37

Disorders of the hypothalamic-pituitary-gonadal axis

37

Disorders of the hypothalamic-pituitary-gonadal axis

37

Disorders of the hypothalamic-pituitary-gonadal axis

37

Disorders of the hypo­thalamic-pituitary-gonadal axis

  37 Disorders of the hypo­thala­mic-pituitary-gonadal axis

Lothar Thomas

37.1 Organization of the hypothalamic-pituitary-gonadal axis

The hypothalamic-pituitary-gonadal axis is a tiered and linearly organized set of endocrine tissues that is dedicated to the regulation and support of reproductive activity /1/. This axis consists of a small subset of hypothalamic neurons that express the decapeptide gonadotropin-releasing hormone (GnRH). The hormone also known as luteinizing-hormone-releasing hormone (LHRH) is delivered to the anterior pituitary via the hypophyseal portal circulation where it binds to the GnRH receptor on the surface of gonadotropic cells, triggering the synthesis and secretion of the gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH) /1/.

37.1.1 Kisspeptin in the regulation of GnRH

The hypothalamic-pituitary-gonadal axis is regulated by GnRH and stimulated by kisspeptins. The gene KISS 1 encodes a family of peptides called kisspeptins which bind to a G protein-coupled receptor (GPR54) /2/. Kisspeptins and its receptor are expressed in the arcuate nucleus and the anteroventral periventricular nucleus of the forebrain. Kisspeptins are expressed as a 145 amino-acid protein that is enzymatically cleaved into a 54 amino-acid peptide, known as kisspeptin-54 or metastasin, as well as shortened peptides of 14, 13, or 10 amino acids. Kisspeptins stimulate the release of GnRH from hypothalamic neurons after binding to its receptor GPR54. GnRH stimulation induces the release of FSH and LH. The gonads respond to gonadotropins by secreting sex steroids, which then feed back to regulate the activity of kisspeptin neurons. These neurons act as central processors for relaying signals from the periphery to GnRH neurons.

37.1.2 Hypothalamic secretion of GnRH

The pulsatile release of GnRH by hypothalamic neurons is required for adequate gonadotropin production by the pituitary. Continuous secretion of GnRH uncouples the gonads from pituitary regulation and leads to decreased synthesis of gonadotropins and to hypogonadism. This strategy forms the basis for the clinical use of GnRH analogs to treat female infertility.

The type of GnRH pulsatility has been shown to be important /2/:

  • In women; both the amplitude and frequency of GnRH pulses are tightly regulated over the course of the reproductive cycle. Different pulse frequencies of GnRH affect the production of gonadotropins. Rapid GnRH pulsatility has been shown to promote the synthesis of LH, while slower GnRH pulsatility favors the production of FSH.
  • In male; fluctuations in GnRH pulsatility play a less significant role in reproductive function.

Regulatory feedback mechanism

Gonadal steroids play an important role in feedback regulation of hypothalamic secretion of GnRH:

  • In men, increasing levels of testosterone exert a negative feedback effect on the release of GnRH from the hypothalamus
  • In women, the feedback effects of gonadal steroids are more complex and depend on the stage of the reproductive cycle. In the preovulatory period, the ovaries respond to GnRH stimulation mainly by producing estradiol. Increasing estradiol levels then feed back to regulate the activity of kisspeptin neurons, inhibiting KISS1 expression in the arcuate nucleus and the anteroventral periventricular nucleus (AVPV). The inductive effect of sex steroids on KISS1 expression in the AVPV contributes the preovulatory LH surge which is the definitive trigger for ovulation. Progesterone exerts a negative feedback effect upon hypothalamic GnRH release and therefore LH synthesis during the post ovulatory phase.

37.1.3 Gonadotropin function in women

Luteinizing hormone (LH)

LH stimulates the production of androgens by the thecal cells that surround the growing ovarian follicle. During the terminal stages of follicular growth, LH also drives the production of progesterone from the granulosa cells of the preovulatory follicle. The LH stimulated biosynthesis of androgens is catalyzed by cytochrome P450c17, an enzyme with both 17-hydroxylase and 17,20 lyase activity. Both types of activity are required for the synthesis of androstenedione, which is then either converted to testosterone by 17β-hydroxy steroid dehydrogenase or to estrone by aromatase (cytochrome P450arom). In women with polycystic ovary syndrome, the conversion of androgens to testosterone by the theca cells is increased.

Follicle stimulating hormone (FSH)

FSH regulates the activity of aromatase enzymes of ovarian granulosa cells and regulates the conversion of thecal androgens to estradiol. Depending on the ratio of LH to FSH, either more estrogens or more androgens are produced preferentially. If there is an excess of LH, androgen synthesis predominates.

