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Gonadotropin’sBioactivity Dr. Vincenzo Volpicelli Fertility Center Cardito Seconda Università degli Studi di Napoli Dipartimento di Scienze della Vita.

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Presentation on theme: "Gonadotropin’sBioactivity Dr. Vincenzo Volpicelli Fertility Center Cardito Seconda Università degli Studi di Napoli Dipartimento di Scienze della Vita."— Presentation transcript:

1 Gonadotropin’sBioactivity Dr. Vincenzo Volpicelli Fertility Center Cardito Seconda Università degli Studi di Napoli Dipartimento di Scienze della Vita SUNfert Seconda Università degli Studi di Napoli

2 Gonadotropins FSH, LH, HCG  glycoproteins  dimers α, β (two peptide chain) α chain aspecific α chain aspecific β chain specific (provides specificity for receptor interaction) β 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.

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4 FSH heterodimeric hormone: 92 amino acids α-chain92 amino acids α-chain 111 amino acids β-chain111 amino acids β-chain Ben-Rafael Z, Levy T, Schoemaker J Pharmacokinetics of follicle-stimulating hormone: clinical significance. Fertil Steril. 63:689–700 Various types of FSH exist according to their sialic acid content The half-life of FSH is 3-4 hours

5 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.

6 LH/HCG bioactivity  LH & HCG: the same amino acids in sequence  LH & HCG both stimulate the same receptor  the 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 minutes  FSH: 3-4 hours  hCG: 24 hours

7 FSH, 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 beta subunits vary

8 Gn secretion estrogens pituitary gland hypothalamus (arcuate nucleus and preoptic area) Gn (Gn-RH pulses) ovary feed-back

9 Estradiol negative feed-back

10 Pituitary gland embryology

11 Pituitary gland by diencephalon (infundibulum) by Rathke pouch (mouth)

12 Pituitary portal system

13 Pituitary gland histology GH HPRL FSHLHTSHACTH

14 Gn mode action activate a PtdIns (phosphatidylinositol)-calcium second messenger system membrane receptors Adenilcyclasi activation

15 Gn mode of action  uterine blood flow: increases the uterine blood flow during the early luteal phase, a periimplantation stage increases the uterine blood flow during the early luteal phase, a periimplantation stage (Index Resistance)

16 Gn 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

17 receptors 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

18 Gn-R expression Its expression requires appropriate hormonal stimulation by FSH and estradiolIts expression requires appropriate hormonal stimulation by FSH and estradiol present on: granulosa cells granulosa cells theca cells theca cells luteal cells luteal cells interstitial cells interstitial cells

19 Extragonadal Gn-Rs Gn-Rs have been found in: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 unexploredphysiologic role largely unexplored.

20 Gn action in ovary  follicular maturation  ovulation  luteal function

21 Gonadotropin’s avverse effects  OHSS  Ovarian volume increased  Multiple pregnancies  Gynecomastia

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

23 FSH in early follicular phase not increase much during a normal ovulatory cyclenot increase much during a normal ovulatory cycle FSH concentrations only 10–30% above the threshold level is sufficient to stimulate normal follicle developmentFSH 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. FSH concentrations reach a maximum in the early follicular phase of the normal menstrual cycle and decrease thereafter

24 FSH 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

25 enzyme of the cytochrome P450 group mediate androgens aromatization:  producing estrogens  sexual development Aromatase

26 FSH in late follicular phase  decrease  due to increased ovarian secretion of: E2E2E2E2  β-inhibin Hotchkiss 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. negative feedback at the hypothalamic-pituitary level

27 Thecal Cell Granulosa Cell LH cAMP ProteinKinaseA cholesterol pregnenolone 17-OH-P DHEA A CYP11 CYP17 3 βHSD A E 1 E 2 FSH R R blood Protein kinase cAMP P βHSD BasementBasementMembraneMembraneBasementBasementMembraneMembrane P4 Aldost Cortisol Steroidogenesis

28 FSH follicular decreasing strict relationship with dominant follicle developmentstrict relationship with dominant follicle development 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 As a consequence, other recruited follicles lack sufficient stimulation by FSH and enter atresiaAs a consequence, other recruited follicles lack sufficient stimulation by FSH and enter atresia Zeleznik 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.

29 FSH follicular decreasing Apparently, the maturing dominant follicle requires less FSH to continue its growth.Apparently, the maturing dominant follicle requires less FSH to continue its growth. It’s due to up-regulated FSH-sensitivity of leading follicle for:It’s due to up-regulated FSH-sensitivity of leading follicle for: 1.induction of locally various growth factors (IGF-I, AMH, inibina B, leptina, ICAM-1, VCAM-1, VEGF ) 2.induction of LH receptors that enhance FSH sensitivity Erickson GF The ovarian connection. In: Adashi EY, Rock JA, Rosenwaks Z, eds. Reproductive endocrinology, surgery, and technology. Philadephia: Lippincott-Raven; 1141–1160.

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31 FSH 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 cycle 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 cycle a cohort of small antral follicles is prevented from undergoing atresia and is stimulated for further development a cohort of small antral follicles is prevented from undergoing atresia and is stimulated for further development Hodgen GD The dominant ovarian follicle. Fertil Steril. 38:281–300

32 LH mode action  With the rise in estrogens, LH receptors are also expressed on the maturing follicle  estrogen rise leads via the hypothalamic interface to the “positive LH feed-back” effect, a release of LH over a hour period  This 'LH surge' triggers ovulation  LH is necessary to maintain luteal function (P4) for the first two weeks  LH supports thecal cells in the ovary that provide androgens and hormonal precursors for estradiol production  In 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

33 FSH gene α-chain gene  α-chain gene  locate in arme 6p  β-chain gene:  locate in 11p13 only in gonadotrope cells of pituitary gland  increased by Gn-RH and activine  decreased by inhibine

34 Deficient gonadotropin’s level hypogonadism and amenorrhoea hypogonadism and amenorrhoea: Kallmann syndrome Hypothalamic suppression Hypothalamic suppression Hypopituitarism Hypopituitarism Eating disorder (leptine) Eating disorder (leptine) Hyperprolactinemia Hyperprolactinemia Gonadotropin deficiency Gonadotropin deficiency Gonadal suppression therapy Gonadal suppression therapy GnRH antagonist GnRH antagonist GnRH agonist (downregulation) GnRH agonist (downregulation)

