Presentation on theme: "Gonadotropin’s Bioactivity Dr. Vincenzo Volpicelli"— Presentation transcript:
1Gonadotropin’s Bioactivity Dr. Vincenzo Volpicelli Seconda Università degli Studi di NapoliSeconda Università degli Studi di NapoliDipartimento di Scienze della VitaSUNfertGonadotropin’sBioactivityFertility Center CarditoDr. Vincenzo Volpicelli
2Gonadotropins FSH, LH, HCG glycoproteins dimers α, β (two peptide chain)α chain aspecificβ chain specific (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.
4FSH heterodimeric hormone: The half-life of FSH is 3-4 hours 92 amino acids α-chain111 amino acids β-chainThe half-life of FSH is 3-4 hoursVarious types of FSH exist according to their sialic acid contentBen-Rafael Z, Levy T, Schoemaker J Pharmacokinetics of follicle-stimulating hormone: clinical significance. Fertil Steril. 63:689–700
5LH The gene for the alpha subunit is located on chromosome 6q12.21. The luteinizing hormone beta subunit gene is localized in the LHB/CGB gene cluster on chromosome 19q13.32
6LH/HCG bioactivity LH & HCG: the same amino acids in sequence LH & HCG both stimulate the same receptorthe hCG β-subunit contains an additional 24 amino acids,both hormones differ in the composition of their sugar moieties.The different composition of these oligosaccharides affects bioactivity and speed of degradation.The biologic half-life:LH: 20 minutesFSH: 3-4 hourshCG: 24 hours
7FSH, LH, HCGThe protein dimer contains 2 polypeptide units, labeled alpha and beta subunits that are connected by two disulfide bridgesThe alpha subunits of LH, FSH, TSH, and hCG are identical, and contain 92 amino acidsThe beta subunits vary
8hypothalamus (arcuate nucleus and preoptic area) Gn secretionhypothalamus (arcuate nucleus and preoptic area)(Gn-RH pulses)pituitary glandGnfeed-backovaryestrogens
14Gn mode action membrane receptors activate a PtdIns Adenilcyclasi activationactivate a PtdIns(phosphatidylinositol)-calcium second messenger system
15Gn mode of action uterine blood flow: (Index Resistance)uterine blood flow:increases the uterine blood flow during the early luteal phase, a periimplantation stage
16Gn mode of actionincrease in the number of receptor in preparation for ovulationAfter ovulation, the luteinized ovary maintains LH-R-s that allow activation in case there is an implantation
17receptors activationbinding LH to the external part of the membrane spanning receptorwith LH attached, the receptor shifts conformation and thusmechanically activates the G proteinand activates the cAMP system~1% receptor sites activatedThe seven transmembrane α-helix structure of a G protein-coupled receptor such as LHCGR
18Gn-R expressionIts expression requires appropriate hormonal stimulation by FSH and estradiolpresent on:granulosa cellstheca cellsluteal cellsinterstitial cells
19Extragonadal Gn-Rs physiologic role largely unexplored. Gn-Rs have been found in:the uterus,sperm,seminal vesicles,prostate,skin,breast,adrenals,thyroid,neural retina,neuroendocrine cells,and (rat) brain.physiologic role largely unexplored.
20Gn action in ovaryfollicular maturationovulationluteal function
22FSH in early follicular phase FSH threshold: FSH serum concentrations needed to stimulate ovarian follicle growth (Brown 1978)At the onset of the menstrual cycle, a cohort of small (2–5 mm) antral follicles is present in each ovaryThis cohort will continue to grow in response to stimulation by FSHa process referred to as follicle recruitmentThe follicle with the highest sensitivity will benefit most from increasing FSH levels and will subsequently gain dominance (leader leader)Scheele F, Schoemaker J The role of follicle-stimulating hormone in the selection of follicles in human ovaries: a survey of the literature and a proposed model. Gynecol Endocrinol. 10:55–66.Brown JB Pituitary control of ovarian function: concepts derived from gonadotropin therapy. Aust NZ J Obstet Gynaecol. 18:47–54
23FSH in early follicular phase FSH concentrations reach a maximum in the early follicular phase of the normal menstrual cycle and decrease thereafternot increase much during a normal ovulatory cycleFSH concentrations only 10–30% above the threshold level is sufficient to stimulate normal follicle development*Brown JB Pituitary control of ovarian function: concepts derived from gonadotropin therapy. Aust NZ J Obstet Gynaecol. 18:47–54.**Messinis IE, Templeton AA The importance of follicle-stimulating hormone increase for folliculogenesis. Hum Reprod. 5:153–156.
24FSH in follicular phase Stimulates:follicular growth,granulosa cell aromatase activity,induction of LH receptors on the granulosa cell membrane,estradiol secretion
25Aromatase enzyme of the cytochrome P450 group mediate androgens aromatization:producing estrogenssexual development
26FSH in late follicular phase decreasedue to increased ovarian secretion of:E2β-inhibinnegative feedback at the hypothalamic-pituitary levelHotchkiss J, Knobil E The menstrual cycle and its neuroendocrine control. In: Knobil E, Neill JD, eds. The physiology of reproduction. New York: Raven Press; 711–750.Groome NP, Illingworth PJ, O’Brien M, et al Measurement of dimeric inhibin B throughout the human menstrual cycle. J Clin Endocrinol Metab. 81:1401–1405.
27Granulosa Cell Thecal Cell blood Steroidogenesis LH FSH R R Basement MembraneFSHcholesterolRCYP11cAMPE2pregnenoloneRCYP17ProteinKinaseA17-OH-P17βHSDCYP17E1DHEAcAMPP43βHSDProtein kinaseP450AldostCortisolAASteroidogenesis
28FSH follicular decreasing strict relationship with dominant follicle developmentAs a consequence, other recruited follicles lack sufficient stimulation by FSH and enter atresiaZeleznik AJ, Hutchison JS, Schuler HM Interference with the gonadotropin-suppressing actions of estradiol in macaques overrides the selection of a single preovulatory follicle. Endocrinology. 117:991–999.Schipper I, Hop J and Fauser B: “The Follicle-Stimulating Hormone (FSH) Threshold/Window Concept Examined by Different 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 & Metabolism Vol. 83, No
29FSH follicular decreasing Apparently, the maturing dominant follicle requires less FSH to continue its growth.It’s due to up-regulated FSH-sensitivity of leading follicle for:induction of locally various growth factors (IGF-I, AMH, inibina B, leptina, ICAM-1, VCAM-1, VEGF)induction of LH receptors that enhance FSH sensitivityErickson GF The ovarian connection. In: Adashi EY, Rock JA, Rosenwaks Z, eds. Reproductive endocrinology, surgery, and technology. Philadephia: Lippincott-Raven; 1141–1160.
