Menstrual Cycle

Ovarian and menstrual cycle

THE HPO AXIS

The hypothalamus

• HT contains neurons secreting GnRH in the ventro-medial nucleus
• These neurones migrated from the olfactory placode in fetal life
• Failure of migration (Kallmann’s syndrome)
  • Congenital absence of gnrh or hypogonadotrophic hypogonadism
  • Usually associated with  anosmia
• HT GnRH centre receives afferent neurons from
  • Limbic system- stress, anxiety
  • Olfactory centre-smell
  • Occipital cortex- visual
  • Other nuclei in HT controlling energy metabolism and the stress response
• During IU life & shortly after birth these neurons secrete GnRH in a pulsatile manner into the hypothalamic hypophyseal portal circulation.
  • Pulsatile secretion of GnRH is essential for maintaining gonadotrophin secretion and ovarian activity
  • Sustained exposure to GnRH (as seen in the use of GnRH analogues) leads to an initial increase of gonadotrophins (flare response) which is followed after 10–14 days by down-regulation and desensitisation of GnRH receptors with subsequent very low levels of gonadotrophins and ovarian steroid hormones (Oestrogen & progesterone)
• It reaches gonadotrophs in the anterior pituitary à synthesis and pulsatile secretion of gonadotrophins (FSH and LH), which intern stimulate ovary
• GnRH pulses cannot be easily measured
  • The amount of GnRH reaching the systemic circulation is very low
  • Its half-life is very short (2 minutes)
• LH pulses can be measured (T ½ – 20 min) & used as indirect measure of GnRH pulses
• Shortly after birth GnRH pulse generator completely suppressed & remains so till puberty
• It is believed that attainment of a critical level of fat content leads to leptin hormone signalling from adipose tissue that releases the GnRH pulse generator from the pre-pubertal inhibition, with initiation of puberty
• During the reproductive years the hypothalamic GnRH pulse centre is under the influence of various endocrine and paracrine regulators (reproductive function)
  • Energy levels (leptin, insulin, ghrelin)
  • Stress (CRH, TRH, opioids)
  • Gonadal signals (oestrogens, progestins, androgens, inhibins, activin)
  • Light/dark signals (melatonin)
    • Some of these exert a stimulatory effect,
      • Leptin
      • Insulin
      • Activin
      • High levels of oestrogens at ovulation
    • Others exert an inhibitory effect,
      • Ghrelin
      • Inhibin
      • CRH, TRH, prolactin
      • Oestrogens, progestins & androgens
      • Opioids and melatonin
• These regulators mediate their actions primarily by neuro-peptide and amine transmitters
  • Stimulatory – Kisspeptin, pro-opiomelanocortin
  • Inhibitory – Neuropeptide Y, dopamine, GABA, serotonin, norepinephrine
• So, pharmacological agents which affect NT levels can also affect reproductive function
• The GnRH pulse frequency & amplitude is dependent on the balance of these signals as well as on the timing, duration and amplitude.
  • Eg: – Rapid rise of oestrogens at the time of ovulation triggers the LH surge

      – Sustained level of GnRH inhibits gonadotrophins

• Other pituitary hormones also have an inhibitory effect on GnRH and gonadotrophin secretions as in hyperprolactinaemia
• Pathological lesions disconnecting or damaging the hypothalamic hypophyseal portal circulation lead to disruptions of the GnRH pulses and a state of hypogonadotrophic hypogonadism (as part of hypopituitarism)

The pituitary gland

• FSH & LH are both glycoprotein hormones
  • Share a common alpha unit with other glycoprotein hormones (TSH & hCG)
  • Contain unique beta subunit
  • All act on transmembrane G-protein receptors à increase in cAMP
  • There is cross-responsiveness of these receptors to the various glycoprotein hormones
  • Gonadotrophins – from pituitary gland mainly in response to GnRH pulses from puberty
• The GnRH receptors are membrane receptors which are down-regulated by sustained exposure of gonadotrophs to GnRH
•  FSH has T 1/2 of 4 hours & is under the influence of both GnRH & gonadal hormones; therefore its pulses are not accurately reflective of GnRH pulses
• LH pulses, with a short half-life, are a more accurate reflection of GnRH pulses
• LH pulses
  • Follicular phase – higher frequency (every 60–90 min) & lower amplitude
  • Luteal phase – lower frequency (120–160 min) & higher amplitude