37.1.4 Gonadotropin function in men

LH stimulates the synthesis of testosterone and sex hormone binding globulin. FSH binds to receptors on the surface of Sertoli cells and acts in combination with testosterone to promote the proliferation of spermatogenesis as well as the meiosis and post mitotic development of germ cells.

37.1.5 Biosynthesis of sex steroids

The biosynthesis of the sex steroids is shown in Fig. 37.1-1 – Biosynthesis of sex steroids. The principal substrate for sex steroid synthesis are cholesterol esters. The initial, rate limiting step in the biosynthesis involves a three stage side chain cleavage. The crucial enzyme, cytochrome P450ssc or 20,22 desmolase, is driven by LH /3/.

Adrenal precursor androgen steroidogenesis

The adrenal cortex produces the three steroids dehydroepiandrosterone (DHEA), androstendione and testosterone /4/ in the order of production shown in Fig. 37.1-1 – Biosynthesis of sex steroids:

  • Cholesterol is metabolized to pregnenolone by P450scc cleaved enzyme, located in the mitochondria, and controlled by anterior pituitary hormones ACTH, FSH and LH.
  • Pregnenolone is converted to the C21 steroid 17-hydroxypregnenolone (also known as 17OH-pregnenolone and 17α-hydroxy pregnenolone) by hydroxylation at the C17α position in presence of 17α-hydroxylase (CYP17A1) contained in the adrenal and gonads. 17-hydroxy pregnenolone is in part released into the circulation and in part converted to androstenedione in the presence of 3β-hydroxy steroid dehydrogenase (3β-HSD).
  • 17-Hydroxy pregnenolone is converted into DHEA by the 17,20 lyase reaction catalyzed by cytochrome P450c17. This enzyme catalyzes both the 17α-hydroxylation reaction converting pregnenolone to 17-OH pregnenolone and the 17,20-lyase reaction converting 17-OH pregnenolone to DHEA. The sulfation of DHEA into its more stable sulfate ester DHEAS is catalyzed by the enzyme hydroxy steroid sulfotransferase, commonly known as DHEA sulfotransferase. DHEAS can be converted back into DHEA by steroid sulfatase.

Effects of FSH

FSH regulates the maturation of ovarian follicles during the follicular phase of the menstrual cycle and, during the preovulatory phase, of the dominant follicle in particular, which produces increasing amounts of estrogens.

Effects of LH

The preovulatory surge in LH secretion triggers ovulation and luteinization of the follicle, which leads to the increased production of progesterone. After ovulation, both progesterone and estradiol interact with the hypothalamus and pituitary as part of a negative feedback inhibition, thus leading to a return to basal concentrations of FSH and LH.

Effects of estrogens

Estrogens are formed by aromatization of androgens in a reaction that involves three hydoxylation steps. The aromatase enzyme complex includes a P450 mixed function oxidase. 17β-estradiol (E2) is primarily formed from testosterone. Estradiol is oxidized to estrone (E1) in the liver and may be further hydrated to estriol (E3). It is believed that LH stimulates androgen production within the theca cells. Androgens are aromatized within the theca cells but are also available to the granulosa cells for aromatization to estrogens. Estrogens of the theca cells origin would be the major source of circulating estrogens. Fig. 37.1-1 – Biosynthesis of sex steroids.

Estrogens, in particular 17β-estradiol, have a number of actions:

  • Activation of endometrial proliferation
  • Induction of progesterone receptor expression
  • Feedback inhibition of GnRH secretion and maintenance of basal blood levels of FSH and LH during the preovulatory phase
  • Just prior to ovulation, the dominant follicle produces so much 17β-estradiol that the negative feedback exerted by 17β-estradiol on LH and FSH transiently transforms into a positive feedback mechanism, resulting in a surge in LH secretion in particular.

Effects of progesterone

Following ovulation, the endometrium undergoes secretory transformation due to the effects of progesterone and provides the embryo with optimal conditions for implantation.

Sex hormones in childhood

In childhood, “secondary gonadal insufficiency” is physiologically present because the hypothalamic-pituitary-gonadal axis has been in a state of functional rest since the neonatal period. Only basal concentrations of gonadotropins and sex steroids are produced due to the absence of pulsatile GnRH secretion.

Sex hormones in puberty in girls

The onset of puberty is regulated by a network of genes that stimulate kisspeptin leading to inductive effects on GnRH neurons. Evidence to date suggests that kisspeptin signaling is an essential component of the hypothalamic-pituitary-gonadal axis and is consistent with the hypothesis that kisspeptin neurons in the forebrain act as gatekeepers to awaking of reproduction at puberty /2/.

In girls puberty begins with increased secretion of gonadotropins. At first, pulsatile GnRH secretion occurs only during sleep, leading to an increase in LH. However, LH fluctuations are not detectable during the daytime. Later in puberty, increased pulsatile activity is also observed in the daytime.