31FSH in late luteal phase At the end of the luteal phase, there is a slight rise in FSH that seems to be of importance to start the next ovulatory cyclea cohort of small antral follicles is prevented from undergoing atresia and is stimulated for further developmentHodgen GD The dominant ovarian follicle. Fertil Steril. 38:281–300
32LH mode actionWith the rise in estrogens, LH receptors are also expressed on the maturing follicleestrogen rise leads via the hypothalamic interface to the “positive LH feed-back” effect, a release of LH over a hour periodThis 'LH surge' triggers ovulationLH is necessary to maintain luteal function (P4) for the first two weeksLH supports thecal cells in the ovary that provide androgens and hormonal precursors for estradiol productionIn case of a pregnancy luteal function will be further maintained by the action of hCG (a hormone very similar to LH) from the newly established pregnancy
33FSH gene β-chain gene: locate in arme 6p21.1-23 locate in 11p13 only in gonadotrope cells of pituitary glandincreased by Gn-RH and activinedecreased by inhibine
35LH-R abnormalities in females can lead to infertility masculinization In 46, XY pseudohermaphroditism,hypospadiasmicropenisAntibodies to LH-R can interfere with LH-R activity
36High Gonadotropin levels 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.Premature menopauseGonadal dysgenesis, Turner syndromeCastrationSwyer syndromePolycystic Ovary SyndromeCertain forms of CAHTesticular failuretypical in the menopause
37FSH in COHmultiple follicle development is induced by elevating FSH concentrations far above the thresholdBy 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 thresholdPolson DW, Mason HD, Saldahna MBY, Franks S 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 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.
38FSH gatethe "FSH-gate" or "FSH-window" concept has been proposed, which adds the element of time to the FSH threshold theory and emphasizes the significance of a transient increase in FSH above the threshold level for single dominant follicle development *Moreover, step-down dose regimen COH, has proven successful in reducing the incidence of multiple follicle development ***Baird DT A model for follicular selection and ovulation: lessons from superovulation. J Steroid Biochem. 27:15–23** van Santbrink EJP, Donderwinkel PFJ, van Dessel HJHM, Fauser BCJM Gonadotrophin induction of ovulation using a step-down dose regimen: single-centre clinical experience in 82 patients. Hum Reprod. 10:1048–1053
39FSH windowthe FSH window concept has been proposed, stressing the significance of the (limited) duration of FSH elevation above the threshold levelrather than the height of the elevation of FSH for single dominant follicle selectionFauser BCJM, van Heusden AM Manipulation of human ovarian function: physiological concepts and clinical consequences. Endocr Rev. 18:71–106.
40Gn dosageFor 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.
47Chinese hamster (Cricetulus griseus), white spotted type
48r-FSHproduced by inserting the genes encoding for α and β subunits of FSH into expression vectors that are transfected into a Chinese hamster ovary cell linePurification by immunochromatographyusing an antibody specifically binding FSHGonal-F, Puregon fl s.c., pen
52HMG vs. r-FSHboth products are probably equally safe and similar in efficacy, based on the available literature to date.Matorras R, Rodriguez-Escudero FG: “Debate. Bye-bye urinary gonadotropins?” . Hum Reprod ;17:1675–1683Suheil J. Muasher, Rony T. Abdallah, Ziad R. Hubayter: “Optimal stimulation protocols for in vitro fertilization”. Fertil Steril 2006; 86,2:
56CC CC alone produces sufficient enhanced follicular recruitment Clomiphene alone had significantly fewer folliclesnecessary gonadotropin support to be continued to prevent atresia of some of the cohort of folliclessupplemental hCG/P4 to corrected short luteal phasesM. M. Quigley: Annals of the New York Academy of Sciences, Vol 442, 1:
57CC + HMG HMG150 IU every other day starting on day 5 CC 100 mg/day on day 3–7 of the menstrual cycleHMG150 IU every other day starting on day 5HCG on leading follicle >18 mm and at least two follicles >15 mmPick-up or IUI hours afterHCG UI 6 days after (staff conversion)P4 50 mg/d i.m. on HCG day or E-T dayM. M. Quigley: Annals of the New York Academy of Sciences, Vol 442, Issue
58CC + delayed HMG CC 100 mg/day on day 1–5 day HMG150 IU every other day starting on day 6HCG on leading follicle >18 mm and at least two follicles >15 mmIUI or Pick-up hours afterHCG UI 6 days after (staff conversion)P4 50 mg/d i.m. on HCG day or E-T day
59CC + HMG best chance of COH minimize the disruption of the subsequent luteal phaseincreased pregnancy rateM. M. Quigley: Annals of the New York Academy of Sciences, Vol 442, Issue
62integrins down regulation Miscarriageintegrins down regulation(markers of endometrial receptivity)endometrialEE/P-r depletionuterine artery flowimpaired endometrial development
63Low Pr in CC/Gn COH desynchronized endometrial development premature LH surgeimmature oocytes
64CC/HMG Pr lower over 38 years old low ovarian reserve poor quality spermendometriosistubal damage or pelvic scar tissueinfertility >3 years
65CC/HMG adverse effects 3,5% twin1/3 of admission in TINTwin/mono mortality 10 +PIH 5 – 10 +placenta previaPlacenta detachment
66CC + E2 CC 100 mg/d on 3° cycle day EE 0.05 mg/d on days 8-12 hCG 10,000 IU at least one follicle was >18 mmA single IUI/Pick-up 24–36 hours afterprogesterone 50 mg daily IMon day of E-Tor3 days after IUI*until β-hCG levels were evaluated* Gerli: Intrauterine insemination. Fertil Steril 2000; 73,1:85-89
