Pratima Mittal1 and Navdeep Kaur Ghuman2
(1)
Department of Obstetrics and Gynaecology, Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi, Delhi, 110016, India
(2)
Department of Reproductive Medicine, Newcastle Fertility Centre at Life, Newcastle upon Tyne Hospitals NHS Trust, Newcastle Upon Tyne, United Kingdom
Pratima Mittal
Email: drpratima@hotmail.com
Abstract
Ovulation comprises of two interlinked processes, folliculogenesis and oogenesis. Folliculogenesis starts soon after the formation of follicles in intra-uterine life. It is a continuous process in which a follicle passes through several stages and ultimately ruptures to release an ovum. Oogenesis is a process by which primary oocyte arrested in diplotene stage of first prophase in embryonic life attains meiotic maturation and undergoes cytoplasmic changes to be finally released as mature ovum during reproductive life, although not all primary oocytes reach the stage of mature ovum and majority of them undergo atresia along this journey.
Anovulation or oligo-ovulation comprises around 21 % of female infertility. Any factor or process that disrupts finely tuned interactions of hypothalamo-pituitary-ovarian axis can potentially lead to anovulation. WHO classifies ovulation disorders in three groups: hypothalamamic pituitary failure, hypothalamic pituitary dysregulation and ovarian failure.
Detection or confirmation of ovulation, although an integral part of infertility workup, can be quite frustrating for clinicians and patients both. Most methods or tests for detection of ovulation are retrospective and demand monitoring over a long duration. Regularity of cycles is a reasonably assuring proof of ovulation, and detection tests are advisable in women with menstrual irregularities. Combining two or more methods for ovulation detection improves efficacy, accuracy and economics involved.
Keywords
OvulationFolliculogenesisOogenesisAnovulationAetiology of anovulationDetection of ovulation
1.1 Introduction
Ovulation is the prime and most important event of menstrual cycle. By definition, it is the occurrence in the menstrual cycle by which a selected mature follicle breaks and releases a viable oocyte from the ovary. Unlike spermatogenesis, which is a continuous process throughout the life of a male, females are born with all the eggs they will ever produce. In humans, by the seventh week of intra-uterine life, primordial germ cells reach the gonadal ridge from yolk sac endoderm. Here, they divide by mitosis to reach the peak level of 6–7 million at 20 weeks. Later on, the germ cells enter the first stage of meiosis and transform into primary oocytes. From mid-gestation onwards, each primary oocyte gets surrounded by a single layer of pre-granulosa cells to form primordial follicle. Within the primordial follicles, primary oocytes remain arrested in the diplotene stage of prophase 1 of meiosis 1, and the number falls dramatically from 6 to 7 million at 20 weeks of gestation to 1–2 million at birth to few lakhs at puberty. Most of the follicles at some point of their journey from primordial follicle to pre-ovulatory follicle undergo atresia thus ultimately leading to exhaustion of pool of oocytes and ovarian senescence.
1.2 Ovulation
The process of ovulation includes two separate but closely interlinked sub-processes – follicullogenesis and oogenesis.
1.2.1 Folliculogenesis
At all times, majority of primordial follicles are in a dormant resting phase. Intra-ovarian autocrine/paracrine factors pump some of these primordial follicles into the growing follicle pool. This initial recruitment of primordial follicles is gonadotrophin independent and occurs in a continuous manner starting soon after the formation of follicles. The follicles in the growing pool undergo a long process of follicullo-genesis and carry on their journey through stages of primordial, primary, secondary (class 1), tertiary (class 2), graffian, early antral (class 3, 4, 5), antral (class 6, 7) and pre-ovulatory (class 8) follicle. These stages can be broadly divided into pre-antral [primordial, primary, secondary and tertiary] and antral [graffian, early antral, antral and pre-ovulatory] type follicles. During this journey, a follicle grows in size (mean diameter of primordial follicle is 25 μm and preovulatory follicles can grow up to 20–30 mm before ovulation), shows mitosis and stratification of granulosa cells, formation of theca cell layer, zona pellucida and development of cavity or antrum along with chromosomal and cytoplasmic changes occurring in oocyte. The pre-antral phase of growth proceeds at a slow rate because of the long doubling time (about 10 days) for the granulosa cells, and it takes 300 days for a follicle to complete it. The growth rate picks up in the antral phase, and in another 50 days an antral follicle reaches the pre-ovulatory stage (Fig. 1.1) [1]. This phase of growth and development (antral phase) is under the influence of gonadotrophins. Atresia can affect follicles at all stages beyond secondary (stage 1) follicle stage but has highest incidence in antral follicles more than 2 mm in diameter [2].