35 LH-R abnormalities in females can lead to infertilityin females can lead to infertility masculinizationmasculinization In 46, XY pseudohermaphroditism,In 46, XY pseudohermaphroditism, hypospadiashypospadias micropenismicropenis Antibodies to LH-R can interfere with LH-R activity

36 High Gonadotropin levels  Premature menopause  Gonadal dysgenesis, Turner syndrome  Castration  Swyer syndrome  Polycystic Ovary Syndrome  Certain forms of CAH  Testicular failure 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. typical in the menopause

37 FSH 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 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.

38 FSH gate the "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 *the "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 **Moreover, step-down dose regimen COH, has proven successful in reducing the incidence of multiple follicle development ** ** 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 * Baird DT A model for follicular selection and ovulation: lessons from superovulation. J Steroid Biochem. 27:15–23

39 FSH window the FSH window concept has been proposed, stressing the significance of the (limited) duration of FSH elevation above the threshold levelthe FSH window concept has been proposed, stressing the significance of the (limited) duration of FSH elevation above the threshold level rather than the height of the elevation of FSH for single dominant follicle selectionrather than the height of the elevation of FSH for single dominant follicle selection Fauser BCJM, van Heusden AM Manipulation of human ovarian function: physiological concepts and clinical consequences. Endocr Rev. 18:71–106.

40 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.

41 FSH initial doses  patient’s age  basal FSH  PCOS

42 HCG HCG Gonasi fl i.m. 1000, 2000, UI HCGHCG pregnant women urinepregnant women urine made by the placentamade by the placenta LH-activity likeLH-activity like > half-life LH (4 h vs. 15 min)> half-life LH (4 h vs. 15 min)

43 hCG in normal pregnancy

44 HCG HCG It is heterodimeric glycoprotein: –α subunit identical to LH, FSH, TSH –β subunit unique to hCG – amino acids

45 HCG mode action interacts with the LHCG receptor Follicle rupture induction maintenance of the corpus luteum during the beginning of pregnancy, causing it to secrete P4 meiosis restarting

46 HMG (Pergonal), (Metrodin), Menogon, Fostimon fl i.m. 75 UI  Menotropin (HMG) (1965s) – Climateric women urine FSH + LH ( ~ 50%) FSH + LH ( ~ 50%) – 5% Gn + 95% urinary proteins  Urofollitropin (1983) — Purified FSH (>95%) — purified by chromatographic techniques

47 Chinese hamster (Cricetulus griseus), white spotted type

48 r-FSH  produced by inserting the genes encoding for α and β subunits of FSH into expression vectors that are transfected into a Chinese hamster ovary cell line Gonal-F, Puregon fl s.c., pen Purification by immunochromatography using an antibody specifically binding FSH

49 r-FSH  1995  European Medicines Evaluation Agency (EMEA) Gonal-F, Puregon fl s.c., pen

50 r-FSH properties  knockdown degradation rate  reduced variability  interblocks firmness  high degree of pureness  diminished immunization

51 r-FSH r-LH (Luveris 75 IU fl s.c.) β -follitropine (Puregon) α -follitropine (Gonal F)

52 HMG vs. r-FSH both products are probably equally safe and similar in efficacy, based on the available literature to dateboth 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. 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:

53 FSH-HMG-HCG target targetFSH/HMGLHHCG Follicles recruitment Oocytes maturation Ovulation trigger E2E2 P4P4 +

54 Gonadotropin75 UI1.500 UI Meropur€ 12,28€ 245,6 Fostimon€ 16,09€ 321,8 Puregon€ 45,83€ 916,6 Gonal-F€ 51,39 € 1027,8 Gn available in the Italy market

55 C O H P R O T O C O L S

56 CC M. M. Quigley: Annals of the New York Academy of Sciences, Vol 442, 1: sufficientCC alone produces sufficient 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

57 CC + HMG CC 100 mg/day on day 3–7 of the menstrual cycle HMGstarting on day 5HMG 150 IU every other day starting on day 5 HCG on leading follicle >18 mm and at least two follicles >15 mm Pick-up or IUI hours after HCG UI 6 days after (staff conversion)HCG UI 6 days after (staff conversion) P 4 50 mg/d i.m. on HCG day or E-T day M. M. Quigley: Annals of the New York Academy of Sciences, Vol 442, Issue

58 CC + delayed HMG CC 100 mg/day on day 1–5 day HMG150 IU every other day starting on day 6 HCG on leading follicle >18 mm and at least two follicles >15 mm IUI or Pick-up hours after HCG UI 6 days after (staff conversion)HCG UI 6 days after (staff conversion) P 4 50 mg/d i.m. on HCG day or E-T day

59 CC + HMG 1. best chance of COH 2. minimize the disruption of the subsequent luteal phase 3. increased pregnancy rate M. M. Quigley: Annals of the New York Academy of Sciences, Vol 442, Issue

60 CC + HMG ovulation outcome 90% for cycle

61 CC/HMG Pregnancy Outcome * USG pregnancy rate/cycle: 25% * * live-birth rates/cycle : 13-17% * * Published overall

62 Miscarriage integrins down regulationintegrins down regulation (markers of endometrial receptivity) endometrialendometrial EE/P-r depletion uterine artery flowuterine artery flow impaired endometrial development

63 Low Pr in CC/Gn COH premature LH surge premature LH surge immature oocytes immature oocytes desynchronized endometrial development desynchronized endometrial development

64 CC/HMG Pr lower over 38 years old low ovarian reserve poor quality sperm endometriosis tubal damage or pelvic scar tissue infertility >3 years

65 CC/HMG adverse effects  3,5% twin  1/3 of admission in TIN  Twin/mono mortality 10 +  PIH 5 – 10 +  placenta previa  Placenta detachment

66 CC + E 2 CC 100 mg/d on 3° cycle day on days 8-12EE 0.05 mg/d on 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

67 CC + E 2 endometrial thickness on the day of hCG administration. = CC only = CC + ethinyl E2

68 CC + E 2 Characteristics and outcome of patients who received CC plus ethinyl E2 (group A) or CC alone (group B) in IUI cycle Characteristic Group A Group B P value No. of patients Mean (±SD) age (y) 28.0 ± ± 4.2 NS Mean (±SD) duration of infertility (mo) 48.1 ± ± 9.6 NS Ongoing Pregnancy 12 (37.5) 2 (6.25) <.05 Miscarried 6 (18.75) <.05 pulsatility index values no difference - -

69 Traditional COH  HMG or r-FSH 300 IU on 2° day cycle  HCG IU on leading follicle >17 mm and at least two follicles >15 mm  Pick-up after h  P 4 50 mg i.m. for luteal supplementation