67CC + E2 endometrial thickness on the day of hCG administration. = CC only= CC + ethinyl E2
68CC + E2Characteristics and outcome of patients who received CC plus ethinyl E2 (group A) or CC alone (group B) in IUI cycleCharacteristicGroup AGroup BP valueNo. of patients32- -Mean (±SD) age (y)28.0 ± 5.626.0 ± 4.2NSMean (±SD) duration of infertility (mo)48.1 ± 18.536.7 ± 9.6Ongoing Pregnancy12 (37.5)2 (6.25)<.05Miscarried6 (18.75)pulsatility index valuesno difference
69Traditional COH HMG or r-FSH 300 IU on 2° day cycle HCG IU on leading follicle >17 mm and at least two follicles >15 mmPick-up after hP4 50 mg i.m. for luteal supplementation
70Traditional COH FSH remain elevated recruitment and growth of ovarian follicles continues throughout treatmentThis FSH serum pattern profoundly divergesfrom the spontaneous menstrual cycle* Filicori M: Characterization of the physiological pattern of episodic gonadotropin secretion throughout the human menstrual cycle . J Clin Endocrinol Metab ;62:1136–1144
71Traditional COHheterogeneous size cohorts of follicles are often found at hCG daythe optimal outcome of COH would be the selective attainment of numerous large mature homogeneous follicles.* Arnot AM , Vandekerckhove P , DeBono MA , Rutherford AJ . Follicular volume and number during in-vitro fertilization (association with oocyte developmental capacity and pregnancy rate) . Hum Reprod ;10:256–261
74Gn-RHGn-RH neurons are inside the medium-basal hypothalamus (arcuate nucleus and median eminence)Lately scientists showed Gn-RH syntesis in pituitary gland too
75Gn-RH biochemistry (1977s) a decapeptide (10 amino acids) in mammals.This chain is represented by: pyroGlu-His-Tyr-Ser-Gly-Leu-Arg-Pro-Gly-NH2The identity of GN-RH1 was clarified by the Nobel Laureates Roger Guillemin and Andrew V. Schally
76Pituitary gland histology Melanocyte-stimulating hormone (MSH)by pars intermedia (part of adenohyphysis)is the predominant hormone secreted
77NEUROHYPOPHYSIS - PARS NERVOSA This region of the pituitary is non secretory. Its cells are neuroglial-like pituicytes.The pars nervosa storesADHandOxytocinwhich were secreted by the hypothalamus.
78Melatonin/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
81Gn-RH secretion males/females in males, in pulses at a constant frequencyin females the frequency of the pulses varies during the menstrual cyclethere is a large surge of GN-RH1 just before ovulation
82Gn-RH frequencyLow frequency FSH releasehigh frequency LH release
83The seven transmembrane α-helix structure of a G protein-coupled receptor
84Gn-Rh analoguesWhile 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
85Effects of Gn-RH analogues agonistantagonistPrevent premature luteinization+ ++ + +Prevent premature ovulationTo synchronize early follicular development+
86Gn-RH agonistis 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 LHAgonists do not quickly dissociate from the Gn-RH receptorAs 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.
87Gn-RH-a Aminoacid sequence 6 10 nameact12345678910forGn-RHPyro-gluHisTrpserotoninTyrLeuArgProGly-NH2ivLeuproreline*15D-LeuN-EtNH2sc, imBuserelin * *20D-Sertriptor * * *D-TripGoserelin* * * *100AzGly-NH2depot sc* Enantone 3.75, 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)* * * Decapeptyl 3.75, mg fl s.c. im; Decapeptyl die 0.1 mg fl s.c.* * * * Zoladex 3.6, 10.8 mg fl s.c. imTriptorelin is an agonist with only a single substitution at position 6
89Gn-RH-a pharmacokinetics two hours: peak serum.It rapidly binds to the LHRH receptor cells in the pituitary glandthus leading to an initial increase in production of LH (flare-up)after 10 days: receptor desensitization and/or down-regulation
90Gn-RH-alysine replacement with ethylamide in 10 → half-time (4 min vs 3 h)
91Gn-RH-alysine replacement with D-amynoacide in 6 → Increase effectiveness ( times)D-aminoacid is hydrophobe chain carrier with enhancement receptor link
92Gn-RH-a effects Follicles synchronization ++++ Fewer small follicles on HCG day++Avoids premature luteinizationMultiple pregnancies≡ ≡ ≡Decreases OHSS frequency
95Goserelin* * Zoladex 3.75 mg, 11.25 mg fl im (FDA, 1989) D-Ser(But)6Azgly10LHRH* Zoladex 3.75 mg, mg fl im (FDA, 1989)
96Goserelin*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 hoursafter a period of about days, production of LH is greatly reduced due to receptor downregulation
97Gn-RH-a protocols long protocol short (“flare-up”) protocol ultrashort protocolmicrodose flare protocol
101PR/transfer in Gn-RH-a Flare-up protocol19.2%Long protocol25.7%Media24.8%without analogues23.2%FIV nel periodo (da FIV-NAT ’97) sec. Barrière et al. 1999
102Gn-RH-a Long protocolGn-Rh-a depot 3.75 mg in one dose on 21st day only of previous cycleGn-Rh-a low-dose daily on the 21st day of previous cicle to HCG day:Buserelin (Suprefact fl 5.5 ml) 0.3 ml fl s.c.Buserelin nasally 1 buff x 3/d (300 μg)Leuproreline (Enantone die fl s.c.) 0.2 ml/dayTriptoreline (Decapeptyl die fl s.c.) 0.2 mloron any day when:LH <0.5E2 <30No ovarian cyst >10 mm8
103Gn-RH-a long protocolr-FSH/HMG IU/day on 2nd cycle day to HCG dayHCG IU on the least two follicles >18 mmPick-up after hoursP4 supplementationHCG IU six days after E-T8
104Short (flare-up) protocol 9Short (flare-up) protocolGn-RH-a 3.75 mg depot ½ fl i.m. on 2° cycle day onlyr-FSH IU/d on 3th day (step-down regimen)HCG IU (18 mm )Pick-up after hHCG (+ P4)poor responder