Fig. 1.1
Chronology of folliculogenesis in human ovaries. Preantral period: It takes 300 days for a recruited primordial to grow and develop to the class 2/3 (0.4 mm) or cavitation (early antrum) stage. Antral period: A class 4 (1–2 mm) follicle, if selected, requires about 50 days to grow and develop to the preovulatory stage. The dominant follicle of the cycle appears to be selected from a cohort of class five follicles, and it requires about 20 days to develop to the ovulatory stage. gc number of granulosa cells, d days (From Gougeon et al. [1]. Image courtesy of Alain Gougeon)
1.2.2 Oogenesis
Process of oogenesis starts with the migration of germ cells from yolk sac to gonadal ridge during intra-uterine life. By birth, all germ cells have initiated their first meiotic division (now called primary oocyte) and remain arrested in prophase stage of meiosis 1 till puberty. After puberty, each month few primary oocytes under the effect of pre-ovulatory surge of FSH and LH resume and complete their first meiotic division and result in formation of secondary oocyte and a polar body. The dominant secondary oocyte enters second meiotic division, gets arrested at second meiotic metaphase and subsequently ovulates. Fertilization triggers the resumption and completion of meiosis resulting in the formation of second polar body.
1.2.3 Physiology of Ovulation
In the luteal phase, corpus luteum is the site of estradiol and progesterone production. Corpus luteum possesses considerable capacity of self-regulation and maintains its function active for 14 days. With the demise of corpus luteum towards late luteal phase, the decreasing estradiol levels trigger rise in plasma FSH levels. This rise in FSH level recruits a cohort of class 5 follicles towards the end of luteal phase and facilitates its growth. One follicle in the recruited cohort of follicles is able to concentrate high levels of FSH in its follicular fluid and show rapid mitosis of granulosa cells to become the dominant follicle. This dominant follicle has most FSH receptors, is most sensitive to FSH and produces maximum oestrogen by FSH-mediated activation of aromatase enzyme. High concentrations of FSH in the micro-environment of dominant follicle, through gap channels between granulosa cells and oocyte, keep the concentration of cAMP and oocyte maturation inhibitor (OMI) high, which in turn keep the oocyte in immature stage. The rising oestrogen level in turn by negative feedback mechanism lowers the plasma FSH level towards the end of the first week in follicular phase of menstrual cycle. This lowering FSH concentration is unable to sustain growth of rest of the follicles of the recruited cohort, which subsequently undergo atresia. The dominant follicle on the other hand by this time becomes less responsive to declining FSH levels and continues to grow. Moreover, the FSH-mediated induction of LH receptors on dominant follicle enables LH to take part in the growth and development of dominant follicle during later follicular phase and also in preparation of dominant follicle for upcoming LH surge. When the rising oestrogen level crosses a critical level, its negative feedback at hypothalamic-pituitary axis turns into a positive feedback giving rise to LH surge. LH surge lasts for 36–48 h. LH surge by dismantling the gap junctions between granulosa cells and oocyte inhibits the flow of maturation-inhibitory factors into ooplasm and causes drop in concentration of cAMP. Decreased concentration of cAMP in turn increases concentration of Ca and maturation-promoting factor (MPF), which are essential for the resumption of meiosis in oocyte and disruption of oocyte-cumulus complex triggering follicular rupture and ovulation about 36 h the LH surge. What enables one follicle of the cohort to concentrate FSH in its micro-environment in preference to others is still not clearly understood, but this selection leads to a single ovum being released by ovaries in each menstrual cycle [2, 3].
