiandrosterone supplementation augments ovarian stimulation in poor Dehydroepiandrosterone supplementation augments ovarian stimulation in poor responders: a case seriesresponders: a case series Human” Reproduction, Vol. 15, No. 10, 2129-2132, October 2000Human” Reproduction, Vol. 15, No. 10, 2129-2132, October 2000

DHEA protocolDHEA protocol

1.Leonidas Mamas, Eudoxia Mamas,: “Premature ovarian failure and dehydroepiandrosterone” Fertil Steril 2008 in press in press

• 75 mg/d for 4-12 months previous COH75 mg/d for 4-12 months previous COHandand• during COHduring COH

•Reduces FSH levelReduces FSH level •release more and better quality eggs prior to IVF release more and better quality eggs prior to IVF •reduces miscarriage rates - especially in older womenreduces miscarriage rates - especially in older women•similar effects of GH increasing IGF-I paracrine effectssimilar effects of GH increasing IGF-I paracrine effects•Increases IGF-I serum levelsIncreases IGF-I serum levels

POFPOFHigh FSHHigh FSH

Testosterone

Balasch J, Fábregues F, Peñarrubia J, Carmona F, Casamitjana R, Creus M, Manau Balasch J, Fábregues F, Peñarrubia J, Carmona F, Casamitjana R, Creus M, Manau D, Casals G and Vanrell JA: “D, Casals G and Vanrell JA: “Pretreatment with transdermal testosterone may improve Pretreatment with transdermal testosterone may improve ovarian response to gonadotrophins in poor-responder IVF patients with normal basal ovarian response to gonadotrophins in poor-responder IVF patients with normal basal concentrations of FSH”. Human Reproduction 2006 21(7):1884-1893concentrations of FSH”. Human Reproduction 2006 21(7):1884-1893

Two IVF treatment cycle cancellations due Two IVF treatment cycle cancellations due to poor follicular response,to poor follicular response, in spite of vigorous gonadotrophin ovarian in spite of vigorous gonadotrophin ovarian stimulation stimulation and having normal basal FSH levelsand having normal basal FSH levels

Testosterone protocol

Balasch J, Fábregues F, Peñarrubia J, Carmona F, Casamitjana R, Creus M, Manau D, Balasch J, Fábregues F, Peñarrubia J, Carmona F, Casamitjana R, Creus M, Manau D, Casals G and Vanrell JA:Casals G and Vanrell JA: “ “Pretreatment with transdermal testosterone may improve Pretreatment with transdermal testosterone may improve ovarian response to gonadotrophins in poor-responder IVF patients with normal basal ovarian response to gonadotrophins in poor-responder IVF patients with normal basal concentrations of FSH”. Human Reproduction 2006 21(7):1884-1893concentrations of FSH”. Human Reproduction 2006 21(7):1884-1893

•Gn-RH-a low dose (Leuprolide 1 mg or triptoreline 0.1 mg) long Gn-RH-a low dose (Leuprolide 1 mg or triptoreline 0.1 mg) long protocol-like protocol-like started in the midluteal phase of the previous started in the midluteal phase of the previous cyclecycle

•at menses start Gn-RH-a is reduced to 0.5 mg and continued at menses start Gn-RH-a is reduced to 0.5 mg and continued until the administration of HCGuntil the administration of HCG

•Gn-RH-a 0.5 mg/day of leuprolide from the midluteal Gn-RH-a 0.5 mg/day of leuprolide from the midluteal phase at menses startphase at menses start•0.25 mg/day thereafter 0.25 mg/day thereafter

1° cycle1° cycle

2° cycle2° cycle

Testosterone


•transdermal testosteronetransdermal testosterone•20 µg/kg per day 20 µg/kg per day •Androderm 2.5 mgAndroderm 2.5 mg daily single patch daily single patch•RemovedRemoved always at 9.00 a.m.always at 9.00 a.m.•0.1 mg/h delivery rate 0.1 mg/h delivery rate (a predetermined number of (a predetermined number of

hours provides the desired daily dose of testosterone [e.g. in a woman weighing 60 kg hours provides the desired daily dose of testosterone [e.g. in a woman weighing 60 kg and needing 1200 µg/day, the patch was used for 12 h (0.1 mg/h delivery rate x 12 h = and needing 1200 µg/day, the patch was used for 12 h (0.1 mg/h delivery rate x 12 h = 1.2 mg or 1200 µg) and thus applied at 21.00 hours].1.2 mg or 1200 µg) and thus applied at 21.00 hours].

•during the 5 days preceding COHduring the 5 days preceding COH

Testosterone COH


•On day 1 of ovarian stimulation, r-FSH 450 IU s.c. On day 1 of ovarian stimulation, r-FSH 450 IU s.c. •On day 2 r-FSH 300 IU On day 2 r-FSH 300 IU •On days 3 and 4 of ovarian stimulation, 150 IU per day On days 3 and 4 of ovarian stimulation, 150 IU per day •From day 5 onwards, r-FSH was administered on an individual basisFrom day 5 onwards, r-FSH was administered on an individual basis

On days 1 and 2 of ovarian stimulation, r-FSH 300 IU per day + HMG 300 IU i.m. On days 1 and 2 of ovarian stimulation, r-FSH 300 IU per day + HMG 300 IU i.m. On days 3 and 4 of ovarian stimulation, 300 IU HMG On days 3 and 4 of ovarian stimulation, 300 IU HMG From day 5 onwards, HMG on an individual basis From day 5 onwards, HMG on an individual basis

1°

1°

cycl

ecy

cle

2°

2°

cycl

ecy

cle

Gonadotrophin ovarian stimulation was started the day following last Gonadotrophin ovarian stimulation was started the day following last testosterone patch application testosterone patch application

