Controllo ormonale dellafollicologenesi

Filippo Maria Ubaldi

M.D. M.Sc.

CLINICA VALLE GIULIA, Roma

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Master in Medicina della Riproduzione

29-30 Maggio 2013

Primordial germ cells migration

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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 1000. There is not yet meiotic activity

Ovary

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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 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 300.000 – 400.000 of

whom only 400-500 will ovulate

Ovarian cortical tissue

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10-year-old10-year-old 20-year-old20-year-old 30-year-old30-year-old

AgeAge

Folliculogenesis

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Primordial follicle

Primary follicle

Preantral follicle

Antral folliclePreovulatory follicle

Corpus luteum

Sviluppo follicolare

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Diverse classi follicolari definite in base al numero di cellule della granulosa osservate:

1) follicoli primordiali: 30-60 m costituiti da un ovoci- ta in diplotene circondato da un singolo strato di cellule non cubiche (pregranulosa)

2) follicoli primari: >60 m costituiti da un ovocita pri- mario circondato da un singolo strato di cellule cubiche della granulosa

3) follicoli secondari: 120 m costituiti da un ovocita primario circondato da diversi strati di cellule della granulosa

FSHFSHLHLHEE22

PreantralePreantrale Antrale precoceAntrale precoce FSH dipendenteFSH dipendenteFSH LH dipendenteFSH LH dipendente

Reclutamento SelezioneReclutamento Selezione10 gg10 gg

MaturazioneMaturazione10 gg10 gg

Crescita tonicaCrescita tonica65 gg65 gg

0,4mm0,4mm5x10 cg5x10 cg33II

0,4mm0,4mm15x10 cg15x10 cg44

IIIIIIIIII

IVIV

0,9mm0,9mm75x10 cg75x10 cg44

2mm2mm37x10 cg37x10 cg55

VV7mm7mm

6619x10 cg19x10 cg

VIVI

VIIVII

VIIIVIII

15mm15mm90x10 cg90x10 cg66

20mm20mm60x10 cg60x10 cg6624%24%

35%35%15%15%24%24%58%58%

77%77%

50%50%

AtresiaAtresia

OvulazioneOvulazioneFOLLICULOGENESISFOLLICULOGENESIS

Menses

ThresholdThreshold

Recruitment Selection DominanceRecruitment Selection Dominance

WindowWindow

Atresia

FSHFSH

MM

PrimordialePrimordialePrimarioPrimario

120 gg120 gg>150 gg>150 gg

0,12mm0,12mm6x10 cg6x10 cg221 strato cg1 strato cg

0,05mm0,05mm

FSH dependent follicular growth

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RecruitmentRecruitment SelectionSelection

SELECTEDSELECTED

LATE EARLY MID LATE LATE EARLY MID LATE LUTEAL FOLLICULAR FOLLICULAR FOLLICULAR LUTEAL FOLLICULAR FOLLICULAR FOLLICULAR

Menses

ThresholdThreshold

Recruitment Selection DominanceRecruitment Selection Dominance

WindowWindow

Atresia

FSHFSH

DOMINANTDOMINANT

Early dominanceEarly dominance

AtresiaAtresia

PREOVULATORYPREOVULATORY

Late Late dominancedominance

AtresiaAtresiaFSHFSH LHLHEE22IVIV

2mm2mm37x10 cg37x10 cg55

VV7mm7mm

6619x10 cg19x10 cg

VIVI

VIIVII

VIIIVIII

15mm15mm90x10 cg90x10 cg66

20mm20mm66

MM

60x1060x10

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FSHFSHLHLHEE22

PrimordialePrimordialePrimarioPrimario PreantralePreantrale Antrale precoceAntrale precoce FSH dipendenteFSH dipendenteFSH LH dipendenteFSH LH dipendente

Reclutamento selezioneReclutamento selezione10 gg10 gg

MaturazioneMaturazione10 gg10 gg

Crescita tonicaCrescita tonica65 gg65 gg120 gg120 gg>150 gg>150 gg

0,12mm0,12mm6x10 cg6x10 cg221 strato cg1 strato cg

0,4mm0,4mm5x10 cg5x10 cg33II

0,4mm0,4mm15x10 cg15x10 cg44

IIIIIIIIII

IVIV

0,05mm0,05mm

0,9mm0,9mm75x10 cg75x10 cg44

2mm2mm37x10 cg37x10 cg55

VV7mm7mm

6619x10 cg19x10 cg

VIVI

VIIVII

VIIIVIII

15mm15mm90x10 cg90x10 cg66

20mm20mm60x10 cg60x10 cg66

MM

PrimordialPrimordial PrimaryPrimary SecondarySecondary

• Oocyte growthOocyte growth

• Granulosa cell proliferationGranulosa cell proliferation

• Theca formationTheca formation

? ? ?

