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Controllo ormonale della

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

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

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

4 Ovarian cortical tissue
Age 10-year-old 20-year-old 30-year-old

5 Preovulatory follicle
Folliculogenesis Primary follicle Antral follicle Primordial follicle Preovulatory follicle Preantral follicle Corpus luteum

6

7 Sviluppo follicolare Diverse classi follicolari definite in base al numero di cellule della granulosa osservate: 1) follicoli primordiali: mm costituiti da un ovoci- ta in diplotene circondato da un singolo strato di cellule non cubiche (pregranulosa) 2) follicoli primari: >60 mm costituiti da un ovocita pri- mario 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

8 Reclutamento Selezione
FOLLICULOGENESIS Ovulazione Menses Threshold Recruitment Selection Dominance Window Atresia FSH 50% Atresia 77% VIII 20mm 58% 24% 60x10 cg 6 35% 15% 24% VII 15mm VI 90x10 cg 6 7mm 19x10 cg 6 V 2mm 37x10 cg 5 IV E2 LH FSH III 0,9mm II 0,4mm 75x10 cg 4 I 0,05mm 0,12mm 0,4mm 15x10 cg 4 M 1 strato cg 6x10 cg 2 5x10 cg 3 Primordiale Primario Preantrale Antrale precoce FSH dipendente FSH LH dipendente Crescita tonica Reclutamento Selezione Maturazione >150 gg 120 gg 65 gg 10 gg 10 gg

9 FSH dependent follicular growth
Threshold Window FSH Menses VIII Recruitment Selection Dominance 20mm PREOVULATORY 60x10 6 VII Atresia DOMINANT 15mm VI 90x10 cg 6 7mm V 19x10 cg 6 2mm SELECTED 37x10 cg 5 Atresia Atresia IV E2 LH FSH M Recruitment Selection Early dominance Late dominance LATE EARLY MID LATE LUTEAL FOLLICULAR FOLLICULAR FOLLICULAR

10 Reclutamento selezione
Primordial Primary Secondary ? ? ? VIII 20mm 60x10 cg 6 VII Oocyte growth Granulosa cell proliferation Theca formation 15mm VI 90x10 cg 6 7mm 19x10 cg 6 V 2mm 37x10 cg 5 IV E2 LH FSH III 0,9mm II 4 0,4mm 75x10 cg I 0,05mm 0,12mm 0,4mm 15x10 cg 4 M 1 strato cg 6x10 cg 2 5x10 cg 3 Primordiale Primario Preantrale Antrale precoce FSH dipendente FSH LH dipendente Crescita tonica Reclutamento selezione Maturazione >150 gg 120 gg 65 gg 10 gg 10 gg

11 FSH Granulosa cell proliferation Antral formation Early antral
Antral formation Early antral ( µm diam) Preantral (<200 µm diam) VIII 20mm 60x10 cg 6 FSH VII 15mm VI 90x10 cg 6 7mm 19x10 cg 6 2mm V 37x10 cg 5 IV E2 LH FSH Granulosa cell proliferation Follicular fluid formation Oocyte growth Thecal cell proliferation LH receptor expression M FSH dependent FSH LH dependent Maturation 10 gg 10 gg

12 LH FSH Antral growth Graffian (terziary) Early antral
Antral growth Graffian (terziary) ( µm diam) Early antral ( µm diam) VIII 20mm 60x10 cg 6 VII FSH 15mm VI 90x10 cg 6 7mm 19x10 cg 6 2mm V LH 37x10 cg 5 IV E2 LH FSH Granulosa cell proliferation Follicular fluid formation Oocyte growth Thecal cell proliferation M FSH dpendent FSH LH dependent Maturation 10 gg 10 gg

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

14 Role of LH What is the importance of LH as an ovarian regulator?
Role of LH What is the importance of LH as an ovarian regulator? Stimulates the enzyme adenylate cyclase synthesis of cAMP activates the enzyme pyruvate kinase and cholesterole transport to the mitochondria where is converted in pre- gnenolone, the rate-determining step in andro- gen biosynthesis Shoham, 1993 14

15 Role of LH What is the importance of LH as an ovarian regulator?
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

16 ? Role of LH The optimal amount of LH The drugs to be used
? The optimal amount of LH The drugs to be used

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

18 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 LH CEILING Normal follicular growth and development Paracrine signaling activated by FSH and LH Adequate granulosa cell proliferation Full follicle and oocyte maturation 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) No full oocyte maturation LH THRESHOLD

19 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 follicular LH receptors need to be occupied to allow normal steroidogenesis (Chappel 1991) LH WINDOW LH THRESHOLD

20 Serum LH “threshold” ? Measurements of serum immunoreactive
Serum LH levels remained below 1.0 IU/L in hypo-hypo patients who received 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 endo- genous LH to respond adequately to FSH