Testosterone

Balasch J, Fábregues F, Peñarrubia J, Carmona F, Casamitjana R, Creus M, Manau D, Casals G and Balasch J, Fábregues F, Peñarrubia J, Carmona F, Casamitjana R, Creus M, Manau D, Casals G and Vanrell JA: “Pretreatment with transdermal testosterone may improve ovarian response to Vanrell JA: “Pretreatment with transdermal testosterone may improve ovarian response to gonadotrophins in poor-responder IVF patients with normal basal concentrations of FSH”. gonadotrophins in poor-responder IVF patients with normal basal concentrations of FSH”. Human Reproduction 2006 21(7):1884-1893Human Reproduction 2006 21(7):1884-1893

•80% showed an increase of over fivefold in the number of recruited follicles, •produced 5.8 ± 0.4 oocytes, •received two or three embryos •pregnancy rate: 30% per oocyte retrieval •cancelled cycles: 20%

LetrozoleLetrozole• Third-generation aromatase inhibitors (AIs) Third-generation aromatase inhibitors (AIs)

• nonsteroidal, reversible, orally administerednonsteroidal, reversible, orally administered • The excellent oral bioavailability (100%) The excellent oral bioavailability (100%)

• relatively short half-life (45 hours)relatively short half-life (45 hours)

• able to effectively block the conversion of :able to effectively block the conversion of :• A E1 • T E2

Letrozole + HMGLetrozole + HMG

Grabia A, Papier S, Pesce R, Mlayes L, Kopelman S, Sueldo C: “Preliminary experience with a low-cost Grabia A, Papier S, Pesce R, Mlayes L, Kopelman S, Sueldo C: “Preliminary experience with a low-cost stimulation protocol that includes letrozole and human menopausal gonadotropins in normal responders stimulation protocol that includes letrozole and human menopausal gonadotropins in normal responders for assisted reproductive technologies”for assisted reproductive technologies” Fertil Steril 2006;86,4:1026-28Fertil Steril 2006;86,4:1026-28

•20%–25% of women are resistant to CC20%–25% of women are resistant to CC •comparable pregnancy results vs.:

•CC/HMG •r-FSH alone

significant saving in the amount of Gonadotropinssignificant saving in the amount of Gonadotropins

CC resistant CC resistant Poor respondersPoor responders

Letrozolo + HMGLetrozolo + HMG

Letrozolo 2.5 mg days 3–7 day cycleLetrozolo 2.5 mg days 3–7 day cycle

HMG 150 IU on day 5 up lead. foll. >18 HMG 150 IU on day 5 up lead. foll. >18

HCG 10.000 IU 36-48 hours afterHCG 10.000 IU 36-48 hours after

Pick-up 33-36 hours after HCGPick-up 33-36 hours after HCG

PP4 4 50 mg/d i.m. on HCG day or E-T day50 mg/d i.m. on HCG day or E-T day

Mohamed F.M Mitwally, Robert F Casper: “Mohamed F.M Mitwally, Robert F Casper: “Use of an aromatase inhibitor for induction of Use of an aromatase inhibitor for induction of ovulation in patients with an inadequate response to clomiphene citrate” Fertil Steril 2001; ovulation in patients with an inadequate response to clomiphene citrate” Fertil Steril 2001; 75,2: 75,2: 305-309305-309

Increasing AAIncreasing AA

• Poor responders: lower expression of FSH receptor in the granulosa cells

• PCOs Patients: hyperexpression of FSH receptor

Increasing AA Increasing AA

• inducing a temporary and reversible PCO-like condition in the ovaries of poor responder patients

• could enhance their follicular recruitment and development

Aromatase Aromatase inhibitorsinhibitors protocol protocol

• aromatase inhibitor which induces a temporary accumulation of intraovarian androgens

ADVANTAGES AIs vs. CCADVANTAGES AIs vs. CCLack of down-regulation of hypothalamic-pituitary Lack of down-regulation of hypothalamic-pituitary

estrogen receptors estrogen receptors Lower FSH doseLower FSH doseHigher number of mature oocytesHigher number of mature oocytesLess adverse effects on endometriumLess adverse effects on endometrium11 and cervix and cervixPregnacy rate: 21%Pregnacy rate: 21%

11 Endometrial thickness <5 mm is usually associated with failure to conceiveEndometrial thickness <5 mm is usually associated with failure to conceive

(Gonen Yand Casper RF: “Sonografic determination of an adverse effect of clomiphene (Gonen Yand Casper RF: “Sonografic determination of an adverse effect of clomiphene citrate on endometrial growth”. Human Reprod 1990;5:670-674).citrate on endometrial growth”. Human Reprod 1990;5:670-674).

Increasing AA Increasing AA

synergistical role of androgens with FSH to promote early follicular recruitment

trophic effects of androgens in small antral follicles

Positive estrogen feed-back on hypotalamic-hypophyseal axis

AA too high reduce follicular healthAA too high reduce follicular health

LetrozoleLetrozole

Mitwally MFM and Casper RF. (2002) Aromatase inhibition improves Mitwally MFM and Casper RF. (2002) Aromatase inhibition improves ovarian response to follicle-stimulating hormone in poor responders. ovarian response to follicle-stimulating hormone in poor responders. Fertil SterilFertil Steril 77:776–780. 77:776–780.

