controllo ormonale della follicologenesi filippo maria ubaldi m.d. m.sc. clinica valle giulia, roma ...
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Controllo ormonale dellafollicologenesi
Filippo Maria Ubaldi
M.D. M.Sc.
CLINICA VALLE GIULIA, Roma
www.generaroma.it
Master in Medicina della Riproduzione
29-30 Maggio 2013
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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
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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
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Ovarian cortical tissue
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10-year-old10-year-old 20-year-old20-year-old 30-year-old30-year-old
AgeAge
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Folliculogenesis
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Primordial follicle
Primary follicle
Preantral follicle
Antral folliclePreovulatory follicle
Corpus luteum
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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
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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
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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
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Role of LH
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? The optimal amount of LH
The drugs to be used
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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
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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
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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)
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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
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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)
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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
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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
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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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Valle Giulia Clinic, Rome, Italy
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