21 Serum LH “threshold” LH <1.0 IU/L: serum and follicular E2 and T (Fleming 1996) LH <0.5 IU/L: no. of oocytes, fertilization and embryo quality rates (Fleming 1998; Janssens 2000) LH <0.5 IU/L: miscarriage rate (Westergaard 2000) LH <0.07 IU/L - <0.5 IU/L: impaired reproductive outcome (Fleming 2000; Esposito 2001; Humaiden 2002) No threshold: (Balash 2001; Cabrera; Bjercke 2005)

22 Role of exogenous LH Clincal results Older poor responder patients
Clincal results Older poor responder patients Some patients treated with FSH and GnRH-a Some patients treated with FSH and GnRH-ant

23 Master in Medicina della Riproduzione
La crioconservazione nelle azoospermie Master in Medicina della Riproduzione La crioconservazione nelle azoospermie Thank you for your attention

24 Demise of germinal cells
Valle Giulia Clinic, Rome, Italy 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 Whats happen now in the early folliculogenesis? …………. It is very important to focus our attention to this point here, the transition between germ cells to primordial follicles. This is a genetic check point that leads to demise of germinal cells in case a correct crhomosomal pairing does not occur. This is important from a clinical view point because the resulting phenotype is …… So me may say that ….. The historical description of female sexual differentiation as the ‘default pathway’ implies that making an ovary is a passive process when, in fact, it simply means that the controlling factors remain unknown. Recently, however, it has been recognized that retinoic acid provides the signal for meiotic entry in the fetal ovary [16]. Germ cells in the male escape this fate because the somatic cells of the differentiating testis produce a testis-specific enzyme (Cyp26b1) that degrades retinoic acid The prophase events of synapsis and recombination that occur in the fetal ovary are essential for germ cell survival and meiotic progression. Early cytological studies in the mouse showed that translocations and other chromosomal aberrations that impede synapsis result in the loss of a significant number of meiocytes in both males and females [20]. Similarly, direct analysis of prophase in human males has revealed a correlation between synaptic and/or recombination defects and cases of azoospermia or oligospermia Regardless of the molecular details of these checkpoint mechanisms, there are apparent sex-specific differences in their efficiency. Specifically, several meiotic mutations affecting synapsis and recombination (including most of those listed above) cause prophase arrest in the male while allowing at least a proportion of oocytes to progress through meiotic prophase. Possibly checkpoint mechanisms are more effective in spermatogenesis than in oogenesis [34,35]. However, some of the discrepancy between the sexes likely reflects differences in the ability to detect the consequences of synaptic or recombination errors. That is, in males, defects during pachytene that cause the elimination of a large number of spermatocytes result in a drop in sperm counts and infertility or reduced fertility. In the female, however, such loss would reduce the reproductive lifespan by decreasing the total follicle reserve. In the human, however, the consequences would be serious; premature ovarian failure and/or accelerated onset of human age-related aneuploidy. 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 24