Follicular cysts develop in the ovaries and polycystic ovaries may be observed even during the early stages of puberty. Under the influence of 17β-estradiol , development of the internal (ovaries, uterus) and external (genital enlargement, breast development) sex characteristics occurs. Endometrial proliferation also occurs, followed by the first uterine bleeding (menarche). The mean age of menarche is 12.8 years. The menarche is usually a hormone withdrawal bleed because ovulation has not yet taken place and therefore, in the absence of corpus luteum formation, the endometrium has not yet been subjected to secretory transformation. Serum 17β-estradiol concentrations of greater than 30 ng/L (110 pmol/L) are required to produce bleeding. Menarche is followed by 5–7 years of relatively long cycles, and then there is increasing regularity as cycles shorten to reach the usual reproductive age pattern.

The interplay between FSH, LH, 17β-estradiol, and progesterone plays an important role in the regulation of the female reproductive cycle. The behavior of these hormones throughout the cycle is shown in Fig. 37.1-2 – Serum levels of FSH, E2, LH, and progesterone synchronized to the day of the LH peak.

37.1.5.1 Female reproductive endocrinology

The ovary is unique among the organs of the endocrine system in that each month a completely new endocrine structure, the Graafian follicle, develops from a microscopic primordial follicle. Under the influence of LH, the androgens androstenedione and testosterone are synthesized in the theca cells localized outside the basement membrane of the maturing follicle. After the androgens diffuse through the basement membrane into the granulosa cell layer of the follicle, aromatization to 17β-estradiol and, to a lesser extent, estrone takes place under the influence of FSH. As the follicle matures, the serum 17β-estradiol concentration increases from basal levels of approximately 30 ng/L (110 pmol/L) to preovulatory concentrations of 200–600 ng/L (0.7–2.2 nmol/L) /5/. The LH peak is triggered by high 17β-estradiol concentration and ovulation occurs approximately one day after this peak (Fig. 37.1 -2 – Serum levels of FSH, E2, LH, and progesterone synchronized to the day of the LH peak).

Simultaneously with the LH peak, the FSH concentration also increases. The progesterone level, which in the preovulatory phase is less than 1 μg/L (3.2 nmol/L), rises continuously with the formation of the corpus luteum, increasing to a concentration of 10–20 μg/L (33–66 nmol/L) by the 8th day after the LH peak. During the luteal phase, 17β-estradiol concentration shows a pattern similar to that of progesterone; however, the values do not usually exceed 100–200 ng/L (370–730 pmol/L).

If pregnancy does not occur, the concentrations of 17β-estradiol and progesterone decrease again to basal levels with the lysis of the corpus luteum. In the event of pregnancy, the corpus luteum graviditatis continuously synthesizes large amounts of progesterone under the influence of hCG, which rises from day 8–10 following conception. The progesterone concentrations are around 10–30 μg/L (33–100 nmol/L) by the 8th gestational week. Subsequently, the placenta takes over the function of the corpus luteum to maintain the pregnancy.

37.1.5.2 Perimenopause and menopause

Perimenopause

The years before menopause that encompass the change from normal ovulatory cycles to cessation of menses are known as the transitional perimenopause period. This period usually lasts five to ten years and is marked by irregularity of menstrual cycles /6/. Anovulatory cycles become more frequent during the fourth decade of life and the length of the menstrual cycle increases five to ten years prior to menopause, primarily due to prolongation of the follicular phase. Perimenopause is characterized by a significant rise in FSH (above 20 IU/L), a normal LH concentration, and a slight rise in 17β-estradiol. Because the corpus luteum may still develop and function, the risk of an unplanned pregnancy is still present. Only when the FSH concentration exceeds 20 IU/L and the LH concentration exceeds 30 IU/L is pregnancy no longer possible.

Menopause

Menopause is defined retrospectively by a 12 month interval free of uterine bleeding. The mean age of menopause in Northern Europe and North America is currently 52 years.

Shortly after menopause, no functional ovarian follicles exist. In the first 1–3 years following menopause, there is a 10–20-fold increase in FSH and a 3-fold increase in LH /6/. This is conclusive evidence of ovarian failure. Gonadotropin levels show a slight decline in subsequent years, however, levels are still higher than those present during the reproductive years. The rise in FSH is more marked than that of LH due to differences in the half life of the hormones (LH 20 min., FSH 3–4 h). The consequence of the arrest of estradiol secretion by the ovary is that the concentration of serum 17β-estradiol decreases from values of at least 80 ng/L in premenopausal women to an average of 4.2 ng/L after menopause with 95% of women having 17β-estradiol levels below 9.2 ng/L. All estrogens and androgens in postmenopausal women are synthesized locally in peripheral target tissues by tissue specific steroidogenic enzymes according to the intracrine process /8/.