•letrozole, 2.5 mg/day from day 1-5 of the menstrual cycleletrozole, 2.5 mg/day from day 1-5 of the menstrual cycle

•FSH (50-225 IU/day) starting on day 6 FSH (50-225 IU/day) starting on day 6

•hCG (10,000 IU) when two leading follicles were hCG (10,000 IU) when two leading follicles were ≥20 mm≥20 mm

• E-P for 15-21 daysE-P for 15-21 days – oror

» E2 <60 pg/mlE2 <60 pg/ml

» absence of cysts >10 mmabsence of cysts >10 mm

Letrozole + AntagonistLetrozole + Antagonist

letrozolo 5 mg on 1° to 5° cycle day letrozolo 5 mg on 1° to 5° cycle day

r-FSH 300 IU + HMG 150 IU on 1° to 5° cycle dayr-FSH 300 IU + HMG 150 IU on 1° to 5° cycle day

On 6° day individual dosages r-FSH/HMGOn 6° day individual dosages r-FSH/HMG

delayed antagonist 0.25 mg/d delayed antagonist 0.25 mg/d

HMG + Antagonist until HCG day HMG + Antagonist until HCG day

r-HCG 250 mg on leading follicle >18 mmr-HCG 250 mg on leading follicle >18 mm

PP44 supplementation with 200 mg of vaginal micronized P (Progeffik) supplementation with 200 mg of vaginal micronized P (Progeffik)

Garcia-Velasco JA. ,Moreno L, Pacheco A, Guillén A, Duque L, Requena A, Pellicer A: Garcia-Velasco JA. ,Moreno L, Pacheco A, Guillén A, Duque L, Requena A, Pellicer A: ““The aromatase inhibitor letrozole increases the concentration of intraovarian androgens The aromatase inhibitor letrozole increases the concentration of intraovarian androgens and improves in vitro fertilization outcome in low responder patients: A pilot study”. Fertil and improves in vitro fertilization outcome in low responder patients: A pilot study”. Fertil Steril 2005;84,1:82-87.Steril 2005;84,1:82-87.

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

E E TT

PP44 PP44 PP44 PP44 PP44

AA AA AA AA AA PP44 PP44 PP44 PP44 PP44

r-FSH

150 IU

HMG HMG

150 UI150 UI Letrozole Letrozole

2,5 mg2,5 mg r-HCG 250 r-HCG 250

mgmgAA

AntagonAntagon

Letrozole + AntagonistLetrozole + Antagonist

Letrozole control

oocytes retrieved 6.1 ± 0.4 4.3 ± 0.3

Fertilization rate 68,2 % 63,3 %

embryos transferred 2 ± 0.1 2.3 ± 0.1

PR/cycle 22.4 % 15.2 %

PR/transfer 41.7 %41.7 % 28.9 %

Implantation rate 25 %25 % 9.4 %

Miscarriage rate 20 %20 % 7.7 %

twins 46.7%46.7% 7.7%

Garcia-Velasco. Letrozole in poor responder JVF patients. Fertil Steril 2005

Assisted hatchingAssisted hatching

a b cM. Carrino, M. Wilding, E. Tosti, V. Volpicelli, B. Dale: “Zona Binding” e “Zona Penetration” come tests predittivi dell’infertlità maschile; Atti “IV GIORNATE ANDROLOGICHE ITALIANE”; Perugia, 10-12 settembre 1998.

E-T on the day 2E-T on the day 2

Microdose flare agonist protocolMicrodose flare agonist protocol

day 2 day 2 day 3

Implantation rate 23.9%23.9% 17.2%

Pregn rate/oocyte 27.7%27.7% 16.2%

Pregnancy rate/E-T 29.029.0 18.3

Luteal supplementation: progesterone 100 mg/day i.m. Luteal supplementation: progesterone 100 mg/day i.m. On oocyte collection through the luteal phaseOn oocyte collection through the luteal phase

Bahceci M.: Fertil Steril 2006;86,1:81-85Bahceci M.: Fertil Steril 2006;86,1:81-85

Ovary deficiencyOvary deficiency

1) gonadic disgenesia

“streak gonad” absence of ovary tissue

2) Ovary

disgenesiaOvary tissue without follicles and without functionality

3) POFOvary tissue without follicles but with past functionality

4) Proof Ovary (Savage Symdrome)

Ovary tissue with hystologic normal follicles

endometriosisendometriosis

adenomiosisadenomiosis

endometriosis

endometriosis

• presence of ovarian endometriomas

• responsiveness to gonadotropins: - 25%- 25%

Long protocol + prednisoneLong protocol + prednisone

• 3.75 mg Gn-Rh-a depot in one dose on 213.75 mg Gn-Rh-a depot in one dose on 21stst day day oror• Low-dose daily on the 21Low-dose daily on the 21stst day of previous cicle to HCG day day of previous cicle to HCG day oror• on any when LH <0.5 and Eon any when LH <0.5 and E22 <30 <30

oror• on the 3th day of menstrual cycleon the 3th day of menstrual cycle

– USGUSG– LH <0.5 UI/mlLH <0.5 UI/ml– E2 <30 pg/mlE2 <30 pg/ml

Long protocol + prednisoneLong protocol + prednisone

Prednisone 15 mg/day on 1° day at HCG dayPrednisone 15 mg/day on 1° day at HCG day (Deltacortene cpr 5 mg)

r-FSH 450 IU on 2° day up 6° dayr-FSH 450 IU on 2° day up 6° day

r-FSH variable dosager-FSH variable dosage

Luteal supplementation: PLuteal supplementation: P4 4 50 mg/d i.m.50 mg/d i.m.

lean womenlean women

• reproductive performance was not poorer

• “inverted U shape theory” applies only to native oocyte conceptions.

• Levens ED, Skarulis MC: “Assessing the role of endometrial alteration among obese patients undergoing assisted reproduction”. Fertil Steril 2008;89,6:1606-8.

