Lecture 7: Menstrual Cycle 2 Flashcards
What does the inter cycle rise and then fall in FSH cause?
- Selection
- & exponential growth of the dominant follicle (dominant follicle -> preovulatory follicle)
How does the switch to positive feedback occur?
- End of FP, E2 feedback becomes +ive
- & persistant - 200pM, 48hrs
= exponential rise in LH that has to exceed a threshold
Once the +ive feedback is achieved, what does it result?
- LH surge
- Lasts for 36-48 hours
- & THEN triggers ovulation (timing varies from species-species)
What does the LH surge cause?
- Ovulation
(E2 300pM, 48hrs –> +ive feedback –> LH surge for 36-48hrs –> OVULATION)
For ovulation to occur, what must happen?
- LH surge must exceed threshold
How quickly is LH cleared from serum compared to hCG?
- LH rapidly cleared from serum,
- hCG cleared slowly & binds with great affinity to LHCGR
Where is hCG produced from?
- Blastocyst
- Only during pregnancy
Where is hCG produced from?
- Blastocyst
What is an indicator of pregnancy?
- hCG
Mid-cycle (during the LH surge/ovulation), where are the LHr found on the follicle?
- Theca
- & Granulosa cells
ONLY ON DOMINANT FOLLICLE
What size should a follicle be to be considered a dominant follicle?
- at least >15mm diameter on ultrasound
How many hours does onset of LH surge precede ovulation?
- 36hrs
How many hours does LH peak precede ovulation?
- 10-12hrs
Does the whole follicle ovulate?
- No
- Only the cumulus-oocyte complex (COC)
As the antral follicles grow, where do they move to in the ovary?
- Middle of the ovary
- Due to rich blood supply
When the selection and growth of dominant follicle occurs, does it move?
- Moves back out to surface of ovary
- Ready to be ovulated
Summarise what needs to happen for ovulation to occur?
- Loss of OSE & breakdown of underlying basal lamina and GC & TC at apex to allow for rupture.
-GC basal lamina disrupted allowing extension of blood vessels into GC layer & for infiltration of TCs & leukocytes into GC compartment. - COC detaches from surrounding GC to expand and move out
Describe in detail the preovulatory follicle prior to LH surge
- Oocyte surrounded by zona pelucida & cumulus GCs that connect to mural GCs that line interior of follicle.
- GC compartment separated from TC compartment by basal lamina.
- TC compartment composed of inner theca interna & outer theca externa. Unlike GC compartment, TC layer is highly vascularized (red).
- Circulating leukocytes present in vessels.
- Theca externa blends into a layer of connective tissue, separated from ovarian surface epithelium by basal lamina.
Describe in detail the preovulatory follicle immediately prior to ovulation
(- Preovulatory follicle following LH stimulation immediately prior to ovulation.)
- Disruption of GC basal lamina allows extension of vessels into GC compartment.
- TC & leukocytes also enter into GC compartment.
- CO Complex detaches from surrounding GCs & undergoes cumulus expansion.
- At follicular apex (top of image), there is loss of ovarian surface epithelium, the breakdown of the underlying basal lamina, & a loss of TCs & GCs.
- Rupture will occur at follicle apex.
Do the GCs typically have a blood supply?
- No
(- blood vessels infiltrate GCs just before ovulation)
Where are the blood vessels typically situated in the follicle?
- Theca externa
Preovulatory follicle prior to the LH surge vs following LH stimulation immediately prior to ovulation
When the blood vessels invade & infiltrate into GC layer just before ovulation, what do they bring in and why?
- Inflammatory markers
- Cytokines
- Leukocytes etc.
- Why? For ovulation to occur & for changes in COC to allow for release of egg which is within the follicle
What happens to the remainder of the follicle after ovulation? i.e. after COC leaves
- lutenised &
- becomes CL
Briefly summarise the state of the oocyte until just before being recruited into the MC (oogenesis)
Mitosis -> Increase in germ cells -> differentiate when at gonadal ridge -> oocytes -> mitosis stops -> enter meiosis 1 -> meiotic arrest
If a female ovulates at the age of 32 years and 3 months, how long had the oocyte ovulated been in meiotic arrest?
-32 years and 3 months (longer if taking gestation into account)
What factors are responsible for holding the oocyte in meiotic arrest?
- High cAMP
- cGMP
- H2O2/NO/calcium
- other cells/ ovarian environment & integrity of the follicle?
How does high cGAMP hold the oocyte in meiotic arrest?
- keep maturation promoting factor (MPF) inactive
How does cGMP hold the oocyte in meiotic arrest?
