Chapter 14

Endocrine Investigations in Gynaecology


The term endocrine investigations is usually used erroneously synonymous to hormonal blood tests. This concept should be changed. The term should be used instead to include all the available means to examine and diagnose endocrine related problems. This is especially so as biochemical results are usually used to complement other diagnostic means, and may be useless as stand alone indices. This chapter will promote this broader concept and address history taking, clinical examination and imaging means, as well as the biochemical tests used in this respect. There are many different clinical scenarios when gynaecologists need to request specific hormonal tests during investigations of certain cases. In other words, hormonal tests are usually requested to verify or exclude a provisional diagnosis. These tests can also be used to monitor patients’ response to treatment, or the progression of the medical condition itself. There is an intricate interrelationship between the normal physiology and pathophysiology related to the pituitary gland, ovaries, adrenals and thyroid gland. Furthermore, many medications can affect the function of these glands, and change their peripheral blood indices. An important role for autoimmune disorders has already been addressed in the previous chapters in this book, especially in relation to the adrenals and the thyroid gland. Accordingly, gynaecologists must be able to appreciate, and address the broader clinical problems, and request the right imaging and biochemical hormonal and non-hormonal investigations. They should also be able to couple these numerical results together, and in relation to the general clinical picture. 

Most endocrine investigations are conducted within a gynaecological unit because of the following reasons:

  1. Abnormal pubertal development, being delayed or precocious;
  2. Menstrual dysfunction being polymenorrhoea, oligomenorrhoea, amenorrhoea, or menorrhagia;
  3. Hyperandrogenisation problems including acne, hirsutism, obesity and androgenic alopecia;
  4. Infertility investigations including ovarian reserve, and monitoring ovulation induction;
  5. Management of abnormal pregnancies including ectopic or molar gestation;
  6. Perimenopausal symptoms including premature ovarian failure.


Medical history and physical examination

As for all medical problems, a thorough medical history forms the platform for all endocrine investigations, depending on the nature of the problem itself. The more usual questions should include the pattern of onset of the problem being sudden or gradual, duration and progression, any relevant related symptoms, history of excessive change in weight, exercise level, medication or surgery, as well as the results of any previous investigations. Symptoms suggestive of specific endocrinopathy should also be explored, including hyperandrogenisation, fatigue, forgetfulness, lethargy, fainting and dizzy spells, increased pigmentation, excessive hair loss, polyurea and polydipsia. Childhood disease and developmental history are important in relation to abnormal pubertal development, whether delayed or precocious. History of malnutrition, chronic anaemia, tuberculosis and other endemic diseases can give good clues to the origin of delayed puberty in developing countries. Any significant family history, especially of maternal age at the menopause should be elucidated. Another good example is history of mental retardation in male siblings, especially in cases of premature ovarian failure and fragile X syndrome.

General examination is important as well. The patient’s general look, height, weight, and arms span are important parameters to be ascertained. Blood pressure measurement is also important, as high levels can be seen in patients with delayed puberty due to 17-hydroxylase deficiency, and in patients with Cushing’s syndrome. Obesity and underweight conditions are very important to ascertain with BMI calculations. It is difficult to give specific scenarios about different physical outlooks, but the following specific signs may indicate an endocrine dysfunction:

  • Facial plethora may be due to alcoholism or increased cortisol levels;
  • Slow speech and reactions, lack of concentration and forgetfulness may indicate severe hypothyroidism;
  • Exophthalmos, warm damp palms, and hand tremors as signs of hyperthyroidism;
  • Very tall or short stature in relation to age, are important clues during investigations of puberty related problems;
  • Discrepancy between height and arms span are important signs to verify; Upper to lower body ratio is also important;
  • Greasy or dry skin can be secondary to hyperandrogenisation or hypothyroidism respectively;
  • Signs of hyperandrogenisation including, acne, hirsutism and alopecia should be recorded;
  • Excessive hair growth should be scored using the Ferriman and Gallwey scoring system (1). A score > 8 is considered abnormal. More information about this system and its limitations can be found in Chapter 6.
  • Enlarged thyroid gland can be a normal sign during puberty and pregnancy;
  • Breast development and staging using Tanner’s stages during investigations of pubertal development problems;
  • Sparse development or absence of axillary and pubic hair in cases of autoimmune adrenal insufficiency. This can also be seen in women with well developed breasts, as seen in cases of androgen insensitivity (testicular feminisation) syndrome;
  • Acanthosis nigricans at the back of the neck, in the axillae and under the breasts have been associated with insulin resistance;
  • The presence of vitiligo, which indicates autoimmune dysfunction, is important when dealing with such cases as premature ovarian failure;
  • Low set ears, webbing of the neck, bone deformities and other abnormal chromosomes signs;
  • Galactorrhoea may be present in about 50% of cases of hyperprolactinaemia, but can be seen in women with normal prolactin levels;
  • Purple striae ≥ 1.0 cm wide can indicate Cushing’s syndrome;
  • Trunk obesity and high waist / hip ratio as signs of androgenic obesity;
  • Enlarged clitoris as a sign of marked increase in androgens production as seen in cases of virilization, together with deepening of the voice, body masculinization and frontal hair recession;
  • Neurological signs especially in cases of precious puberty.