Donna magra come un treno (Mango)Donna magra come un treno (Mango)

OverweightOverweight

• Levens ED, Skarulis MC: “Assessing the role of endometrial alteration among obese patients undergoing assisted reproduction”. Fertil Steril 2008;89,6:1606-8.

according to body mass index (BMI):

•lean (<20 kg/m2), •normal (20.0–24.9 kg/m2), •overweight (25.0–29.9 kg/m2), •obese (≥30 kg/m2).

Overweight in PMAOverweight in PMA

**Fedorcsáck P, Storeng R, Dale PO, Tanbo T, Abyholm T. Fedorcsáck P, Storeng R, Dale PO, Tanbo T, Abyholm T. Obesity is Obesity is associated with early pregnancy loss after IVF or ICSIassociated with early pregnancy loss after IVF or ICSI. . Acta Obstet Gynecol Acta Obstet Gynecol ScandScand. 2000;79:43–48.. 2000;79:43–48.* * the effect of recipient body weight on reproductive performancethe effect of recipient body weight on reproductive performance

•Higher cancellation rate Higher cancellation rate •Lower pregnancy rates Lower pregnancy rates •higher miscarriage rates higher miscarriage rates •lower live-birth rates in natural and PMAlower live-birth rates in natural and PMA

•more frequent complications in pregnancymore frequent complications in pregnancy

Poor reproductive performance:Poor reproductive performance:


•Norman RJ, Clark AM. Obesity and reproductive disorders: a review. Norman RJ, Clark AM. Obesity and reproductive disorders: a review. Reprod Reprod Fertil DevFertil Dev. 1998;10:55–63. . 1998;10:55–63.

Extraovum effects of obesity on FIVET Extraovum effects of obesity on FIVET outcome:outcome:

•insulin resistance insulin resistance •hyperandrogenism hyperandrogenism

•elevated leptin levelselevated leptin levels


**Beliver J: “Obesity and poor reproductive outcome: the potential role of the endometrium “. Fertil Steril Beliver J: “Obesity and poor reproductive outcome: the potential role of the endometrium “. Fertil Steril 2007;88:446-451 2007;88:446-451 * * Levens ED,Levens ED, Skarulis MC: “ Skarulis MC: “Assessing the role of endometrial alteration among obese patients undergoing assisted Assessing the role of endometrial alteration among obese patients undergoing assisted reproduction”. Fertil Steril 2008;89,6:1606-8. reproduction”. Fertil Steril 2008;89,6:1606-8.

* * Loveland JB, McClamrock HD, Malinow AM, Sharara FI. Increased body mass index has a deleterious effect on in Loveland JB, McClamrock HD, Malinow AM, Sharara FI. Increased body mass index has a deleterious effect on in vitro fertilization outcome. J Assist Reprod Genet. 2001;18:382–386.vitro fertilization outcome. J Assist Reprod Genet. 2001;18:382–386.

* * * * Gambineri A, Pelusi C, Vicennati V, Pagotto U, Pasquali R. Obesity and polycystic ovary syndrome. Int J Obes Gambineri A, Pelusi C, Vicennati V, Pagotto U, Pasquali R. Obesity and polycystic ovary syndrome. Int J Obes Relat Metab Disord. 2002;26:883–896.Relat Metab Disord. 2002;26:883–896.

Obesity could impair reproduction by acting on:Obesity could impair reproduction by acting on:1.1. the ovary the ovary 2.2. and/or the endometrium (and/or the endometrium (unfavorable unfavorable

intrauterine milieu)intrauterine milieu) ** 3.3. 35-50% PCOS are overweight or obese 35-50% PCOS are overweight or obese ****

Discrepancies in miscarriage rates mainly due to statistical flaws Discrepancies in miscarriage rates mainly due to statistical flaws caused by small sample sizescaused by small sample sizes

* * Fedorcsáck P, Storeng R, Dale PO, Tanbo T, Abyholm T. Fedorcsáck P, Storeng R, Dale PO, Tanbo T, Abyholm T. Obesity is associated with early Obesity is associated with early

pregnancy loss after IVF or ICSIpregnancy loss after IVF or ICSI. . Acta Obstet Gynecol ScandActa Obstet Gynecol Scand. 2000;79:43–48. . 2000;79:43–48. MEDLINEMEDLINE

Figure 1 Ongoing pregnancy rate per cycle (%) in each BMI group.

CI: confidence interval(women undergoing ovum donation)


**Norman RJ, Noakes M, Wu R, Davies MJ, Moran L, Wang JX. Norman RJ, Noakes M, Wu R, Davies MJ, Moran L, Wang JX. Improving Improving reproductive performance in overweight/obese women with effective weight reproductive performance in overweight/obese women with effective weight managementmanagement. . Hum Reprod UpdateHum Reprod Update. 2004;10:267–280. . 2004;10:267–280. MEDLINEMEDLINE | | CrossRefCrossRef

•low-calorie diet low-calorie diet •for a short period (4-6 week)for a short period (4-6 week)•before IVF cyclebefore IVF cycle andand•during IVF cycleduring IVF cycle

weight loss can improve weight loss can improve spontaneous ovulation spontaneous ovulation **

•Positive correlation between weight loss and ovulation and Positive correlation between weight loss and ovulation and pregnancy outcome: **pregnancy outcome: **

** ** Clark AM: Human Reprod 1998; 13:1502-1505Clark AM: Human Reprod 1998; 13:1502-1505


BMI > 25 :BMI > 25 :•Gn-RH-a : PR 26.7%Gn-RH-a : PR 26.7%•Gn-RH antagonists: 22%Gn-RH antagonists: 22%

BMI < 25: BMI < 25: •Gn-RH-a: Pr 29.9%Gn-RH-a: Pr 29.9%•Gn-RH antagonists: Pr 17.5%Gn-RH antagonists: Pr 17.5%

•Robinson J: Gn-RH-a vs. Gn-RH antagonist in ovarian stimulation: the influence Robinson J: Gn-RH-a vs. Gn-RH antagonist in ovarian stimulation: the influence of BMI on in vitro fertilization outcome”. Fertil Steril 2008;89,2:472-474. of BMI on in vitro fertilization outcome”. Fertil Steril 2008;89,2:472-474.