In the menopause, androstenedione is the main hormone secreted by the ovary, however, the majority is derived from the adrenal gland. The serum concentration decreases to around half of the level seen during the reproductive years. Although more testosterone is produced post menopausally the serum testosterone concentration is around 25% lower than before menopause. Dehydroepiandrosterone (DHEA) and its sulfate originating by the adrenal glands are about 70% less than concentrations seen in young women. A large series of problems are associated with the deficiency of sex steroids and decline in DHEA accompanying menopause, osteoporosis being the best defined example. For production rates of sex hormones and their serum levels refer to:

37.1.6 Sex hormone effects in males

As with girls, pubertal development in boys begins with the re-awaking of pulsatile GnRH secretion and the subsequent secretion of gonadotropins, initially with a predominantly FSH output. This then reverses to primarily nocturnal LH pulses, extending to the whole daytime in adulthood. A circadian rhythm of testosterone exists, with a secretory peak in the morning and a nadir in the late afternoon and evening. Testosterone plays a key role in the development of male habitus, the promotion of musculoskeletal development, and the growth of pubic and axillary hair /3/. By the end of puberty, growth ceases and epiphyseal fusion occurs through mechanisms not yet completely understood and mediated via aromatase conversion of testosterone to 17β-estradiol (Fig. 37.1-1 – Biosynthesis of important sex steroids).

The testes are glands with dual functions:

  • Spermatogenesis, the prerequisite for reproduction, takes place in the seminiferous tubules
  • Production of androgens (testosterone, androstenedione, dehydroepiandrostenedione, dehydroepiandrosterone sulfate) in the Leydig cells.

Spermatogenesis and androgen production are regulated by the secretion of gonadotropins in response to active GnRH stimulation throughout a man’s life. Prostatic function is maintained by local conversion of testosterone to dihydrotestosterone (Fig. 37.1-1 – Biosynthesis of important sex steroids). Adult men produce 3.7 ± 2.2 mg testosterone per day. The critical enzyme involved in testosterone synthesis is the LH-dependent enzyme 20,22-desmolase (cytochrome P450scc).

37.1.6.1 Gonadotropic hormones in aging men

In older men, adaptations occur in the gonadotropic system that lead to changes in hormonal and reproductive function /7/. This generally leads to reduced testosterone synthesis. Hypoandrogenemia is thought to be responsible for muscle weakness, sarcopenia, osteopenia, decreased psychological problems, erectile dysfunction, systolic hypertension, carotid artery wall thickness, abdominal visceral fat mass, insulin resistance, low HDL concentrations, postprandial somnolence, impaired quality of life, depressive mood, diminished working memory, and decreased executive cognitive function /7/.

Testosterone depletion in aging men has been documented in /7/:

  • The 15-year, longitudinal New Mexico Aging Process Study, according to which testosterone concentrations in men over the age of 60 years decline by 1.1 μg/L (3.8 nmol/L) every 10 years
  • The Baltimore Longitudinal Study of Aging, which showed an annual decrease of 4.9 nmol testosterone/nmol sex hormone binding globulin
  • The Massachusetts Male Aging Study, a cohort study that evaluated an annual decline in free testosterone of 0.8–1.3%.

Data suggest the following etiological factors for testosterone depletion in older men /7/:

  • Ageing attenuates testis responses to LH
  • Age diminishes hypothalamic GnRH secretion
  • Age impairs testosterone mediated negative feedback on brain GnRH secretion and/or pituitary LH secretion.

Literatur

1. Bliss SP, Navratil AM, Xie J, Roberson MS. GnRH signaling, the gonadotrope and endocrine control of fertility. Front Neuroendocrinol 2010; 31: 322–40.

2. Duncan HM, Clifton DK, Steiner RA. Minireview: Kisspeptin neurons as central processors in the regulation of gonadotropin-releasing hormone secretion. Endocrinology 2006; 147: 1154–8.

3. Puttabyatappa M, Padmanalkan V. Developmental programming of ovarian functions and dysfunctions. Vitam Horm 2018; 107: 377-422.

4. Belchetz PE, Barth JH, Kaufman JM. Biochemical endocrinology of the hypogonadal male. Ann Clin Biochem 2010; 47: 503–15.

5. South SA, Yankov VI, Evans WS. Normal reproductive neuroendocrinology in the female. Endocrinol Metab Clin North Am 1993; 22: 1–28.

6. Speroff L. The perimenopause: definitions, demography, and physiology. Obstet Gynecol Clin North Am 2002; 29: 397–410.

7. Veldhuis JD. Ageing and hormones of the hypothalamo-pituitary axis: gonadotrope axis in men and somatotropic axes in men and women. Ageing Res Rev 2008; 7: 189–208.

