Ovulation is a very complex phenomenon and its clear understanding is essential to understand the abnormalities of the same. Ovulation will be discussed here under following heads:
- Physiology of ovulation
- Endocrinal control of ovulation
- Tests to detect ovulation
- Causes of anovulation.
PHYSIOLOGY OF OVULATION
Detailed understanding of physiology of ovulation is very essential to manage ovulation dysfunction, which is one of the important causes of fertility problems. Ovulation occurs from the ovaries. Each oocyte is surrounded by a number of cells to create a follicle. When the menstrual cycle begins one, or maybe even a few, primary oocytes begin to grow larger and the follicle cells increase in number and cause the follicle to grow larger too. Usually, some of the developing oocytes will degenerate and only leave one follicle that will mature, but every once in a while two, or even more, follicles will mature. As a follicle reaches maturity, the primary oocyte completes its first meiotic division and becomes a secondary oocyte. Very soon after that the follicle ruptures, and the secondary oocyte is released into the fallopian tube, even though the second meiotic division has not occurred yet. The release of a secondary oocyte from the ovaries is called ovulation.1
During intrauterine life of a woman, there are 7 million oogonia that reduce to only 1 million at the time of birth.2
This number is further reduced to only 300–400 thousands at the menarche. At the time of menopause, only 1,500 ova are available. During reproductive age only 400 ova reach up to ovulation, i.e. only 0.1% of the total available ova at menarche, reach up to ovulation and 99.9% become atretic.3
Ovulation occurs once in a month in majority of the normal women. It is the result of the integrated and synchronized succession of hormonal action and morphological changes in hypothalamus, pituitary and ovary. Autocrine and paracrine factors also take part in ovulation induction. So neuroendocrine complex system regulates ovulation and menstrual cycle.
Figure 1.1 explains the influence of different hormones on ovaries and endometrium. Grossly the gonadotropin-releasing hormones (GnRHs) released from the hypothalamus, control the gonadotropin [luteinizing hormone (LH) or follicle-stimulating hormone (FSH)] secretion from the pituitary and these gonadotropins finally control the estrogen and progesterone secretion from the ovaries. These steroids are responsible for the cyclical changes in ovaries and uterus. The entire control system is thus called hypothalamic-pituitary-ovarian axis. This hypothalamic-pituitary-ovarian axis should be intact and synchronized for normal ovulation (Fig. 1.2).
ROLE OF INDIVIDUAL HORMONE AND ITS CLINICAL APPLICATION
A decapeptide is synthesized and secreted from the neuronal endings in anterior and mediobasal part of the hypothalamus. It is released from hypothalamus and acts on the pituitary. GnRH traverses through hypothalamic-hypophyseal portal system. This is a compact blood vessel system and so it is not detected in circulation and it releases FSH and LH from the pituitary. Its secretion is pulsatile in nature and stimulates pituitary to secrete more LH than FSH. It is always correlated with LH pulse. Each pulse is of 60–90 minutes. It is chiefly influenced by ovarian steroids.
Fig. 1.1: Hormonal and morphological variations during the menstrual cycle—diagrammatic representation.4 (LH: luteinizing hormone; FSH: follicle-stimulating hormone).
Fig. 1.2: Diagrammatic representation of hypothalamic-pituitary-ovarian (HPO) axis.5 (GnRH: gonadotropin-releasing hormone; LH: luteinizing hormone; FSH: follicle-stimulating hormone).
Opiates, catecholamines and neuropeptides, all have an influence on GnRH secretion. There is enormous release of GnRH before the LH surge and then under the effect of progesterone, the frequency of the GnRH pulse decreases to only one in 8–12 hours.6 GnRH is suppressed by inhibitory factors till puberty.
Clinical Application for Ovulation Induction
- It is used for ovulation induction in hypogonadotropic hypogonadism. But unfortunately it is not yet widely available.
- GnRH agonist (GnRHa) can be used for downregulation in in vitro fertilization (IVF).
- GnRHa as short protocol is used for endogenous FSH and LH surge for ovulation induction.
- GnRHa is used for ovulation trigger to elicit endogenous LH surge.
- GnRH antagonist is used for prevention of LH surge in intrauterine insemination (IUI) and IVF cycles.
Follicle-stimulating hormone is secreted from anterior pituitary. FSH secretion is controlled by the feedback system though estradiol (E2) and progesterone. When corpus luteum undergoes atresia or regression just before the menstruation, the FSH secretion increases markedly. It acts on the preantral and antral follicles and helps in recruitment and stimulation of the graafian follicle to make it dominant. It also enhances the conversion of androgens to estrogen though the pathway of aromatase enzyme. Secretion of FSH is governed by estrogen. It frees the oocyte from follicular attachment and converts plasminogen into plasmin. It helps in development of LH receptors for ovulation and for the luteal phase action. It has a definite minimum value, below which it is not sensed by the follicles. This is called FSH threshold. This value is different for every individual. Second peak of FSH is in the midcycle. It has been observed in animals that this second peak of FSH is must for successful ovulation. If this peak is absent, it may cause luteinized unruptured follicle (LUF). But its role in human is not clear. This is so because in humans when surrogate trigger is used in form of LH or human chorionic gonadotropin (hCG), it acts successfully even in absence of FSH. Though the second FSH surge may select the follicles for the next cycle.
- It is responsible for LH expression after the follicle size of 10–12 mm. This means FSH rescues the follicle to maturity. So there is no need to increase the dose of FSH for follicular growth after 10–12 mm size. Instead supplementation of LH will grow the follicle.7
- Follicle-stimulating hormone secretion decreases during periovulatory period because of progesterone, but bioactivity is highest during this period.
- Granulosa cells have androgen receptors. Androgen acts on the immature cells and potentiates action of FSH, e.g. aromatase inhibitor that increases androgen and dehydroepiandrosterone (DHEA) also potentiates the action of FSH.
- Only 1% of the LH receptors are required for folliculogenesis and so in spite of downregulation with GnRHa in long protocol, the follicle continues to grow.
- Follicle-stimulating hormone can stimulate paracrine signal that sustains thecal androgen synthesis. So FSH is capable of ovarian androgen synthesis.7
Luteinizing hormone is another hormone secreted from anterior pituitary. In the first half of the cycle, its level is low and secretion is pulsatile. High levels of LH are detrimental to ova. This can be explained by the LH threshold and LH ceiling concept.
Luteinizing hormone ceiling and LH threshold:8 Figure 1.3 indicates that threshold level, i.e. certain minimum amount is required for growth of follicle. But if the level goes beyond a certain level, it hampers the growth of the follicle. This is known as ceiling effect and that level of LH is called the LH ceiling level. So LH level required for follicle maturation should be between the threshold and the ceiling level. This is known as LH window9 (Fig. 1.4).