These are just examples of the physical signs seen in patients presenting with problems which may have gynaecological endocrinology background or origin. Gynaecological pelvic assessment should be performed, when indicated.


Imaging tests

Different imaging examinations are available, depending on the condition being investigated. Transabdominal and transvaginal ultrasound scan examinations are widely used to ascertain the presence and normality of the internal genital organs. Testicles can be detected within the inguinal canals in patients with testicular feminization syndrome. Diagnosis of polycystic ovaries is a common objective, depending on the patient’s presentation. The presences of 12 cysts in one or both ovaries, or an ovarian size ≥10 cc are the two ultrasonic criteria adopted for the diagnosis of polycystic ovaries (2). Diagnosis of functional ovarian cysts, and endocrinologically active solid ovarian masses can be helpful in certain cases. Adrenal masses can also be detected ultrasonically, but are better diagnosed with MRI. Pituitary adenomas are diagnosed with MRI which superseded the use of plain skull X-ray and CT scanning, even as a first line imaging technique. Patients with high prolactin levels are a prime target for this technique within the gynaecology clinic. MRI of the brain is also indicated in cases with abnormal pubertal development to exclude brain tumours, especially in the presence of neurological symptoms or signs. The role of left hand and wrist X-ray in the investigation and monitoring of abnormal pubertal development has already been addressed in Chapter 2. Monitoring ovulation with transvaginal ultrasound scan examination is important both for natural cycle tracking and during induction of ovulation. A monitored natural cycle can also reveal the length of the follicular and luteal phases, especially in patients with short cycles. Other useful parameters include endometrial thickness and texture, and Doppler assessment of uterine and endometrial blood flow. The superior role of ultrasound scanning for monitoring induction of ovulation in comparison to serial oestradiol estimations has already been discussed in Chapter 7.


Hormonal tests

A thorough account has been given about biological testing of oestrogens and progestogens effects and potency in Chapter 5. In a clinical setup, the most practical similar bioassay test is the progestogen challenge test, which is a diagnostic test for oestrogen exposure. The most commonly used version of the test is to administer oral progestogens for 5-7 days, before being withdrawn. An oestrogen primed endometrium will bleed following the withdrawal of the progestogen. This positive response is used to indicate good oestrogen exposure, and to exclude hypoestrogenism. Negative results can occur in well oestrogenised patients with endometrial adhesions, or other causes of genital tract obstruction, and in cases of endometrial tuberculosis. Accordingly, many authorities advised against the routine use of the test, and recommended the use of blood hormone tests instead. Pregnancy is a physiological cause for negative tests.

Following an appropriate clinical assessment, a hormone test may be necessary to verify or refute a provisional diagnosis. It is not uncommon for a hormonal test to be contradictory with a well reached clinical impression. This may be due to different causes including:

1. Most hormones are produced in pulses, and one test may not be a true representation of the hormonal milieu. Examples of such difficulty can be seen with FSH, LH, testosterone, oestradiol and insulin which are the more commonly requested tests. The coefficients of variation (SD/Mean%) for a single estimation of these hormones relative to multiple samples examined within 6 hours have been reported as 14.7 % for FSH, 26.8 % for LH, 31.9 % for testosterone, 15.4 % for oestradiol and 31.3 % for insulin by Abdel-Gadir et al in 1990 (3). In the same year, serial screening with basal FSH estimations in different cycles has been suggested by Scott et al (4), to compensate for the limited diagnostic and predictive values of single FSH estimations. 