8. Santoro N. Update in hyper- and hypogonadotropic amenorrhea. J Clin Endocrinol Metab 2011; 96: 3281–8.

37.2 Ovarian dysfunction

Disorders of ovarian function are a major reason for conducting endocrine investigations and functional tests in addition to clinical examination. Ovarian dysfunction often occurs sporadically or during periods of intense physical activity (competitive sports) or psychological stress (marital problems, exams). Menstrual disturbances are common during perimenopause and the early years of reproductive life. For investigations and functional tests refer to:

Female patients present for endocrine investigations because of the following symptoms and concerns:

  • Menstrual abnormalities i.e., too few menses (oligomenorrhea), absence of menses (amenorrhea) or a decrease in time interval between menstruation (polymenorrhea)
  • Anovulatory cycles and corpus luteum insufficiency. Both disorders are especially relevant in women who desire pregnancy. These disorders are detectable only by basal temperature controls or by hormone determinations.
  • Signs and symptoms of hyper androgenism. In conjunction with oligomenorrhea, these patients frequently have polycystic ovary syndrome. This ovarian peculiarity is detectable by sonography; LH/FSH ratio ≥ 2, and elevated androgen level. Refer to Section 34.6.5.2 – Polycystic ovary syndrome.
  • Infertility; it represents the most frequent reason for consultation. According to estimates, 10–15% of couples of reproductive age suffer from infertility. Even though the causes are in about equal parts attributable to both partners, hormonal diagnostic investigations and hormone therapy in women are also frequently performed in cases of infertility due to underlying adrogenital syndrome causes.
  • Climacteric symptoms. They are the presenting complaint primarily at 45–55 years of age.
  • Uterine bleeding occurring outside the reproductive age range (i.e., either before puberty or after the menopause).
  • Signs of precocious puberty during childhood.

Blood collection

In the presence of a menstrual cycle, the blood collection for hormone determination should be performed between the 3rd and 7th day of the cycle since the results in this time period are most likely to be comparable.

37.2.1 Female infertility

A couple that has never conceived despite at least 1 year of unprotected coitus experiences primary infertility /1/. Infertile couples with previous conception have secondary infertility /2/. The causes are in about equal parts attributable to both partners and should be investigated in both partners.

37.2.1.1 Cycle disorders

Ovarian dysfunction is the most common cause of female infertility. A review of infertile patients presenting to a university women’s hospital /1/ revealed that more than 80% were eumenorrhoeic, while 12% and 4% were oligomenorrhoeic and amenorrheic, respectively. Approximately 60% of the eumenorrhoeic, infertile females displayed no endocrine peculiarities during the early follicular phase. Elevated androgen concentrations were measured in approximately 30%.

Apart from hyperandrogenemia, hyperprolactinemia is another important cause of infertility. Refer to Tab. 36-4 – Clinical symptoms of hyperprolactinemia.

Laboratory findings in menstrual disturbances are shown in Tab. 37.2-3 – Clinical and laboratory findings in menstrual disorders.

Simultaneous determination of prolactin, FSH, LH, 17β-estradiol, testosterone, DHEAS, and TSH is recommended whenever eumenorrhoeic, oligomenorrhoeic, or amenorrhoeic patients with infertility present for the first time.

In the presence of a menstrual cycle, the hormone determination should be performed between the 3rd and 7th day of the cycle since the results in this time period are most likely to be comparable.

Elevated testosterone and/or DHEAS levels suggest that the infertility is due to hyperandrogenism. If testosterone and/or DHEAS as well as LH are elevated, underlying polycystic ovary syndrome (PCOS) should be suspected.

Cycle disorders may be present despite regular menstruation and unremarkable hormonal findings. It is therefore recommended to monitor at least one cycle (cycle monitoring) using repeat sonographic and endocrine examinations (Fig. 37.2-1 – Example of cycle monitoring in a 28-day cycle).

During the first half of the cycle, the increase in serum 17β-estradiol concentration is usually proportional to follicular growth. In untreated cycles, preovulatory 17β-estradiol levels reach 200–600 ng/L. In stimulated cycles during treatment with clomiphene, human menopausal gonadotropin (hMG), or FSH, 17β-estradiol increases up to 2,000–3,000 ng/L are not unusual.

Progesterone determinations during the late follicular phase may be useful when premature luteinization of follicles is suspected. LH determinations in the late follicular phase are used to identify the endogenous LH increase. About 30 h after LH level starts to rise in serum and 24 h after it starts to rise in urine, ovulation occurs.

In addition to hormone determinations during the follicular phase, evaluation of the luteal phase after ovulation by means of repeated progesterone determinations is also recommended. Two determinations should be performed, approximately 5 and 10 days after ovulation. If both progesterone levels are ≥ 10 μg/L, corpus luteum insufficiency is unlikely. Approximately 14 days after ovulation, pregnancy may be detected by means of hCG determination. However, in stimulated cycles, it must be remembered that hCG injected for induction of ovulation and possibly for luteal phase support may still be detectable at serum levels above 5 IU/L for more than a week after the last administration. In such cases, only a new rise in hCG is a reliable indicator of endogenous hCG production. A very early abortion (“biochemical pregnancy”) can be misinterpreted by a transient rise in hGC, possibly in conjunction with delayed onset of menses.