70 Traditional COH FSH remain elevatedFSH remain elevated recruitment and growth of ovarian follicles continues throughout treatmentrecruitment and growth of ovarian follicles continues throughout treatment * Filicori M: Characterization of the physiological pattern of episodic gonadotropin secretion throughout the human menstrual cycle. J Clin Endocrinol Metab. 1986;62:1136–1144 This FSH serum pattern profoundly diverges from the spontaneous menstrual cycle

71 Traditional COH heterogeneous size cohorts of follicles are often found at hCG dayheterogeneous size cohorts of follicles are often found at hCG day the optimal outcome of COH would be the selective attainment of numerous large mature homogeneous follicles.the 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. 1995;10:256–261

72 Traditional protocol Long protocol n° ampules Mature oocytes 816 Fertilization rate 83%78% PR/ET28%31% Cost-saving — — MF Stress — — + + +

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74 Gn-RH Gn-RH neurons are inside the medium-basal hypothalamus (arcuate nucleus and median eminence)Gn-RH neurons are inside the medium-basal hypothalamus (arcuate nucleus and median eminence) Lately scientists showed Gn-RH syntesis in pituitary gland tooLately scientists showed Gn-RH syntesis in pituitary gland too

75 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-NH 2 The identity of GN-RH 1 was clarified by the 1977 Nobel Laureates Roger Guillemin and Andrew V. Schally

76 Pituitary gland histology Melanocyte-stimulating hormone (MSH) is the predominant hormone secreted by pars intermedia (part of adenohyphysis)

77 stores The pars nervosa stores ADHADH and OxytocinOxytocin hypothalamus which were secreted by the hypothalamus. NEUROHYPOPHYSIS - PARS NERVOSA This region of the pituitary is non secretory. Its cells are neuroglial-like pituicytes.

78 Melatonin/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

79 Melatonin/steroidogenesis + P4P4P4P4

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81 Gn-RH secretion males/females in males, in pulses at a constant frequency in males, in pulses at a constant frequency in females the frequency of the pulses varies during the menstrual cycle in females the frequency of the pulses varies during the menstrual cycle there is a large surge of GN-RH 1 just before ovulation there is a large surge of GN-RH 1 just before ovulation

82 Gn-RH frequency Low frequency  FSH releaseLow frequency  FSH release high frequency  LH releasehigh frequency  LH release

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

84 Gn-Rh analogues While Gn-RH 1 has been synthesized and become available, its short half-life requires infusion pumps for its clinical use. Modifications of the decapeptide structure of Gn-RH 1 have led to Gn-RH 1 analog medications that either stimulate (Gn-RH 1 agonists) or suppress (Gn-RH 1 antagonists) the gonadotropins

85 Effects of Gn-RH analogues agonistantagonist Prevent premature luteinization Prevent premature ovulation To synchronize early follicular development

86 Gn-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.

87 Gn-RH-a Aminoacid sequence nameact for Gn-RH1 Pyro -glu Hi s TrpserotoninTyr Leu Le u Arg Pr o Gly- NH 2 iv Leuproreline * 15 D- Leu N- EtNH 2 sc, im Buserelin * * 20D-Ser N- EtNH 2 sc, im triptor * * * D- Trip sc, im Goserelin * * * * 10 0 D-Ser AzGly -NH 2 depot sc * Enantone 3.75, mg fl s.c. im; Enantone die 1 mg/die (0.2 ml) fl s.c.; * 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. im Triptorelin is an agonist with only a single substitution at position 6

88 Gn-RH-a brand name Injectionmg/ml Leuproreline acetate Enantone die 1 fl 1.6 ml 8 doses 1 mg (0.2 ml) s.c Triptoreline Decapeptyl depot 1 fl i.m mg Decapeptyl die 14 fl pre-filled 0.2 ml (0.1 mg) s.c. daily Buserelin Suprefact fl 5.5 ml 0.5 ml/d spray 1 flac 1 buff = 100 μg triptorelin, buserelin and goserelin are equally effective

89 Gn-RH-a pharmacokinetics two hours: peak serum. It rapidly binds to the LHRH receptor cells in the pituitary gland flare-up thus leading to an initial increase in production of LH ( flare-up ) receptor desensitization down-regulation after 10 days: receptor desensitization and/or down-regulation

90 Gn-RH-a lysine replacement with ethylamide in 10 → half-time (4 min vs 3 h)

91 Gn-RH-a lysine replacement with D- amynoacide in 6 → Increase effectiveness ( times) lysine replacement with D- amynoacide in 6 → Increase effectiveness ( times) D-aminoacid is hydrophobe chain carrier with enhancement receptor link D-aminoacid is hydrophobe chain carrier with enhancement receptor link

92 Gn-RH-a effects Follicles synchronization++++ Fewer small follicles on HCG day++ Avoids premature luteinization++++ Multiple pregnancies≡ ≡ ≡ Decreases OHSS frequency≡ ≡ ≡

93 Triptoreline depot serum levels

94 Triptorelin [d-Trp6]GnRH

95 Goserelin* * Zoladex 3.75 mg, mg fl im (FDA, 1989) 610 D-Ser(But) 6 Azgly 10 LHRH

96 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 days, production of LH is greatly reduced due to receptor downregulation

97 Gn-RH-a protocols long protocol short (“flare-up”) protocol ultrashort protocol microdose flare protocol

98 Long protocol: 1. Avoid pre-menses FSH surge 2. Follicles timing 3. Avoid premature LH surge 4. Higher follicular recruitment (synchronization) 5. Improvement immune attitude 6. Expensive cost High responders PCOS

99 short protocols 1. follicles timing 2. avoid premature LH surge 3. lower follicular recruitment 4. make procedures easier Poor responders

100 Serum levels Short protocol Long protocol E2E2 idemIdem D4 D depression ++++ Pregnancy rate/cicle9.2% 16. 5% PR/transfer9..9% 23. 5% patients «poor responders» «High responders» PCOS > 40 years hyrsutism HMG ampules Cancelled cycles Short/long protocol (Volpicelli V. 2003)

101 PR/transfer in Gn-RH-a FIV nel periodo (da FIV-NAT ’97) sec. Barrière et al Flare-up protocol 19.2% Long protocol 25.7% Media24.8% without analogues 23.2%