  1. Hormones have a circadian pattern. Few hormones have higher levels in the morning, and timing the test for an afternoon slot will give an erroneous result. Good examples are 17-hydroxyprogesterone which is used for the diagnosis of 21-hydroxylase deficiency, androstenedione and testosterone. These hormones are usually higher in morning blood samples following the adrenal circadian rhythm. The circadian variations in cortisol levels are well known.
  2. Hormone levels vary during the different stages of the menstrual cycle and give different results at different times. Early follicular phase assessment reflects the basic gonadotrophins levels, before the expected rise during the follicular phase and midcycle. High LH and testosterone blood levels during the early follicular phase are usually associated with the polycystic ovary syndrome, while higher levels of the same hormones during the midcycle are normal physiological findings indicating ovulation. A further example is FSH which needs to be tested on the 2nd or 3rd days of the cycle, as the blood levels increase toward the middle of the follicular phase. One further example is mid luteal phase serum progesterone which needs to be tested about 7-9 days before the next period. Accordingly, conducting the test arbitrarily on day 21 of a 35 day cycle is not be useful, and gives a wrong diagnosis.
  3. Hormone blood tests are affected by many drugs, and the patient should be asked about such medication. This is especially so for hormonal treatment including contraceptives, which can interfere with the endogenous endocrine milieu. This is even valid for a progestogen challenge test which may affect gonadotrophins blood levels, mainly LH. Induction of ovulation with clomid can have a similar effect. It is a well known fact that LH level is lower in cases of PCOS after induction of ovulation, because of the modulatory effects of progesterone. Accordingly, such hormonal treatment needs to be stopped for a month or two before conducting meaningful hormonal tests. This statement is not valid for patients on treatment for hyperprolactinaemia and hypo or hyperthyroidism, when a hormonal test is performed to monitor response to treatment and compliance.
  4. Certain blood tests need to be performed after overnight fasting such as fasting insulin and glucose levels, and the fasting lipid profile when investigating women for insulin resistance.
  5. Serum prolactin level can be affected even by the venepuncture used to collect the blood sample itself. Furthermore, marginal elevations of TSH are not uncommon, and usually settle with time. This can be a reflection of mild transient thyroiditis, or even laboratory errors due to cross reaction with other glycoproteins. In both these scenarios the blood test needs to be repeated.
  6. Heterotypic antibodies can affect immunoassays, and give spurious results. Repeating the test using a different method will be necessary.
  7. Marginally elevated hormonal results are inconclusive, and should be repeated. Examples of minor elevations of TSH and prolactin levels have already been given. Dynamic tests may be necessary to reach a definitive diagnosis in some cases. The mostly used test within a gynaecological setup is the short synacthen test (Alliance Pharmaceuticals), which is used for confirmation or exclusion of adrenal enzymatic deficiency. In this scenario 17-hydroxyprogesterone level is measured before and 60 minutes after a bolus dose of synthetic ACTH (synacthen). An exaggerated response of 17-hydroxyprogesterone indicates adult onset 21-hydroxylase deficiency. The result should be read according to the laboratory normal ranges, as there are overlaps between normal responses and heterozygous enzymatic deficiency. This test is also useful within a general endocrinology setup, as a test for adrenal insufficiency. Different cortisol cut-off levels have been used to indicate normal and abnormal responses after a synacthen test (5). Studies using 1 µg synacthen showed good reproducibility and higher sensitivity compared to the standard test with 250 µg as reported by Abdu and Clayton in 2000 (6). The same authors reported that post synacthen cortisol levels <400 nmol/L were diagnostic of adrenal insufficiency and Levels >600 nmol/L practically excluded the condition. Conversely, levels between 400 and 600 nmol/L were doubtful and should be interpreted in the light of the clinical data. The appropriate timing for the post synacthen blood test has also been investigated, and 60 minutes tests were found to be essential to avoid false results retrieved after 30-minute short synacthen tests (7).


Specific clinical examples

1.  Hormonal tests for delayed and precocious pubertal development include FSH, LH, and oestradiol. In these cases LH is more important than FSH to detect the initiation of puberty, as the upper and lower levels of the pulse increase with the progressive pubertal stages. In case of FSH, it is only the upper limit of the range which increases during the initial stages of puberty. It is sometimes difficult to ascertain whether normal pubertal development has started or not. This is the case with isolated premature breast development (thelarche). In such cases there is exaggerated FSH response to the GnRH challenge test. There is also a tendency toward lower basal and post GnRH test LH levels than in other girls with precocious puberty as shown by Della Manna et al in 2002 (8). In contrast, girls with central precocious puberty had higher LH peak levels after GnRH injection, compared to prepubertal girls as shown by the same authors. Nonetheless, this dynamic test is not usually necessary, as baseline LH assessments are adequate to make a diagnosis, and to monitor response after initiation of treatment. This is especially so as Pescovitz et al in 1988 (9) showed that girls with early central precocious puberty frequently had LH and FSH responses to GnRH stimulation similar to the FSH predominant response of girls with isolated thelarche. Measurement of testosterone and 17-hydroxyprogesterone blood levels is necessary in cases of precocious heterosexual puberty. This is mainly to detect 21-hydoxylase deficiency, which makes more than 90% of all cases of adrenal enzymatic deficiencies. In all cases bone age assessment is an integral part of the work scenario as discussed in Chapter 2. Furthermore, ultrasound scan examination can show increased uterine and ovarian volumes, in addition to breast enlargement in cases of normal or central precocious puberty (8).

2. The range of hormonal investigations in patients with anovulatory menstrual dysfunction depends on the presence of collateral symptoms. Basic investigations should include gonadotrophins, oestradiol, thyroid stimulating hormone (TSH), free thyroxine (T4) and prolactin estimations. In the presence of hyperandrogenic symptoms or signs, adrenal and ovarian precursors and androgens should be investigated. This should include morning blood tests for 17-hydroxyprogesterone, androstenedione, testosterone and sex hormone binding globulin (SHBG). This will allow calculation of the free androgen index (the level of testosterone divided by SHBG). With mild elevation of 17-hydroxprogesterone level, a synacthen test is necessary. With adult onset 21-hydroxylase deficiency, there is an exaggerated increase in the level of 17-hydroxyprogesterone. The result should be compared to the values set by the laboratory for normal, heterozygous and homozygous response, as mentioned before. Basic morning 17-hydroxyprogesterone blood levels should also be used to monitor adequate response to glucocorticoids replacement therapy in patients with 21 hydroxylase deficiency. In all cases of suspected anovulation, transvaginal ultrasound scan examination should be performed to ascertain the presence of polycystic ovaries, as mentioned before.