1.2.4 Recent Research
Recent research work has indicated the possibility of presence of renewable oogonia in the lining of female ovaries of humans, primates and mice. These studies have discovered that some mitotically active germ cells may migrate to ovaries from bone marrow and act as extra genial source of stem cells. Researchers have discovered these renewable germ cells as these were identified positive for several essential oocyte markers. If further studies support these findings, it could revolutionise treatment of infertility [4–6].
1.3 Aetiology of Anovulation
Ovulation is the result of complex and finely tuned interactions between hypothalamus, pituitary and ovary (Fig. 1.2). Any aetiology leading to the disruption of this fine tuning can cause anovulation. These can be broadly categorized as
Fig. 1.2
Hormonal regulation of ovulation. Solid arrows: positive feedback. Dotted arrows: negative feedback
1.3.1 Hypothalamic Factors
Hypothalamic hormones particularly gonadotrophin-releasing hormone (GnRH) are an important factor responsible for functional hypothalamo-pituitary-ovarian axis. GnRH hormone is a decapeptide which is synthesised and released by specialised neuronal endings of nucleus arcuate of hypothalamus. Any factor hindering pulsatile release of GnRH hormone leads to anovulation.
1.3.1.1 Functional Hypothalamic Dysfunction
Excessive strenuous exercise, stress, anxiety, under-nutition, eating disorders like anorexia nervosa by inhibiting normal GnRH pulsatility due to excessive release of corticotrophin-releasing hormone and stimulation of beta-endorphins can lead to amenorrhoea and anovulation. Drug abuse (cocaine, marijuana) and psychiatric disorders (schizophrenia) can also cause anovulation by suppression of GnRH.
1.3.1.2 Structural Hypothalamic Dysfunction
Infiltrative disorders of the hypothalamus (e.g. Langerhans cell granulomatosis, lymphoma, sarcoidosis, TB), tumours of hypothalamus, irradiation to the hypothalamus, chemo-toxic agents and traumatic brain injury by destruction of arcuate nucleus or distortion of hypothalamic-pituitary axis can lead to anovulation.
1.3.1.3 Genetic Disorders
Less commonly, genetic disorders like Kallmaan syndrome (defective migration of GnRH neurons), Prader-Willi syndrome and GnRH receptor gene mutation can be a cause of anovulation and infertility [7].
1.3.2 Pituitary Factors
GnRH from hypothalamus via portal circulation is transported to anterior pituitary where it leads to the release of gonadotrophins (LH and FSH). The amplitude and frequency of GnRH pulse determines the release of FSH or LH.
1.3.2.1 Structural Pituitary Dysfunction
Infiltrative conditions of pituitary (TB, sarcoidosis, hemochromatosis), space-occupying lesions of pituitary (microadenomas, macroadenomas, aneurysms), tumours of brain (meningioma, gliomas, craniopharngiomas), trauma to brain, irradiation to brain or postpartum pituitary necrosis by causing destruction of pituitary leads to anovulation.
1.3.2.2 Genetic Disorders
Idiopathic hypogonadotrophic gonadism, isolated gonadotrophin deficiency and gene mutation of beta subunit of FSH and LH are genetic disorders which can result in anovulation and infertility.
1.3.3 Ovarian Factors
The site of the final step in the process of ovulation is ovaries.
1.3.3.1 Iatrogenic Causes
Irradiation to pelvis, chemotherapy and surgical removal of ovaries are some of the iatrogenic factors that can lead to anovulation and infertility.
1.3.3.2 Genetic Factors
Chromosomal abnormalities like Turner syndrome, fragile X syndrome, idiopathic accelerated ovarian follicular atresia and gonadal dysgenesis are genetic causes of absent ovulation.