37.2.1.2 Nonclassical congenital adrenal hyperplasia

Congenital adrenal hyperplasia (CAH) is one of the most common congenital endocrine disorders and has an autosomal recessive mode of inheritance /3/. V281L is the most frequent mutation of the CYP21A2 gene.

The mutation in the gene which encodes 21-hydroxylase, causes disruption of aldosterone and cortisol synthesis, which leads to androgen excess (Fig. 34.1-2 – Biosynthesis of adrenocortical steroids).

Depending on the severity of the deficiency, three phenotypes can be distinguished (Tab. 34.6-2 – Enzyme deficiencies in congenital adrenal hyperplasia):

  • The classical salt wasting form
  • The classical virilizing form
  • The late onset nonclassical form (NCCAH).

While the classical forms of CAH are usually diagnosed shortly after birth, the clinical symptoms of NCCAH do not appear until childhood or puberty.

In NCCAH, there is a partial deficiency of 21-hydroxylase. It occurs in approximately 1 in 1,000 of the general population depending on the ethnic group, and up to 6% of hirsute women. This late form is characterized by a variety of late onset symptoms, including hirsutism, menstrual disturbances, and infertility. The concentration of 17-hydroxy progesterone is elevated and the size of the stimulation observed in the ACTH test indicates a partial deficiency of 21-hydroxylase.

A correlation exists between the molecular genotype and the clinical symptoms that show that the NCCAH phenotype is encoded by mutations that allow a fairly high degree of residual enzyme activity, unlike in other forms of CAH. According to a study /3/, in 63.7% of patients with the NCCAH phenotype, the 21-hydroxylase deficiency is caused by a serious mutation that can be passed from parent to child. The most common mutation found is V281L. Post ACTH stimulation the 17-hydroxy progesterone concentration is more than 10 μg/L. Because the frequency of heterozygous carriers is high and the ACTH test is not always positive in these carriers, partners with a family history of NCCAH should undergo genetic testing.

A suggested approach to the diagnosis of androgenization is shown in Fig. 37.2-2 – Diagnostic approach to female androgenization.

References

1. Puttabyatappa M, Padmanalkan V. Developmental programming of ovarian functions and dysfunctions. Vitam Horm 2018; 107: 377-422.

2. Illions EH, Valley MT, Kaunitz AM. Infertility. A clinical guide for internist. Med Clin North Am 1998; 82: 271–95.

3. Bidet M, Bellane-Chantelot C, Galand-Portier MB, Tardy V, Billaud L, Laborde K, et al. Clinical and molecular characterization of a cohort of 161 unrelated women with nonclassical congenital adrenal hyperplasia due to 21-hydroxylase deficiency and 330 family members. J Clin Endocrinol Metab 2009; 94: 1570–8.

4. Speroff L. The perimenopause: definitions, demography, and physiology. Obstet Gynecol Clin North Am 2002; 29: 397–410.

5. Nelson LM. Primary ovarian insufficiency. N Engl J Med 2009; 360: 606–14.

6. Jirecek S, Kink E, Wenzl R, Vytiska-Binsdorfer E, Huber J. Die hormonellen Ursachen der sekundären Amenorrhoe. Wien Klin Wochenschr 1998; 110/12: 441–5.

7. Yen SSC. Female hypogonadotropic hypogonadism. Endocrinol Metab Clin North Am 1993; 22: 29–57.

8. Ehrmann DA. Polycystic ovary syndrome. N Engl J Med 2005; 352: 1223–36.

9. Rothman MS, Wierman ME. How should postmenopausal androgen excess be evaluated? Clin Endocrinol 2011; 75: 160–4.

10. Sarfati J, Bachelot A, Coussieu C, Meduri G, Touraine P. Impact of clinical, hormonal, radiological, and immunohistochemical studies on the diagnosis of postmenopausal hyperandrogenism. Eur J Endocrinol 2011; 165: 779–88.

37.3 Male hypogonadism

Male hypogonadism, which results from failure of the testes to produce adequate testosterone and spermatozoa, is caused by disruption of the hypothalamic-pituitary-gonadal axis /1/. Klinefelter syndrome, the most common congenital cause of hypogonadism, has an incidence of 1 in 400 to 1 in 1,000 live births. In a cross sectional survey on 3369 community-dwelling men aged 40–79 years the frequency of secondary, primary, and compensated hypogonadism has been found to be 11.8%, 2.0%, and 9.5%, respectively /2/. Up to 20% of men who experience hip fractures have low levels of testosterone.