102 Gn-RH-a Long protocol Gn-Rh-a depot 3.75 mg in one dose on 21 st day only of previous cycleGn-Rh-a depot 3.75 mg in one dose on 21 st day only of previous cycle Gn-Rh-a low-dose daily on the 21 st day of previous cicle to HCG day:Gn-Rh-a low-dose daily on the 21 st day of previous cicle to 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 μg)Buserelin nasally 1 buff x 3/d (300 μg) 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 or or on any day when:on any day when: »LH <0.5 »E 2 <30 »No ovarian cyst >10 mm 8

103 Gn-RH-a long protocol r-FSH/HMG IU/day on 2 nd cycle day to HCG dayr-FSH/HMG IU/day on 2 nd cycle day to HCG day HCG IU on the least two follicles >18 mmHCG IU on the least two follicles >18 mm Pick-up after hoursPick-up after hours P4 supplementationP4 supplementation HCG IU six days after E-THCG IU six days after E-T 8

104 Short (flare-up) protocol Gn-RH-a 3.75 mg depot ½ fl i.m. on 2° cycle day onlyGn-RH-a 3.75 mg depot ½ fl i.m. on 2° cycle day only r-FSH IU/d on 3 th day (step-down regimen) HCG IU (18 mm ) Pick-up after h HCGHCG (+ P4) poor responder 9

105 Gn-RH-a flare low dose protocol on 1 st cycle day at HCG day:on 1 st cycle day at HCG day: »Triptoreline (decapeptyl die) 0.2 ml (0.1 mg) s.c. daily »Leuproreline acetate (enantone die) 0.2 ml (1 mg) s.c. daily »Buserelin (Suprefact flac 5.5 ml) 0.3 ml s.c. »Buserelin nasally 3 buff/day (300 μg) or or on any day when:on any day when: »LH <0.5 »E 2 <30 »No ovarian cyst >10 mm r-FSH/HMG UI/d on 3 rd cycle dayr-FSH/HMG UI/d on 3 rd cycle day After 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

106 Gn-RH-a ultrashort protocol on 2 nd cycle day for three days:on 2 nd cycle day for three days: »Triptoreline 0.2 ml s.c »Leuproreline 0.2 ml s.c. »Buserelin 0.5 ml s.c. »Buserelin nasally 3 buff/day or or on any day when:on any day when: »LH <0.5 »E 2 <30 »No ovarian cyst >10 mm r-FSH/HMG on the 2 nd cycle dayr-FSH/HMG on the 2 nd cycle day 11

107 11UltrashortLong HMG 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

108 HCG low-dose long protocol in ovarian follicles of largerGranulosa 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. LH appears capable of exerting virtually all the physiologic actions of FSHAt this time LH appears capable of exerting virtually all the physiologic actions of FSH on GCs (2) Zeleznik AJ, Hillier SG. The role of gonadotropins in the selection of the preovulatory follicle. Clin Obstet Gynecol. 1984;27:927– 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

109 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 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

110 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 (200IU/d), corresponding to roughly 1,200 IU/d of LHhCG alone (200 IU/d), corresponding to roughly 1,200 IU/d of LH The hCG is also drastically less expensive than recombinant FSH or hMG.12 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– 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

111 HCG low-dose in long protocol Gn-RH-a long protocol r-FSH/hMG (1:1/2) IU on 2° day at least six follicles >12 mm and E 2 >300 pg/ml hCG 250 IU/day alonehCG 250 IU/day alone until the end of COH Filicori M: Fertil Steril 2005: 84, 2: 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 12 or variable amounts of r-FSH and low-dose (10-50) IU hCG

112 gonadotropin and steroid at HCG day Group A Group B P value (no hCG) (hCG) LH (IU/L) 0.6± ±0.3 NS 11.3±1.24.3±0.6FSH (IU/L) 11.3± ±0.6 <.001 hCG (IU/L) 0.4± ±0.5 < ± ±317E2 (pg/mL) 2.358± ±317 <.05 P (ng/mL) 1.1± ±0.1 NS T (ng/mL) 0.9± ±0.1 <.05 Filicori M: Fertil Steril 2005: 84, 2:

113 Clinical outcome no-HCGHCG COH days 11.6± ±0.1NS r-FSH/hMG days 11.6±0.28.6±0.1<.001 Daily hCG duration (days) —3.3±0.1— r-FSH/hMG dose (IU) 2,779±1601,960±99<.001 Immature oocytes (n) 1.4±0.21.6±0.3NS Mature oocytes (n) 8.0±0.78.2±0.6NS Fertilization rate (%) 48±4% (0–100) 74±3% (36–100) <.001 Good quality embryos (%) 86±6%84±5%NS Embryos transferred (n) 2.3±0.22.5±0.1NS Implantation rates (%) 11%12%NS Pregnancy rates (%) 21%25%NS Filicori M: Fertil Steril 2005: 84, 2:

114 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 UI, step-down regimen, on 2 nd at 8 th cycle day HMGuntil HCG day (if LH < 1 mUI/ml) HMG on 9 th until HCG day (if LH < 1 mUI/ml) r-FSH continueduntil HCG day (if LH ≥5 mUI/ml)r-FSH continued until HCG day (if LH ≥5 mUI/ml) or Ye H: Fertil Steril 2006;86,3S:S420-S

115 r-FSH/HMG Long protocol  LH >5 mIU/ml   LH <1 mIU/ml  r-FSH HMG  HMG  Gn-RH-a low dose 21° Gn-RH-a depot or low dose long protocol 13

116 r-FSH/HMG Long protocol Normal LH Low LH r-FSH r- & HMG r-FSH Oocytes MII Oocyt fert Embryos Implant % 35.8% 31.4%40.7% 32.3% Pregn rate 55.2% 43.8%61.7% 54.1% miscarriage07.1%18.9%3.0%13

117 Antagonists (1990s) * Orgalutran, Cetrotide 0.25 mg fl 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”

118 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 method early administration (very high- responders) On 1° COH day until leading follicle ≥18 mm and at least two follicles ≥ 15 mm Ovulation triggering with Gn- RH-a long-acting

119 on 1° days Gn stimulation on 5°-6° days on leading follicle ≥14 mm HMG or r-FSH + LH addedHMG or r-FSH + LH added Antagonists protocol Fixed and early start of the antagonist is probably more effective than an individualized and late start.