Luteinizing hormone causes luteinization of granulosa cells. LH acts on theca cells to produce androgen. With the rising estrogen levels with developing follicle, when the FSH gets a negative feedback, LH gets a positive feedback. LH permits final maturation of follicle forming secondary oocyte and first polar body. It causes disruption of cumulus-oocyte complex and rupture of the follicle. LH surge should be between 14 hours and 27 hours after E2 peak for ovum maturation for rupture and LH surge lasts for 48–50 hours. The two-cell-two hormone theory explains the role of LH in ovarian steroidogenesis10,11 (Fig. 1.5).
Both the gonadotropins—FSH and LH as well as both the cells, i.e. granulosa cells and theca cells are required for maturation of ova. LH stimulates theca cells to produce androgen from cholesterol.
Androgen from theca cells go to granulosa cells where under the influence of FSH, androgen is converted into estrogen by aromatase activity. So both gonadotropins and both the cells are required for folliculogenesis.
- Luteinizing hormone in clomiphene citrate cycle: Clomiphene citrate stimulates FSH and LH from the hypothalamus. This LH may reach the ceiling level especially in polycystic ovarian syndrome (PCOS) and is detrimental to ova. The level of LH is therefore checked on day 8–10 of the cycle and if it is more than 10–12 IU/mL, it is less likely that clomiphene can give pregnancy as the follicle quality deteriorates due to high LH.12
- Luteinizing hormone in PCOS: There is tonically high level of LH that causes abnormal oocyte maturation. There is also premature luteinization that decreases implantation because of progesterone secretion. So chronic low-dose protocol and antagonist protocol in PCOS gives very good pregnancy rate. High level of LH causing ceiling effect is clearly seen in PCOS.
- Luteinizing hormone in downregulated cycle: In a downregulated cycle, there is low level of LH. When it reaches below the threshold level, the growth of the follicle is hampered. Low level of LH causes poor luteal conversion and so luteal phase defect (LPD) is very common. To compensate this loss, hCG works very well as luteal support but ovarian hyperstimulation syndrome (OHSS) should be kept in mind in PCOS patients.
- Luteinizing hormone and ovarian drilling: By drilling, we are destroying stroma, i.e. theca cells. So androgen production is decreased and this will have negative feedback to hypothalamus and pituitary to decrease LH secretion. This decrease in LH will revert the LH/FSH ratio and spontaneous ovulation is established.
- Luteinizing hormone in hypogonadotropic hypogonadism: LH is must for follicular development as it is discussed in two-cell-two gonadotropin theory. Without LH follicular growth is present but estrogen level is low.
- Luteinizing hormone in poor responder: In poor responder rather than increasing dose of FSH, LH or hCG can be added for rescue of the follicle after 10 mm size. Here LH receptors have developed and it can rescue the follicular growth. But dose of more than 150 IU can have a LH ceiling effect.
It is secreted by granulosa cells of the follicles under the influence of FSH as explained in the two-cell-two hormone theory. It is also produced from androgen by aromatization as discussed earlier. This means for the production of estrogen, androgen is essential and androgen is produced in theca cells under the effect of LH.
High level of estrogen causes negative feedback mechanism and decreases FSH and LH. But when estrogen reaches to certain peak, it causes positive feedback mechanism and causes rise of FSH and LH. This rise causes LH peak and second FSH peak (surge). But LH peak is more intense than FSH peak.
Estrogen prepares the endometrium and is essential for development of progesterone receptors. Estrogen concentration is at their lowest at menstruation and rises to maximum at the preovulatory phase. Following ovulation, the estrogen level falls and then again rises in the luteal phase with the activity of corpus luteum.
- Because of negative feedback mechanism, FSH level decreases after rise of estrogen from developing follicle. This decrease in FSH causes monofollicular development of the follicle. Letrozole does not abolish this negative feedback unlike clomiphene citrate and so letrozole gives monofollicular development.
- Exogenous estrogen is given for preparation of endometrium in premature ovarian failure (POF) patients.
Inhibin (Fig. 1.6)
Inhibin is also produced by granulosa cells. It has two dimers, inhibin A and inhibin B and both differ in their secretion pattern. Inhibin B rises in follicular phase and is inhibitory to FSH (similar to estrogen). Inhibin A rises in periovulatory period and luteal phase.
Clinical application: Inhibin B is assessed for measuring ovarian reserve but anti-Müllerian hormone (AMH) and antral follicle count (AFC) are more reliable.
Activin and Follistatin (Fig. 1.6)
These increase secretion and promotes action of FSH, and lead to follicular growth and inhibit androgen production. Follistatin being an activin-binding protein, and neutralizes activin bioactivity.
Insulin-like growth factor (IGF), IGF-2, IGF-binding protein (BP) 1–6, transforming growth factor (TGF), TGF-α, TGF-β, epithelial growth factor (EGF), etc. are the growth factors for the follicle growth. Activity of IGFs can be counteracted by IGF-BPs. Receptors to these are present in ovaries and promote androgen production. All these factors passively help the growth of the follicle (Fig. 1.6).
Ovulation occurs after 34–36 hours of onset of LH surge. Onset of LH surge correlates with perifollicular blood flows. Pre-LH surge perifollicular resistance index (RI) between 0.4 and 0.48 and peak systolic velocity (PSV) of 10 cm/sec are the indicators of a mature follicle on Doppler ultrasound13 (Fig. 1.7). Ovulation occurs 10–12 hours after LH peak (Fig. 1.8). Estrogen peak initiates LH peak and ovulation occurs after 24–26 hours of estrogen peak. Inhibitory activities are high before and after ovulation. But it is important to remember that apart from these hormones, normal levels of prolactin, thyroid and androgen are also essential for normal ovulation.
Human chorionic gonadotropin plays a major role in inducing influx of blood within follicles. Follicular PSV rises markedly with the rise in the LH levels at the time of preovulatory LH surge.15 Because of this physiology, when PSV of the follicle is high, it means that the follicle is likely to rupture early, IUI should be done earlier than the conventional protocol of 34–38 hours.
Luteinizing hormone induces the secretion of progesterone from luteinized granulosa cells (Fig. 1.9). Progesterone rises before ovulation and this rise is because of rise of FSH. It has a triggering role for high GnRH which may give LH surge.
Fig. 1.8: Estradiol (E2) and LH fluctuations around ovulation.14
It helps in increasing proteolytic enzymes for rupture. Progesterone production from corpus luteum keeps on rising till the midluteal period (7–8 days postovulation), when its level is at its peak. Rising progesterone has a negative feedback on the pituitary gonadotropins and decreases the frequency of LH pulse. During this phase, FSH is synthesized but not secreted. It is stored for release when corpus luteum fails and estrogen and progesterone levels decrease markedly. Decrease in LH causes demise of corpus luteum and decrease in progesterone secretion towards the end of the cycle, just before menstruation.
Progesterone prepares endometrium for implantation by expressing genes needed for implantation. hCG of pregnancy rescues corpus luteum and progesterone secretion is maintained till the trophoblastic and placental hormones take over.
- Preparation of endometrium occurs with progesterone along with estrogen in POF patients.
- Human chorionic gonadotropin can be used to rescue corpus luteum as a luteal phase support.