3.   The presence of insulin resistance may be explored in cases of PCOS, especially in obese women and those with history of gestational diabetes or family history of type II diabetes mellitus. Fasting insulin level on its own is not a satisfactory method to test for insulin resistance, because of the wide variability in the results, and an isolated fasting glucose measurement is utterly useless in this respect unless the patient is already diabetic. The oral glucose tolerance test (OGTT) is generally recommended for that purpose. However, for practical clinical purpose the fasting glucose /insulin ratio can be used instead, as it had good predictive value of insulin response during OGTT, and highly correlated with insulin sensitivity (10, 11). A fasting glucose (mg/dL) / insulin (µU/mL) ratio <4.5 had positive and negative predictive values of 87% and 94% respectively for diagnosing insulin resistance (11). So, it is more useful in excluding the diagnosis than confirming it, which is still important information for patients’ management. The homeostasis model assessment of insulin resistance (HOMA-IR) is another method which also proved to be useful in this respect (12). It correlated highly with estimates obtained by using the euglycaemic and hyperglycaemic clamps and fasting insulin concentration (13). A cutoff level >2.5 was considered to be diagnostic of insulin resistance in adults. HOMA-IR proved to be more reliable than fasting glucose/insulin ratio and quantitative insulin sensitivity check index as shown by Keskin et al in 2005 (14). A higher cutoff point of 3.16 was considered to be diagnostic for insulin resistance in adolescents by the same authors. HOMA-IR can be calculated by multiplying fasting insulin level in µU/mL by fasting glucose level in mmol/l, and divide the outcome by 22.5. The concept of homeostasis model assessment is extended to include HOMA-B which measures changes in pancreatic B-cell function. It is mostly used in research projects and is calculated by the equation (20 x insulin level in µU/mL) / (glucose level in mmol/L – 3.5). High HOMA-IR and Low HOMA-B were found to be independently associated with increased diabetic risk in a multiethnic cohort of women, reflecting the value of HOMA indexes in epidemiologic studies (15).

4.  Infertility hormone investigations depend on the age of the patients, and their mode of presentation. Patient with irregular menstruation should be investigated for anovulation as discussed in section 2 above. Patients with regular periods need only mid luteal serum progesterone assessment to document adequate ovulation. The length of the cycle should be taken into consideration, as day 21 progesterone assessment will give wrong results in patients with regular cycles longer than 28 days. The length of the follicular and luteal phases can be accurately documented by ultrasound scan monitoring of the cycle. Women in their mid or late 30s need to have their ovarian reserve assessed, irrespective of their menstrual pattern. A recent study by Gleicher et al (16) investigated the aetiology of premature ovarian aging, which is another name for reduced ovarian reserve. They reported that 16.2% of women had genetic, 38.8% autoimmune and 12.2% combined causes, whereas 33.8% were idiopathic with no identifiable cause. They concluded that premature ovarian aging and premature ovarian failure should be considered as continuum, and should be investigated accordingly. In a different context, it should be appreciated that such testing is not only necessary during infertility investigations, but is indicated also to screen women in their 30s who would like to delay childbearing.


Assessment of ovarian reserve

Follicle stimulating hormone

Over the years great efforts have been made to assess the number and quality of oocytes prior to assisted reproduction treatment cycles. The emphasis has now moved to include assessment of infertile women during basic hormonal investigations. Age as a single parameter is weak predictor of ovarian reserve and response to controlled ovarian stimulation with gonadotrophins (17). For many years, FSH assessment on the 3rd day of the cycle was the only available investigation to assess ovarian reserve. The value of high basal FSH level during assisted reproductive treatment cycles has been reflected by a high cancellation rate due to poor response. As a single indicator, it was shown to have better predictive value than age alone in this respect (18). On the other hand, chronological age was associated with lower implantation rates owing to poor oocyte quality (19, 20). Such reduced embryos implantation capacity with age has been related to increased aneuploidy rate (21). This pattern was shown by other studies as well (22, 23). At the same time, Levi et al in 2001 (24) related high FSH levels to increased rate of pregnancy loss, regardless of the woman’s age. They advised that patients should be counselled regarding the low probability of conception, and low live birth rates. This view has not been supported by other articles when young women with high FSH blood levels have been examined. This was shown by a study published by van Rooij et al in 2003 (20) which examined young women with high FSH blood levels, against women older than 40 years of age with normal FSH levels. The young group with high FSH had more cycle cancellation than the older group with normal FSH. Nevertheless, the young group with high FSH had better implantation rate per embryo, and higher ongoing pregnancy rate per cycle and embryo transfer, than the older age group with normal FSH levels. Poor responders in both groups had lower pregnancy rate than good responders.

It is an established observation that normal FSH levels are unreliable predictors of ovarian reserve. This can be a reflection of the following points:

1.  FSH is produced in pulses, and timing of the blood sample may coincide with the peak or the bottom of the pulse, giving different results. As discussed previously, the coefficient of variation (SD/Mean%) for a single FSH reading relative to multiple samples examined within 6 consecutive hours on the same day has been reported as 14.7 % by Abdel-Gadir et al in 1990 (3).