120 Gn-RH Antagonist advantages: Prevention surge LHPrevention surge LH larger cohort of follicleslarger cohort of follicles Avoidance of adverse effects of agonistsAvoidance of adverse effects of agonists More friendly stimulation protocolMore friendly stimulation protocol  OHSS  OHSS disavantages LDP  peak E 2 on HCG day  mature follicles  oocytes  embryos  PR

121

122 LH 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? Fertil Steril 20003;79:253–5.

123 LH added Granulosa cells in ovarian follicles of larger size (>10– 12 mm) normally express the LH/hCG receptor and become sensitive to LH activity stimulation *Granulosa 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 **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 * *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. 1984;27:927–940 * * Campbell BK, Dobson H, Baird DT, Scaramuzzi RJ.: J Reprod Fertil. 1999;117:355–367

124 − LH <1 UI/ml at the start of Gn stimulation − Gn-RH-a flare protocol (LH suppression) − Gn-RH antagonist during stimulation − >35 years − Poor responders − High responders (LH prevalence activity decrease n. small follicles and OHSS risk) LH ADDED target

125 LH added target

126 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

127 LH added target Cycle’s phase FSH recLH rec Prevalence activity of early follicolar (G)+ (T)FSH late follicolar + + (G) (T&G) LH luteal (CL)LH

128 LH added target * 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

129 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

130 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. theca cells produce androstenedione and testosterone under LH stimulationFSH induces granulosa cell aromataseIn 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). (vedi iperandrogenismo in PCOS) 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)

131 LH Added protocol 15 r-LH 375 IU twice a day (7.30 and h) for the last 2 days of COH or  Leading 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. 1999;84:228–232. leuprolide acetate 1 mg daily, sc, from menstrual day 21 for 14 days (+ 7 days) LH <2.5 IU/L and E 2 <20 pg/mL r-FSH starting at 150 IU sc daily at 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 maintained at 150 IU/dayIf serum E2 levels were greather than 100pg/mL, the r-FSH was maintained at 150 IU/day

132 LH added vs. HMG in over 38 * * * Gomez-Palomares J. L. ; Acevedo-Martin B. ; Andres L. ; Ricciarelli E. ; Hernandez E. R.; Reproductive biomedicine online ISSN ; 2008 r-FSH + HMG 75 UI (group I) and r-FSH + r-LH 75 UI (group II) HMG group LH group n. follicles on 6 day 6.72   1.29 COH days 10.5   1.8 M II oocytes75.3%93.1% Pregnancy rate26%47%

133

134 Luteal supplementation in agonists/antagonists protocols Pituitary depletion Pituitary desensitization Negative estrogen feed-back Compulsory supplementation E/P HCG supplementation absolutely necessary !!!

135 P 4 secretion Follicular phase Luteal phase * Ovary48%95% Adrenal gland 48%4% from pregnenolone 4%1% *P 4 serum level:4 ng/ml is low level; 40 ng/ml is high *P 4 serum level: 4 ng/ml is low level; 40 ng/ml is high

136 luteal P 4 supplementation ☻Few studies in the last 20 years ☻Currently, no reliable method for specific diagnosis of P 4 deficiency in luteal phase ☻Regimens often determined by clinical experience The rationale for P 4 supplementation: 1.Aspiration of the granulosa cells 2.Presence of high levels of E 2 3.Analogues  poor luteal function (due to residual suppression of pituitary LH secretion) ASRM Practice Committee: “Exogenous progesterone supplementation” Fertil Steril 2008;89,4:

137 luteal P 4 supplementation  P 4 50 mg/d i.m. Or  mg/day vaginally  Starting:  3 days after IUI or  at E-T day  Prontogest fl im 100 mg

138 luteal P 4 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

139 P4P4P4P4 * Posaci C, Smitz J, Camus M, Osmanagaoglu K, Devroey P: “Progesterone for the luteal support of ART: clinical options”. Human Reprod 2000; 15,S1: Orally: bioavailabilty diminished by the liver first pass bioavailabilty diminished by the liver first pass the serum level typically returns to baseline level by 6 hours the serum level typically returns to baseline level by 6 hours dizziness and somnolencedizziness and somnolence fatigue, headache, urinary frequencyfatigue, headache, urinary frequency vaginal route: the level remains elevated for up 48 hoursthe level remains elevated for up 48 hours Crinone gel 8% once a day and contains 90 mg of P 4Crinone gel 8% once a day and contains 90 mg of P 4 Progeffik gel 200 mg 1-3/dayProgeffik gel 200 mg 1-3/day Uterine tissue higher level P 4 despite a lower serum P 4Uterine tissue higher level P 4 despite a lower serum P 4 Vaginal irritationVaginal irritationIntramuscular: P4 in oil result in higher plasma concentrationP4 in oil result in higher plasma concentration and longer duration *and longer duration * Severe allergic reactionSevere allergic reaction Adult respiratory distress syndromeAdult respiratory distress syndrome Eosinophilic pneumonitisEosinophilic pneumonitis

140 luteal supplementation in agonist/antagonist protocols Pituitary desensitization for 2-3 w after last administration * 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: Worldwide standard practice *

141 luteal suppl agonist/antagonist protocols P4P4P4P4 * Martinez F: “ Human Corionic Gonadotropin and intravaginal natural progesterone are equally effective for luteal phase support in IVF”. Gynecol Endocrinol 2000; 14: HCG : more effectivemore effective Increased production of E 2 and P 4Increased production of E 2 and P 4 Better endocrine profileBetter endocrine profile No differences in pregnancy outcomeNo differences in pregnancy outcome OHSS risk (E 2 peak at HCG day)OHSS risk (E 2 peak at HCG day) or/and

142 Luteal E 2 supplementation E 2 orally 2-6 mg/d (Progynova cpr 2 mg) * Start on: »E-T day or »7 days after E-T Increases implantation rate Increases pregnancy rate In IVF cycles, the levels of E 2 and P 4 drop in the mid-late luteal phase Lower E 2 at 11 days after pick-up is associated with lower pregnancy rate * Lukaszuk K: Fertil Steril 2005;83:

143 PROTOCOLS:

144 Poor 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 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

145 Poor responders diminished ovarian reserve A lower expression of FSH receptor in the granulosa cells Advanced maternal age E 2 < 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* * Keay et al., 1997 ; Karande and Gleicher, 1999 ; Fasouliotis et al., 2000 ; Tarlatzis et al., 2003 “occult ovarian failure”

146 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 16 Murat Arslan: Fertil Steril 2005; 84,3:

147 the 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: Stop to pre-menstrual FSH and, subsequently, stop to size discrepancy in the developing follicles 17