The ovary

Embryologically ovarian tissue is formed from three main types of cells:

1. Primordial germ cells derived from endodermal cells of the yolk sac à migrate at 6 weeks of intrauterine life to the genital ridge. Primordial germ cells grow & begin to differentiate into oogonia by 6 – 8 week of development
2. Coelomic cells (sex cord) which develop into granulosa cells that surround the oogonia to form the primordial follicles
3. Mesenchymal (sex stromal) cells which later form theca cells & other ovarian stromal cells

• Until 6-8 (10) weeks of gestation the germ cells and surrounding sex cord and sex stromal cells are not differentiated, and the gonadal ridges are bi-potential gonads
• These gonads develop into ovaries by lack of paracrine factors transcripted by the genes in the sex determining region on the Y chromosome (SRY).

THE OVARIAN CYCLE

Gonadotrophin-independent phase

• Once PGCs have arrived in the gonad of a genetic female, they differentiate into oogonia
• These cells undergo a number of mitotic divisions, and by the end of the third month, they are arranged in clusters surrounded by a layer of flat epithelial cells
• The majority of oogonia continue to divide by mitosis
• Some of them enter into meiosis & arrest in prophase I by 12 weeks & form primary oocytes
• During the next few months, oogonia increase rapidly in number, and by the fifth month, total number reaches its maximum – 7 million
• At this time, cell death begins, and many oogonia as well as 1ry oocytes degenerate & become atretic.
• By the seventh month, all surviving primary oocytes (prophase I) individually surrounded by a layer of flat follicular epithelial cells à A primary oocyte, together with its surrounding flat epithelial cells, is known as a primordial follicles (started to appears around 6 weeks intrauterine life, and complete by about 6 months after birth)
• Near the time of birth, all primary oocytes instead of proceeding into metaphase, they enter the diplotene stage – a resting stage during prophase
• Primary oocytes do not finish their first meiotic division before puberty is reached
• This arrested State is produced by oocyte maturation inhibitor (OMI), a small peptide secreted by follicular cells
• The total number of primary oocytes at birth is estimated to vary from 7 lakhs.
• During childhood, most oocytes become atretic; only approximately 40,000 are present by the beginning of puberty, and fewer than 500 will be ovulated.

• The first step in follicular growth is that a primordial follicle becomes a primary follicle
• 1ry follicle – oocyte enlarges and spindle cells become cuboidal cells à1ry follicle contains a larger primary oocyte that is surrounded by a single layer of cuboidal granulosa cells
• 11ry follicle – primary oocyte surrounded by several layers of cuboidal granulosa cells. In addition, stromal cells differentiate, surround the follicle and become the theca cells. These theca cells are on the outside of the follicle’s basement membrane.
• As the developing follicle increases in size, the number of granulosa cells increases to about 600, and the theca cells show increasing differentiation. The progression to secondary follicles also entails the formation of capillaries and an increase in the vascular supply to developing follicular units.
• The increasingly abundant granulosa cells secrete fluid into the centre of the follicle creating a fluid-filled space called the antrum. At this stage, the follicle is now called a tertiary follicle.