1.3.3.3 Ovarian Failure
Premature ovarian failure and resistant ovarian syndrome are other causes of anovulation [8].
1.3.4 Endocrine Causes
1.3.4.1 Polycystic Ovarian Syndrome
Polycystic ovarian syndrome is a heterogeneous group of disorders with a prevalence of 5–10 % in reproductive age-group [9]. Hyper-androgenism, oligo-ovulation or anovulation, oligo- or amenorrhoea, insulin resistance and obesity are the common clinical presentation of this syndrome complex. Abnormal endocrine environment with unopposed oestrogen and excess of LH leads to suppression of FSH release and hits the process of ovulation at the stage of follicular recruitment [10].
1.3.4.2 Hyperprolactinemia
Hyperprolactinemia of any cause can lead to anovulation by affecting the hypothalamo-pituitary axis at multiple sites. The important ones are impaired pulsatility of GnRH release and interference with the positive feedback effect of oestrogen on LH surge [7].
1.3.4.3 Hyper-androgenism
Other causes of hyper-androgenism like congenital adrenal hyperplasia, Cushing syndrome, androgen-secreting tumours and drug-induced virilization can lead to anovulatory infertility.
1.3.4.4 Thyroid Dysfunction
Severe untreated thyroid dysfunction, both hyper- or hypothyroidism, can cause menstrual irregularities and anovulatory infertility. The anovulatory effect of severe hypothyroidism is partly mediated by hyper-prolactinemia because of the fact that elevated TSH acts as a release factor for prolactin.
1.3.5 Systemic Causes
1.3.5.1 Renal Disease
Chronic and end-stage renal disease causes hypothalamic anovulation and menstrual acyclicity probably due to absence of positive feedback effect of oestrogen on hypothalamus and thus absence of LH surge. Women with uremia usually show high LH and high prolactin level [11].
1.3.5.2 Liver Disorders
Anovulatory infertility is common in women with end-stage liver disease. These women usually show decreased levels of gonadotrophins and oestrogen. These patients usually do not respond to GnRH stimulation or clomiphene, but successful liver transplant can result in restoration of ovulation.
Testicular feminising syndrome and other intersex conditions are unrelated conditions which can present with anovulatory infertility.
Anovulatory infertility accounts for 21 % of female infertility [12]. The World Health Organization classifies ovulation disorders into three groups (Table. 1.1) [13].
Table 1.1
World Health Organization Classification of ovulation disorders
|
Term |
Definition |
|
Group 1 Hypothalamic pituitary failure (hypogonadotrophic hypogonadism) |
This group accounts for approximately 10 % of ovulatory disorders. This type is characterised by low gonadotrophins, low oestrogen and normal prolactin |
|
Group 2 Hypothalamic pituitary dysfunction |
This group includes anovulatory disorders characterised by gonadotrophin disorder and normal oestrogen. It accounts for 85 % of ovulatory disorders. Polycystic ovarian syndrome and hyperprolactinaemic amenorrhoea constitute majority of cases falling in this group |
|
Group 3 Ovarian failure |
This group is characterised by high gonadotrophins and low oestrogen. Around 5 % of women with anovulatory infertility have group 3 ovulation disorders |
Adapted from Dhont [13]
1.4 Detection of Ovulation
Anovulation or ovulation disorders are the cause of infertility in 25 % of couples who have difficulty to conceive. In day-to-day clinical practice, it often becomes necessary to confirm ovulation either as a part of infertility workup or on a woman’s request, who is facing difficulty in conceiving. Medical literature describes several methods to test ovulation, but majority of these tests are based on subjective symptoms and thus are not reliable or are cumbersome. The list includes
1.4.1 Regularity of Menstrual Cycle
Menstrual charting involves the recording of onset of menstruation over successive cycles. Regular menstrual cycles ranging from 26 to 36 days are usually an indicator of ovulation [9]. Longer or shorter cycles warrant further investigations to detect ovulation. Although regularity of cycles is a reasonably reassuring indicator of ovulation for a clinician, this may not be sufficient to reassure a woman facing difficulties with conceiving.