- cGMP enters oocytes from CC via gap junctions to inhibit oocyte cAMP phosphodiesterase PDE3A activity
(PDE3A normally degrades cAMP)
What enzyme degrades cAMP?
- cAMP phosphodiesterase (PDE3A)
What inhibits phosphodiesterase 3A (PDE3A)
- cGAMP
How do the cumulus cells and oocyte communicate?
- Projections between cumulus cells and oocyte
What is the effect of the LH surge within 3-12hrs?
- Detachment of COC from surrounding mural GC
- Cumulus cell expansion: formation of unique extracellular matrix between cumulus cells (aka “mucification”)
-↓cGMP production and closure of gap junctions - Activation of PDE3A → ↓cAMP → activation of pathways leading to breakdown of nuclear membrane in primary oocyte aka germinal vesicle breakdown (GVBD)
- Resumption of meiosis in oocyte → completion of Meiosis I & release of 1st polar body
- Arrests again in Metaphase II
What is mucification comprised of?
- long chains of hyaluronan
- Visco-elastic properties of CC matrix important for successful ovulation, ovum pick up by oviducts and penetration of sperm
What properties does the CC matrix have and what is its importance in the MC?
- Visco-elastic properties of CC matrix
- important for successful ovulation, ovum pick up by oviducts and penetration of sperm
What is contained in the first polar body?
- 23 chromosomes (that are chromatids)
- small amount of cytoplasm
What is CC expansion?
- formation of unique extracellular matrix between cumulus cells (aka “mucification”)
What are early oocytes classified as?
- Immature
- ie at germinal vesicle (GV) or metaphase 1 stage
The cAMP is produced endogenously in oocyte through the stimulation of the Gs G-protein by the GPR3, transported into oocyte from adjacent cumulus cells, and/or held by PDE3A inhibitor(s) in the follicular environment.
1b: A proposed model for maintenance of meiotic arrest at diplotene stage in follicular oocyte. The NO produced through nitric oxide synthases in cumulus-granulosa cells stimulate generation of cGMP through GCs pathway. The cGMP from encircling somatic cells is transferred though gap junctions to the oocyte. An increased level of intraoocyte cGMP level may inactivate PDE3A in the oocyte. The NO is also produced by oocyte itself through iNOS-mediated pathway and possibly inhibits PDE3A through cGMP pathway. The inhibition of oocyte PDE3A prevents cAMP hydrolysis and increase intraoocyte cAMP level. The increased cAMP level may activate PKA which in turn inactivate CDC25B phosphatase and thereby MPF. The inactive MPF does not induce meiotic resumption and diplotene arrest is maintained. The reduced production of H2O2 and Ca2+ release from mitochondria may also maintain meiotic arrest at diplotene arrest.
2b: A proposed model of LH/hCG-induced meiotic resumption from diplotene arrest in in preovulatory oocyte. LH/hCG reduces iNOS activity and induces disruption of gap junctions between cumulus-granulosa cells and oocyte. The interruption of communication between cumulus-granulosa cells and oocyte may block the transfer of cGMP produced through NO–GCs pathway. The reduced iNOS activity and thereby decreased intraoocyte NO level further decreases oocyte cGMP level. The net reduction in cGMP level may activate PDE3A that reduces cAMP level generated by oocyte itself through GPR3/AC pathway. The decrease in the cAMP level results in the inactivation of PKA activity, which in turn stimulates CDC25B phosphatase in the oocyte. The activated CDC25B phosphatase induces MPF activity that finally induces resumption of meiosis. Generation of tonic level of ROS and Ca2+ release from mitochondria may also be associated with the induction of meiotic resumption from diplotene arrest.
When meiosis 1 is completed, what does the secondary oocyte contain?
- half chromosomes
- nearly all cytoplasm
What is in the discarded polar body?
- other half of the chromosomes
- small bag of cytoplasm
What does the breakdown of the germinal vesicle indicate?
- resumption of meiosis
What does the the extrusion of the first polar body (1 PB) indicate?
- completion of the first meiotic division in human oocytes
Why the unequal division of cytoplasm at the end of meiosis 1?
- Need to conserve for the oocyte all the materials synthesised earlier –> takes fertilized zygote forward in growth and implantation
What happens to the secondary oocyte once it is formed?
- Immediately enter 2nd meiotic division
- Form 2nd metaphase spindle and arrest
How is the arrest maintained in the secondary oocyte?
- cytostatic factor (protein complex)
In what state is the egg ovulated?
- Secondary oocyte (arrested at Metaphase II)
What does the LH surge induce in GCs and DF cells?
- GC: PR expression in all species
- DF: luteinisation (both GCs & TCs)
What occurs to the levels of E2 and P during the LH surge?