2.  There is intercycle variability of basal FSH blood levels, being higher in few but not all cycles. Normally cycling women over the age of 40 years who had a normal day 3 FSH level have 50% chance of having an elevated day 3 FSH level in a subsequent cycle (25).

3. FSH levels rise late in comparison to inhibin B and antimullerian hormone, following the decline in the number of follicles.

4.  FSH level can be negatively affected by the level of oestradiol in the same blood sample. A high oestradiol level >200 pmol/l on the 3rd day of the cycle carries similar bad prognosis as high FSH. This can be a reflection of rapid recruitment, with a follicle reaching a larger size than usual on day 3 of the cycle. This is usually seen in patients with polymenorrhoea and short follicular phases, as the follicle has started growing earlier during the luteal phase of the previous cycle. This is a reflection of the early rise in FSH level at that time of the previous cycle. Alternatively, the high oestradiol level follows recruitment of multiple intermediate size follicles by day 3 of the cycle; each producing its share of oestradiol. Transvaginal scan examination will help in making a diagnosis, and can differentiate between these two possibilities.

The following techniques have been used to improve the predictive value of FSH:

·      Repeat the test during successive cycles, which is time wasting for patients who need to start fertility treatment;

·      Take 3 samples within a short period of time on the same day to cover for the pulse variability;

·   The clomiphene citrate challenge test has been used as well. It entails FSH estimation on the 3rd day of the cycle, followed by 100 mg clomid every day from day 5 to 9 of the cycle. FSH level should be examined again on day 10 of the cycle. A level >10 IU/L indicates a bad prognosis with reduced ovarian reserve, as it is expected to be suppressed by the rising oestradiol level. Higher cut-off levels of FSH have been suggested in the literature. The test has no additional value in women who already showed high basal FSH levels. Few studies have shown significant intercycle variability of the clomiphene citrate challenge test results in the same patients (4, 26). Furthermore, the predictive or clinical value of the test was not superior to that of a basal FSH level in combination with antral follicles count (27).

·    Both intranasal and subcutaneous GnRH stress tests have been used to check the ovarian reserve. The injectable test entails measuring FSH blood levels before and one hour after a bolus dose of subcutaneous or intravenous dose of GnRH. An exaggerated increase in the level of FSH relative to the basal measurement indicates reduced ovarian reserve. The intranasal test entails 6 hourly administration of GnRH after measuring the basal levels of FSH and oestradiol. These same hormones should be measured 24 hours later. Similarly exaggerated increase in FSH level, and reduced oestradiol response are taken as markers of reduced ovarian reserve. These tests are hardly used in clinical practice nowadays, and will not be discussed any further.

It is evident that high early follicular phase FSH blood levels are more reliable than normal levels in predicting the ovarian reserve. Certain conditions should be excluded from this general statement. FSH blood levels can be increased physiologically during puberty, after using oral contraceptives and during lactation. Other conditions associated with elevated FSH levels include excessive smoking, during recovery from hypothalamic amenorrhoea, and after unilateral oophorectomy (28). Patients with hyperthyroidism can also have mild elevation of both FSH and oestradiol, as discussed in Chapter 11.

High FSH may be caused by direct ovarian problems related to several granulosa cells FSH receptors polymorphisms, but 2 of them located at codon 307 and 680 are more frequent. The amino acid asparagine (Asn) is replaced by serine (Ser) at position 680 (N680S), and threonine (Thr) is replaced by alanine (Ala) at position 307. The two most common allelic combinations are Thr307/Asn680 and Ala307/Ser680 as reported by Théron-Gérard et al in 2007 (29). Patients are usually classified as homozygous (Ser/Ser or Asn/Asn) or heterozygous (Asn/Ser). Homozygous patients for the Ser680/Ser680 variant (N680S) have longer follicular phase of the cycle and higher basal FSH blood levels (29, 30). The later being a natural compensation to stimulate normal follicular growth despite reduced FSH receptors sensitivity. These patients also need higher doses of gonadotrophins and produce lower levels of oestradiol during induction of ovulation, though they have normal number of follicles (31). Accordingly, Greb et al (31) suggested more studies to investigate the role of routine genotyping for N680S polymorphism to help with tailoring ovarian stimulation protocols to individual patient’s needs.

Mothers of familial dizygotic twins showed high basal FSH blood levels and pulse frequency, not related to the normal age-induced reduction of negative feedback mechanism, as reported by Lambalk et al in 1998 (33). Hypothalamic and/or pituitary neuroendocrine factors not related to GnRH were suggested as possible causes. There was no change in FSH pulse amplitude or response to GnRH stimulation, compared to control groups. At the same time, there were no differences in basal or GnRH induced LH levels, oestradiol, inhibin A or inhibin B levels between mothers of dizygotic twins and controls. Genetic studies so far failed to pinpoint the exact mutations which may lead to dizygotic twining. No linkage was found in correlation to mutations in the gene coding transmembrane FSH receptors (FSHR) as reported by Montgomery et al in 2001 (34). Furthermore, rare mutations in growth differentiation factor 9 (GDF9) may affect twining chances, but dizygotic twining is not associated with common variations in GDF9 (35).