• In tertiary follicles, gap junctions are formed between theca and granulosa cells.
• Gap junctions may also exist between the oocyte and the granulosa cells closest to the oocyte and may function as channels to transport nutrients and paracrine signals from the granulosa cells to the oocyte and vice versa
• In addition, tight junctions and desmosomes exist between adjacent cells
• The granulosa cells closest to the oocyte also secrete a layer of mucopolysaccharides (the zona pellucida).
• These stages occur independent of gonadotrophin stimulation and under the effect of local autocrine and paracrine factors such as growth differentiation factor (GDF) and anti-Müllerian hormone (AMH)
• AMH produced from the granulosa cells and reaches the systemic circulation in levels proportional to the secondary follicle pool. In the absence of further gonadotrophin stimulation the secondary follicles undergo apoptosis and atresia.
• This process of gonadotrophin-independent recruitment to secondary follicles and apoptosis in absence of gonadotrophins is continuous during intrauterine, pre-pubertal and reproductive life till depletion of the follicular pool at the age of menopause.
• Unlike spermatogenesis, the atretic follicles cannot be replenished; therefore the ovarian reserve of follicles is a finite pool.
• During childhood, most oocytes become atretic; only approximately 40,000 are present by the beginning of puberty, and fewer than 500 will be ovulated.
• The rate of loss of the primordial follicle pool is variable among individual females
• It is believed that natural fertility is lost around 10 years earlier than the age of menopause (fixed-interval hypothesis).
• The duration of the gonadotrophin-independent phase is around 74–80 days

Gonadotrophin-dependent phase

• In the absence of pituitary gonadotrophins, the growing follicles in the ovary will be destined to atresia (apoptosis). The rise of pituitary gonadotrophins during the reproductive years, as a result of release of hypothalamic GnRH pulse centre from pre-pubertal inhibitory signals, leads to rescue of the preantral/antral follicles.
• The number of rescued and recruited follicles depends on the pool of secondary follicles available at the time of the rise of FSH, which is indirectly related to the total pool of primordial follicles in the ovary (ovarian reserve). It also depends on the level of FSH and duration of rise (selection window).
• A critical step of this rescue process is the induction of aromatase enzyme activity in the granulosa cells which converts the androgens synthesised in the theca cells into oestrogen under the effect of LH.
• Therefore the two gonadotrophins, FSH and LH, act referentially on the two main steroid-producing cells (granulosa and theca cells) in the ovary.
• The main effect of FSH is the rescue of granulosa cells (and oocytes) from atresia and induction of aromatase activity.
• The main role of LH is the stimulation of steroidogenesis by acting on theca cells to synthesise androgenic substrates that are converted into oestrogens in the granulosa cells.
• This is called the two cells, two gonadotrophins theory.
• The effect of this synchronised action of FSH and LH is conversion of the microenvironment of the secondary pre-antral follicles from one dominated by androgens into one dominated by oestrogens
• The effect of the latter is further proliferation of the granulosa and theca cells with accelerated production of oestrogen and peptide hormones (inhibins A B, activin, follistatin) and formation of a cavity (antrum) in which the oocyte is surrounded by a few layers of granulosa cells projecting into the cavity
• The follicular cavity fluid contains a myriad of growth factors and signalling molecules involved in bidirectional communication between the oocyte and surrounding granulosa.

Inhibins/activin/follistatin and insulin-like growth factors

• The granulosa cells of the growing follicles secrete a group of proteins which are important for regulating the HPO axis.
• In the luteo-follicular transition and early follicular phase, inhibin B is secreted by the granulosa cells, recruited from the secondary follicles pool, to enter the gonadotrophin-dependent phase
• Each month, 15 to 20 follicles selected from this pool begin to mature.
• In the early follicular phase FSH induces granulosa cells to secrete activin, inhibin A and follistatin.
  • Activin
    • Augments FSH action by increasing its receptors
    • Increase granulosa cell proliferation
    • Increase aromatase enzyme production
    • Inhibits the theca cells, androgen production
  • Inhibin A
    • Exerts a negative feedback effect on the pituitary synthesis and secretion of FSH
    • Stimulates theca cell LH receptors and androgen production
  • Follistatin
    • Combines activin and inhibits its action
    • Directly suppresses FSH synthesis and secretion by the pituitary.
• As the follicular phase progresses inhibin A activity predominates over activin
• This facilitates the selection of the dominant follicle which is able to maintain steroidogenesis in the face of declining FSH levels
• Concurrent with these effects the insulin-like growth factor-2 (IGF-2) and its binding protein (IGF2 BP) modulate intra-ovarian activity of FSH and LH
• In androgen-dominant follicles there is a higher concentration of IGF2 BP and lower concentration of bioavailable IGF-2. This leads to decreased FSH action on the granulosa and less steroidogenic activity with less oestradiol production and follicular atresia.
• This could be one of the mechanisms for arrest of follicles and anovulation in polycystic ovary syndrome in the presence of hyperinsulinaemia and hyper-androgenaemia