Testicular function is regulated by the pulsatile release of GnRH by the hypothalamus, which in turn stimulates the release of FSH and LH by the anterior pituitary /3/. GnRH pulses occur at a mean rate of 3.8 pulses every 6 hours. At a lower pulse rate, FSH is released preferentially; at a higher pulse rate, LH release is predominant. Circulating FSH binds to Sertoli cells and stimulates them to produce spermatozoa and proteins, in particular inhibin. The Leydig cells are stimulated by LH to produce testosterone. Testosterone and inhibin regulate the secretion of FSH and LH via a negative feedback control. Rising levels of testosterone inhibit the secretion of FSH and LH, while inhibin primarily inhibits the secretion of FSH. Inhibin induces and maintains spermatogenesis in conjunction with testosterone.

The clinical symptoms of hypogonadism depend on the time of onset of the testosterone deficiency /4/:

  • Testosterone deficiency during embryogenesis can lead to intersexuality
  • If testosterone deficiency exists at the time of normal onset of puberty, it can delay puberty and lead to eunuchoid gigantism
  • Onset of testosterone deficiency after puberty does not result in a change of body proportions, but can lead to sparse secondary body hair, muscle atrophy, increased body fat, osteoporosis, anemia, loss of libido, impotence, and infertility.

37.3.1 Male primary hypogonadism

Primary hypogonadism results from testicular failure and affects:

  • The production of testosterone by the Leydig cells
  • The seminiferous tubules. The testes are composed of numerous thin, tightly coiled tubules (seminiferous tubules). The sperm cells are produced within the walls of the tubules that contain Sertoli cells with the function to support and nourish the sperm cells.

For functional tests in hypogonadism and the causes of primary hypogonadism refer to:

Laboratory findings

Primary hypogonadism is diagnosed based on a reduced testosterone concentration, elevated FSH and LH, and reduced spermatogenesis. In some cases it is important to determine whether a mild or moderate dysfunction is present e.g., due to a varicocele, since the LH concentration can still be normal in these cases. An excessive response in the GnRH test indicates that the Leydig cells are also affected /3/.

37.3.2 Male secondary hypogonadism

Secondary hypogonadism, also known as hypogonadotropic hypogonadism, can be the result of congenital defects or acquired disorders. The clinical features shown by these patients depend on whether the onset of the disorder is before or after puberty. The causes of secondary hypogonadism are listed in Tab. 37.3-3 – Secondary and mixed acquired hypogonadism.

Laboratory findings

Suspicion of secondary hypogonadism is aroused if testosterone production and sperm count are reduced while FSH and LH are elevated.

37.3.3 Late-onset hypogonadism

Age related decline in testosterone starting in early adulthood and continuing into old age in men is associated with a decline in testicular reserve and reduced drive of the hypothalamo-pituitaty-gonadal axis. This leads to late-onset hypogonadism (LOH). Older men with conditions such as obesity, type 2 diabetes, chronic obstructive pulmonary disease, chronic inflammation, liver disease or chronic renal or cardiac failure have a higher prevalence of borderline low serum testosterone levels /56/.

Symptoms of LOH

Common clinical symptoms are /5/:

  • The easily recognized features of diminished sexual desire (libido), and erectile quality and frequency, particularly nocturnal erections
  • Changes in mood, with concomitant decreases in intellectual activity, cognitive functions, spatial orientation ability, fatigue, depressed mood, and irritability
  • Sleep disturbances
  • Decrease in lean body mass with associated diminution in muscle volume and strength
  • Increase in visceral fat.

Biochemical changes and risk factors of LOH

  • Dyslipidemia, metabolic syndrome, type 2 diabetes, reduced bone density, hypertension, cardiovascular risk
  • Decreased testosterone level. According to a study /2/, testosterone concentration in serum declines progressively at a rate of 0.4–2% per year after the age of 30 and a significant proportion of men over the age of 60 years have serum testosterone levels that are below the lower limits of young adult (age 20–30 years).

Diagnosis of LOH

Diagnosis of LOH requires /17/:

  • Clinical features of androgen deficiency
  • Low testosterone concentration on at least two occasions: less than 3.2 μg/L (11 nmol/L) of total testosterone or less than 64 ng/L (220 pmol/L) for free testosterone (determined using GC-MS/MS)
  • Exclusion of other causes of hypogonadism

37.3.4 Testosteron body composition and sexual function in men

Low testosterone as a risk factor

Especially in young men aged 20–39 years, low testosterone might be directly associated with metabolic syndrome. In the Study Health of Pomerania /8/ the relative risk of incident metabolic syndrome for men with total testosterone levels in the lowest quartile was 2.06 μg/L (7.0 nmol/L) compared to those with testosterone levels in the highest quartile. Low serum total testosterone levels were also associated with an increased risk of mortality. For example, serum total testosterone levels of less than 2.5 μg/L (8.7 nmol/L) were associated with a hazard ratio for death from cardiovascular disease of 2.56 and for death from cancer of 3.46 /9/.