False high FSH results can also follow the presence of heterotypic antibodies in a patient’s blood, which can interfere with the immunoassay. Spurious high FSH level was reported in a 33 year old women who had regular cycles by Cahill et al in 1992 (36). She proved to have normal levels when an alternative laboratory method was used. Such antibodies are not species specific. They can be found in patients regularly exposed to animals or their products. Blood transfusions and autoimmune diseases, especially the rheumatoid factor, have also been mentioned as possible causes.


Antral follicles count

To overcome the limitations of FSH in predicting ovarian reserve, many other parameters have been introduced as alternatives, or to complement its role in this respect. Transvaginal ultrasound scan examination is one such parameter. Ovarian size and antral follicles count during the early follicular phase have been used.

Reduction of ovarian size, irrespective of parity, after the age of 40 years has been documented by Andolf et al in 1987 (37). As expected, a substantial decline in ovarian size has also been noticed after the menopause (38). These points were taken further by Lass et al in 1997 (39), who assessed ovarian response to induction of ovulation with gonadotrophins in relation to ovarian volume. Women with ovaries <3.0 cc in volume needed higher dosage of gonadotrophins, had higher cancellation rate, and produced less follicles than women with larger ovaries. Accordingly, the authors recommended that assessment of ovarian size to be an integral part of the infertility evaluation. This concept was taken even further by Tomás et al in the same year (40). They found that patients with <5 antral follicle (2 - 5 mm in diameter) in each ovary had lower response to gonadotrophins than patients with more antral follicles. This parameter was found to be more sensitive than ovarian volume or patients’ age alone. Accordingly, they recommended antral follicles count, rather than ovarian volume, for counselling patients regarding their expected response to induction of ovulation. Using the same parameter, Frattarelli et al in 2003 (41) confirmed the high cancellation (41%) and low pregnancy (23%) rates in women with £ 4 antral follicles. The more important finding was that no antral follicles count absolutely predicted pregnancy or cycle cancellation. Nevertheless, a meta-analysis published by Hendriks et al in 2005 (42) showed a superior predictive value of antral follicles count over basal FSH toward poor response. These points put together support the recommendation made earlier by Bancsi et al in 2002 (43) that antral follicles count should be used together with other endocrine parameters for the assessment of ovarian reserve.

It is evident that patients with small ovaries and those with low antral follicles need higher gonadotrophins doses to secure a response, and to reduce the risk of cycle cancellation, during assisted reproduction treatment cycles. This should be taken into consideration especially with the increased cost involved. A step-down induction protocol with initial high dosage may be a better option to secure the initial recruitment of follicles before reducing the dose, if that proved to be necessary.


Inhibin B

Inhibin B is another product of the granulosa cells. A substantial decline in its blood levels with no significant changes in inhibin A or oestradiol has been reported in early perimenopausal women with regular cycles (44, 45). This was followed after a period of time, and changes in menstrual cyclicity, by marked fall in inhibin A and oestradiol levels, and a rise in FSH level, without any further changes in inhibin B. This is another sign that basal FSH and oestradiol levels are not reliable markers of the early decline in ovarian reserve. The value of inhibin B as a measure of ovarian reserve has been shown by many studies. Women with blood levels less than 45 pg/ml on day 3 of the cycle had lower oestradiol levels after induction of ovulation, higher cancellation rate and lower number of oocytes collected, in comparison to women with higher blood levels (46). This finding was contradicted by a study published by Corson et al in 1999 (47), who failed to find any clinical value for testing inhibin B. This was not a common observation, as many other articles confirmed its value in predicting ovarian response to induction of ovulation. On the other hand, it has the same drawback as basal FSH, because of the physiological variability during the different stages of the same menstrual cycle. Accordingly, it must be assessed on days 2 or 3 of the cycle.

In a different approach, Kwee et al in 2004 (48) tested the intercycle variability of basal inhibin B and oestradiol levels before and after injecting 300 IU of recombinant FSH subcutaneously. There were no significant differences in the increment of either hormone, when the test was performed during different cycles. Because of the reproducibility of the results, they considered this test to be more reliable than basal FSH assessment and the clomiphene citrate challenge test. Both tests gave significantly variable results when performed during different cycles. Nevertheless, like all other invasive dynamic tests, it did not attract much interest, and has not commanded popular use in routine clinical setups.