- E2 falls
- P stimulated (& 17alpha-OHP)
What are the changes that occur in the ovary after the LH surge?
- GC: PR expression in all species
- DF: luteinisation (both GCs & TCs)
- E2 falls
- P stimulated (& 17alpha-OHP)
- Blood flow to follicle increases
- New vessels appear in avascular GC
- Increased prostaglandins & proteolytic enzymes e.g. collagenase & plasmin (in response to LH & P)
- Appearance of apex or stigma on ovary wall
What do the prostaglandins and proteolytic enzymes do?
- Digest collagen in follicle wall
Give 2 examples of prostaglandins and proteolytic enzymes?
- Collagenase
- Plasmin
What is the appearance of the stigma on ovary wall?
- Point of the dominant follicle closest to the ovarian surface where digestion occurs
When does ovulation occur?
- 12-18 hours after LH surge peak
How does ovulation occur?
- Cascade of events → release of COC → Ovulation
Is ovulation a type of inflammatory response?
- Yes
What type of inflammatory response is ovulation?
- acute inflammatory response
How is an acute inflammatory response triggered?
Increased secretion of chemokine/cytokines from GC & TC
→ triggers massive infiltration of leukocytes from circulation
= acute inflammatory response
What ovary does ovulation occur?
- In humans – ovulation occurs randomly from either ovary during a given cycles, some indication more common from right ovary
Which sex steroid hormone is essential for ovulation?
- Progesterone
What suppresses ovulation?
- Progesterone inhibitor (RU486)
Other than P, what else is essential during ovulation?
- Prostaglandins-E and -F and hydroxyeicosatetraenoic acid (HETE - metabolite of arachidonic acid) reach a peak level in follicular fluid just prior to ovulation
What is the role of Prostaglandins-E and -F and hydroxyeicosatetraenoic acid?
- stimulate proteolytic enzymes (proteases)
What do HETEs stimulate?
- HETEs (other types of prostaglandins - metabolite of arachidonic acid), HETEs may stimulate angiogenesis and hyperemia (↑blood flow)
Does astral pressure increase before ovulation?
- No
Summary of Follicle
- LH surge causes:
- Release of prostaglandins
- Proteases and enzymes digest wall
- Resumption of meiosis
- Cumulus expansion
- Release/Ovulation of expanded COC
What is the structure of the ovarian wall?
- OSE=simple layer of epithelieal cells (squamous/cuboidal/columnar depending on location),
- OSE supported by basement membrane that lies over the TA (held together by desmosomes and gap/tight junctions).
- Preferential growth of the DF brings it in close apposition with the OSE.
How does the COC overcomes the ovarian wall barrier?
- LH from circulation (acts on follicle of course to resume meiosis etc) but also binds to LHRs on OSE
-> release of plasminogen activator, which converts Plasminogen to Plasmin.
-> Activates collagenase via MMP-1 formation -> disrupts fibril network in tunica albuginea & also digest basement membranes of follicle & OSE.
-> Also get cytokine production, primarily TNFalpha = induces proteolysis via MMP-2 production & promotes apoptosis, stigma formation & eventual rupture.
= digestion of layers = release of COC
What occurs once oocyte ovulates?
- Secondary oocyte (arrested in metaphase II) with cumulus cells is extruded from the ovary
- follicular fluid may pour into Pouch of Douglas
- egg ‘collected’ by fimbria of uterine tube
- egg progresses down tube by peristalsis and action of cilia
- Residual part of follicle collapses into space left by fluid – clot forms & whole structure becomes CL
How is the COC collected?
- Uterine tube moves around
(unless adhesions present)
Ciliated cells are controlled by which hormones?
- E2
– also important is oestrogen:progesterone ratio
How do we know inflammation is associated with ovulation?
- The follicular fluid is “inflammatory”
- Inflammation definitely present, but too much is detrimental
- Gram
Is high inflammation beneficial when trying for pregnancy?
- Inflammation definitely present, but too much is detrimental…
- Higher “inflammation markers” in FF associated with decreased pregnancy rate (specifically C Reactive Protein, CRP)
- Gingivitis associated with poorer IVF outcomes!
How does the ovulatory wound heal?
(- Ovary faces monumental task of repairing damage caused by follicle rupture after each ovulation)
- Basic steps are known but the underlying mechanisms are still unknown
- Maybe steroidogenic environment helps – mitogenic (high in E2 etc which may help)
- Recently identified stem cell/progenitor population that may contribute to maintenance of OSE
Does ovulation wound scar?
Yes, but not for long - quick resolution
What are the signs of ovulation?