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FSHFSHLHLHEE22

FSH dependentFSH dependent FSH LH dependentFSH LH dependent

10 gg10 ggMaturationMaturation

10 gg10 gg

2mm2mm37x10 cg37x10 cg55

VV7mm7mm

6619x10 cg19x10 cg

VIVI

VIIVII

VIIIVIII

15mm15mm90x10 cg90x10 cg66

20mm20mm60x10 cg60x10 cg66

MM

IVIV

FSHFSH

• Granulosa cell proliferation

• Follicular fluid formation• Oocyte growth• Thecal cell proliferation• LH receptor expression

Antral formation

PreantralPreantral(<200 µm diam)(<200 µm diam)

Early antralEarly antral(200-300 µm diam)(200-300 µm diam)

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FSHFSHLHLHEE22

FSH dpendentFSH dpendent FSH LH dependentFSH LH dependent

10 gg10 ggMaturationMaturation

10 gg10 gg

2mm2mm37x10 cg37x10 cg55

VV7mm7mm

6619x10 cg19x10 cg

VIVI

VIIVII

VIIIVIII

15mm15mm90x10 cg90x10 cg66

20mm20mm60x10 cg60x10 cg66

MM

IVIV

FSHFSH

Antral growth

Graffian (terziary)Graffian (terziary)(500-700 µm diam)(500-700 µm diam)

• Granulosa cell proliferation• Follicular fluid formation• Oocyte growth• Thecal cell proliferation

Early antralEarly antral(200-300 µm diam)(200-300 µm diam)

LH

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The ‘two-cell, two-gonadotrophin’ theory

Theca cellsTheca cellsCholesterols ►androgens

Granulosa cellsGranulosa cells

Androgens transferred to granulosa

cells

AA

AA

AA

AAAA

AA

Androgens ► estradiol

FSHFSH

Oocyte Oocyte maturationmaturation

Follicular Follicular growthgrowth

EE

EEEE

EE

EE

EE

EstradiolEstradiol

Levy 2000; Hillier 1994; Kobayashi 1990; Fevold JCEM,1941

LH-activityLH-activity

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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

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

Role of LHRole of LH

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

Role of LHRole of LH

•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

Role of LH

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? The optimal amount of LH

The drugs to be used

HMG or CC/HMGHMG or CC/HMG(PCO Patient)(PCO Patient)

Flare-up GnRHa ProtocolFlare-up GnRHa ProtocolLH CeilingLH Ceiling (Hillier, 1994)(Hillier, 1994)

Role of LH in Role of LH in follicular developmentfollicular development

LH ThresholdLH ThresholdLH ThresholdLH Threshold

OUCH !OUCH !OUCH !OUCH !

WatchWatchthe bottom,the bottom,darling....darling....

WatchWatchthe bottom,the bottom,darling....darling....

OUCH !OUCH !OUCH !OUCH !WatchWatch

the ceiling,the ceiling,darling....darling....

WatchWatchthe ceiling,the ceiling,darling....darling....

Over exposure to LHOver exposure to LH

Rec-FSH with GnRH antag. Rec-FSH with GnRH antag.

Rec- FSH with GnRH agon.Rec- FSH with GnRH agon. LH deficiency: Hypo-LH deficiency: Hypo-Hypo & older patientsHypo & older patients

Role of LH in follicular developmentRole of LH in follicular development

Suppression of granulosa cell proliferationSuppression of granulosa cell proliferation

Follicular atresia of non dominant folliclesFollicular atresia of non dominant follicles

Premature luteinisation of pre-ovulatory foll.Premature luteinisation of pre-ovulatory foll.