105Gn-RH-a flare low dose protocol EE-P for 1-2 cycleson 1st cycle day at HCG day:Triptoreline (decapeptyl die) 0.2 ml (0.1 mg) s.c. dailyLeuproreline acetate (enantone die) 0.2 ml (1 mg) s.c. dailyBuserelin (Suprefact flac 5.5 ml) 0.3 ml s.c.Buserelin nasally 3 buff/day (300 μg)oron any day when:LH <0.5E2 <30No ovarian cyst >10 mmr-FSH/HMG UI/d on 3rd cycle dayAfter administration s.c. enantone die reachs a serum peak of 32.3 mg/ml in 0.6 h
106Gn-RH-a ultrashort protocol 11on 2nd cycle day for three days:Triptoreline 0.2 ml s.cLeuproreline 0.2 ml s.c.Buserelin 0.5 ml s.c.Buserelin nasally 3 buff/dayoron any day when:LH <0.5E2 <30No ovarian cyst >10 mmr-FSH/HMG on the 2nd cycle day
10711 ~ ~ ~ Ultrashort Long + + + HMG ampoules cancelled cycles n. oocytesfertilization rateembryo cleavage ratesupernumerary embryosSamuel 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
108HCG low-dose long protocol 12HCG low-dose long protocolGranulosa cells in 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 the physiologic actions of FSH on GCs (2).1. Zeleznik AJ , Hillier SG . The role of gonadotropins in the selection of the preovulatory follicle . Clin Obstet Gynecol ;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 ;117:355–367
109HCG low-dose in a-long protocol 12HCG low-dose in a-long protocolBased 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.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 ;78:414–416
110HCG low-dose long protocol 12HCG low-dose long protocolThe 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 (200 IU/d), corresponding to roughly 1,200 IU/d of LHThe hCG is also drastically less expensive than recombinant FSH or hMG .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 ;60:175–178Sullivan 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 ;84:228–232
111HCG low-dose in long protocol Gn-RH-a long protocolr-FSH/hMG (1:1/2) IU on 2° day at least six follicles >12 mm and E2 >300 pg/mlhCG 250 IU/day alone until the end of COHorvariable amounts of r-FSH and low-dose (10-50) IU hCGreduced r-FSH/hMG consumptionoutcome comparable to traditional COH regimens;reduced number of small preovulatory follicles;did not cause premature luteinization;more estrogenic intrafollicular environment12Filicori M: Fertil Steril 2005: 84, 2:
112gonadotropin and steroid at HCG day 12gonadotropin and steroid at HCG dayGroup A Group B P value(no hCG) (hCG)LH (IU/L) ± ± NSFSH (IU/L) 11.3± ± <.001hCG (IU/L) 0.4± ± <.001E2 (pg/mL) ± ± <.05P (ng/mL) 1.1± ± NST (ng/mL) ± ± <.05Filicori M: Fertil Steril 2005: 84, 2:
11413FSH/HMG long protocolGn-RH-a depot on 21° day of previous cycle onlyor Gn-RH-a low dose on 21° day up HCG dayr-FSH UI, step-down regimen, on 2nd at 8th cycle day8r-FSH continued until HCG day (if LH ≥5 mUI/ml)orHMG on 9th until HCG day (if LH < 1 mUI/ml)Ye H: Fertil Steril 2006;86,3S:S420-S421
115r-FSH/HMG Long protocol 13123456789101112LH >5 mIU/mlLH <1 mIU/ml r-FSH HMG Gn-RH-a low dose21° Gn-RH-a depot or low dose long protocol
117Antagonists (1990s) They bind immediately to the receptor Receptor targetthis leads to immediate pituitary down-regulationand do not activate classic postreceptor events;no “flare-up”*Orgalutran, Cetrotide 0.25 mg fl s.c
118Gn-RH AntagonistsLubecca Method,delayed somministration0.25 mg s.c. on 6° COH day or leading follicle >14 mm until HCG dayCalifornia 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
119on 1° days Gn stimulation on 5°-6° days on leading follicle ≥14 mm Antagonists protocolon 1° days Gn stimulationon 5°-6° dayson leading follicle ≥14 mmHMG or r-FSH + LH addedFixed and early start of the antagonist is probably more effective than an individualized and late start.
120Gn-RH Antagonist disavantages LDP advantages: peak E2 on HCG day mature follicles oocytes embryos PRadvantages:Prevention surge LHlarger cohort of folliclesAvoidance of adverse effects of agonistsMore friendly stimulation protocol OHSS
122LH addedThe 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? Fertil Steril 20003;79:253–5.
123LH addedGranulosa cells in ovarian follicles of larger size (>10–12 mm) normally express the LH/hCG receptor and become sensitive to LH activity stimulation *Campbell et al. showed that pulsatile LH administration in sheep maintained elevated ovulatory rates despite FSH withdrawal **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 * ** Zeleznik AJ , Hillier SG .: Clin Obstet Gynecol ;27:927–940* * Campbell BK , Dobson H , Baird DT , Scaramuzzi RJ .: J Reprod Fertil ;117:355–367
124LH ADDED target LH <1 UI/ml at the start of Gn stimulation Gn-RH-a flare protocol (LH suppression)Gn-RH antagonist during stimulation>35 yearsPoor respondersHigh responders (LH prevalence activity decrease n. small follicles and OHSS risk)
126LH added targetthe 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
129Rationale 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
130Rationale 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 testosterone under LH stimulation, and FSH induces granulosa cell 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 iperandrogenismo in PCOS)
131LH Added protocol15leuprolide acetate 1 mg daily, sc, from menstrual day 21 for 14 days (+ 7 days)excluded from further treatment if E2 >20 pg/ml and/or LH >2,5 IU after 21 days leuprolideLH <2.5 IU/L and E2 <20 pg/mLr-FSH starting at 150 IU sc daily at h. for 4 daysOn 5° dayIf serum E2 levels were less than 100 pg/mL, the r-FSH dose was increased to 225 IUIf serum E2 levels were greather than 100pg/mL, the r-FSH was maintained at 150 IU/dayr-LH 375 IU twice a day (7.30 and h) for the last 2 days of COHorLeading follicle ≥14 mm*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” J Clin Endocrinol Metab ;84:228–232 .