25 Primordial to primary follicle transition:
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 BMP15, are critical for regulation of ovulation rate and result in high multifollicular rate or ovarian failure (McNatty et al. 2003) Whats happen in the transition between primordial to primary follicle? …… The pool of tho oocytes that reach the primary stage, inhibit via AMH secreted by the granulosa cells the transition of other primordial follicles. In fact genetic alteration….. In particolare fattori prodotti dall’ovocita nel follicolo primario (GDF9 e BMP15) sono ritenuti essere responsabili di indurre nelle cellule della Granulosa la produzione dei segnali necessari per il reclutamento delle cellule della teca e per l’inibizione dei follicoli primordiali circostanti (AMH). Infatti AMH prodotto dai follicoli primari esercita un’azione repressiva sui follicoli primordiali, bloccandone la transizione a follicolo primario. L’AMH sembra essere prodotto dalle cellule della granulosa in risposta a fattori secreti dall’ovocita, regolando così il quantitativo di follicoli che vengono reclutati nella fase di cresita. Infatti alterazione nei genei ovocitari coinvolti in questa transizione ……. GDF-9 is expressed by the oocyte throughout folliculogenesis and is required for progression beyond the primary stage of development (Dong et al., 1996; Carabatsos et al., 1998; Elvin et al., 1999a). Female mice that are homozygous for a targeted deletion of exon 2 of the gdf-9 gene are infertile These findings, together with the immunisation results with BMP15 and GDF9 peptides, demonstrate that by altering the bioavailability of GDF9 and BMP15 in vivo, it is possible by exogenous means to enhance ovulation rate and increase lamb production or to induce infertility. In the human female, the first germ cells initiate meiosis at 11–12 weeks of gestation [18], 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 synapsis and recombination, and then enter a protracted arrest phase in late prophase Genes critical in early folliculogenesis are important determinants of reproductive life span and represent candidate genes for human ovarian failure. Ovaries that lack aromatase can develop primordial, primary, secondary and antral follicles. Estrogen deficiency is therefore not critical for early folliculogenesis. However, a closer look at aromatase knockouts revealed that the number of primordial follicles was approximately 40% less than in the wild-type ovaries [152, 153]. The number of primary follicles was statistically not significantly different between aromatase knockout and wild-type animals. Primordial follicle numbers were also reduced in the newborns of pregnant baboons treated with an aromatase inhibitor [154]. It is unclear if the aromatase inhibitor effect is due to the direct effect on the developing ovary or whether aromatase inhibitors exert subtle effects on the embryonic vasculature resulting in the smaller endowment of germ cells. The rapid decline in estrogen and progesterone concentrations after birth was hypothesized to be in part responsible for the breakdown of germ cell clusters and formation of primordial follicles in mammals. It is interesting to note, however, that primordial follicle formation occurs in humans during the time of continual rise in estrogen and progesterone levels, around 17–19 weeks of gestation [157–159]. In summary, the effects of estrogen and progesterone on early folliculogenesis are not critical, as shown by mouse knockout models, and further research is necessary to determine whether the observed effects documented by in vitro and in vivo approaches are physiologically relevant. Formazione dei follicoli nel secondo trimestre di vita fetale, ma sono sconosciuti i meccanismi che lo determinano, probabilmente guidati dallo stato di dyctate dell’ovocita (Albertini 2007 oocentric view of folliculogenesis). Sicome le pgc prima dell’entrata in meiosi vanno incontro ad una serie di mitosi caratterizzate da incompleta citocinesi con formazione di gruppi di cellule interconnesse(germ cell cysts), la formazione del follicolo richiede due fasi: 1 la rottura dei ponti intercellulari tra ovociti e 2 l’avvolgimento(enclousure) dell’ovocita da parte di cellule somatiche precursori di GC. (anche qui fattori ambientali interagiscono con la formazione del follicolo portando alla formazione di follic con 2 o+ ovociti, polyovular follicle). Esposizione nei topi a sostanze estrogeniche aumentano i multioocyte follicle e mortalità embrionale. Ci sono evidenze che questi multioo. Foll siano selezionati negativamente durante la selezioone follicolare, cmq questo può avere riperscussioni sul potenziale riproduttivo in termini quantitativi e qualitativi.(studi wildlife) Tuttavia i follicoli multinucleati sono stati riportati anche in conseguenza ad alterazioni genetiche (BMP15-GDF-9-dmrt4)…esposizione neonatale in topid estrogeni risulta in una marcata riduzione di mRNA e livelli proteici della famiglia delle tgf-b. CCs possess highly specialized trans-zonal cytoplasmic projections which penetrate through the zona pellucida and form gap junctions at their tips with the oocyte, forming an elaborate structure called the cumulus–oocyte complex (COC) During the course of antral follicular development, the oocyte gradually and sequentially acquires meiotic and developmental competence. It is during this phase of oogenesis that the oocyte acquires the molecular and cytoplasmic machinery it requires to fully support embryo development (Brevini-Gandolfi and Gandolfi, 2001; Sirard et al., 2006), and as such this process has been termed ’oocyte capacitation’. a central regulator of follicular cell function and thereby plays a critical role in the regulation of oogenesis, ovulation rate and fecundity Animals heterozygous for null mutations in these genes have higher ovulation rates than wild-type contemporaries, while homozygotics display ovarian failure 25

26 Follicular antral growth:
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 depen- dent 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 follicu- lar growth, including the process of follicle selection, steroidogenesis and maturation OSFs Regulate the granulosa cell activin- follistatin-inhibin system Modulate FSH-induced P and E2 synthesis by mural and cumulus granulosa cells Modulate GCs AMH secretion ↓ LHR mRNA and ↑aromatase mRNA in CCs Oocyte secreted factors orchestrate the two cells function, and so doing coordinate follicular growth, steroidogenesis and its own maturation and possible the process of follicle selection The antral stages of follicle development, including the process of follicle selection, are also dependent on appropriately timed endocrine signals, notably, pituitary gonadotrophins and metabolic hormones, which act on receptors on the two somatic cell types and interact with a myriad of locally produced factors operating in an autocrine/paracrine manner to coordinate and control cell function. Once again, members of the TGF-b superfamily feature prominently amongst these locally produced factors this oocyte mitogen(s) interacts with key known granulosa cell regulators such as FSH, IGF-I and androgens, augmenting their growth-promoting activities (Armstrong et al., 1996; Lanuza et al., 1998; Li et al., 2000; Hickey et al., 2004). Simultaneously, oocytes potently regulate granulosa/cumulus cell differentiation (reviewed - Eppig, 2001). For example, oocytes modulate FSH-induced progesterone and oestradiol synthesis by mural and cumulus granulosa cells (Vanderhyden et al., 1993; Coskun et al., 1995; Vanderhyden and Tonary, 1995; Li et al., 2000) and suppress FSH-induced luteinizing hormone receptor (LHR) mRNA expression Importantly, oocytes also regulate the granulosa cell activin-follistatin-inhibin system intriguingly, because inhibin acts primarily on the pituitary, the oocyte may indirectly regulate secretion of key endocrine hormones such as FSH. Regulation of steroidogenesis Intriguingly, because inhibin acts primarily on the pituitary, the oocyte may indirectly regulate secretion of key endocrine hormones such as FSH 26


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