Antimullerian hormone

Antimullerian hormone (AMH) is produced by the granulosa cells in close proximity to the oocytes, and by few cells surrounding the antrum of 4 – 6 mm antral follicles. These cells continue producing AMH till further recruitment and development of the follicles into dominant ones or their ultimate atresia. Human AMH is a dimeric glycoprotein with a molecular weight of 140 kdaltons, and it is a member of the transforming growth factor beta (TGF-b) family of growth and differentiation factors (49). At the ovarian level, AMH is involved with follicular steroidogensis, and regulation of ovarian activity. It reduces granulosa cells aromatase activity and the number of LH receptors in cultured granulosa cells, and regulates testosterone production by the theca cells (50). Reduction of the aromatase enzymatic activity affects the intraovarian androgen / oestrogen ratio, hence oocyte function. A high ratio leads to follicular degeneration, whereas a low ratio causes germinal vesicles rupture (51-53). On the other hand, AMH reduces follicular sensitivity to FSH. Furthermore, oocytes upregulate AMH expression in granulosa cells, depending on their developmental stage (54). This led to the hypothesis that oocytes in growing follicles control primordial follicles recruitment and development through the inhibitory effects of AMH.

AMH level has a direct correlation with the number of antral follicles; hence it is a good marker of ovarian reserve. It was more consistently correlated with the degree of follicular depletion than inhibin B and antral follicles count, even in young patients with high FSH levels (55). Furthermore, it has relatively stable serum concentration within one year in premenopausal women, and can be measured with good reproducibility using commercial kits (56). Additionally, blood levels do not change during the menstrual cycles, and the test can be performed at any time irrespective of the stage of the cycle. A meta-analysis published by Broer et al in 2009 (57) showed that AMH is at least as accurate as the antral follicle count in predicting poor response and non-pregnancy during IVF treatment cycles. However, most of the present data stress the fact that AMH is the best currently available test for ovarian reserve. Nevertheless, like all other parameters it does not reflect the quality of the eggs, which is controlled by the patient's age. Furthermore, it was not a better predictor of pregnancy when compared to the other available tests (49). It is interesting that a recent report showed that AMH levels were affected by ethnicity. Its blood levels were found to be 25.2% and 24.6% lower in Afro-Caribbean and Hispanic women respectively, when compared to White women, after adjusting for age, body mass index and smoking (58). This raises the need to establish different cut-off levels for different ethnic groups, which allows better utilisation of the test results in modern day cosmopolitan societies.

A recent publication showed that AMH level was significantly reduced during oral contraception use, with a trend toward lower levels during metformin therapy (59). The question here would be what is the recovery time to normal levels once such mediation is stopped? This is necessary to know if patients who come off the pill need to have their ovarian reserve established. The same authors came to the conclusion that AMH is an accurate marker of antral follicles pool in WHO-2 / PCOS women, but its measurement is not likely to be helpful in the management of these patients (59). This may not be true when dealing with PCOS cases, as patients with high AMH blood levels (≥ 7.7 ng/ml) are less likely to respond to ovarian drilling (60). Accordingly, AMH can be used together with LH to select PCOS patients who are more likely to respond to this procedure.


Figure 46 shows a small right ovary with 3 antral follicles (2-5 mm in diameter) marked by asterisks on day 3 of the cycle. 

Figure 47 shows a very small left ovary with no antral activity at all. These two pictures belong to a 24 years old woman who had regular monthly cycles. Her day 3 FSH level was 7.3 IU/L, but her AMH level was only 4.2 pmol/L. She needed 450 IU of human menopausal gonadotrophin every day for 13 days to produce 3 follicles. Three oocytes were collected and injected with her husband’s sperm, during intracytoplasmic sperm injection treatment cycle. Two oocytes were fertilized, and were replaced on day 2 of the procedure. She conceived a single intrauterine pregnancy and delivered a healthy baby at term. This case reflected the unreliability of a normal day 3 FSH blood level in predicting the ovarian response during an assisted reproduction treatment cycle. It also showed the value of both transvaginal ultrasound scan examination and AMH in this respect.

Figure 48 shows two small ovaries with a single antral follicle in the left one. This 37-year old patient had regular menstrual cycles with normal day 3 FSH blood levels (<10 IU/L). Her AMH was only 1.73 pmol/L. She failed to respond to controlled ovarian hyperstimulation with high doses of gonadotrophins injections. It was a further example of how normal FSH blood levels failed to predict her ovarian response.


Other clinical conditions

Human chorionic gonadotrophin is another glycoprotein hormone which is used for the diagnosis of normal and abnormal pregnancies, and as a tumour marker. The total molecule cross-reacts with other glycoproteins due to the similarity of their a chains. Accordingly, the ß subunit is used because of its specificity. The three more common pathological conditions related to high human ßhCG blood levels are ectopic pregnancies, gestational trophoblastic tumours, and ovarian teratomas.