Fig. 1.9: Ovarian steroids genesis.6
CLINICOPATHOLOGY OF OVULATION
For the diagnosis of ovulatory dysfunction, it is extremely important to advice for precise investigations depending on the history of the patient. It helps the clinician to reach to the correct diagnosis. Unnecessary investigations may give no relevant information but may create more confusions.
Profiles and packages are useless in infertility and are expensive in clinical practice. In infertility management, specific investigations are required at specific times for correct interpretation. So one has to be very precise when requesting for investigations. The routinely required investigations for infertility and assisted reproductive techniques (ART) are discussed here.
Serum Follicle-stimulating Hormone
- Day 2–3 serum FSH and serum LH for diagnosis of PCOS: High LH indicates anovulation and it is very evident from the history. Therefore, ratio of LH/FSH is not required for diagnosis of PCOS. Both high LH and PCOS can be diagnosed by an ultrasound scan done on day 2–3 of the menstrual cycle.
- Serum FSH for diagnosis of menopause: FSH of greater than 40 IU/mL indicates ovarian failure. Oral contraceptive (OC) pills can be used to induce withdrawal bleed in these patients to confirm menopause. When the patient does not have a withdrawal bleed after giving progesterone for 3–5 days, it indicates she is menopausal. Ultrasound shows hardly any follicles and ovaries are small.
- Follicle-stimulating hormone for assessment of ovarian reserve: Day 2–3 FSH is done to know the ovarian reserve and also to predict the possibility of pregnancy. Higher the FSH, lesser is the chance for development of good quality follicles/ova, thus resulting in a lesser chance to achieve conception. FSH of less than 8 IU/mL is considered normal. Between 8 IU/mL and 12 IU/mL FSH, the values are considered high. But when these values are greater than 12 IU/mL, there is very little chance of retrieving good quality ova and therefore decreased chance of achieving pregnancy. Chances of pregnancy beyond FSH level of 15 IU/mL is rare.16 Though currently age, AFC and AMH are used for assessment of ovarian reserve and thought to be much more accurate and reliable for the purpose, than FSH. Utility of FSH as a marker for ovarian reserve has significantly reduced. This is because, AFC indicates the number of follicle available in that cycle for recruitment, whereas serum FSH level gives overall impression about the availability of the ova.
If the patient with low ovarian reserve is given DHEA for 3 months, the AFC may rise, but the FSH still remains high. In these cases, it would be worthwhile to initiate ovulation induction. AFC thus may be a better marker for patient counseling too. Moreover, repeated FSH assessment is of no significance, because it is the highest level of FSH that acts as the decision maker for the possibility of pregnancy. AFC is so far considered the most feasible and reliable parameter for assessment of ovarian reserve.17,18
- Low FSH: Less than 5 IU indicates pituitary or hypothalamic cause for anovulation.
Serum Luteinizing Hormone
- Serum LH on day 8–10 in clomiphene citrate cycle: Clomiphene citrate stimulates hypothalamus to secrete both FSH and LH as it blocks estrogen receptors. This high LH is detrimental to ova and gives poor pregnancy rate in spite of good ovulation rate. Therefore, when LH is more than 10–12 IU/mL on day 8–10 of a clomiphene cycle, the subsequent cycles should not be continued with clomiphene citrate, instead the ovulation induction should then be done with gonadotropins in these patients. This is more common scenario in PCOS patients.
- Serum LH in downregulated cycle: In downregulated patients on long protocol, the LH on day 2–3 of the treatment cycle should be between 0.5 IU and 2 IU to confirm the adequacy of downregulation. Actually 0.1 IU/mL LH is sufficient for the development of follicle and so assessment of LH levels at this phase of cycle is questionable.
- Serum LH less than 5 IU/L indicates pituitary or hypothalamic cause for anovulation, especially with low FSH.
- Follicular maturity: When the follicle is mature, serum E2 will be 150 pg/mL/follicle. If E2 is normal and endometrium is poor, it is a local cause for poor endometrium. But if the E2 level is low, the endometrium is usually poor, and may grow to normal when E2 level rises. Using color Doppler for the assessment of follicular quality can be a useful tool for the same because the flow parameters of the follicle can be correlated with the functional maturity of the follicle which indirectly indicates E2 levels. So E2 can differentiate between the causes of poor endometrium.
- For poor responder: In IVF cycles, E2 is assessed on day 2 of the cycle to confirm downregulation. If it is greater than 80 pg/mL, it indicates a possibility of poor response to stimulation. This high E2 is because of high FSH that is present in poor responders and causes early recruitment of the follicle.
- To assess ovarian suppression: In long agonist protocol IVF cycles, GnRHa is started from day 21 of the previous cycle. This downregulates pituitary and FSH and LH levels become very low. This also leads to low E2 levels (30–50 pg/mL). The stimulation is started only thereafter. If E2 level is more than 50 pg/mL, it is advisable to continue downregulation for 1 day or 2 days more before starting the stimulation. In patients on OC pills, E2 should be checked after 12–14 days after starting OC pills.
- For suspected risk of hyperstimulation: In IUI cycles or in non-IVF gonadotropin cycles, hyperstimulation is suspected if E2 is more than 1,600 pg/mL on the day of trigger. This is common in PCOS patients. In IVF cycles, if E2 is more than 4,000 pg/mL on the day of trigger, hyperstimulation is suspected. Now with chronic low-dose protocol in IUI cycles and ultrasound-based scoring systems for deciding the stimulation protocols,21,22 extremely low OHSS rates are claimed. Though in IVF cycles with antagonist protocol and agonist trigger, OHSS can be almost completely prevented. Therefore, the strategy in PCOS patients should be antagonist protocol with agonist trigger.
Ovarian hyperstimulation syndrome can also be predicted by calculating the ovarian volume. If total ovarian volume of both the ovaries together is more than approximately 180 cc, OHSS should be suspected,23 whereas if it is less than approximately 110 cc the risk of OHSS is extremely low. We depend more on ovarian volume than on the E2 levels and withhold hCG trigger when combined ovarian volume of two ovaries is more than 180 cc.
Progesterone is chiefly secreted by corpus luteum in 2nd half of the cycle. Otherwise it is an intermediate hormone for other steroid hormones during their production after conversion from cholesterol.
: 1 ng/mL
: 1–2 ng/mL
: 3 ng/mL
Midluteal serum progesterone, i.e. a week before menstruation is approximately 10 ng/mL. A level of greater than 3 ng/mL indicates ovulation. Therefore, if only ovulation is to be documented, repeated ultrasound scans are not required. In the same way, endometrial biopsy is not required to confirm secretory changes in endometrium and serum progesterone estimation suffices for the same.
Ultrasound with color Doppler is a better modality to assess the secretory phase of the cycle. It can diagnose luteal phase problems viz. LPD/LUF. So it is an inevitable conclusion that random serum progesterone has little value beyond documenting ovulation.