- A slight rise in basal body temperature, typically 0.5 to 1 degree, measured by a thermometer
- Tender breasts
- Abdominal bloating
- Light spotting
- Changes in cervical mucus
- Slight pain or ache on one side of the abdomen
How would one know that their basal temp has changed? and why do they do that?
- Need to keep a chart of basal body temp from day 1 of Last Menstrual Period
Why?
- Temperature varies every day
Why can some feel pain?
- Pre-ovulatory follicle can grow to about 20um
- Ovary only about 2-3cm
- Thus, pre-ov follicle may cause ovary to twist = pain
Does the cervical mucus/fluid change during MC?
- Yes
Describe the cervical mucus immediately after menstruation (EFP)
- scant and viscous
Describe the cervical mucus in late follicular phase
- In LFP, ↑ E2 levels, cervical mucous becomes clear, copious & elastic.
Is there a difference in quantity of the mucous between LFP and EFP?
- Yes
- Quantity ↑ 30 fold in LFP compared to EFP
How to evaluate the elasticity of the cervical mucous?
- The stretchability/elasticity of cervical mucous evaluated between two glass slides and recorded as the spinnbarkeit
Why does the mucous change throughout MC?
- Sperm penetration
- either to make it easy for sperm to reach egg
- or to prevent it
How is the mucous after ovulation?
- ↑progesterone levels
= cervical mucous again becomes thick, viscous, opaque & ↓ quantity produced
How may one check whether they are ovulating other than basal temp increase etc?
- MC apps
- Ovulation prediction kits
Can MC apps accurately predict ovulation?
- Ovulation day varies considerably for any given MC length, thus not possible for calendar/app methods that use cycle-length information alone to accurately predict the day of ovulation.
- To identify fertile period, must track physiological parameters such as basal body temperature & not just cycle length.
Study of MC length and Covid-19
- Not worrisome
- Just shows variation
Which phase is fixed?
- Luteal Phase (14 days)
Which phase is variable each month?
- Follicular Phase
What is measured in ovulation prediction kits?
- LH (LH surge)
- Some may even measure E2 (E2 high for 48 hrs before LH surge)
How long is the fertile period?
- 6 days approx
What affects the fertile period?
- Lifespan of the egg → up to 24h after ovulation (in the tract)
- Lifespan of sperm → median=1.5days but sperm can survive up to 5 days in sperm supportive mucus of fertile days of cycle -> sperm survival is dependent on the type & quantity of mucus within cervix & quality of the sperm
What is the main function of the CL?
- To support a potential pregnancy
How would the CL support a pregnancy? i.e. what hormones does it produce?
- Progesterone
- Inhibin A
- Oestrogens
- Androgens
How is the CL formed?
- After ovulation
- remaining granulosa enlarge, become vacuolated in appearance & accumulate lutein (yellow pigment)
- Massive angiogenesis to form new capillaries
- Luteinized GCs combine with newly formed theca-lutein cells & surrounding stroma in ovary = becomes CL
What is the CL made up of?
- mixture of large luteal cells (formerly GCs)
- and small luteal cells (formerly TCs)
What determines the life-span of the CL?
- continued LH support
- or hCG from pregnancy
(luteotrophic support predominately)
How high is LH support post-ovulation?
- LH levels are very low however even small amounts of LH can allow CL to continue to thrive
What determines CL degradation in humans and higher primates?
- loss of luteotrophic support (i.e. below threshold levels of LH enough for maintenance for a whilst)
NOT due to luteolytic agents!!!
How does CL undergo degradation?
- CL undergoes luteolysis if no pregnancy and forms a scar tissue called the corpus albicans.
- Cell death occurs, vasculature breakdown, CL shrinks
What does pregnancy do to the CL?
- Prevents CL from degrading
- hCG keeps it alive
Why does CL shrink and die if no pregnancy?
- removal of CL essential to initiate new cycle
What maintains the endometrium?
- P and E2
- Endometrium built up in response to P and E2 for pregnancy
- No pregnancy = Decrease in P (and E2) etc.
How does menstruation occur?
- P withdrawal -> increased coiling & constriction of spiral arterioles
- Endometrium releases prostaglandins -> contractions of uterine smooth muscle & sloughing of degraded endometrial tissue
= Menstruation
How do we know that prostaglandins cause menstruation?
- Use of prostaglandin synthetase inhibitors decreases amount of menstrual bleeding
Average duration of menstrual flow?
- 4-6 days (range 2-8 days)
Average amount of menstrual blood loss?
- 30ml with >80ml abnormal
What is the most common cause of infertility in women?