148 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 dayson 6°-8° stimulation days or leader follicle ≥ 14 mm if very few folliclesor leader follicle ≥ 14 mm if very few follicles HCG UI on day dominant follicle ≥18 mm 18 Luteal supplementation: HCG IU/d (+ P 4 )Luteal supplementation: HCG IU/d (+ P 4 )

149 E-P Antagonist protocol FarmakonDosageTimelength E-P pill previous cycle days FSH/HMG ° cycle dup HCG d CC150 mg/d1° cycle d5-7 days Antagon 0.25 mg/d 7-8° dup HCG d HCG IU>18 mm 19

150 Age >40 Long protocol CC + HMG + Antagonist n° ampules5083 follicles > E 2 on day E 2 on day ( ) E 2 on HCG day ( ) Cancellation rate34%4.8% total oocytes Mature oocytes n° embryo PR15.3%22.2% Implant rate7.6%13.5% Weghofer, 2004

151 Luteal estradiol protocol Oral micronized E 2 2 mg twice a day »On luteal day 21 »At 3 days of COH r-FSH IU/d down regimen on 2° day  microdose flare Gn-RH-a on 3° COH day or  delayed Gn-RH antagonist Dragisic KG Fertil Steril 2005;84: HCG low-dose (10-50 IU/d) on 8° day lowering FSH levels with estrogen, the ovary will respond when high doses of FSH are added in COH protocol

152 Luteal estradiol protocol * outcomeAll cycles Luteal Estradiol Standard protocol Clinical Pr38,3%40,9%31,3% Miscarriag e rate 43,5%38,9%60,0% Delivery rate 20.0%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:

153 AACEP 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 days of E-P pills until onset of mensesGn-RH-a low-dose in a standard long protocol overlapping the last 5 to 7 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 valerate * 2 mg/d on 1° to 10° cycle dayEstradiol valerate * 2 mg/d on 1° to 10° cycle day Estrogen suppositories ** were used to maintain the endometrium until at last one follicle measured 15 mmEstrogen suppositories ** were used to maintain the endometrium until at last one follicle measured 15 mm r-FSH in initial doses of 600 or 750 IU/day, decreasing to 225 IU/day of r-FSH.r-FSH in initial doses of 600 or 750 IU/day, decreasing to 225 IU/day of r-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: * Progynova cpr 2 mg ** Vagifem cpr vaginali mg

154 AACEP Protocol E-PE-PppIIllllssE-PE-PppIIllllsspIlls*agonist …                                                                                                     ETET P4P4P4P4 P4P4P4P4 P4P4P4P4 AAAAAAAAAAA P4P4P4P4 P4P4P4P4 P4P4P4P4 EEEEEEEEEE HCGHCGHCGHCG  r-FSH 150 IU A antagonist

155 Androgens 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 oocyte insulin-like growth factor I expression and initiation of follicle development in the primate ovary. Biol Reprod 1999; 61:353–

156 Androgens TTTT  DHT  DHEA  Letrolozole

157 DHEA For women under 50, DHEA levels of less than 150 ng/dL are considered low DHEA is the cornerstone to all sex hormones Casson PR: “Dehydroepiandrosterone supplementation augments ovarian stimulation in poor responders: a case series Human” Reproduction, Vol. 15, No. 10, , October 2000

158 DHEA protocol in press 1.Leonidas Mamas, Eudoxia Mamas,: “Premature ovarian failure and dehydroepiandrosterone” Fertil Steril 2008 in press 75 mg/d for 4-12 months previous COH 75 mg/d for 4-12 months previous COHand during COH during COH Reduces FSH levelReduces FSH level release more and better quality eggs prior to IVFrelease 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 POF High FSH

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

160 Testosterone protocol 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): Gn-RH-a low dose (Leuprolide 1 mg or triptoreline 0.1 mg) long protocol-like started in the midluteal phase of the previous cycleGn-RH-a low dose (Leuprolide 1 mg or triptoreline 0.1 mg) long protocol-like started in the midluteal phase of the previous cycle at menses start Gn-RH-a is reduced to 0.5 mg and continued until the administration of HCGat menses start Gn-RH-a is reduced to 0.5 mg and continued until the administration of HCG Gn-RH-a 0.5 mg/day of leuprolide from the midluteal phase at menses startGn-RH-a 0.5 mg/day of leuprolide from the midluteal phase at menses start 0.25 mg/day thereafter0.25 mg/day thereafter 1° cycle 2° cycle

161 Testosterone 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): transdermal testosteronetransdermal testosterone 20 µg/kg per day20 µg/kg per day Androderm 2.5 mg daily single patchAndroderm 2.5 mg daily single patch Removed always at 9.00 a.m.Removed 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].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 COHduring the 5 days preceding COH

162 Testosterone COH 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): 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 IUOn day 2 r-FSH 300 IU On days 3 and 4 of ovarian stimulation, 150 IU per dayOn 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 3 and 4 of ovarian stimulation, 300 IU HMG From day 5 onwards, HMG on an individual basis 1° cycle 2° cycle Gonadotrophin ovarian stimulation was started the day following last testosterone patch application

163

164 Testosterone 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): % 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%

165

166 Letrozole 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  E 1 T  E 2

167 Letrozole + HMG 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 for assisted reproductive technologies” Fertil Steril 2006;86,4: %–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 Gonadotropins CC resistant Poor responders

168 Letrozolo + HMG Letrozolo 2.5 mg days 3–7 day cycle HMG 150 IU on day 5 up lead. foll. >18 HCG IU hours after Pick-up hours after HCG P 4 50 mg/d i.m. on HCG day or E-T day Mohamed 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:

169 Increasing AA Poor responders: lower expression of FSH receptor in the granulosa cells PCOs Patients: hyperexpression of FSH receptor

170 Increasing AA inducing a temporary and reversible PCO-like condition in the ovaries of poor responder patients could enhance their follicular recruitment and development

171 Aromatase inhibitors protocol aromatase inhibitor which induces a temporary accumulation of intraovarian androgens ADVANTAGES AIs vs. CC Lack of down-regulation of hypothalamic-pituitary estrogen receptors Lower FSH dose Higher number of mature oocytes Less adverse effects on endometrium 1 and cervix Pregnacy 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: ).