This investigation is done in all patients with hirsutism. It is increased due to 21-hydroxylase deficiency. Because of 21-hydroxylase deficiency in patients of heterogeneous carrier, late onset of congenital adrenal hyperplasia is observed. 17-hydroxyprogesterone (17-OHP) is weak androgen and manifests as hirsutism, acne, clitoral enlargement and menstrual irregularity at puberty.
Normal level of 17-OHP is less than 200 ng/dL. Whenever the level is between 200 ng/dL and 800 ng/dL, one should get the adrenocorticotropic hormone (ACTH) levels assessed in serum. Level of greater than 800 ng/dL is diagnostic of 21-hydroxylase deficiency. These patients are treated with dexamethasone 0.5 mg daily at bedtime.
Normal value of serum testosterone is 20–80 ng/dL. Its normal production is 0.2–0.3 mg/day. Out of this 80% binds to sex hormone-binding globulin (SHBG) and 19% to albumin. Only 1% is free testosterone. Testosterone may be high in patients with PCOS and tumors. Tumor is suspected if there is sudden onset of symptoms due to increased testosterone. PCOS patients show gradual rise in testosterone levels. Routine estimation of serum testosterone is not required, as it does not change the line of treatment.
Androgens and their sources have been shown in Table 1.1.
Serum Dehydroepiandrosterone Sulfate
Dehydroepiandrosterone sulfate (DHEA-S) is exclusively secreted from adrenal glands. The normal value is 350 ng/dL. It increases with hyperprolactinemia. In patients with PCOS, prolactin and DHEA-S are a little high and do not require any specific treatment except the treatment of PCOS. This rise is secondary to estrogen because of anovulation.
Whenever DHEA-S is very high, it is due to adrenal tumor. DHEA-S greater than 700 µg/dL is accepted as a marker for adrenal dysfunction. This is rarely found and does not change the management. And ultrasound is a better guide to diagnose adrenal tumor. Serum testosterone and ultrasound suffices to rule out adrenal tumors. If 17-OHP is normal, there is no need to search for adrenal enzyme defect. Therefore, routine testing of DHEA-S is not required.
Inhibin A and B
Inhibin B is predominantly secreted by antral follicles. Low day 3 inhibin B level less than 45 pg/mL indicates poor response to superovulation and patient is less likely to conceive. Same thing can be judged by serum FSH, serum AMH or AFC and so routine use of inhibin B estimation is not justified. Inhibin A is secreted from preovulatory follicles. Both inhibin A and B are secreted from granulosa cells and regulate FSH by negative feedback mechanism. Routine estimation of inhibin A does not add any information to change the management of the patient.
Serum Anti-Müllerian Hormone
It is a member of TGF. It is produced from granulosa cells of small antral follicles. The expression of AMH is localized in granulosa cells of primary preantral and small antral follicles and has important role in human folliculogenesis. AMH expression in follicle decreases in antral follicle greater than 8 mm in size. It is now well established that serum AMH concentrations reflect the number of preantral and small antral follicles in the ovary, this would account for raised AMH levels found in both polycystic ovarian morphology (PCOM) and PCOS.24 In follicles undergoing atresia and in corpus luteum also, the AMH expression is completely lost.11 AMH levels in women are low until the age of 8, rise rapidly until puberty and decline steadily from the age of 25 until menopause, when AMH production ceases.
Physiology of Anti-Müllerian Hormone
Anti-Müllerian hormone has an inhibiting role in the ovary, contributing to follicular arrest.19 It lowers the sensitivity of follicles to FSH, which is required for normal folliculogenesis. In vitro studies have shown that the action of FSH in promoting follicular growth is counteracted by AMH.25 It has a steady level throughout the cycle except for a slight dip just after LH peak.26
Though some investigators have recorded cyclical fluctuations in AMH with rapid decrease in early luteal phase.27 This can be correlated with the follicular atresia before recruitment in the late luteal phase.
It can predict ovarian response. It can predict menopause by low levels. High AMH is very high in PCOS, and it predicts OHSS.
Anti-Müllerian hormone values and its interpretation for ovarian response have been shown in Table 1.2.
Anti-Müllerian Hormone and PCOS
The reported property of AMH to counteract the actions of FSH imply that the high production of AMH by polycystic ovary may have an important role in the pathophysiology of the syndrome.28 In anovulatory PCOS, the failure of follicle development is due to an intrinsic inhibition of FSH action and that this inhibition is due to an intrinsic inhibition of FSH action and due to the high concentration of AMH.29
Increased ovarian stromal blood flow in PCOS may be because of over expression of vascular endothelial growth factor (VEGF) that modulates the permeability of theca cells and increased IGF-130,31 which in turn enhances gonadotropin-stimulated steroid production in granulosa cells and theca cells resulting in increased ovarian androgen production and subsequently increased AMH production.32
Anti-Müllerian hormone levels are two to three times higher in PCOS, than in healthy controls.33 This has been attributed not only to increased number of antral follicles but also to higher production of AMH per follicle in patients with PCOS as compared to size-matched counterparts from normal ovary.34 In addition, a positive correlation between AMH and both LH and testosterone serum concentrations in PCOS has been reported.35
Each individual follicle in women with PCOS produces significantly more AMH than its size-matched counterpart from a patient with normal ovary.34 Moreover, it has also been shown that metformin administration in PCOS patients is associated with reduction in AMH concentration in follicle and serum, suggesting that the measurement of AMH can be used to evaluate the treatment efficacy with insulin sensitizers.36
Anti-Müllerian Hormone and IVF
In IVF patients, serum AMH value can predict the response of ovary and so it is useful for counseling the patient. When calculated optimal AMH cutoff of less than 1.26 ng/mL was used to predict responses to controlled ovarian stimulation (COS), it was found to have a 97% sensitivity for predicting poor responses (<4 oocytes retrieved) and 98% accuracy in predicting a normal COS response.37
Nelson et al. suggested the AMH-based strategy for deciding the COS protocol.38
But we have found AFC as equally effective and more precise for prediction of ovarian response. With extremely low-serum AMH levels, moderate, but reasonable pregnancy and live birth rates are still possible. Extremely low AMH levels do not seem to represent an appropriate marker for withholding fertility treatment. Younger women are likely to have better pregnancy rates than their older counterparts with equally low AMH.39 Constant AMH and inhibin B levels suggest that neither AMH nor inhibin B is an accurate marker of ovarian response after low-dose gonadotropins ovulation induction in patients with PCOS.40 Mashiach et al. have also shown a relationship between follicular fluid AMH concentrations and the quality of embryos in patients with PCOS.41
All these references indicate that routine use of AMH does not help in managing non-ART patients. Only 1–2 mature follicles are required for superovulation with IUI and AMH will not help us in change the line of treatment. Poor responding ovary or low reserve ovary can be very precisely diagnosed by ultrasound.
Serum AMH estimation is indicated in:42
- Predicting both over and under response in COS
- Determining the most appropriate treatment regimen
- Pretreatment counseling for couples to make an appropriate and informed choice
- Predicting long-term fertility
- Predicting the age of menopause
- Predicting ovarian aging prior to or following chemotherapy/surgery
- Screening for polycystic ovaries.