The dominant follicle

• In the mid-follicular phase (usually on day 7–8 of a 28-day cycle) selection of a dominant follicle occurs by the effect of rising oestrogen and inhibin A levels produced by actively growing follicles
• This results in a negative feedback effect on the pituitary gonadotrophin (FSH/LH) secretion, and starvation of most of the follicles of the necessary FSH to support granulosa cell proliferation and aromatase activity
• The dominant follicle expands in size with an exponential rise of oestradiol levels. This further accentuates the decline in FSH levels and leads to atresia of the rest of the follicles
• In natural cycles selection of a single dominant follicle & monofollicular ovulation is the rule
• In ovarian stimulation cycles the prolonged exogenous FSH stimulation leads to support and survival of more than one follicle
• The dominant follicle accumulates more fluid in the follicular cavity and its granulosa cells become organised into three compartments:
  • Mural granulosa cells surrounding the antrum
  • Cumulus oophorus (which is a stalk of granulosa cells connecting the oocyte to the mural granulosa)
  • Corona radiata (which is a layer of granulosa cells in direct contact with the oocyte)

• The oocyte with its surrounding corona radiata and cumulus oophorus are bathed within follicular cavity fluid
• The latter separates it from the mural granulosa and outer theca cells. The follicle is now is known as the pre-ovulatory or Graafian follicle.
• The Graafian follicle continues to produce oestrogen, independent of FSH stimulation, and has the highest number of granulosa cells and oestradiol levels with the lowest androgen-to-oestrogen ratio
• This follicle also develops LH receptors in the granulosa cells, which helps with maturation of the oocyte and prepares the follicle for the ovulatory stimulus of the LH surge. The LH receptors also ensure adequate progesterone production by the Luteinised granulosa cells from the corpus luteum after ovulation.

Ovulation

• When estradiol (E2) levels peak to 300–400 pg/ml (coincides with a follicle size of 18–20 mm), the pituitary gland responds by a surge of LH levels to about 15-30 IU/L
• This leads to a cascade of changesin the Graafian follicle and leads to ovulation within 36 hours (34–39 hours) of the onset of the LH surge
• The LH surge initiates the following changes in the Graafian follicle and ovary:
  • Resumption of meiosis in the oocyte with extrusion of the first polar body ( oocyte becomes haploid), and the oocyte becomes arrested into the metaphase-11 which is completed at fertilisation with extrusion of second polar body
  • Induction of angiogenesis and increased vascularity and capillary permeability in the theca cell layers with increased production of follicular fluid and increase in intra-follicular pressure
  • Synthesis and secretion of various prostaglandins that help increase blood flow in the follicular wall and stimulate smooth muscle cells within the ovarian stroma that help expel the oocyte
  • Activation of matrix metalloproteinases and other proteolytic enzymes that digest the follicular wall and ovarian capsule at the site of the follicle to facilitate follicular rupture and oocyte release the i.e. ovulation
  • Stimulates progesterone synthesis by the granulosa and theca cells shortly before ovulation. This further accentuates the LH surge and ensures adequate luteinisation of the theca and granulosa cells and adequate corpus luteum function later
• The resulting effect of these changes is follicular wall rupture with release of follicular fluid and the oocyte and its surrounding cumulus cells
• Usually picked up by the fimbrial end of the tube, and is transported to the ampullary part of the fallopian tube where fertilisation may occur
• In a regular 28-day cycle the gonadotrophin-dependent phase (follicular phase of the ovarian cycle) lasts about 14 days
• However, this is variable amongst individuals and leads to variable lengths of the follicular phase and subsequent variable menstrual cycle lengths as the corpus luteum lifespan is nearly fixed at about 14 days
• The timing of ovulation is therefore difficult to predict prospectively; however a fertile period when ovulation is likely to occur can be predicted using the woman’s menstrual history and cycle lengths.