172 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 health

173 Letrozole Mitwally MFM and Casper RF. (2002) Aromatase inhibition improves ovarian response to follicle-stimulating hormone in poor responders. Fertil Steril 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 ( IU/day) starting on day 6FSH ( IU/day) starting on day 6 hCG (10,000 IU) when two leading follicles were ≥20 mmhCG (10,000 IU) when two leading follicles were ≥20 mm E-P for daysE-P for days –or »E2 <60 pg/ml »absence of cysts >10 mm

174 Letrozole + Antagonist letrozolo 5 mg on 1° to 5° cycle day r-FSH 300 IU + HMG 150 IU on 1° to 5° cycle day On 6° day individual dosages r-FSH/HMG delayed antagonist 0.25 mg/d HMG + Antagonist until HCG day r-HCG 250 mg on leading follicle >18 mm P 4 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.

175 …      ETETETET         P4P4P4P4 P4P4P4P4 P4P4P4P4 P4P4P4P4 P4P4P4P4 AAAAA P4P4P4P4 P4P4P4P4 P4P4P4P4 P4P4P4P4 P4P4P4P4     r-FSH 150 IU  HMG 150 UI Letrozole 2,5 mg  Letrozole 2,5 mg r-HCG 250 mg  r-HCG 250 mg A Antagon

176 Letrozole + Antagonist Letrozolecontrol oocytes retrieved 6.1 ± ± 0.3 Fertilization rate 68,2 %63,3 % embryos transferred2 ± ± 0.1 PR/cycle 22.4 %15.2 % PR/transfer 41.7 % 28.9 % Implantation rate 25 % 9.4 % Miscarriage rate 20 % 7.7 % twins 46.7%7.7% Garcia-Velasco. Letrozole in poor responder JVF patients. Fertil Steril 2005

177 Assisted hatching a bc M. 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.

178 E-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-T Luteal supplementation: progesterone 100 mg/day i.m. On oocyte collection through the luteal phase Bahceci M.: Fertil Steril 2006;86,1:81-85

179 Ovary deficiency 1) gonadic disgenesia “streak gonad” absence of ovary tissue 2) Ovary disgenesia Ovary tissue without follicles and without functionality 3) POF Ovary tissue without follicles but with past functionality 4) Proof Ovary (Savage Symdrome) Ovary tissue with hystologic normal follicles

180

181

182

183

184 endometriosis

185 adenomiosis

186 endometriosis

187 presence of ovarian endometriomas - 25%responsiveness to gonadotropins: - 25%

188 Long protocol + prednisone 3.75 mg Gn-Rh-a depot in one dose on 21 st day3.75 mg Gn-Rh-a depot in one dose on 21 st day or or Low-dose daily on the 21 st day of previous cicle to HCG dayLow-dose daily on the 21 st day of previous cicle to HCG day or or on any when LH <0.5 and E 2 <30on any when LH <0.5 and E 2 <30 or or on the 3th day of menstrual cycleon the 3th day of menstrual cycle –USG –LH <0.5 UI/ml –E2 <30 pg/ml

189 Long protocol + prednisone Prednisone 15 mg/day on 1° day at HCG day Prednisone 15 mg/day on 1° day at HCG day (Deltacortene cpr 5 mg) r-FSH 450 IU on 2° day up 6° day r-FSH 450 IU on 2° day up 6° day r-FSH variable dosage r-FSH variable dosage Luteal supplementation: P 4 50 mg/d i.m. Luteal supplementation: P 4 50 mg/d i.m.

190 lean 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: Donna magra come un treno (Mango)

191

192 Overweight Levens ED, Skarulis MC: “Assessing the role of endometrial alteration among obese patients undergoing assisted reproduction”. Fertil Steril 2008;89,6: 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).

193 Overweight in PMA *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 Higher cancellation rateHigher cancellation rate Lower pregnancy ratesLower pregnancy rates higher miscarriage rateshigher 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:

194 Overweight Norman RJ, Clark AM. Obesity and reproductive disorders: a review. Reprod Fertil Dev. 1998;10:55–63.Norman RJ, Clark AM. Obesity and reproductive disorders: a review. Reprod Fertil Dev. 1998;10:55–63. Extraovum effects of obesity on FIVET outcome: insulin resistanceinsulin resistance hyperandrogenismhyperandrogenism elevated leptin levelselevated leptin levels

195 Overweight * 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. Obesity could impair reproduction by acting on: 1.the ovary 2.and/or the endometrium (unfavorable intrauterine milieu) * % PCOS are overweight or obese ** Discrepancies in miscarriage rates mainly due to statistical flaws caused by small sample sizes

196 * 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 MEDLINE Figure 1 Ongoing pregnancy rate per cycle (%) in each BMI group. CI: confidence interval (women undergoing ovum donation)

197 Overweight *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 MEDLINECrossRefMEDLINECrossRef low-calorie dietlow-calorie diet for a short period (4-6 week)for a short period (4-6 week) before IVF cyclebefore IVF cycle and and during IVF cycleduring IVF cycle weight loss can improve spontaneous ovulation * Positive correlation between weight loss and ovulation and pregnancy outcome: **Positive correlation between weight loss and ovulation and pregnancy outcome: ** ** Clark AM: Human Reprod 1998; 13:

198 Overweight BMI > 25 : Gn-RH-a : PR 26.7%Gn-RH-a : PR 26.7% Gn-RH antagonists: 22%Gn-RH antagonists: 22% 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 of BMI on in vitro fertilization outcome”. Fertil Steril 2008;89,2: 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:

199 Stimulation protocol Nichols, Jr. BMI extremes and IVF pregnancy rates. Fertil Steril 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 continued daily until the day of hCG 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 R-FSH or HMG on cycle day 2–4 at a dose of IU daily 10,000 IU of hCG on the leading follicle >18 mm Women not undergoing the standard protocol received a modified microdose 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 bleeding.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 dayr-FSH or HMG IU daily on 2 days after Lupron at HCG day II° Protocol Two 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. P 4 supplementation: 50 mg im or 90 mg vaginally daily

200 TABLE 1. Distribution of variables and outcomes by BMI group. BMI < 20 BMI BMI ≥ 28 ampules FSH 29.5 (18.7) * 27.8 (13.9) * 30.5 (16.7) * COH days 9.2 (1.3) * 9.0 (1.3) * thickness 10.8 (1.9) * 10.7 (2.2) * 11.6 (2.5) * pregnancy rate 35.6%52.1%35.2% abortion abortion0%5.9%4.0% * ± SD Nichols, Jr. BMI extremes and IVF pregnancy rates. Fertil Steril 2003.