Clinical Applications of AMH, Apart from Fertility Assessment
- To confirm presence of testicular tissue in children with low testosterone levels.
- Differential diagnosis of intersex disorders.
- In patients with bilateral nonpalpable gonads.
- Females with granulosa cell tumors.
- The inhibitory effect of AMH on folliculogenesis may in future be used for hormonal contraception.
Now it has been proved that in patients of PCOS there is high androgen because of increased insulin level and insulin resistance. It indicates the severity of PCOS. Clinically waist circumference of 35 inches or 90 cm is predictive of abnormal endocrinology and metabolic function.
There are various methods to diagnose insulin resistance. But 2-hour glucose and insulin response is the one of the most reliable ones.
Two-hour glucose tolerance test (75 gm of glucose):
: <140 mg/dL
: 140–190 mg/dL
: >200 mg/dL
Two-hour insulin response:
: 100–150 µU/mL
: 151–300 µU/mL
: >300 µU/mL
In clinical practice, patients with high insulin resistance are the ones who have tonically high LH levels require ovarian drilling. Alternatively they may also be benefitted by GnRH antagonist. Antagonist decreases LH levels, prevents premature luteinization and improves oocyte quality.
Patients who do not want immediate pregnancy are the patients in whom insulin sensitizer alone is the recommended line of treatment. It has been proved beyond doubts that insulin sensitizers do not have any direct effect on ovulation induction, though when given for 3–6 months, the improved insulin sensitivity, improved the hormonal milieu of PCOS patient, thus improving the chance of spontaneous ovulation and conception.
Three-dimensional (3D) ultrasound is a novel way to diagnose insulin resistance, based on assessment of stromal volume.43 But when volume ultrasound is unavailable, 2-hour glucose and 2-hour insulin test is the best alternative.
So for chronic anovulation only three investigations are required:
- Serum prolactin
- Serum TSH
- Insulin resistance.
Prolactin is the hormone secreted by anterior pituitary and is regulated by prolactin inhibitory factor that is secreted from hypothalamus.
- Normal level of prolactin is 10–25 ng/mL.
- In microadenoma, it is between 100 ng/mL and 200 ng/mL.
- In macroadenoma, it is greater than 200 ng/mL.
- Immunoassay for prolactin does not reflect its bioassay levels always.
- Many a times patients have no symptoms in spite of high prolactin level because of presence of macromolecules which are big prolactin molecules that account for 10–12% of hyperprolactin in a symptomatically normal patient.
Galactorrhea with Normal Prolactin Levels
Patients with galactorrhea present with normal serum prolactin levels. This may be mucoid discharge and not actual galactorrhea. Any discharge therefore from the breast should be seen under microscope. If it contains fat globules, only then it is galactorrhea.
When ovulation induction is done, in many patients estrogen leads to rise in prolactin levels in first half of the menstrual cycle. These patients have night spikes of high prolactin level and are known as spikers. This is transient hyperprolactinemia. The clinical presentation is poor endometrium in spite of good follicles. This condition is difficult to diagnose, but bromocriptine can be given in a dose of 1.25 mg twice a day, in these patients in first half of the cycle. It is a therapeutic test. If endometrium improves, it is continued in the subsequent cycle, otherwise it is stopped. Apart from bad oocyte quality, the spikers also have a high chance of LPD.
As discussed earlier, marginally high prolactin levels are also found in PCOS patients due to high estrogen and high DHEA-S. In these cases, bromocriptine or cabergoline is not required, PCOS is to be treated and this corrects hyperprolactinemia.
DIAGNOSIS OF OVULATION
Detection of ovulation is extremely important to decide the further line of treatment in patients for treatment of infertility. There are various tests to detect ovulation and current status of each test is discussed here.
- “Mittelschmerz”: This is a mid-cycle pain in an ovulatory cycle because of contraction of smooth muscles surrounding the follicle about 12–24 hours prior to ovulation. This contraction helps the follicle to rupture and ovulation occurs.
- Cervical mucus feel: Patient feels watery discharge at the introitus which becomes thin and copious prior to ovulation. This indicates a mature follicle and not ovulation.
- Pregnancy is the only surest sign of ovulation.
- Painful menstruation is also an indicator of the previous cycle being ovulatory.
Basal Body Temperature
The principle of basal body temperature (BBT) measurement is that progesterone secretion after ovulation raises the body temperature. BBT is between 97°F and 98°F during the follicular phase. After ovulation, it starts rising and goes high by 0.4–0.8°F, as the progesterone rises. BBT is at its lowest just before ovulation which is known as nadir. The temperature falls before the period or when progesterone level decreases due to regressing corpus luteum. The rise in temperature persists, in case the patient conceives and patient feels her body temperature as much as low-grade fever. This is because of persistent and high levels of progesterone from the corpus luteum of pregnancy, stimulated by hCG.
Basal body temperature is taken before rising from bed, with glass thermometer placed in oral cavity. The calibration on thermometer is 96–100°F. The change pattern in thermometer is known as biphasic pattern. The next period in normal cycle starts after 12 days of rise in temperature if pregnancy does not occur. It is a low cost, noninvasive and simple method and indicates abnormalities of follicular and luteal phase. But the temperature may be altered with smoking and disturbed sleep. It may be stressful for the patient to take temperature daily. There may not be shift in temperature in spite of ovulation and in this case, only after a few days of ovulation it may be diagnosed. This is not clinically useful. BBT is not commonly used in clinical practice.
Midcycle LH Surge Diagnosed by LH Kit
Luteinizing hormone surge occurs for 48–50 hours. LH has short half-life. LH rise can be detected by urinary LH kits. When the threshold level of 40 mIU/mL is crossed, test becomes positive on the kit and the color is seen. The intensity of the color increases as LH level increases. LH level normally starts rising early in the morning and may be misinterpreted as negative if urine is tested in the morning as it appears in the urine after 6–8 hours. Therefore, LH assessment in the afternoon is more reliable.
The test is done daily. But doing it twice daily decreases the false negatives. Too much of fluid intake is avoided few hours before the test to prevent dilution of the urine and LH in urine.
Ovulation occurs 20 hours ±6 hours after detection of urinary LH surge.44–47 Patient is advised intercourse for 2 days following detection of LH surge as these days are the most fertile ones. If artificial insemination of donor (AID) is planned, it should also be done for 2 days. The specificity and sensitivity for LH surge are 90% and 96% respectively and if test is done twice a day, sensitivity reaches to 99%.47
So LH detection kits are noninvasive, self-monitored and indicate fertile period, so it is useful clinically for planning timed intercourse, IUI and AID. It can diagnose abnormality of follicular phase and luteal phase. But the disadvantage is that it is tedious, may be false negative and restricts patients from fluid intake. False positive results may occur with OC pills, clomiphene citrate, hCG, human menopausal gonadotropin (hMG) and danazol. But easy availability of ultrasound has reduced the utility of LH kits.