Oocyte development compromisedOocyte development compromised

Normal follicular growth and developmentNormal follicular growth and development

Paracrine signaling activated by FSH and LHParacrine signaling activated by FSH and LH

Adequate granulosa cell proliferationAdequate granulosa cell proliferation

Full follicle and oocyte maturationFull follicle and oocyte maturation

Follicular growth-granulosa cell prolife. (FSH action)Follicular growth-granulosa cell prolife. (FSH action)

Induction of granulosa cell aromatase activityInduction of granulosa cell aromatase activity

No paracrine signals between gran. and theca layersNo paracrine signals between gran. and theca layers

No androgen synthesis (no estrogens)No androgen synthesis (no estrogens)

No full oocyte maturationNo full oocyte maturation

LH CEILINGLH CEILING

LH THRESHOLDLH THRESHOLD

LH WINDOWLH WINDOW

Role of LH in follicular developmentRole of LH in follicular development

LH CEILINGLH CEILING

LH THRESHOLDLH THRESHOLD

LH WINDOWLH WINDOW

The amount of LH activity actually necessary The amount of LH activity actually necessary

for normal follicle and oocyte development isfor normal follicle and oocyte development is

unknown, but it is likely to be low, as <1% ofunknown, but it is likely to be low, as <1% of

follicular LH receptors need to be occupied tofollicular LH receptors need to be occupied to

allow normal steroidogenesis allow normal steroidogenesis (Chappel 1991)(Chappel 1991)

Serum LH “threshold”

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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 endo-

genous LH to respond adequately to FSH

Serum LH “threshold”

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LH <0.07 IU/L - <0.5 IU/L: impaired reproductive

outcome (Fleming 2000; Esposito 2001; Humaiden 2002)

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)

No threshold: (Balash 2001; Cabrera; Bjercke 2005)

Role of exogenous LH

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Clincal resultsClincal results

• Older poor responder patientsOlder poor responder patients

• Some patients treated with FSH and GnRH-aSome patients treated with FSH and GnRH-a

• Some patients treated with FSH and GnRH-antSome patients treated with FSH and GnRH-ant

La crioconservazione nelle azoospermieLa crioconservazione nelle azoospermieMaster in Medicina della RiproduzioneMaster in Medicina della Riproduzione

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Thank you Thank you

for your attentionfor your attention

Valle Giulia Clinic, Rome, Italy

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 prophaseAround the time of arrest, oocytes become surrounded by somatic cells (pregranulosacells), forming primordial follicles

Genetic check-Genetic check-point control point control meccanismmeccanism

Demise of germinal

cells

Early folliculogenesis:Early folliculogenesis: implication for human reproductive life span and

ovarian failure

Premature ovarian failure and/or accelerated onset of human age-related aneuploidy.

The genetic quality of the oocyte meiotic prophase is critical during The genetic quality of the oocyte meiotic prophase is critical during this developmental window for the formation of primordial folliclethis developmental window for the formation of primordial follicle

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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

Genetic alteration in oocytes Genetic alteration in oocytes

secreted factors OSFs secreted factors OSFs

governing this transition, in governing this transition, in

particular particular GDF-9GDF-9 and and BMP15BMP15, ,

are critical for regulation of are critical for regulation of

ovulation rate and result in ovulation rate and result in

high multifollicular rate or high multifollicular rate or

ovarian failureovarian failure (McNatty et al. McNatty et al.

2003)2003)

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

Animals heterozygous for null mutations in these genes have higher ovulation rates than wild-type contemporaries, while homozygotics display ovarian failure

Primordial to primary follicle Primordial to primary follicle transition:transition: regulation of ovulation rateregulation of ovulation rate

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Follicular antral growth:Follicular antral growth: OSFs regulate the two cells / two gonadotropins model of OSFs regulate the two cells / two gonadotropins model of steroidogenesissteroidogenesis

OSFOSFss

Regulation of Regulation of steroidogenesissteroidogenesis

Intriguingly, because inhibin acts primarily on the pituitary, the Intriguingly, because inhibin acts primarily on the pituitary, the

oocyte may indirectly regulate secretion of key endocrine oocyte may indirectly regulate secretion of key endocrine

hormones such as FSHhormones such as FSH

• Regulate the granulosa cell activin-Regulate the granulosa cell activin- follistatin-inhibin systemfollistatin-inhibin system• Modulate FSH-induced P and E2 Modulate FSH-induced P and E2 synthesissynthesis by mural and cumulus granulosa by mural and cumulus granulosa cellscells• Modulate GCs AMH secretionModulate GCs AMH secretion• ↓ ↓ LHR mRNA and LHR mRNA and ↑↑aromatase mRNA aromatase mRNA in CCsin CCs

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 volumeOnce again oocyte secreted factors regulate the two cells function

coordinating follicu- lar growth, including the process of follicle selection,

steroidogenesis and maturation