201

202 Low dose aspirin protocol Patients with autoimmune disorders Suppressed tromboxane A 2 but Decreases PG I 2 too Improves number mature folliclesImproves number mature follicles Improve size folliclesImprove size follicles Not improve pregnancy rate/ETNot improve pregnancy rate/ET

203 Low dose aspirin protocol Start in previous cycle of COH 100 mg/day until pick-up day r-FSH 450 IU/d HCG 20 IU/d Microdose flare on 2° day of COH or Delayed antagonist Frattarelli JL et al: “Low-dose aspirin use does not improve in vitro fertilization outcomes in poor responders”. Fertil Steril 2008;89,5:

204

205 OHSS physiopathology Ovary hyperstimulation Multiple follicle recruitment Luteal cysts Neovascularisation Ovary enlargement Abdomen distension Abdomen pain Nausea Vomiting Massive luteinization histamine prostaglandins citochine renin permeability vascular alteration AscitesHypovolemiaOliguriaCID

206 OHSS Classification (Volpicelli V. CIC Roma 1998) SLIGHTYMODERATESEVERE I°II°III°IV°V°VI°  ovary (cm) < > 20 >20 Abdomen distension Abdomen pains  Peritoneal flogosis  Vomiting Nausea Diarrhoea ++++ Hydrothorace            Ascites [1] [1] +             Electrolytic Imbalance +++ Hypovolemia +++++ Venous central pressure                    Hypovolemic shock +++ Acidosis +++ Kidney perfusion                    Oliguria +                   Hyperazotemia +++++ [1] [1] Key symptom to hypersevere syndrome [1]

207 High 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 UI on leading follicle ≥18 mm

208 High 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° day up HCG dayantagonist 0.25 mg/d on 2° day up HCG day Doxycycline* 80 mg/Kg/day (inhibits vascular leakage) * Folkman HJ: fertil Steril 2007;88,S1:O14 * Folkman HJ: fertil Steril 2007;88,S1:O14 *Bassado cpr 100 mg

209 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.

210 AA 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 E 2 ≥ pg/ml (range ) 0% OHSS0% OHSS

211 Agonist vs. HCG as trigger Gn-RH-a: HCG UI mature oocytes premature oocytes implantation rate clinical pregnancy ongoing pregnancy OHSS  

212 OHSS/withholding E 2 >4.000 pg/ml and/or Follicles >10 in each ovary term ≤3 day PCOS Young High responders 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:

213 OHSS/Coasting Until drop of estrogen level <3.000 pg/mlUntil drop of estrogen level <3.000 pg/ml Coasting >3 days no affects on PrCoasting >3 days no affects on Pr Egbase 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. 2000;15:2082–

214 OHSS/Coasting  inverse relationship  duration of coasting/number of mature oocytes retrieved  Pregnancy rate * 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. 2004;82:338–342 M. 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

215 Coasting long protocol Gn-RH-a long protocol HMG or r-FSH 225 IU step-down regimen On 2 nd cycle day at HCG or coasting dayOn 2 nd cycle day at HCG or coasting day 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  Coasting  HCG IU when E2 <3.000 pg/ml) 34

216 Ganirelix Orgalutran fl s.c mg

217 “Ganirelix salvage” Gn-RH-a long protocol on late luteal phase previousGn-RH-a long protocol on late luteal phase previous r-FSH 225 IU/d starting dose (down regimen)r-FSH 225 IU/d starting dose (down regimen) Antagonist 0.25 mg daily dose at E 2 > and or >10 follicles in each ovary daily measurement of serum E 2 HCG IU at E2 ≤3.000 pg/ml 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 Syndrome (OHSS)”. Fertility and Sterility 2005, 84,S318 35

218 “Ganirelix salvage” reduce E 2 levels (4.219  2.613/24 h)reduce E 2 levels (4.219  2.613/24 h) and avoid cycle cancellationand avoid cycle cancellation 24 oocytes mean/cycle24 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 (11):

219 PCOS Protocol Pre-treatment with metformin ≥6 months mg/day Improvment in menstrual cyclicity Long-protocol agonist Higher pregnancy outcome  Essah et al Fertil Steril 2006;86,1:

220 IVF cycle cost (ASRM’s average) total IVF cycle $ 12,500 with “coasting” were $ 400/day the cost per day of “ganirelix salvage” $ 553 cycle cancellation prior to retrieval $ 6379 plus the costs associated with either “coasting” or “ganirelix salvage M.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 M.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

221 OHSS SUPPLEMENTATION Micronized progesterone vaginally* mg/dMicronized progesterone vaginally* mg/d E 2 orally** 4 mg/dE 2 orally** 4 mg/d No HCG ! ! !No HCG ! ! ! * crinone 8% vaginal gel; prometrium cps 100 mg ** progynova cpr 2 mg; sprediol spray 1.5 mg; sandrena gel

222

223 Natural 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 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 mg/d of im P4 in oil after oocyte retrieval

224 Luteal 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

225 SUMMARY Controversies on gonadotropinsControversies 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: “Controlled ovarian hyperstimulation protocols for in vitro fertilization : two decades of experience after the birth of Elizabeth Carr”Fertil Steril 2005;84,3: 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:

226 HMG or r-FSH higher clinical pregnancy rate with hMGhigher clinical pregnancy rate with hMG but no significant differences in ongoing pregnancy rates or live birthsbut no significant differences in ongoing pregnancy rates or live 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.

227 intermediate 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 must be performed to achieve optimized results.but 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: 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:

228 High Responders High responders perform favorably with gentler stimulation that minimizes the occurrence of OHSS The number of oocytes retrieved is predictive of clinical pregnancy only in patients over 40 years of age M. Luna-Rojas, B. Sandler, M. Duke, A.B. Copperman, L. Grunfeld, J. Barritt Fertility and Sterility September 2004 (Vol. 82, Page S206)

229 Low 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: outcome suboptimal: poor ovarian responsepoor ovarian response poor oocyte/embryo qualitypoor oocyte/embryo quality in spite of stimulation regimens used

230 LH ADDED Suheil J. Muasher, Rony T. Abdallah, Ziad R. Hubayter: “Optimal stimulation protocols for in vitro fertilization” Fertil Steril 2006; 86,2: Adding LH should be considered in severe situations of LH suppression: 1.use of potent GnRH-agonists 2.Gn-RH-antagonists 3.Over 35 years

231 Conclusion(s) Ovarian stimulation is a critical step in in vitro fertilization therapy.Ovarian stimulation is a critical step in in vitro fertilization therapy. A variety of controlled ovarian hyperstimulation regimens are available and efficacious,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.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.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: 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:

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