132LH added vs. HMG in over 38 *r-FSH + HMG 75 UI (group I) and r-FSH + r-LH 75 UI (group II)HMG groupLH groupn. follicles on 6 day6.72 2.225.87 1.29COH days10.5 1.712 1.8M II oocytes75.3%93.1%Pregnancy rate26%47%* Gomez-Palomares J. L. ; Acevedo-Martin B. ; Andres L. ; Ricciarelli E. ; Hernandez E. R.; Reproductive biomedicine online ISSN ; 2008
135P4 secretion Follicular phase Luteal phase * Ovary 48% 95% Adrenal gland4%from pregnenolone1%*P4 serum level: 4 ng/ml is low level; 40 ng/ml is high
136luteal P4 supplementation Few studies in the last 20 yearsCurrently, no reliable method for specific diagnosis of P4 deficiency in luteal phaseRegimens often determined by clinical experienceThe rationale for P4 supplementation:Aspiration of the granulosa cellsPresence of high levels of E2Analogues poor luteal function (due to residual suppression of pituitary LH secretion)ASRM Practice Committee: “Exogenous progesterone supplementation” Fertil Steril 2008;89,4:
137luteal P4 supplementation P4 50 mg/d i.m. Ormg/day vaginallyStarting:3 days after IUIorat E-T dayProntogest fl im 100 mg
138luteal P4 supplementation Higher pregnancy rate *Lack of evidence in literature * *Increased of hypospadias (progestins derived from androgens and that bind to androgen receptors) * * ** Yovich JL et al: “Early luteal serum progestyerone concentration are higher in pregnancy cycles”. Fertil Steril 1985;44:** Ziad R. Hubayter: “luteal supplementation in in vitro fertilization: more question than answers”. Fertil Steril 2008; 89,4:***ASRM Practice Committee: “Exogenous progesterone supplementation” Fertil Steril 2008;89,4:Carmichael SL et al: “Maternal progestin intake and risk of hypospadias”. Arch Pediatr Adolesc Med 2005;159:957
139P4Orally:bioavailabilty diminished by the liver first passthe serum level typically returns to baseline level by 6 hoursdizziness and somnolencefatigue, headache, urinary frequencyvaginal route:the level remains elevated for up 48 hoursCrinone gel 8% once a day and contains 90 mg of P4Progeffik gel 200 mg 1-3/dayUterine tissue higher level P4 despite a lower serum P4Vaginal irritationIntramuscular:P4 in oil result in higher plasma concentrationand longer duration *Severe allergic reactionAdult respiratory distress syndromeEosinophilic pneumonitis*Posaci C, Smitz J, Camus M, Osmanagaoglu K, Devroey P: “Progesterone for the luteal support of ART: clinical options”. Human Reprod 2000; 15,S1:
140luteal supplementation in agonist/antagonist protocols Pituitary desensitization for 2-3 w after last administrationWorldwide standard practice ** 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:
141luteal suppl agonist/antagonist protocols or/andHCG :more effectiveIncreased production of E2 and P4Better endocrine profileNo differences in pregnancy outcomeOHSS risk (E2 peak at HCG day)*Martinez F: “ Human Corionic Gonadotropin and intravaginal natural progesterone are equally effective for luteal phase support in IVF”. Gynecol Endocrinol 2000; 14:
142Luteal E2 supplementation In IVF cycles, the levels of E2 and P4 drop in the mid-late luteal phaseLower E2 at 11 days after pick-up is associated with lower pregnancy rateE2 orally 2-6 mg/d (Progynova cpr 2 mg) *Start on:E-T dayor7 days after E-TIncreases implantation rateIncreases pregnancy rate* Lukaszuk K: Fertil Steril 2005;83:
144Poor response — the devil is in the definition The original definition of low response to ovarian response by Garcia et Acosta was based on low peak E2 concentrations aloneThey stimulated patients with hMG (150 IU IM daily) and defined low responders as patients with a peak E2 concentration of <300 pg/mL
145Poor responders diminished ovarian reserve A lower expression of FSH receptor in the granulosa cellsAdvanced maternal ageE2 < 500 pg/mL on day of hCG<4 de Graaf follicles on HCG daylower fertilization rateslower cleavage rateslower resulting embryosLower implantation ratelower pregnancy rates“occult ovarian failure”10–25% of the ART population** Keay et al., 1997 ; Karande and Gleicher, 1999 ; Fasouliotis et al., 2000 ; Tarlatzis et al., 2003
146increase Gn dose 16 first and simplest approach limited benefit to 450 IU per day300 IU r-FSH + hMG 150 IUbeyond this amount little or no improvementMurat Arslan: Fertil Steril 2005; 84,3:
147the stop Gn-RH-a protocol 17Gn-RH-a low dose on 21° day until the beginning of menstruation.Stop analoguesgonadotropins from day 2 of the cycle until HCG dayTarget of this protocol:Stop to pre-menstrual FSHand, subsequently,stop to size discrepancy in the developing follicles
14818 CC + HMG + Antagonist CC 150 mg/d on 1°-5° days HMG (r-FSH) large dosage ( IU/d) on 2-3° cycle day Antagonist delayed administration:on 6°-8° stimulation daysor leader follicle ≥ 14 mm if very few folliclesCGHCG UI on day dominant follicle ≥18 mmLuteal supplementation: HCG IU/d (+ P4)
150CC + HMG + Antagonist Long protocol n° ampules 50 83 follicles >14 Age >40Long protocolCC + HMG + Antagonistn° ampules5083follicles >143.75.8E2 on day 53674E2 on day 9169945 ( )E2 on HCG day744833 ( )Cancellation rate34%4.8%total oocytes3.35.5Mature oocytes2.64.29n° embryo1.41.6PR15.3%22.2%Implant rate7.6%13.5%Weghofer, 2004
151Luteal estradiol protocol 20lowering FSH levels with estrogen, the ovary will respond when high doses of FSH are added in COH protocolOral micronized E2 2 mg twice a dayOn luteal day 21At 3 days of COHr-FSH IU/d down regimen on 2° daymicrodose flare Gn-RH-a on 3° COH dayordelayed Gn-RH antagonistHCG low-dose (10-50 IU/d) on 8° dayDragisic KG Fertil Steril 2005;84:
152Luteal estradiol protocol * outcomeAll cyclesLuteal EstradiolStandard protocolClinical Pr38,3%40,9%31,3%Miscarriage rate43,5%38,9%60,0%Delivery rate20.0%25.0%12.5%* Frattarelli J, et al: “A luteal estradiol protocol for expected poor-responders improves embryo number and quality” Fertil Steril 2008;89,5:
153AACEP Protocol E-P pills for 1 to 3 weeks Gn-RH-a low-dose in a standard long protocol overlapping the last 5 to 7 days of E-P pills until onset of mensesGn-Rh antagonist low-dose (0.125 mg/day) on cycle day 2Estradiol valerate* 2 mg/d on 1° to 10° cycle dayEstrogen suppositories** were used to maintain the endometrium until at last one follicle measured 15 mmr-FSH in initial doses of 600 or 750 IU/day, decreasing to 225 IU/day of r-FSH.* Progynova cpr 2 mg** Vagifem cpr vaginali mgFisch 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:
154* AACEP Protocol a g o n i t E-P p I l s E T A E 12345678910121314151617… E TP4AEHCG r-FSH 150 IUA antagonist
15522Androgensandrogens may influence the responsiveness of ovaries to gonadotrophinspositive regulators of follicular developmentaugments follicular FSH-receptor expression in granulosa cellsIGF-I oocyte expressionpromotes initiation of primordial follicle growthincreases the number of growing preantral and small antral folliclesVendola 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 primate ovary. Biol Reprod 1999; 61:353–357.