- Anovulation (women do not ovulate properly or not enough ovulation) due to many reasons
- affecting up to 40% of infertile women
What may cause anovulation?
- Non-ovarian causes eg obesity, thyroid disorders
- Ovarian causes e.g. - primary ovarian insufficiency (POI) aka premature ovarian failure due to loss of follicles OR due to disorders that prevent ovulation
What disorders prevent ovulation?
- Luteinized unruptured follicle syndrome (LUF)
- Effect of non-steroidal anti-inflammatory drugs (NSAIDs)
- Polycystic Ovary Syndrome (PCOS)
What is lutenised unraptured follicle syndrome (LUF)?
- Normal follicle growth in follicular phase & normal hormonal profile
- but absence of DM rupture & no release of oocytes
- Form a CL with trapped oocyte
- Linked to dysregulation of ovulation associated inflammatory changes
How is LUF diagnosed?
- Diagnose using repeated transvaginal ultrasound
What are the rates of occurrence of LUF in normal MC women and infertile women?
- normal menstrual cycle at rate of 5%
- infertile women at rate of >25%
Is there a change in Luteal phase length in women with LUF?
- No
- LP length is normal
Does ovulation occur in women with LUF?
- No
How is LUH linked dysregulation of ovulation associated inflammatory changes?
Examples:
- reduction in prostaglandin synthesis/action. EVIDENCE: Patients treated with high dose prostaglandin synthetase inhibitors (eg Indomethacin) → block in prostaglandin production and follicular rupture
- The lack of cytokine - Granulocyte colony-stimulating factor 3 (CSF3) - has links to LUF formation in infertile women. In anovulatory women, single injection of CSF3 during LF phase = ovulation in most women
What are NSAIDs commonly used for?
- relieving pain,
- lowering fever
- reducing swelling.
How do NSAIDs work?
- by suppressing prostaglandins (essential stimulators of inflammation)
How does the use of NSAIDs cause anovulation?
- Ovarian follicle expresses 2 types of prostaglandin synthase: PTGS1 (constitutive) & PTGS2 (inducible)
- ↑PTSG2 expression just before ovulation
- Administration of NSAID that specifically inhibit PTGS2 → delayed follicle rupture & oocyte release
At what dosage do studies of NSAID inhibition of ovulation use?
- at or above the maximum dose prescribed
What is a limitation of the studies of NSAID inhibition of ovulation use?
- Used very high dosage (normally not prescribed dosage)
How can we prove that prostaglandins are involved in follicle rupture?
What is the most common cause of death from gynaecological malignancy in the developed world? (ovarian cancer)
- Epithelial ovarian cancer (EOCs)
- EOCs comprise heterogenous group, most lethal form is high-grade serous cancer (HSGC)
What are 3 main cellular origins of ovarian cancer?
- epithelium (90%),
- germ cells (5%)
- stromal cells (5%)
How are ovarian cancers deadly?
- Malignant cells of the primary tumor are shed into the peritoneal cavity where they are disseminated throughout the abdominal cavity.
- These malignant cells often aggregate & form spheroid-like structures.
- Conversely – women on OCP and who have had numerous pregnancies have lower risk.
Which gene mutations are associated with a higher chance of breast and ovarian cancers?
- BRCA1 or BRCA2 mutations
- 10-25% of OVCa associated with these gene mutations
What WAS the most accepted hypothesis of Epithelial ovarian cancers (EOC) carcinogenesis for many years?
INCESSANT OVULATION THEORY
- Ovulation traumatises the OSE, hence increasing error during cell replication
- Epidemiological evidence that women with high number of life-time ovulations at increased risk of EOC eg. nulliparous women, those with early menarche and late menopause
- Long-term use of oral contraception reduces OvCa risk
What does new evidence indicate regarding EOC?
- HSGC arises from fimbria of fallopian tube rather than the ovaries!
- This study came from the BRCA studies whereby they took the ovaries and tubes from the women with the mutations and did histology analysis looking for early markers of cancer and found the markers in the fallopian tube rather than the ovaries
How was the new evidence regarding EOC carcinogens found?
- Removal of ovary & tube as prophylactic surgery for BRACA-1,-2 mutations
– histological examinations shown that premalignant lesions on fimbria region of tube -> NOT ovary. - Serous carcinomas of the ovary share many similarities & biochemical markers with the Fallopian tube epithelium.
- While this can be explained by the common embryonic origin of the ovarian surface epithelium & Mullerian epithelium of the tube,
- it has recently raised the possibility that the fimbrial end of the Fallopian tube may be an alternative source or main source of ovarian serous carcinoma
Link the menstrual, ovarian and uterine cycle.