Stimulation protocolStimulation protocol

Nichols, Jr. BMI extremes and IVF pregnancy rates. Fertil Steril 2003.Nichols, Jr. BMI extremes and IVF pregnancy rates. Fertil Steril 2003.

starting with luteal phase leuprolide acetate 1 mg or 0.25 mg starting with luteal phase leuprolide acetate 1 mg or 0.25 mg

Gn-RH-a was decreased to 0.25 or 0.5 mg at the start of gonadotropins Gn-RH-a was decreased to 0.25 or 0.5 mg at the start of gonadotropins and continued daily until the day of hCG continued daily until the day of hCG

R-FSH or HMG on cycle day 2–4 at a dose of 150-300 IU dailyR-FSH or HMG on cycle day 2–4 at a dose of 150-300 IU daily10,000 IU of hCG on the leading follicle >18 mm 10,000 IU of hCG on the leading follicle >18 mm

Women not undergoing the standard protocol received a modified microdose Women not undergoing the standard protocol received a modified microdose flare protocol:flare protocol:•After at least 21 days of oral contraceptives, After at least 21 days of oral contraceptives, •40 μg of Lupron twice daily beginning on the second day of withdrawal 40 μg of Lupron twice daily beginning on the second day of withdrawal bleeding. bleeding.

•r-FSH or HMG 300-450 IU daily on 2 days after Lupron at HCG dayr-FSH or HMG 300-450 IU daily on 2 days after Lupron at HCG day

II° ProtocolII° Protocol

Two days before ET, 16 mg of Two days before ET, 16 mg of methylprednisolonemethylprednisolone daily for 5 days. daily for 5 days.

On the day of ET, On the day of ET, assisted hatchingassisted hatching was performed on all 3- and 4-day embryos was performed on all 3- and 4-day embryos through the use of a diluted Tyrodes acid solution. through the use of a diluted Tyrodes acid solution.

PP4 4 supplementation: 50 mg im or 90 mg vaginally dailysupplementation: 50 mg im or 90 mg vaginally daily

TABLE 1. Distribution of variables and outcomes by BMI group.

BMI < 20 BMI < 20 BMI 20-28 BMI 20-28 BMI ≥ 28BMI ≥ 28

ampules FSHampules FSH 29.5 (18.7)29.5 (18.7)** 27.8 (13.9)27.8 (13.9) ** 30.5 (16.7) 30.5 (16.7) **

COH daysCOH days 9.2 (1.3) 9.2 (1.3) ** 9.0 (1.3) 9.0 (1.3) ** 9.0 (1.3) 9.0 (1.3) **

thickness thickness 10.8 (1.9) 10.8 (1.9) ** 10.7 (2.2) 10.7 (2.2) ** 11.6 (2.5) 11.6 (2.5) **

pregnancy ratepregnancy rate 35.6%35.6% 52.1%52.1% 35.2%35.2%

abortionabortion 0%0% 5.9%5.9% 4.0%4.0%

* * ± SD± SD

Nichols, Jr. BMI extremes and IVF pregnancy rates. Fertil Steril 2003.Nichols, Jr. BMI extremes and IVF pregnancy rates. Fertil Steril 2003.

Low dose aspirin protocolLow dose aspirin protocol

• Patients with autoimmune disorders

• Suppressed tromboxane A2

but

• Decreases PG I2 too

•Improves number mature folliclesImproves number mature follicles•Improve size folliclesImprove size follicles•Not improve pregnancy rate/ETNot improve pregnancy rate/ET

Low dose aspirin protocolLow dose aspirin protocol

Start in previous cycle of COHStart in previous cycle of COH

100 mg/day until pick-up day100 mg/day until pick-up day

r-FSH 450 IU/d

HCG 20 IU/d

Microdose flare on 2° day of COH or

Delayed antagonistFrattarelli JL et al: “Low-dose aspirin use does not improve in vitro Frattarelli JL et al: “Low-dose aspirin use does not improve in vitro fertilization outcomes in poor responders”. Fertil Steril 2008;89,5:1113-17 fertilization outcomes in poor responders”. Fertil Steril 2008;89,5:1113-17

OHSS physiopathologyOHSS physiopathology

Ovary hyperstimulationOvary hyperstimulation•Multiple follicle recruitment•Luteal cysts•Neovascularisation

Ovary Ovary enlargementenlargement

•Abdomen distension•Abdomen pain•Nausea•Vomiting

Massive luteinization

•histamine•prostaglandins•citochine•renin

permeability vascular alteration

AscitesAscitesHypovolemiaHypovolemiaOliguriaOliguriaCIDCID

OHSS Classification (Volpicelli V. CIC Roma 1998)OHSS Classification (Volpicelli V. CIC Roma 1998) SLIGHTYSLIGHTY MODERATEMODERATE SEVERESEVERE