157DHEA DHEA is the cornerstone to all sex hormones For women under 50, DHEA levels of less than 150 ng/dL are considered lowCasson PR: “Dehydroepiandrosterone supplementation augments ovarian stimulation in poor responders: a case series Human” Reproduction, Vol. 15, No. 10, , October 2000
158DHEA protocol and during COH POF High FSH 75 mg/d for 4-12 months previous COHandduring COHReduces FSH levelrelease more and better quality eggs prior to IVFreduces miscarriage rates - especially in older womensimilar effects of GH increasing IGF-I paracrine effectsIncreases IGF-I serum levelsLeonidas Mamas, Eudoxia Mamas,: “Premature ovarian failure and dehydroepiandrosterone” Fertil Steril 2008 in press
159TestosteroneTwo IVF treatment cycle cancellations due to poor follicular response,in spite of vigorous gonadotrophin ovarian stimulationand having normal basal FSH levelsBalasch 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 gonadotrophins in poor-responder IVF patients with normal basal concentrations of FSH”. Human Reproduction (7):
160Testosterone protocol Gn-RH-a low dose (Leuprolide 1 mg or triptoreline 0.1 mg) long protocol-like started in the midluteal phase of the previous cycleat menses start Gn-RH-a is reduced to 0.5 mg and continued until the administration of HCG1° cycleGn-RH-a 0.5 mg/day of leuprolide from the midluteal phase at menses start0.25 mg/day thereafter2° cycleBalasch 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 gonadotrophins in poor-responder IVF patients with normal basal concentrations of FSH”. Human Reproduction (7):
161Testosterone transdermal testosterone 20 µg/kg per day Androderm 2.5 mg daily single patchRemoved always at 9.00 a.m.0.1 mg/h delivery rate (a predetermined number of 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 = 1.2 mg or 1200 µg) and thus applied at hours].during the 5 days preceding COHBalasch 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 gonadotrophins in poor-responder IVF patients with normal basal concentrations of FSH”. Human Reproduction (7):
162Testosterone COH 1° cycle 2° cycle Gonadotrophin ovarian stimulation was started the day following last testosterone patch application1° cycleOn day 1 of ovarian stimulation, r-FSH 450 IU s.c.On day 2 r-FSH 300 IUOn days 3 and 4 of ovarian stimulation, 150 IU per dayFrom day 5 onwards, r-FSH was administered on an individual basis2° cycleOn 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 HMGFrom day 5 onwards, HMG on an individual basisBalasch 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 gonadotrophins in poor-responder IVF patients with normal basal concentrations of FSH”. Human Reproduction (7):
164Testosterone80% showed an increase of over fivefold in the number of recruited follicles,produced 5.8 ± 0.4 oocytes,received two or three embryospregnancy rate: 30% per oocyte retrievalcancelled cycles: 20%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 gonadotrophins in poor-responder IVF patients with normal basal concentrations of FSH”. Human Reproduction (7):
166Letrozole A E1 T E2 Third-generation aromatase inhibitors (AIs) nonsteroidal, reversible, orally administeredThe excellent oral bioavailability (100%)relatively short half-life (45 hours)able to effectively block the conversion of :A E1T E2
167Letrozole + HMG CC resistant Poor responders 20%–25% of women are resistant to CCcomparable pregnancy results vs.:CC/HMGr-FSH alonesignificant saving in the amount of GonadotropinsGrabia 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 for assisted reproductive technologies” Fertil Steril 2006;86,4:
168Letrozolo + HMG Letrozolo 2.5 mg days 3–7 day cycle HMG 150 IU on day 5 up lead. foll. >18HCG IU hours afterPick-up hours after HCGP4 50 mg/d i.m. on HCG day or E-T dayMohamed F.M Mitwally, Robert F Casper: “Use of an aromatase inhibitor for induction of ovulation in patients with an inadequate response to clomiphene citrate” Fertil Steril 2001; 75,2:
169Increasing AAPoor responders: lower expression of FSH receptor in the granulosa cellsPCOs Patients: hyperexpression of FSH receptor
170Increasing AAinducing a temporary and reversible PCO-like condition in the ovaries of poor responder patientscould enhance their follicular recruitment and development
171Aromatase inhibitors protocol aromatase inhibitor which induces a temporary accumulation of intraovarian androgensADVANTAGES AIs vs. CCLack of down-regulation of hypothalamic-pituitary estrogen receptorsLower FSH doseHigher number of mature oocytesLess adverse effects on endometrium1 and cervixPregnacy rate: 21%1 Endometrial thickness <5 mm is usually associated with failure to conceive(Gonen Yand Casper RF: “Sonografic determination of an adverse effect of clomiphene citrate on endometrial growth”. Human Reprod 1990;5: ).
172Increasing AAsynergistical role of androgens with FSH to promote early follicular recruitmenttrophic effects of androgens in small antral folliclesPositive estrogen feed-back on hypotalamic-hypophyseal axisAA too high reduce follicular health
173Letrozole E-P for 15-21 days or E2 <60 pg/ml absence of cysts >10 mmletrozole, 2.5 mg/day from day 1-5 of the menstrual cycleFSH ( IU/day) starting on day 6hCG (10,000 IU) when two leading follicles were ≥20 mmMitwally MFM and Casper RF. (2002) Aromatase inhibition improves ovarian response to follicle-stimulating hormone in poor responders. Fertil Steril 77:776–780.
174Letrozole + Antagonist letrozolo 5 mg on 1° to 5° cycle dayr-FSH 300 IU + HMG 150 IU on 1° to 5° cycle dayOn 6° day individual dosages r-FSH/HMGdelayed antagonist 0.25 mg/dHMG + Antagonist until HCG dayr-HCG 250 mg on leading follicle >18 mmP4 supplementation with 200 mg of vaginal micronized P (Progeffik)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 and improves in vitro fertilization outcome in low responder patients: A pilot study”. Fertil Steril 2005;84,1:82-87.
175A Antagon E T A r-FSH 150 IU HMG 150 UI 23456789101213141516171819… E TP4A r-FSH150 IU HMG150 UI Letrozole 2,5 mg r-HCG 250 mgA Antagon
177Assisted hatchingabcM. 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, settembre 1998.