The luteal phase

• After the release of the oocyte-cumulus complex the follicular antrum is filled with blood and new blood vessels forms
• The theca-lutein cells become full of cholesterol (luteinised) and the resulting structure is called a corpus luteum
• Corpus luteum produces oestrogen, progesterone (P4) & inhibin A in response to LH pulses
• These in turn suppress FSH and LH secretion by the pituitary
• In the face of declining FSH and LH levels the corpus luteum functions for only about 10 days, with peak activity at about 7 days after ovulation (mid-luteal peak of progesterone on day 21 of a 28-day cycle)
• It then enters into apoptosis & regression phase of about 4 days if pregnancy doesn’t occur
• In the absence of pregnancy, the corpus luteum has a fairly predictable life span of 14 days
• The falling oestradiol and progesterone levels lead to apoptosis and shedding of the endometrium
• The falling ovarian steroid levels release the hypothalamus and pituitary from the negative feedback effect, with a subsequent increase in FSH levels and ensuring a new cycle of recruitment of secondary follicles
• The luteo-follicular transition phase is characterised by increasing FSH levels, low oestradiol and progesterone levels and high inhibin B secreted by the granulosa cells of recruited follicles
• If pregnancy occurs the hCG produced by the trophoblast of the implanting embryo rescues the corpus luteum from apoptosis and atresia, enabling the corpus luteum to function and produce progesterone till 10–12 weeks’ gestation when the placenta takes over this function.

THE MENSTRUAL CYCLE

• The endometrium undergoes cyclic changes which mirror the effect of the hormones produced by the growing follicles and corpus luteum in the ovary
• In the early follicular phase the endometrium is rebuilt from the basalis layer after its superficial layer has been shed in the menses of the previous cycle
• Oestradiol secreted from the ovary leads to active mitosis &  proliferation of the endometrial glands and stroma
• This leads to an increase in the thickness of the endometrium from 2–3 mm to about 6–8 mm by the end of this proliferative phase
• Following ovulation progesterone hormone leads to more functional maturation of the endometrial glands and decidualisation of the endometrial stroma
• Histologically
  • Endometrial glands
    • enlarged tortuous endometrial glands that are full of secretions; hence this phase is called the secretory phase
    • The effect of the epithelial changes is secretion of adhesion molecules, such as integrins and glycodelins, which mediate the attachment of the blastocyst (in the case of successful fertilisation) that initiates the implantation process.
  • Endometrial stroma
    • Appears oedematous with an increased number and coiling of the spiral arteries and pericapillary leukocytic and cellular infiltrates, a process called decidualisation
    • The effect of stromal decidualisation is recruitment of immunological cells (natural killer cells, dendritic cells) that help regulate the trophoblastic invasion and effect changes in the spiral arteries that lead to the development of the early placenta
• The peak of secretory changes in the endometrium is 7–9 days after ovulation when the endometrium is most receptive to implantation of the blastocyst, the so called implantation window.
• If fertilisation occurs and a blastocyst successfully implants in the endometrium the trophoblast of the implanting embryo secretes hCG which rescues the corpus luteum
• This prolongs the lifespan of the corpus luteum and maintains progesterone secretion till about 10 weeks’ gestation when the developing placenta takes over the hormonal production (luteo-placental shift)
• As a result of maintenance of oestrogen and progesterone production and stability of the decidua, pregnant women will experience the physiological amenorrhoea of pregnancy
• If there was no fertilisation or a blastocyst could not successfully implant then the declining corpus luteum function (starting around day 10–12 after ovulation and almost complete by day 14) is associated with falling  estrogen and progesterone levels
• This in turn initiates apoptosis in the endometrium with release of prostaglandins and lysosomal enzymes from the cells, setting waves of vasoconstriction leading to ischaemia
• There is further breakdown of blood vessels and cells followed by vasodilatation, with escape of blood cells (erythrocytes, leukocytes, platelets) as well as various proteolytic enzymes (metalloproteinases, enkephalinases, fibrinolysins) into the endometrial stroma
• The effect of these waves of vasoconstriction and vasodilatation with apoptosis and proteolysis culminates into endometrial sloughing at the junction of superficial and basal layers forming the menstrual bleeding
• In concert with these changes the released prostaglandins from the endometrium, as well as the myometrial cells under the effect of falling progesterone levels, lead to myometrial contractions. This leads to expulsion of the menstrual blood from the uterus.