I° I° II° II° III° III° IV° IV° V° V° VI° VI°

ovary (cm)ovary (cm) < 5< 5 5-85-8 8-118-11 12-2012-20 > 20> 20 >20>20

Abdomen distensionAbdomen distension ++ ++ ++ ++++ ++++++ ++++++++

Abdomen painsAbdomen pains ++ ++ ++++ ++++++ ++++++++

Peritoneal flogosisPeritoneal flogosis ++ ++ ++ ++++ ++++++

VomitingVomiting ++ ++ ++ ++++ ++++ ++++++

NauseaNausea ++ ++ ++ ++ ++ ++++

DiarrhoeaDiarrhoea ++ ++ ++++

HydrothoraceHydrothorace Ascites Ascites [1][1]

++

Electrolytic Imbalance Electrolytic Imbalance ++ ++++

HypovolemiaHypovolemia ++++ ++++++

Venous central pressure Venous central pressure

Hypovolemic shockHypovolemic shock ++ ++++

AcidosisAcidosis ++ ++++

Kidney perfusionKidney perfusion

OliguriaOliguria ++

HyperazotemiaHyperazotemia ++++ ++++++

[1][1] Key symptom to hypersevere syndrome Key symptom to hypersevere syndrome

High responders protocol IHigh responders protocol I

• CC 100 mg/d 3°-7° days• r-FSH 150 UI s.c. on cycle day 9 at HCG day• antagonist 0.25 mg/d delayed regimen• Aspirin 100 mg/d on 1° at 45° cycle day

• HCG 10.000 UI on leading follicle ≥18 mm

High responders protocol IIHigh responders protocol II

• Gn 225 UI/d on 2° cycle daysGn 225 UI/d on 2° cycle days

• step-down regimenstep-down regimen

• antagonist 0.25 mg/d on 2° antagonist 0.25 mg/d on 2° day up HCG dayday up HCG day

DoxycyclineDoxycycline** 80 mg/Kg/day 80 mg/Kg/day (inhibits vascular (inhibits vascular leakage)leakage)

* Folkman HJ: fertil Steril 2007;88,S1:O14* Folkman HJ: fertil Steril 2007;88,S1:O14

*Bassado cpr 100 mg*Bassado cpr 100 mg

FSH – Antagonist – Agonist + HCG

• received triptorelin 0.2 mg in addition to the hCG. The GnRH-a dose was administered at the same time as the hCG; this was devised to achieve the induction of an endogenous LH surge that would coincide with the LH-like

• 34–36 hours before oocyte retrieval.

AA high responders IIIAA high responders III

• FSH 225 IU/d on the 2° cycle day (step-down regimen)

• antagonist 0.25 mg/d on the 2° cycle at HCG day

• Agonist (3.75 mg) as HCG trigger to achieve an endogenous LH surge

• when E2 ≥ 3.700 pg/ml (range 3.000-7.500)

• 0% OHSS0% OHSS

Agonist vs. HCG as triggerAgonist vs. HCG as trigger

Gn-RH-a:Gn-RH-a: HCG 10.000 UIHCG 10.000 UI

mature oocytespremature oocytesimplantation rateclinical pregnancyongoing pregnancyOHSS

OHSS/withholdingOHSS/withholding

EE22 >4.000 pg/ml >4.000 pg/ml

and/or and/or

Follicles >10 in each Follicles >10 in each ovaryovary

term term ≤≤3 day3 day

PC

OS

Yo

un

gH

igh

res

po

nd

ers

Yorie Ohata, Tasuku Harada, Masayuki Ito, Souichi Yoshida, Tomio Iwabe, Naoki Terakawa: “Coasting May Reduce the Severity of the Ovarian Hyperstimulation Syndrome in Patients with Polycystic Ovary Syndrome”. Gynecol Obstet Invest 2000;50:186-188

3434

OHSS/CoastingOHSS/Coasting

• Until drop of estrogen level Until drop of estrogen level <3.000 pg/ml<3.000 pg/ml

• Coasting >3 days no affects on Coasting >3 days no affects on PrPr

Egbase PE , Al Sharhan M , Berlingieri P , Grudzinskas JG . Egbase PE , Al Sharhan M , Berlingieri P , Grudzinskas JG . Serum oestradiol and Serum oestradiol and progesterone concentrations during prolonged coasting in 15 women at risk of progesterone concentrations during prolonged coasting in 15 women at risk of ovarian hyperstimulation syndrome following ovarian stimulation for assisted ovarian hyperstimulation syndrome following ovarian stimulation for assisted reproduction treatment reproduction treatment . . Hum Reprod Hum Reprod . 2000;15:2082–2086 . 2000;15:2082–2086

3434

OHSS/CoastingOHSS/Coasting

inverse relationship inverse relationship duration of coasting/number duration of coasting/number of mature oocytes retrievedof mature oocytes retrievedPregnancy ratePregnancy rate

* * Ulug U , Ben Shlomo I , Bahceci M . Predictors of success during the coasting period in high-Ulug U , Ben Shlomo I , Bahceci M . Predictors of success during the coasting period in high-responder patients undergoing controlled ovarian stimulation for assisted conception . Fertil responder patients undergoing controlled ovarian stimulation for assisted conception . Fertil Steril . 2004;82:338–342 Steril . 2004;82:338–342

M. Aygun, F. Vanlioglu, G. Karlikaya, H. Karagozoglu, B. Kumbak, S. Kahraman: “M. Aygun, F. Vanlioglu, G. Karlikaya, H. Karagozoglu, B. Kumbak, S. Kahraman: “Coasting Coasting may effect endometrial thickness and outcome”. Fertil Steril 2004; may effect endometrial thickness and outcome”. Fertil Steril 2004; 82, S 2, S21182, S 2, S211

CoastingCoasting

• Gn-RH-a long protocollong protocol• HMG or r-FSH 225 IU step-down regimen • On 2On 2ndnd cycle day at HCG or coasting day cycle day at HCG or coasting day