It was used to document secretory changes in the endometrium after ovulation. Endometrial biopsy differentiates follicular phase from secretory phase. An experienced pathologist can date the endometrium after ovulation. Two days out of phase endometrium is regarded as LPD.48 This test is not as preferred as ultrasound or serum progesterone levels as it is inaccurate and highly invasive.
Luteal Serum Progesterone Levels
Progesterone level is below 1 ng/mL during follicular phase. Before ovulation it rises up to 1–2 ng/mL. The peak reaches after a week of ovulation. Level more than 3 ng/mL indicates ovulation. Normally on 7th postovulatory day, progesterone level is greater than 10 ng/mL.49 Serum progesterone level is only used to document ovulation as it is simple, fairly accurate, noninvasive and reliable.
Daily assessment of progesterone for diagnosis of LPD is not used as is inconclusive and multiple levels are required for accuracy.
Clinically estrogen peak will give copious mucus discharge and when dried on a slide will show a “fern” pattern. Before ovulation it shows tertiary branches. Normal preovulatory mucus strand can be stretched to 8–10 cm between two ends of artery forceps. Both these tests indicate high estrogen level, mostly due to mature follicle but do not indicate ovulation.
A follicle that is of greater than 10 mm in diameter, grows at a rate of 2–3 mm/day has no internal echogenicity and has thin (pencil line like) walls is not only more likely to become the leading follicle but will also give mature healthy ovum. The growing follicle can be assessed by transvaginal sonography. A mature follicle is 16–18 mm, has thin walls, regular round shape and no echogenicity in the lumen. When functionally mature, on color Doppler, the follicle shows blood vessels covering at least three-fourths of the follicular circumference (see Fig. 1.7). On pulse Doppler, these blood vessels show an RI of 0.4–0.4813 and PSV of greater than 10 cm/sec. The endometrium starts appearing multilayered on ultrasound as early as mid-proliferative phase with rising estrogen. On transvaginal scan, endometrial thickness of 6 mm is considered minimum that is required on the day of ovulation or on the day of hCG trigger for a successful outcome, although 8 mm is generally considered optimum.50 Segmental uterine artery perfusion demonstrates significant correlation with hormonal and histological markers of uterine receptivity, reaching the highest sensitivity for subendometrial blood flow.51
Endometrial vessels on pulse Doppler if have RI of less than 0.6, it have been reported to be a good prognostic factor for implantation.52 Rupture of the follicle is documented as disappearance of the follicle and it may be replaced by a cystic structure with thick shaggy walls and echogenicity in the lumen. But it is known to have variable appearances (Fig. 1.10) like ground glass echogenicity in lumen or lace-like echogenicities. This structure is known as corpus luteum.
Corpus luteum can be seen as low resistance vascular structure with thick walls and internal echogenicities (Fig. 1.11).
Secretory changes are seen in the endometrium in the form of echogenicity of the endometrium which starts from outside in the early luteal phase, proceeding to the central line making a ring sign of the endometrium.53 Posterior wall of the uterus also appears more echogenic in this phase due to acoustic enhancement by the endometrium due to fluid accumulation (Fig. 1.12).
Serial ultrasound for documenting ovulation should not be done if intervention like ovulation trigger or IUI is not planned. Whether ovulation has occurred or not can be known by postovulatory serum progesterone level only.
Summarizing Diagnosis of Ovulation
- Clinical history suggests ovulation in most cases.
- Basal body temperature is inconclusive and not used routinely.
- Urinary LH kit is useful only for patients who cannot visit clinic off and on. It is not very commonly used.
- Serum progesterone greater than 3 ng/mL postovulatory indicates ovulation and is a most reliable means to document ovulation.
- Daily ultrasound for just confirming that ovulation has occurred or not is not required unless some intervention is intended.
- Endometrial biopsy is not required to assess the secretory phase changes.
- Fern pattern and spinnbarkeit test indicate high estrogen level and not ovulation.
CAUSES OF ANOVULATION (Fig. 1.13)
The incidence of infertility is 15–17% after 1 year of unprotected intercourse. After 2nd year, only 7–8% of couples remain infertile. After 2.5 years almost 7% will still remain infertile. Out of these infertile couples, in 20–40%, anovulation or oligo-ovulation is the causative factor.
The classification of causes of anovulation is very well described by the World Health Organization (WHO). It is classified as:
- WHO group I: Hypothalamic-pituitary failure
- WHO group II: Hypothalamic-pituitary dysfunction
- WHO group III: Ovarian failure
- WHO group IV: Hyperprolactinemia
- WHO group V: Outflow defects (not associated with anovulation).
Hypothalamic-Pituitary Failure (WHO Group I)
It is also known as hypogonadotropic hypogonadism.
In this situation, gonadotropin (FSH and LH) concentrations are so low that follicle is not stimulated and therefore there is extremely low estrogen level. These patients have anovulation and amenorrhea.
- Anorexia nervosa: It is weight related. It may be due to crash diet or frank anorexia nervosa.
- Stress: It may be stress-related anovulation and amenorrhea. Severe exercises like marathon running can cause amenorrhea.
- Kallmann syndrome: It is associated with anosmia.
- Debilitating systemic diseases.
- Severe postpartum hemorrhage (PPH) causing Sheehan's syndrome.
- Radiotherapy for pituitary tumors.
In these conditions, GnRH replacement therapy is very useful. But gonadotropins are usually given for ovulation induction.
Hypothalamic-Pituitary Dysfunction (WHO Group II)
As contrast to WHO group I, in patients of this group, FSH is present that stimulates follicle and so estrogen is also present. Clinically this condition presents as oligo-ovulation or anovulation causing amenorrhea. As endogenous estrogen is present in these patients, 5 days of oral progesterone (10 mg hydroxyprogesterone acetate daily) will give withdrawal bleeding usually within 10 days and this is confirmatory of WHO type II anovulation. PCOS is the most common condition amongst WHO group II. Diagnosis of PCOS is made by Rotterdam criteria (2003). These are as follows:
- Clinical and/or biochemical hyperandrogenism
- Polycystic ovaries on ultrasound.
Any two of these above mentioned three criteria establishes the diagnosis of PCOS. These criteria are defined as mentioned below:
- Oligo-ovulation/anovulation: Oligo-ovulation is ovulation occurring once in 35–180 days. Anovulation is defined as no ovulation in 6 consecutive months.
- Hyperandrogenism: clinical or biochemical: Clinical signs include hirsutism, acne, alopecia (male type) and female virilization. Biochemical indicators include raised total testosterone, androstenedione and free androgen index.
- Polycystic ovaries on ultrasound: Presence of 12 or more follicles in either ovary, 2–9 mm in size and/or ovarian volume greater than 10 cc. The new cut off for follicle number is 20 per ovary.