1.4.2 Pre-menstrual Symptoms
Pre-menstrual symptoms like menstrual cramps, breast tenderness, fluid retention and mood swings are useful clinical indicators of normal hormonal cyclicity and thus indirect indicators of ovulation. Though the symptoms are subjective and thus not very reliable in terms of an indicator for ovulation, their importance in a fertility workup cannot be over-emphasised.
1.4.3 Basal Body Temperature (BBT)
Van de Velde in 1904 observed the biphasic pattern of basal body temperature during menstrual cycle. Progesterone production after ovulation causes increase in basal body temperature with a minimum increase of 0.5° Fahrenheit above the follicular-phase basal temperature. This increase in basal body temperature is a retrospective indicator of ovulation. Measurement of basal body temperature is best done by specially calibrated thermometer and done as a first thing in the morning before leaving bed. Guermandi and associates in their study concluded that BBT coincides with ultrasonographic detection of ovulation in 74 % of cases with a sensitivity of 0.77 and specificity of 0.33 taking ultrasonography as standard for ovulation detection [14]. Another study has however shown that BBT agreed with ultra-sonographic ovulation only in 30.4 % cases [15]. Although a relatively inexpensive and self-administered method, studies have shown that this method is less accurate, not sufficiently reliable for detection of ovulation [16, 17]. Moreover, BBT can be affected by many factors other than hormonal changes.
1.4.4 Cervical Mucus Changes
Cervical mucus changes can be used alone or in combination with basal body temperature as an indicator of ovulation. Near the time of ovulation under the effect of oestrogen, cervical mucus becomes copious, thin and stretchy. These changes in cervical mucus do not indicate ovulation per se and are rather an index of optimum circulatory oestrogen levels before ovulation and may be seen in anovulatory cycles also. Alliende and co-workers in their study found that when adequately instructed, women can perceive ovulation by recording changes in their self-aspirated upper vaginal fluid in 76 % of cycles within ±1 day of ultrasonographic ovulation detection [18]. Other studies have quoted 48.3 % correlation with ultrasonographic detection of ovulation [15]. Insler and associates (based on quantity, spinnbarkeit, ferning and appearance of external os) [19] and Moghissi (based on amount of mucus, spinnbarkeit, ferning, viscosity and cellularity of cervical mucus) [20] had devised different cervical scoring systems. A value of 10–12 on Insler score and a value of 13–15 on Moghissi score are taken as indicator of pre-ovulatory cervical mucus. Although not very reliable, still this method is an inexpensive indicator of ovulation and helps a woman to identify her fertile days. Presence of a vaginal and cervical infection nullifies the utility of this method as an ovulation indicator [14, 15, 21].
1.4.5 LH Surge Detection Kits
LH surge causes luteinisation of mature follicle and disruption of oocyte-cumulus complex triggering follicular rupture and ovulation. The LH surge can be measured in serum and blood and indicate imminent ovulation [22]. Urinary LH testing has the advantage of being simpler and being less affected by episodic fluctuations of LH levels than serum LH measurement. Studies have quoted sensitivity, specificity and accuracy of urinary LH test for detection of ovulation as 1.0, 0.25 and 0.97 respectively taking ultrasonographic detection of ovulation as standard [14, 15]. Several urinary LH surge detection kits are available in market which use test strips to detect changes in urinary LH levels. Urine testing is commenced 2–4 days prior to the expected ovulation and is continued till LH surge is detected. With irregular menstrual cycles, urine testing has to be timed according to the earliest and latest possible dates of ovulation. Success rate of detection of ovulation is usually quoted as 80 % with 5 days of testing and 90 % with 10 days of testing, with majority of these commercially available kits [23]. The main advantage of this test is that it can predict ovulation. On the other hand, the major disadvantage of this method is high false-negative rate which can be due to short LH surge or incorrect use of kit [24]. False positives can result in case of premature ovarian failure, peri-menopausal period and some cases of PCOS because of high basal LH level. LH surge in blood usually lasts for 36–48 h. Detection of LH surge in blood is impractical due to wide variation of normal LH levels during menstrual cycle and also is invasive and expensive. Six cohort studies evaluating the use of basal body temperature and urinary LH kits as indicators of ovulation to time intercourse did not find improvement in chance of natural conception [9].