Owj , E . Tehrani Negad , E . Amirchaghmaghi , Z . Ezabadi , A . Baghestani: Owj , E . Tehrani Negad , E . Amirchaghmaghi , Z . Ezabadi , A . Baghestani: “The Evaluation of Withholding Gonadotropins (Coasting) Effects on the “The Evaluation of Withholding Gonadotropins (Coasting) Effects on the Outcome of In-Vitro Fertilization Cycles”. Fertil Steril 2005;84,S254Outcome of In-Vitro Fertilization Cycles”. Fertil Steril 2005;84,S254

Coasting Coasting

HCG 10.000 IU when E2 <3.000 pg/ml)HCG 10.000 IU when E2 <3.000 pg/ml)

3434

GanirelixGanirelix

Orgalutran fl s.c. 0.25 mgOrgalutran fl s.c. 0.25 mg

““Ganirelix salvage” Ganirelix salvage”

• Gn-RH-a long protocol on late luteal phase Gn-RH-a long protocol on late luteal phase previousprevious

• r-FSH 225 IU/d starting dose (down regimen)r-FSH 225 IU/d starting dose (down regimen)

Antagonist Antagonist 0.25 mg daily dose 0.25 mg daily dose at Eat E22 > 4.000 > 4.000

and orand or>10 follicles in each ovary>10 follicles in each ovary

daily measurement of serum daily measurement of serum EE22

HCG 10.000 IU at E2 HCG 10.000 IU at E2 ≤3.000 pg/ml≤3.000 pg/ml

M . Wittenberger , R . Gustofson , A . Armstrong , J . Segars: “A Cost Comparison of “Ganirelix M . Wittenberger , R . Gustofson , A . Armstrong , J . Segars: “A Cost Comparison of “Ganirelix Salvage” Protocol Versus “Coasting” Strategy for Patients at Risk for Ovarian Hyperstimulation Salvage” Protocol Versus “Coasting” Strategy for Patients at Risk for Ovarian Hyperstimulation Syndrome (OHSS)” . Fertility and Sterility 2005, 84 ,S318Syndrome (OHSS)” . Fertility and Sterility 2005, 84 ,S318

3535

““Ganirelix salvage”Ganirelix salvage”

•reduce Ereduce E22 levels (4.219 levels (4.219 2.613/24 h)2.613/24 h)•and avoid cycle cancellationand avoid cycle cancellation

•24 oocytes mean/cycle 24 oocytes mean/cycle •79.2% MII79.2% MII

Gustofson RL,, Segars JH and Larsen FW: “Ganirelix acetate causes a rapid reduction in estradiol levels without adversely affecting oocyte maturation in women pretreated with leuprolide acetate who are at risk of ovarian hyperstimulation syndrome”. Human Reproduction 2006 21(11):2830-2837

3535

PCOS ProtocolPCOS Protocol

• Pre-treatment with metformin ≥6 months• 2.000 mg/day• Improvment in menstrual cyclicity• Long-protocol agonist• Higher pregnancy outcome

Essah et al Fertil Steril 2006;86,1:230-232 Essah et al Fertil Steril 2006;86,1:230-232

3636

IVF cycle cost (ASRM’s average)

total IVF cycle total IVF cycle $ 12,500 $ 12,500

with “coasting” werewith “coasting” were $ 400/day $ 400/day

the cost per day of the cost per day of “ganirelix salvage”“ganirelix salvage” $ 553 $ 553

cycle cancellation prior to cycle cancellation prior to retrievalretrieval

$ 6379 $ 6379

plus the costs associated with plus the costs associated with either “coasting” or “ganirelix either “coasting” or “ganirelix salvagesalvage

M.D. Wittenberger, R.L. Gustofson, A. Armstrong, J.H. SegarsM.D. Wittenberger, R.L. Gustofson, A. Armstrong, J.H. Segars : “ : “A Cost Comparison of A Cost Comparison of “Ganirelix Salvage” Protocol Versus “Coasting” Strategy for Patients at Risk for Ovarian “Ganirelix Salvage” Protocol Versus “Coasting” Strategy for Patients at Risk for Ovarian Hyperstimulation Syndrome (OHSS)”. Fertil Steril 2995; 84Hyperstimulation Syndrome (OHSS)”. Fertil Steril 2995; 84,S1:S318

OHSS OHSS SUPPLEMENTATIONSUPPLEMENTATION• Micronized progesterone vaginally* 90-Micronized progesterone vaginally* 90-

100 mg/d100 mg/d

• EE2 2 orally** 4 mg/dorally** 4 mg/d

• No HCG ! ! !No HCG ! ! !

* * crinone 8% vaginal gel; prometrium cps 100 mgcrinone 8% vaginal gel; prometrium cps 100 mg ** ** progynova cpr 2 mg; sprediol spray 1.5 mg; progynova cpr 2 mg; sprediol spray 1.5 mg;

sandrena gelsandrena gel

Natural cycle modificationNatural cycle modification• when dominant follicle ≥ 14 mm (8°-9°

days)• r-FSH 75 UI/d up HCG day • ± antagonist 0.25 mg/d up HCG day• HCG 5.000 UI (leading follicle ≥18 mm

and at least two follicles ≥15 mm)

• Women aged 40 yearsWomen aged 40 years• FSH elevatedFSH elevated• Poor respondersPoor responders• Cost-saving alternativeCost-saving alternative

3737

•50 mg/d of im P4 in oil after oocyte retrieval

Luteal supplementationLuteal supplementation

• The luteal phase was supported with 50 mg/d of IM P in oil initiated immediately (?) after oocyte retrieval.