Though the concepts on the number of follicles and ovarian volume for the diagnosis of PCOS are changing. An average value of 26 or more follicles per ovary is a reliable threshold for detecting polycystic ovaries in women with frank manifestation of PCOS. Sensitivity and specificity for diagnosis of PCOS for follicle number per ovary (FNPO) (26) were 85% and 94% and for ovarian volume (10 cc) were 81% and 84%. But the same study has also quoted that the lower follicle threshold may be required to detect milder variants of the syndrome.54
Hyperinsulinemia and high androgen levels are very useful for diagnosis. Postprandial insulin levels (2 hours after 75 g of glucose) are increased in patients with insulin resistance that is one of the major diagnostic features for PCOS. Along with this, increased stromal volume on 3D ultrasound is diagnostic of PCOS. Management includes lifestyle changes, oral ovulogens, gonadotropins and ovarian drilling.
Ovarian Failure (WHO Group III)
This group presents with high FSH along with low estrogen and amenorrhea. The patient will have symptoms of hypoestrinism. Physiologically this condition is found with the onset of menopause. But menopause may set in prematurely.
Causes of Ovarian Failure
- Menopause (>40 years of age)
- Premature menopause (<40 years of age):
- Familial tendency
- Autoimmune disease
- Infections like tuberculosis
- Chromosomal—Turner's syndrome.
Diagnosis is confirmed by high FSH greater than 25 IU/mL with extremely low AMH. The only available treatment option to achieve a pregnancy in these patients is ovum donation. Hormone replacement therapy (HRT) is required in these patients, to support their physiological needs.
Hyperprolactinemia (WHO Group IV)
Anovulation because of hyperprolactinemia is present when prolactin levels are double the upper normal limits. This leads to anovulation. Marginally high prolactin levels do not require any treatment as it does not lead to anovulation. Mildly high prolactin levels are found in PCOS but in these cases no therapy is required to decrease prolactin levels. Prolactin is regulated by “prolactin-inhibiting factor”, dopamine. Therefore, any hypothalamic lesion or drugs suppressing hypothalamic activity will increase prolactin level and LH will be low.
Outflow Defects (WHO Group V)
- Primary: Imperforate hymen, absent uterus.
- Secondary: Asherman's syndrome.
So chiefly three tests: (1) progesterone withdrawal bleeding, (2) serum prolactin and (3) serum FSH level assessment can give accurate diagnosis of anovulation for the management.
CARRY HOME MESSAGE
- In all patients of anovulation/amenorrhea, always assess the prolactin levels as the first investigation to exclude hyperprolactinemia. (WHO IV)
- Progesterone withdrawal bleeding indicates presence of endogenous estrogen and diagnosis of hypothalamic-pituitary dysfunction. (WHO II)
- Low FSH indicates hypogonadotropic hypogonadism. (WHO I)
- High FSH indicates ovarian failure. (WHO III)
- Normal FSH with no withdrawal bleed indicates absent uterus or outflow tract obstruction or Asherman's syndrome. (WHO V)
- Clinical examination:
- Patient's height and weight are assessed for calculation of body mass index (BMI). (BMI = weight in kg/height2 in meters.) Normal BMI is 20–25, less than 20 is considered underweight and more than 30 is considered frank obesity. These values are decided according to the western standards, but may differ marginally for the Indian standards. Anovulation is common in patients with abnormal BMI.
- With obesity, PCOS must be considered, which is characterized by hirsutism and/or acne, acanthosis nigricans, dark discoloration of skin in axilla and neck and waist circumference more than 35 inches or 90 cm. But it is important to remember here that these features will be seen only in full blown PCOS and there is a much larger percentage of infertile population that has silent forms or milder forms of PCOS.
- Body mass index less than 20 may be seen in thin lean PCOS, malnourishment or anorexia nervosa. Causes of primary amenorrhea and ovarian failures must also be ruled out by excluding the clinical presentation of estrogen deficiencies.
- Oogenesis: how the female reproductive system produces eggs, Chapter 16. CLEP Biology: Study Guide and Test Prep/Science Courses. Instructor: Joshua Anderson.
- Lobo RA. Early ovarian ageing: a hypothesis. What is early ovarian ageing? Hum Reprod. 2003;18(9):1762–4.
- Gilbert SF. The number of ova therefore decides the reserve of the ovary. In: Gilbert SF (Ed). Developmental Biology, 6th edition. Sunderland (MA): Sinauer Associates; 2000.
- Chaudhary S, Deshpande A. Physiology of ovulation. In: Deshpande H (Ed). Practical Management of Ovulation Induction. New Delhi: Jaypee Brothers Medical Publishers (P) Ltd; 2016. p. 15
- Chaudhary S, Deshpande A. Physiology of ovulation. In: Deshpande H (Ed). Practical Management of Ovulation Induction. New Delhi: Jaypee Brothers Medical Publishers (P) Ltd; 2016. p. 7.
- Homburg R. Ovulation Induction and Controlled Ovarian Stimulation. Switzerland: Springer International Publishing; 2014. pp. 7–23.
- Hiller SG. Current concepts of the roles of follicle stimulating hormone and luteinizing hormone in folliculogenesis. Hum Reprod. 1994;9:188–91.
- Balasch J, Fabregues F. Is luteinizing hormone needed for optimal ovulation induction? Curr Opin Obstet Gynecol. 2002;14(3):265–74.
- Shoham Z. The clinical therapeutic window for luteinizing hormone in controlled ovarian stimulation. Fertil Steril. 2002;14:265–74.
- Kobayashi M, Nakano R, Ooshima A. Immunohistochemical localization of pituitary gonadotropins and gonadal steroids confirms the two cells two gonadotropins hypothesis of steroidogenesis in the human ovary. J Endocrinol. 1990;126:483–8.
- Yamoto M, Shima K, Nakano R. Gonadotropin receptors in human ovarian follicles and corpora lutea throughout the menstrual cycle. Horm Res. 1992;37 Suppl 1:5–11.
- Hughes E, Collins J, Vandekerckhove P. Gonadotropin-releasing hormone analogue as an adjunct to gonadotropin therapy for clomiphene-resistant polycystic ovarian syndrome. Cochrane Database Syst Rev. 2000;(2):CD000097.
- Kupesic S, Kurjak A. Uterine and ovarian perfusion during the periovulatory period assessed by transvaginal colour Doppler. Fertil Steril. 1993;3:439–43.
- Regulation of menstrual cycle. In: Speroff L, Fritz MA (Eds). Clinical Gynecologic Endocrinology and Infertility. Baltimore: Lippincott, Williams and Wilkins; 2012. p. 213.
- Bourne T, Jurkovic D, Waterstone J, et al. Intrafollicular blood flow during human ovulation. Ultrasound Obstet Gynecol. 1991;1:53–9.
- Scott RT, Hofmann GE. Prognostic assessment of ovarian reserve. Fertil Steril. 1995;63:1.
- Krishnakumar J, Agarwal A, Nambiar D, et al. Comparison of antral follicle count, anti-Mullerian hormone and day 2 follicle-stimulating hormone as predictor of ovarian response and clinical pregnancy rate in patient with an abnormal ovarian reserve test. Int J Reprod Contracept Obstet Gynecol. 2016;5(8):2762–7.
- Panchal S, Nagori CB. Comparison of anti-Mullerian hormone and antral follicle count for assessment of ovarian reserve. JHRS. 2012;5(3):274–8.