1.4.6 Serum Progesterone Level
In practice, ovulation is confirmed retrospectively by measuring serum progesterone level in the mid-luteal phase, for example, day 21 of 28-day menstrual cycle, produced by luteinised ruptured follicle after ovulation. In women with longer cycles, the test needs to be performed later in the cycle and repeated weekly till the next menstrual bleed. Values ranging from 16 to 28 nmol/L (5–8.8 ng/ml) are taken as the lowest limit indicative of ovulation [9, 25–27]. Studies comparing the efficacy of different methods of ovulation have quoted 90 % concordance of ovulation detection by this method with ultrasonographic ovulation detection [14]. Major drawbacks of this method are lack of well-defined cutoff limits for serum ovulatory progesterone levels and the need for repeated testing especially with irregular menstrual cycles.
1.4.7 Transvaginal Ultrasonography
Follicle growth can be monitored through the menstrual cycle by using ultrasonography, ideally by transvaginal ultrasonography. Ovulation usually occurs when the follicle measures about 18–25 mm in size. Presence of free fluid in cul-de-sac, visualisation of collapsed and smaller follicle with internal echoes instead of previously visualised dominant follicle or visualisation of corpus luteum are ultrasonographic indicators of follicular rupture and ovulation. Ecochard and co-workers in their study compared different ultrasonographic indices for detection of ovulation and found the sensitivity and specificity to be 84 and 89.2 for disappearance or sudden decrease in follicle size, 61.6 and 87.1 for irregularies of follicular walls, 71 and 88.2 for free fluid in Pouch of Douglas and 38.4 and 79.7 for appearance of internal echoes in the follicle [28]. Ultrasonography is helpful in planning timed intercourse or insemination. This method is also helpful to precisely monitor follicular growth and detect multi-follicular development in women undergoing ovulation induction treatment along with providing additional information about endometrium and pelvic organs. Although serial ultrasounds through the menstrual cycles can detect ovulation, yet this method is not very accurate in predicting ovulation as follicles can grow up to varying sizes before rupture. This is an expensive method in terms of instrument cost, requirement of skilled personnel and multiple visits required by the patient. Combining ultrasonography with other methods, for example, menstrual dating and serum hormone levels, can reduce the number of visits required making it more economical.
1.4.8 Endometrial Biopsy
Histological examination of small amounts of endometrium in late luteal phase showing progesterone-induced changes in the endometrium can provide indirect and retrospective indication of ovulation. Because of its invasive nature and risk of dislodging an implanted potential gestational sac, this method is not recommended for this purpose [23].
Conclusion
In conclusion, majority of methods used for ovulation detection are retrospective. Moreover, majority of these methods need monitoring over a long duration and therefore can be frustrating for both patient and clinician. In clinical practice, regularity of menstrual cycles is a reasonably adequate proof of ovulation, and ovulation detection tests should be resorted to in women with irregularities of cycles. Measurement of mid-luteal progesterone level is the most commonly used method in practice to detect ovulation. Transvaginal ultrasonography is useful for precise follicular growth monitoring and detection of multi-follicular development in women receiving fertility drugs especially in combination with serum hormone level measurement. Timed intercourse has been suggested to be stressful by a plethora of medical literature. Therefore, regular sexual intercourse (every 2–3 days) instead of using ovulation prediction methods for timed intercourse should be recommended. However, for minority of couples who find it difficult to have regular intercourse or couples who use some form of artificial insemination for conception, prediction of ovulation by LH kits can be useful. Also, using two or more methods in combination can improve efficacy, accuracy and economics involved.
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