Prontogest, AMSA fl i.m. 100 mgProntogest, AMSA fl i.m. 100 mg

SUMMARYSUMMARY

• Controversies on gonadotropins Controversies on gonadotropins

• Controversies on analoguesControversies on analogues

• Controversies on E-P pillsControversies on E-P pills

• Controversies on LH addedControversies on LH added

Arslan MA, Bocca S, Mirkin S, Barroso G, Stadtmauer L, Oehninger S: “Arslan MA, Bocca S, Mirkin S, Barroso G, Stadtmauer L, Oehninger S: “Controlled ovarian hyperstimulation protocols for in Controlled ovarian hyperstimulation protocols for in vitro fertilization : two decades of experience after the birth of Elizabeth Carr”vitro fertilization : two decades of experience after the birth of Elizabeth Carr” Fertil Steril 2005;84,3: 555-569Fertil Steril 2005;84,3: 555-569

HMG or r-FSHHMG or r-FSH

• higher clinical pregnancy higher clinical pregnancy rate with hMGrate with hMG

• but no significant but no significant differences in ongoing differences in ongoing pregnancy rates or live pregnancy rates or live births births

Van Wely M , Westergaard L , Bossuyt P , Van Der Veen M . Effectiveness of human menopausal gonadotropin versus recombinant follicle-stimulating hormone for controlled ovarian hyperstimulation in assisted reproductive cycles (a meta-analysis) . Fertil Steril . 2003;80:1086–1093 .

intermediate respondersintermediate responders

excellent outcome:excellent outcome:

• with either a Gn-RH-a (long protocol) with either a Gn-RH-a (long protocol)

• or a GnRH antagonistor a GnRH antagonist• but tailoring of gonadotropin dose but tailoring of gonadotropin dose

must be performed to achieve must be performed to achieve optimized results.optimized results.

Arslan MA, Bocca S, Mirkin S, Barroso G, Stadtmauer L, Oehninger S: “Arslan MA, Bocca S, Mirkin S, Barroso G, Stadtmauer L, Oehninger S: “Controlled ovarian hyperstimulation protocols Controlled ovarian hyperstimulation protocols for in vitro fertilization : two decades of experience after the birth of Elizabeth Carr”for in vitro fertilization : two decades of experience after the birth of Elizabeth Carr” Fertil Steril 2005;84,3: 555-569Fertil Steril 2005;84,3: 555-569

High RespondersHigh Responders

High responders perform favorably High responders perform favorably with gentler stimulation that minimizes with gentler stimulation that minimizes the occurrence of OHSSthe occurrence of OHSS

The number of oocytes retrieved is The number of oocytes retrieved is predictive of clinical pregnancy only predictive of clinical pregnancy only in patients over 40 years of age in patients over 40 years of age

M. Luna-Rojas, B. Sandler, M. Duke, A.B. Copperman, L. Grunfeld, J. Barritt M. Luna-Rojas, B. Sandler, M. Duke, A.B. Copperman, L. Grunfeld, J. Barritt Fertility and SterilityFertility and Sterility September 2004 (Vol. 82, Page S206) September 2004 (Vol. 82, Page S206)

Low RespondersLow Responders

Arslan MA, Bocca S, Mirkin S, Barroso G, Stadtmauer L, Oehninger S: “Controlled ovarian hyperstimulation protocols for in vitro fertilization : two decades of experience after the birth of Elizabeth Carr” Fertil Steril 2005;84,3: 555-569

outcome suboptimal: outcome suboptimal:

•poor ovarian response poor ovarian response •poor oocyte/embryo quality poor oocyte/embryo quality

in spite of stimulation regimens usedin spite of stimulation regimens used

LH ADDEDLH ADDED

Suheil J. Muasher, Rony T. Abdallah, Ziad R. Hubayter: “Optimal stimulation protocols for in Suheil J. Muasher, Rony T. Abdallah, Ziad R. Hubayter: “Optimal stimulation protocols for in vitro fertilization” Fertil Steril 2006; 86,2:267-273vitro fertilization” Fertil Steril 2006; 86,2:267-273

Adding LH should be considered in Adding LH should be considered in severe situations of LH severe situations of LH suppression:suppression:

1.1. use of potent GnRH-agonists use of potent GnRH-agonists

2.2. Gn-RH-antagonistsGn-RH-antagonists

3.3. Over 35 yearsOver 35 years

Conclusion(s)Conclusion(s)

• Ovarian stimulation is a critical step in in vitro Ovarian stimulation is a critical step in in vitro fertilization therapy. fertilization therapy.

• A variety of controlled ovarian hyperstimulation A variety of controlled ovarian hyperstimulation regimens are available and efficacious, regimens are available and efficacious,

• but but individualization of management is essentialindividualization of management is essential and depends on assessment of the ovarian and depends on assessment of the ovarian reserve. reserve.

• Identification of the etiologies of poor ovarian Identification of the etiologies of poor ovarian response constitutes a formidable challenge response constitutes a formidable challenge facing reproductive endocrinologists.facing reproductive endocrinologists.

Arslan MA, Bocca S, Mirkin S, Barroso G, Stadtmauer L, Oehninger S: “Arslan MA, Bocca S, Mirkin S, Barroso G, Stadtmauer L, Oehninger S: “Controlled ovarian Controlled ovarian hyperstimulation protocols for in vitro fertilization : two decades of experience after the birth hyperstimulation protocols for in vitro fertilization : two decades of experience after the birth of Elizabeth Carr”of Elizabeth Carr” Fertil Steril 2005;84,3: 555-569Fertil Steril 2005;84,3: 555-569

Gonadotropin’sBioactivity Dr. Vincenzo Volpicelli Fertility Center Cardito Seconda Università...

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