178E-T on the day 2 Microdose flare agonist protocol day 2 day 3 Implantation rate23.9%17.2%Pregn rate/oocyte27.7%16.2%Pregnancy rate/E-T29.018.3Luteal supplementation: progesterone 100 mg/day i.m.On oocyte collection through the luteal phaseBahceci M.: Fertil Steril 2006;86,1:81-85
179Ovary deficiency 1) gonadic disgenesia “streak gonad” absence of ovary tissue2) OvarydisgenesiaOvary tissue without follicles and without functionality3) POFOvary tissue without follicles but with past functionality4) Proof Ovary (Savage Symdrome)Ovary tissue with hystologic normal follicles
187endometriosis presence of ovarian endometriomas responsiveness to gonadotropins: - 25%
188Long protocol + prednisone 3.75 mg Gn-Rh-a depot in one dose on 21st dayorLow-dose daily on the 21st day of previous cicle to HCG dayon any when LH <0.5 and E2 <30on the 3th day of menstrual cycleUSGLH <0.5 UI/mlE2 <30 pg/ml
189Long protocol + prednisone Prednisone 15 mg/day on 1° day at HCG day(Deltacortene cpr 5 mg)r-FSH 450 IU on 2° day up 6° dayr-FSH variable dosageLuteal supplementation: P4 50 mg/d i.m.
190lean women reproductive performance was not poorer Donna magra come un treno (Mango)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:
192Overweight 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).Levens ED, Skarulis MC: “Assessing the role of endometrial alteration among obese patients undergoing assisted reproduction”. Fertil Steril 2008;89,6:
193Overweight in PMA Higher cancellation rate Lower pregnancy rates Poor reproductive performance:Higher cancellation rateLower pregnancy rateshigher miscarriage rateslower live-birth rates in natural and PMAmore frequent complications in pregnancy*Fedorcsáck P, Storeng R, Dale PO, Tanbo T, Abyholm T. Obesity is associated with early pregnancy loss after IVF or ICSI. Acta Obstet Gynecol Scand. 2000;79:43–48.* the effect of recipient body weight on reproductive performance
194Overweight Extraovum effects of obesity on FIVET outcome: insulin resistancehyperandrogenismelevated leptin levelsNorman RJ, Clark AM. Obesity and reproductive disorders: a review. Reprod Fertil Dev. 1998;10:55–63.
195Overweight Obesity could impair reproduction by acting on: the ovary and/or the endometrium (unfavorable intrauterine milieu) *35-50% PCOS are overweight or obese **Discrepancies in miscarriage rates mainly due to statistical flaws caused by small sample sizes*Beliver J: “Obesity and poor reproductive outcome: the potential role of the endometrium “. Fertil Steril 2007;88:* Levens ED, Skarulis MC: “Assessing the role of endometrial alteration among obese patients undergoing assisted reproduction”. Fertil Steril 2008;89,6:* 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.* * 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.
196Figure 1 Ongoing pregnancy rateper cycle (%)in each BMI group.CI: confidence interval(women undergoingovum donation)* Fedorcsáck P, Storeng R, Dale PO, Tanbo T, Abyholm T. Obesity is associated with early pregnancy loss after IVF or ICSI. Acta Obstet Gynecol Scand. 2000;79:43–48. MEDLINE
197Overweight low-calorie diet for a short period (4-6 week) before IVF cycleandduring IVF cycleweight loss can improve spontaneous ovulation *Positive correlation between weight loss and ovulation and pregnancy outcome: ***Norman RJ, Noakes M, Wu R, Davies MJ, Moran L, Wang JX. Improving reproductive performance in overweight/obese women with effective weight management. Hum Reprod Update. 2004;10:267–280. MEDLINE | CrossRef** Clark AM: Human Reprod 1998; 13:
198Overweight BMI < 25: Gn-RH-a: Pr 29.9% Gn-RH antagonists: Pr 17.5% 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:
199Stimulation protocolstarting with luteal phase leuprolide acetate 1 mg or 0.25 mgGn-RH-a was decreased to 0.25 or 0.5 mg at the start of gonadotropins and continued daily until the day of hCGR-FSH or HMG on cycle day 2–4 at a dose of IU daily10,000 IU of hCG on the leading follicle >18 mmII° ProtocolWomen not undergoing the standard protocol received a modified microdose flare protocol:After at least 21 days of oral contraceptives,40 μg of Lupron twice daily beginning on the second day of withdrawal bleeding.r-FSH or HMG IU daily on 2 days after Lupron at HCG dayTwo days before ET, 16 mg of methylprednisolone daily for 5 days.On the day of ET, assisted hatching was performed on all 3- and 4-day embryos through the use of a diluted Tyrodes acid solution.P4 supplementation: 50 mg im or 90 mg vaginally dailyNichols, Jr. BMI extremes and IVF pregnancy rates. Fertil Steril 2003.
200TABLE 1. Distribution of variables and outcomes by BMI group. ampules FSH29.5 (18.7)*27.8 (13.9) *30.5 (16.7) *COH days9.2 (1.3) *9.0 (1.3) *thickness10.8 (1.9) *10.7 (2.2) *11.6 (2.5) *pregnancy rate35.6%52.1%35.2%abortion0%5.9%4.0%* ± SDNichols, Jr. BMI extremes and IVF pregnancy rates. Fertil Steril 2003.
203Low dose aspirin protocol Start in previous cycle of COH100 mg/day until pick-up dayr-FSH 450 IU/dHCG 20 IU/dMicrodose flare on 2° day of COH orDelayed antagonistFrattarelli JL et al: “Low-dose aspirin use does not improve in vitro fertilization outcomes in poor responders”. Fertil Steril 2008;89,5:
206OHSS Classification (Volpicelli V. CIC Roma 1998) SLIGHTYMODERATESEVEREI°II°III°IV°V°VI° ovary (cm)< 55-88-1112-20> 20>20Abdomen distension++++++++++Abdomen painsPeritoneal flogosisVomitingNauseaDiarrhoeaHydrothorace Ascites  Electrolytic ImbalanceHypovolemiaVenous central pressure Hypovolemic shockAcidosisKidney perfusionOliguria Hyperazotemia Key symptom to hypersevere syndrome
207High responders protocol I CC 100 mg/d 3°-7° daysr-FSH 150 UI s.c. on cycle day 9 at HCG dayantagonist 0.25 mg/d delayed regimenAspirin 100 mg/d on 1° at 45° cycle dayHCG UI on leading follicle ≥18 mm
208High responders protocol II Gn 225 UI/d on 2° cycle daysstep-down regimenantagonist 0.25 mg/d on 2° day up HCG dayDoxycycline* 80 mg/Kg/day (inhibits vascular leakage)* Folkman HJ: fertil Steril 2007;88,S1:O14*Bassado cpr 100 mg
209FSH – 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-like34–36 hours before oocyte retrieval.