- Balen A, Conway G, Homburg R, et al. Polycystic Ovary Syndrome: A Guide to Clinical Management. Boca Raton: Taylor and Francis Group; 2007.
- Panchal S, Nagori CB. Ultrasound based decision making on stimulation protocol for superovulated IUI cycles. IJIFM. 2016;7(1):7–13.
- Panchal S, Nagori CB. Ultrasound Based decision making on stimulation protocol in IVF. DSJUOG. 2016;10(3):330–7.
- Oyesanya OA, Parsons JH, Collins WP, et al. Total ovarian volume before human chorionic gonadotropin administration for ovulation induction may predict the hyperstimulation. Hum Reprod. 1995;10:3211–2.
- Fanchin R, Maria SL, Righini C, et al. Serum AMH is more strongly related to ovarian follicle status that serum inhibin B, oestradiol, FSH and LH on the day 3. Hum Reprod. 2003;18:323–7.
- Durlinger AL, Gruijters MJ, Kramer P, et al. Anti-Mullerian hormone attenuates the effect of FSH on follicle development in the mouse ovary. Endocrinology. 2001;142:4891–9.
- La Marca A, Stabile G, Artenisio AC, et al. Serum anti-Mullerian hormone throughout the human menstrual cycle. Hum Reprod. 2006;21(12):3103–7.
- Streuli J, Fraisse T, Chapron C, et al. Clinical uses of anti-Mullerian hormone assays: pitfalls and promises. Fertil Steril. 2009;91(1):226–30.
- Pellat L, Rice S, Mason HD. Anti-Mullerian hormone and polycystic ovarian syndrome: a mountain too high? Reproduction. 2010;139:825–33.
- Pellat L, Rice S, Dilaver N, et al. Anti-Mullerian hormone reduces follicle sensitivity to follicle-stimulating hormone in human granulosa cells. Fertil Steril. 2011;96:1246–51.
- El Behery MM, Diab AE, Mowafy H, et al. Effect of laparoscopic ovarian drilling on vascular endothelial growth factor and ovarian stromal blood flow using three-dimensional power Doppler. Intern J Gynecol Obstet. 2011;112:119–21.
- Abd El, Aal DE, Mohamed SA, et al. Vascular endothelial growth factor and insulin like growth factor 1 in polycystic ovary syndrome and their relation to ovarian blood flow. Eur J Obstet Gynecol Reprod Biol. 2005;118:219–24.
- Willis D, Mason H, Gilling-Smith C, et al. Modulation by insulin of follicle-stimulating hormone and luteinizing hormone actions in human granulosa cells of normal and polycystic ovaries. J Clin Endocrinol Metab. 1996;81:302–9.
- Pigny P, Merlen E, Robert Y, et al. Elevated serum level of anti-Mullerian hormone in patients with polycystic ovary syndrome: relationship to the ovarian follicle excess and to the follicular arrest. J Clin Endocrinol Metab. 2003;88:5957–62.
- Pellat L, Hanna L, Brincat M, et al. Granulosa cell production of anti-Mullerian hormone is increased in polycystic ovaries. J Clin Endocrinol Metab. 2007;92:240–5.
- Carlsen SH, Vanky E, Fleming R. Anti-Mullerian hormone concentrations in androgen suppressed women with polycystic ovary syndrome. Hum Reprod. 2009;24:1732–8.
- Piltonen T, Morin-Papunen L, Koivunen R, et al. Serum anti-Mullerian hormone levels remain high until late reproductive age and decrease during metformin therapy in women with polycystic ovary syndrome. Hum Reprod. 2005;20(7):1820–6.
- Gnoth C, Schuring AN, Friol K, et al. Relevance of anti-Mullerian hormone measurement in a routine IVF program. Hum Reprod. 2008,23:1359–65.
- Nelson SM, Yates RW, Lyall H, et al. Anti-Mullerian hormone-based approach to controlled ovarian stimulation for assisted conception. Hum Reprod. 2009;24:867–75.
- Weghofer A, Dietrich W, Barad DH, et al. Live birth chances in women with extremely low serum anti-Mullerian hormone levels. Hum Reprod. 2011;26(70):1905–9.
- Fong SL, Schippe I, de Jong FH, et al. Serum anti-Mullerian hormone and inhibin B concentrations are not useful predictors of ovarian response during ovulation induction treatment with rFSH in women with polycystic ovary syndrome. Fertil Steril. 2011;96(2):459–63.
- Loh JS, Maheshwari A. Anti-Mullerian Hormone—is it a crystal ball for predicting ovarian ageing? Human Reprod. 2011;26(11): 2925–32.
- Panchal S, Nagori CB. Correlation of ovarian and stromal volumes to fasting and postprandial insulin levels in polycystic ovarian syndrome patients. Int J Infertil Fetal Med. 2014;5(1):12–4.
- Filicori M, Butler JP, Crowley WF. Neuroendocrine regulation of the corpus luteum in the human. Evidence for pulsatile progesterone secretion. J Clin Invest. 1984;73:1638–47.
- Syrop CH, Hammond MG. Diurnal variations in midluteal serum progesterone measurements. Fertil Steril. 1987;47:67–70.
- Jordan J, Craig K, Clifton DK, et al. Luteal phase defect: the sensitivity and specificity of diagnostic methods in common clinical use. Fertil Steril. 1994;62:54–62.
- Miller PB, Soules MR. The usefulness of a urinary LH kit ovulation prediction during menstrual cycles of normal women. Obstet Gynecol. 1996;87:13–7.
- Duggan MA, Brashert P, Ostor A, et al. The accuracy and interobserver reproducibility of endometrial dating. Pathology. 2001;33:292–7.
- Wathen NC, Perry L, Lilford RJ, et al. Interpretation of single progesterone measurement in diagnosis of anovulation and defective luteal phase: observations on analysis of the normal range. Br Med J. 1984;288:7–9.
- Dickey RP, Olar TT, Taylor SN, et al. Relationship of biochemical pregnancy to preovulatory endometrial thickness and pattern in patients undergoing ovulation induction. Hum Reprod. 1993;8:327–30.
- Kupesic S, Kurjak A. Prediction of IVF outcome by three-dimensional ultrasound. Hum Reprod. 2002;17:950–5.
- Kupesic S, Bekavac I, Bjelos D, et al. Assessment of endometrial receptivity by transvaginal colour Doppler and three-dimensional power Doppler ultrasonography in patients undergoing in vitro fertilization procedures. J Ultrasound Med. 2001;20(2):125–34.
- Bald R, Hackeloer BJ. Ultraschall-darstellung verschiendener endometrium for men. In: Otto R. Jan FX (Eds). Ultraschalldiagnostik 1982. Stuttgart: Thieme; 1983. pp. 187.
- Lujan ME, Jarrett BY, Brooks ED, et al. Updated ultrasound criteria for polycystic ovary syndrome: reliable thresholds for elevated follicle population and ovarian volume. Hum Reprod. 2013;28(5):1361–8.