Ectopic pregnancies

With ectopic pregnancies, a positive ßhCG test is associated with an empty uterus, and occasionally a positive ultrasonic identification of an ectopic mass outside the uterine cavity. Transvaginal ultrasound scan examination can give a very high positive predictive value and helps with the diagnosis in 90% of the cases, when performed by experienced personnel. An empty uterus with ßhCG level of 1500-2000 IU/ml indicates an ectopic pregnancy, as an intrauterine sac should be seen at such levels. The most common ultrasound finding is the presence of a mass beside the uterus on the same side as the corpus luteum, in almost 80% of the cases. Viable ones with fetal heart activity are the least common type. Depending on the stage at diagnosis, variable amounts of fluid with floating particles can be seen in the pouch of Douglas indicating intra-peritoneal leak of blood from the ectopic pregnancy. ßhCG blood levels are usually lower than those expected for the period of amenorrhoea, and the rate of increase over a period of time is also lower than expected for normal intrauterine pregnancies. The level of ßhCG usually doubles, or at least increases by 1.66 fold every 2 – 3 days in 85% of normal intrauterine pregnancies. In recent years, serum progesterone has been used as an extra parameter for the diagnosis of ectopic pregnancies, as it has a constant blood level during the whole of the first trimester. A blood level ≥ 25 ng/ml is usually diagnostic of an intrauterine pregnancy. It excludes an ectopic pregnancy with 97.4% certainty, in spontaneously conceived cycles. Conversely, a level < 15 ng/ml has been reported in 81% of ectopic and 93% of abnormal intrauterine pregnancies. It is also seen in 11% of normal intrauterine pregnancies. It is evident that the higher the progesterone blood level, the stronger its negative predictive value will be in excluding a diagnosis of ectopic pregnancy. Accordingly, all three parameters (clinical, scan examination and hormonal tests) should be combined to reach a diagnosis. The use of hormonal tests in the follow up of cases managed conservatively, and in the management of pregnancies of unknown location has reduced the need for unnecessary surgery in many cases.


Gestational trophoblastic tumours

Very high blood ßhCG levels are suggestive of gestational trophoblastic tumours (GTT), which can be benign or cancerous. The group includes molar pregnancy, persistent trophoblastic disease, placental site trophoblastic tumours (PSTT), and choriocarcinomas. The intrauterine contents can show a snowstorm appearance in cases of a complete mole, during transvaginal ultrasound scan examination. Conversely, partial molar pregnancy may look almost normal during ultrasound scanning. The ovaries are usually enlarged with multiple corpora luteal cysts. Serial estimations of ßhCG levels are used for monitoring response to treatment. Patients usually need other investigation modalities including X-ray chest, CT scans, MRI, and even lumbar puncture. A complete account about this subject is beyond the remit of this chapter.

The use of ßhCG as a tumour marker is not limited to trophoblastic diseases, as the level can also be elevated in patients with other gynaecological neoplasms. Teratomas, dysgerminomas and germinal cell tumours are such examples. In these cases, the diagnosis is usually made with the help of ultrasound scan examinations or MRI. Paradoxically, high ßhCG levels have been reported in patients with vulvovaginal and cervical cancers. The prognosis was found to be worse for patients with higher than normal ßhCG levels (61). Other malignant and benign non gynaecological conditions were also associated with high blood ßhCG levels. The list included melanomas, colonic, breast and renal tact carcinomas (62-64). Despite the high ßhCG levels, trophoblastic cells were not present in biopsies retrieved from these tumours. Benign conditions associated with high ßhCG levels included liver cirrhosis, duodenal ulcer, and inflammatory bowel disease. Accordingly, these conditions should be taken into consideration when a high ßhCG level is detected in non pregnant women. However, ßhCG is not a recognised method for the diagnosis or monitoring of any of these conditions. 

Many other hormones can also be used electively for detecting pelvic tumours, or are chance findings in correlation to certain gynaecological problems. AMH can be a biomarker of increased breast cancer risk (65), and a marker of granulosa cell tumours which also produce very high levels of oestradiol. This can cause long periods of amenorrhoea followed by excessive breakthrough uterine bleeding. Such tumours also result in precocious isosexual pubertal development. On the other hand, case reports of parasitic ovarian leiomyomas presenting with endocrine related problems have been published. Hyperandrogenic skin and hormonal changes have been reported in postmenopausal women with ovarian leiomyoma due to theca cell reaction (66), and hilus cells hyperplasia (67). Similarly a case report of secondary amenorrhoea caused by high inhibin B level secondary to a parasitic ovarian leiomyoma has been published by Abdel-Gadir et al in 2010 (68). This patient resumed menstruating within one month after excision of the ovarian fibroid.


Summary

This chapter has been written to complement the information given in the previous chapters, with gynaecologists in mind. It is meant to give a general overview of the different endocrine investigations used in gynaecological practice. In many cases clinical history, physical examination and imaging techniques provide the necessary information to reach a diagnosis, with minimal need for elaborate hormone tests. Special efforts have been made to emphasise the strength and limitations of the common hormone tests used in gynaecological practice. The need for proper timing of blood samples collection within the day, and in relation to the stage of the menstrual cycle has been stressed. Any medication taken by the patient should be taken into consideration when requesting a hormone test, and during the interpretation of the results. Such tests should be used to complement the clinical impression, and to extend the clinical judgement necessary for the management of any particular case. Many results can be spurious for different reasons, and may not agree with the general clinical picture. In such cases the examination should be repeated for confirmation purpose, using a different method if possible, before changing the patient’s management plan. Adequate knowledge of the relevant basic Reproductive Endocrinology will allow better utilisation of the available diagnostic means, facilitates proper patients’ care, and eliminates misuse of resources.

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