210AA high responders III 0% OHSS FSH 225 IU/d on the 2° cycle day (step-down regimen)antagonist 0.25 mg/d on the 2° cycle at HCG dayAgonist (3.75 mg) as HCG trigger to achieve an endogenous LH surgewhen E2 ≥ pg/ml (range )0% OHSS
211Agonist vs. HCG as trigger Gn-RH-a:HCG UImature oocytespremature oocytesimplantation rateclinical pregnancyongoing pregnancyOHSS
21234 OHSS/withholding High responders Young PCOS E2 >4.000 pg/ml and/orFollicles >10 in each ovaryterm ≤3 dayHigh respondersYoungPCOSYorie 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:
21334 OHSS/Coasting Until drop of estrogen level <3.000 pg/ml Coasting >3 days no affects on PrEgbase PE , Al Sharhan M , Berlingieri P , Grudzinskas JG . Serum oestradiol and progesterone concentrations during prolonged coasting in 15 women at risk of ovarian hyperstimulation syndrome following ovarian stimulation for assisted reproduction treatment . Hum Reprod ;15:2082–2086
214inverse relationship OHSS/Coasting duration of coasting/number of mature oocytes retrievedPregnancy rateM. Aygun, F. Vanlioglu, G. Karlikaya, H. Karagozoglu, B. Kumbak, S. Kahraman: “Coasting may effect endometrial thickness and outcome”. Fertil Steril 2004; 82, S 2, S211* 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 Steril ;82:338–342
21534 Coasting Gn-RH-a long protocol HMG or r-FSH 225 IU step-down regimenOn 2nd cycle day at HCG or coasting dayCoastingHCG IU when E2 <3.000 pg/ml)Owj , E . Tehrani Negad , E . Amirchaghmaghi , Z . Ezabadi , A . Baghestani: “The Evaluation of Withholding Gonadotropins (Coasting) Effects on the Outcome of In-Vitro Fertilization Cycles”. Fertil Steril 2005;84,S254
217“Ganirelix salvage”35Gn-RH-a long protocol on late luteal phase previousr-FSH 225 IU/d starting dose (down regimen)Antagonist 0.25 mg daily doseat E2 > 4.000and or>10 follicles in each ovarydaily measurement of serum E2HCG IU at E2 ≤3.000 pg/mlM . Wittenberger , R . Gustofson , A . Armstrong , J . Segars: “A Cost Comparison of “Ganirelix Salvage” Protocol Versus “Coasting” Strategy for Patients at Risk for Ovarian Hyperstimulation Syndrome (OHSS)” . Fertility and Sterility 2005, 84 ,S318
21835 reduce E2 levels (4.219 2.613/24 h) and avoid cycle cancellation “Ganirelix salvage”reduce E2 levels (4.219 2.613/24 h)and avoid cycle cancellation24 oocytes mean/cycle79.2% MIIGustofson 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 (11):
21936 PCOS Protocol Pre-treatment with metformin ≥6 months 2.000 mg/day Improvment in menstrual cyclicityLong-protocol agonistHigher pregnancy outcomeFEssah et al Fertil Steril 2006;86,1:
220IVF cycle cost (ASRM’s average) total IVF cycle$ 12,500with “coasting” were$ 400/daythe cost per day of “ganirelix salvage”$ 553cycle cancellation prior to retrieval$ 6379plus the costs associated with either “coasting” or “ganirelix salvageM.D. Wittenberger, R.L. Gustofson, A. Armstrong, J.H. Segars : “A Cost Comparison of “Ganirelix Salvage” Protocol Versus “Coasting” Strategy for Patients at Risk for Ovarian Hyperstimulation Syndrome (OHSS)”. Fertil Steril 2995; 84,S1:S318
223Natural cycle modification 37Natural cycle modificationwhen dominant follicle ≥ 14 mm (8°-9° days)r-FSH 75 UI/d up HCG day± antagonist 0.25 mg/d up HCG dayHCG UI (leading follicle ≥18 mm and at least two follicles ≥15 mm)50 mg/d of im P4 in oil after oocyte retrievalWomen aged 40 yearsFSH elevatedPoor respondersCost-saving alternative
224Luteal 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 mg
225SUMMARY Controversies on gonadotropins Controversies on analogues Controversies on E-P pillsControversies on LH addedArslan 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:
226higher clinical pregnancy rate with hMG HMG or r-FSHhigher clinical pregnancy rate with hMGbut no significant differences in ongoing pregnancy rates or live birthsVan 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 ;80:1086–1093 .
227intermediate responders excellent outcome:with either a Gn-RH-a (long protocol)or a GnRH antagonistbut tailoring of gonadotropin dose must be performed to achieve optimized results.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:
228High RespondersHigh responders perform favorably with gentler stimulation that minimizes the occurrence of OHSSThe number of oocytes retrieved is predictive of clinical pregnancy only in patients over 40 years of ageM. Luna-Rojas, B. Sandler, M. Duke, A.B. Copperman, L. Grunfeld, J. BarrittFertility and Sterility September 2004 (Vol. 82, Page S206)
229Low Responders outcome suboptimal: poor ovarian response poor oocyte/embryo qualityin spite of stimulation regimens usedArslan 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:
230LH ADDEDAdding LH should be considered in severe situations of LH suppression:use of potent GnRH-agonistsGn-RH-antagonistsOver 35 yearsSuheil J. Muasher, Rony T. Abdallah, Ziad R. Hubayter: “Optimal stimulation protocols for in vitro fertilization” Fertil Steril 2006; 86,2:
231Conclusion(s)Ovarian stimulation is a critical step in in vitro fertilization therapy.A variety of controlled ovarian hyperstimulation regimens are available and efficacious,but individualization of management is essential and depends on assessment of the ovarian reserve.Identification of the etiologies of poor ovarian response constitutes a formidable challenge facing reproductive endocrinologists.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: