www generaroma it CLINICA VALLE GIULIA Roma Controllo

www. generaroma. it CLINICA VALLE GIULIA, Roma Controllo ormonale della follicologenesi Filippo Maria Ubaldi M. D. M. Sc. Master in Medicina della Riproduzione 29 -30 Maggio 2013

www. generaroma. it Primordial germ cells migration ü Primordial germ cells originate from the entoderm of the yolk sac around the III week of gestation. ü At VI week of gestation the number of the oogonia is about 1000. There is not yet meiotic activity

www. generaroma. it Ovary ü At VIII week of gestation the meiotic divisions lead to 600. 000 oogonia and three activities are present: mithosis, meiosis and atresia. At XX week of gestation the number of germ cells is 3 -6 million ü At birth 1 -2 million of germ cells are present and at puberty there are left only 300. 000 – 400. 000 of whom only 400 -500 will ovulate

www. generaroma. it Ovarian cortical tissue Age 10 -year-old 20 -year-old 30 -year-old

www. generaroma. it Folliculogenesis Primordial follicle Primary follicle Antral follicle Preovulatory follicle Preantral follicle Corpus luteum

www. generaroma. it Sviluppo follicolare ü Diverse classi follicolari definite in base al numero di cellule della granulosa osservate: 1) follicoli primordiali: 30 -60 mm costituiti da un ovocita in diplotene circondato da un singolo strato di cellule non cubiche (pregranulosa) 2) follicoli primari: >60 mm costituiti da un ovocita primario circondato da un singolo strato di cellule cubiche della granulosa 3) follicoli secondari: 120 mm costituiti da un ovocita primario circondato da diversi strati di cellule della granulosa

FOLLICULOGENESIS Window 50% Threshold Atresia 77% FSH Menses Recruitment Selection Dominance 24% VIII 20 mm 60 x 106 cg 58% 35% 15% 24% VII Atresia IV 0, 05 mm 0, 12 mm 1 strato cg 6 x 102 cg Primordiale. Primario >150 gg 120 gg Ovulazione III 0, 9 mm II 0, 4 mm 75 x 104 cg I 0, 4 mm 15 x 104 cg 5 x 103 cg Preantrale Antrale precoce Crescita tonica 65 gg 15 mm VI 90 x 106 cg 7 mm 19 x 106 cg V 2 mm 37 x 105 cg LH E 2 FSH M FSH dipendente FSH LH dipendente Reclutamento Selezione Maturazione 10 gg

www. generaroma. it FSH dependent follicular growth Window VIII 20 mm 60 x 106 Threshold FSH Mense s Recruitment Selection Dominance VII 15 mm VI 90 x 106 cg 7 mm 19 x 106 cg V 2 mm 37 x 105 cg IV LH E 2 FSH Atresia PREOVULATORY DOMINANT SELECTED Atresia M Recruitment LATE LUTEAL Selection Early dominance Late dominance EARLY MID LATE FOLLICULAR

www. generaroma. it Primordial Primary ? Secondary ? ? VIII 20 mm 60 x 106 cg VII • Oocyte growth • Granulosa cell proliferation • Theca formation IV 0, 05 mm 0, 12 mm 1 strato cg 6 x 102 cg III 0, 9 mm II 0, 4 mm 75 x 104 cg I 0, 4 mm 15 x 104 cg 5 x 103 cg Primordiale. Primario Preantrale >150 gg 120 gg Antrale precoce Crescita tonica 65 gg 15 mm VI 90 x 106 cg 7 mm 19 x 106 cg V 2 mm 37 x 105 cg LH E 2 FSH M FSH dipendente FSH LH dipendente Reclutamento selezione Maturazione 10 gg

www. generaroma. it Antral formation Early antral Preantral (<200 µm diam) VIII 20 mm 60 x 106 cg (200 -300 µm diam) FSH VII IV 15 mm VI 90 x 106 cg 7 mm 19 x 106 cg V 2 mm 37 x 105 cg LH E 2 FSH • Granulosa cell proliferation • • Follicular fluid formation Oocyte growth Thecal cell proliferation LH receptor expression M FSH dependent FSH LH dependent 10 gg Maturation 10 gg

www. generaroma. it Antral growth Early antral Graffian (terziary) VIII 20 mm 60 x 106 cg (500 -700 µm diam) (200 -300 µm diam) VII FSH LH • • Granulosa cell proliferation Follicular fluid formation Oocyte growth Thecal cell proliferation IV 15 mm VI 90 x 106 cg 7 mm 19 x 106 cg V 2 mm 37 x 105 cg LH E 2 FSH M FSH dpendent 10 gg FSH LH dependent Maturation 10 gg

13 www. generaroma. it The ‘two-cell, two-gonadotrophin’ theory FSH A A A Theca cells Cholesterols ►androgens A Androgens transferred to granulosa cells E Granulosa cells Oocyte Androgens maturation ► estradiol E LH-activity E E E Follicular growth Estradiol E Levy 2000; Hillier 1994; Kobayashi 1990; Fevold JCEM, 1941

www. generaroma. it Role of LH What is the importance of LH as an ovarian regulator? Stimulates the enzyme adenylate cyclase synthesis of c. AMP pyruvate kinase and activates the enzyme cholesterole transport to the mitochondria where is converted in pregnenolone, the rate-determining step in androgen biosynthesis Shoham, 1993

www. generaroma. it Role of LH What is the importance of LH as an ovarian regulator? • Tonic stimulation of thecal androgen production • Mainteins normal follicular oestradiol biosynthesis • Selection of the dominant follicle • Triggers ovulatory LH surge • Luteinisation of granulosa cells • Mainteins corpus luteum

www. generaroma. it Role of LH ? v The optimal amount of LH v The drugs to be used

Role of LH in follicular development Over exposure to LH LH Ceiling (Hillier, 1994) OUCH ! Watch the ceiling, darling. . HMG or CC/HMG (PCO Patient) Flare-up Gn. RHa Protocol LH Threshold Rec-FSH with Gn. RH antag. LH deficiency: Hypo & older patients Rec- FSH with Gn. RH agon. OUCH ! Watch the bottom, darling. .

Role of LH in follicular development Suppression of granulosa cell proliferation Follicular atresia of non dominant follicles Premature luteinisation of pre-ovulatory foll. Oocyte development compromised Normal follicular growth and development Paracrine signaling activated by FSH and LH Adequate granulosa cell proliferation Full follicle and oocyte maturation LH CEILING LH WINDOW Follicular growth-granulosa cell prolife. (FSH action) Induction of granulosa cell aromatase activity No paracrine signals between gran. and theca layers No androgen synthesis (no estrogens) LH THRESHOLD No full oocyte maturation

Role of LH in follicular development LH CEILING The amount of LH activity actually necessary for normal follicle and oocyte development is unknown, but it is likely to be low, as <1% of LH WINDOW follicular LH receptors need to be occupied to allow normal steroidogenesis (Chappel 1991) LH THRESHOLD

www. generaroma. it Serum LH “threshold” Serum LH levels remained below 1. 0 IU/L in hypo-hypo patients who received 75 -225 IU every day of rec-LH together with rec-FSH (The European Rec LH Study group, JCEM, 1998) ? Measurements of serum immunoreactive LH to identify patients with sufficient endogenous LH to respond adequately to FSH

www. generaroma. it Serum LH “threshold” ü LH <1. 0 IU/L: serum and follicular E 2 and T (Fleming 1996) ü LH <0. 5 IU/L: no. of oocytes, fertilization and embryo quality rates (Fleming 1998; Janssens 2000) ü LH <0. 5 IU/L: miscarriage rate (Westergaard 2000) ü LH <0. 07 IU/L - <0. 5 IU/L: impaired reproductive outcome (Fleming 2000; Esposito 2001; Humaiden 2002) ü No threshold: (Balash 2001; Cabrera; Bjercke 2005)

www. generaroma. it Role of exogenous LH Clincal results • Older poor responder patients • Some patients treated with FSH and Gn. RH-ant

www. generaroma. it Master in Medicina della La crioconservazione nelle. Riproduzione azoospermie Thank you for your attention

Early folliculogenesis: implication for human reproductive life span and ovarian failure The first germ cells initiate meiosis at 11– 12 weeks of gestation, with subsequent groups of cells entering meiosis over the course of the next several weeks. Oocytes progress through meiotic prophase, undergoing the complex events of synapsi and recombination, and then enter a protracted arrest phase in late prophase Around the time of arrest, oocytes become surrounded by somatic cells (pregranulosa cells), forming primordial follicles Genetic check-point control meccanism Demise of germinal cells Premature ovarian failure and/or accelerated onset of human age-related aneuploidy. The genetic quality of the oocyte meiotic prophase is critical during this developmental window for the formation of primordial follicle Valle Giulia Clinic, Rome, Italy

Primordial to primary follicle transition: regulation of ovulation rate The pre-antral phase of folliculogenesis is characterized by zona pellucida formation, granulosa cell proliferation, which is at first slow, the recruitment of thecal cells to the follicular basal lamina and a dramatic increase in oocyte volume Pre-antral follicle growth is hormonal independent and its regulation predominantly involves direct interactions between granulosa cells and oocytes. The local production of growth factors (TGF-b superfamily) regulate this transition Genetic alteration in oocytes secreted factors OSFs governing this transition, in particular GDF 9 and BMP 15, are critical for regulation of ovulation rate and result in high multifollicular rate or ovarian failure (Mc. Natty et al. 2003) Animals heterozygous for null mutations in these genes have higher ovulation rates than wild-type contemporaries, while homozygotics display ovarian failure Valle Giulia Clinic, Rome, Italy

Follicular antral growth: OSFs regulate the two cells / two gonadotropins model of steroidogenesis Follicle progression through the antral stage of development is a gonadotrophins dependent phase associated with intense proliferation of granulosa and theca cells, increased thecal vascularisation, further oocyte enlargement and increase in diameter and volume Once again oocyte secreted factors regulate the two cells function coordinating follicular growth, including the process of follicle selection, steroidogenesis and maturation OSFs • Regulate the granulosa cell activin- follistatin-inhibin system • Modulate FSH-induced P and E 2 synthesis by mural and cumulus granulosa cells • Modulate GCs AMH secretion • ↓ LHR m. RNA and ↑aromatase m. RNA in CCs Regulation of steroidogenesis Intriguingly, because inhibin acts primarily on the pituitary, the oocyte may indirectly regulate secretion of key endocrine hormones such as FSH Valle Giulia Clinic, Rome, Italy