A normal menstrual phase

• Expected to last 2–6 days and the amount of bleeding is generally 20–80 ml
• The rising oestradiol level caused by the recruited follicles leads to rapid repair and rebuilding of the endometrium and the end of the menstrual phase
• Menstrual bleeding in ovulatory cycles (oestrogen/ progesterone withdrawal bleeding) is characterised by being synchronised and less prolonged and associated with menstrual cramps due to higher levels of prostaglandins
• Menstrual bleeding in anovulatory dysfunctional menses are usually prolonged, erratic, heavy and painless
• Cases of dysfunctional menorrhagia  (prolonged and/or heavy menses due to mainly anovulation and/or dysfunctional molecular changes in the endometrium in the absence of significant pathological changes) may benefit from progestins only or sequential oestrogens/progestin treatment to mimic the oestrogen/ progesterone withdrawal bleeding in normal cycles
• The prostaglandin synthetase inhibitors (such as non-steroidal anti-inflammatory agent mefenamic acid) are also used as a treatment of dysfunctional menorrhagia

NORMAL UTERINE BLEEDING

• It is necessary to define normal menses before being able to accurately define AUB
• Menstruation should be described in terms of:
  • Frequency
    • Measured by the number of days from the first day of one menstrual period to the first day of the next
    • Women often mistakenly describe the gap between bleedsà could lead to a false impression of increased frequency of menses.
  • Regularity
    • Quantifies the cycle to cycle variation in frequency over a 12 month time frame
    • The large degree of variation within the normal population should be noted and women reassured accordingly
  • Duration
    • Self-explanatory and usually quantified easily in the clinical setting
  • Volume or flow
    • Average blood loss is 40 ml, with 90% of women having a blood loss <80 ml
    • Therefore, HMB is objectively defined as a loss of > 80 ml per cycle
    • These objective measurements less important in the clinical setting, where a woman’s perception of heaviness and its impact on her social/material/ economic quality of life are of much greater importance.

SUGGESTED NORMAL LIMITS FOR UTERINE BLEEDING IN MID-REPRODUCTIVE YEARS

OUT OF DATE TERMINOLOGY

• Dysfunctional uterine bleeding/Functional uterine bleeding
• Menorrhagia (including idiopathic menorrhagia, essential menorrhagia, ovulatory menorrhagia, anovulatory menorrhagia, polymenorrhagia, epimenorrhagia)
• Menorrhoea (including epimenorrhea, hypermenorrhea, hypomenorrhoea, polymenorrhea)
• Menometrorrhagia
• Metorrhagia
• Metropathia hemorrhagica
• Oligomenorrhea
• Uterine haemorrhage

NEW NOMENCLATURE AND CLASSIFICATION

AUB

Acute AUB

An episode of bleeding in a woman of reproductive age, who is not pregnant, that is of sufficient quantity to require immediate intervention to prevent further blood loss.

Chronic AUB

Bleeding from the uterus that is abnormal in frequency, duration and/or volume and has been present for the majority of the previous six months

Inter-menstrual bleeding

Bleeding between clearly defined cyclic and predictable menses and includes random episodes as well as predictable episodes occurring at the same time each month.

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