Obesity

The most powerful predictor of low testosterone is obesity, whereby co morbidities have an additive effect. Obese men are the youngest group of men with late-onset hypogonadism and are predisposed to secondary hypogonadism. Hypothalamic-pituitary deregulation without physiological compensation occurs in this group. Increased conversion of testosterone to estradiol in adipose tissue, insulin resistance, synthesis of pro- inflammatory cytokines (TNF-α, IL-6) by adipocytes, and reduced sexual hormone binding globulin all affect the gonadotropic system /2/.

Erectile dysfunction

Erectile dysfunction is defined as a persistent inability to achieve or maintain penile erection sufficient for satisfactory sexual performance. Erectile dysfunction lasting for 3 months is considered a reasonable length of time to warrant evaluation and consideration of treatment. The prevalence of low testosterone levels in men with erectile dysfunction varies depending on the cutoff value used. Total testosterone levels < 2.0 μg/L (6.9 nmol/L) indicate hypogonadism. In cases of testosterone levels between 2.0 and 4.0 μg/L (6.9 and 13.9 nmol/L), measurement should be repeated and supplemented by determination of free testosterone. The prevalence of low testosterone levels defined as < 2.88 μg/L (9.9 nmol/L) for total testosterone or < 9 ng/L (31.2 pmol/L) for free testosterone in men with erectile dysfunction varied from 12.5% to 35% depending on the study /10/.

37.3.4.1 Testosterone and the aging phenotype in hypogonadism

The association between declining testosterone levels and the phenotype of elderly men is weak. Late-onset hypogonadism can be classified to a certain extent based on the coupling of testosterone concentration and LH levels. In some older men, testosterone levels are normal and LH is elevated a condition called compensated hypogonadism.

In the European Male Aging Study (EMAS) /2/ a total of 3,369 men aged 40–79 years were invited for an interviewer-assisted questionnaire, anthropometric measurements, and testosterone determination. The testosterone concentration was determined using GC-MS/MS. The majority of subjects (76.6%) were eugonadal, whereas 11.8%, 2% and 9.5% had secondary, primary, and compensated hypogonadism, respectively.

Primary hypogonadism

In the EMAS /2/ a small minority of elderly men (2%) were at risk for primary hypogonadism which may be a consequence of the age-related attrition of Leydig cells. Their relationship with age was strong and they probably represented the extreme end of the physiological spectrum of the hypothalamic-pituitary-gonadal axis encountered in aging. Their hypogonadal status and testosterone levels correlated with a number of variables (low sexual thoughts, limited mobility, inability to engage in vigorous activity).

For instance, mean total testosterone levels among men with sexual symptoms were 3.68 (1.44–9.42) μg/L if decreased frequency of sexual thoughts was the only symptom, 1.76 (0.93–3.32) μg/L in the absence of morning erections, and 1.38 (0.71–2.70) μg/L if erectile dysfunction was present /2/.

Secondary hypogonadism

Secondary hypogonadism is associated with obesity and co morbid conditions at any age and appears to be associated with hypothalamic-pituitary dysregulation for which there are no physiological compensatory mechanisms /2/. The prevalence of secondary hypogonadism does not increase with age. Testosterone levels are mainly within the range of 1–2 μg/L (3.5–6.9 nmol/L).

Compensated hypogonadism

The negative effects of aging alone on testicular function can be moderated by increased LH compensation for a number of years /2/. About 9.5% of the men studied had compensated hypogonadism, forming the largest LOH category (21.1%). Their testosterone levels were ≥ 3.6 μg/L (10.5 nmol/L) and their LH levels were > 9.4 IU/L.

Men with compensated hypogonadism complain mainly of physical symptoms (inability to engage in vigorous activity, difficulty walking more than 1 kilometer, bending difficulty). Elevated LH levels indicate a decline in testosterone within the reference interval in the same way as elevated TSH levels indicate latent hypothyroidism when FT4 levels are within the reference interval. An inverse relationship exists between LH and muscle strength independent of testosterone. It is suggested that elevated LH levels in compensated hypogonadism are not an isolated laboratory finding but significantly associated with physical symptoms /2/.

Eugonadal men

Among eugonadal men, the prevalence of physical symptoms was 5–21% and the prevalence of sexual symptoms was 25–35% /2/.

References

1. Bhasin S, Cunningham GR, Hayes FJ, Matsumoto AM, Snyder PG, Swerdloff RS, et al. Testosterone therapy in adult men with androgen deficiency syndromes: an Endocrine Society clinical practice guideline. J clin Endocrinol Metab 2010; 95: 2536–59.

2. Tajar A, Forti G, O’Neill TW, Lee DM, Silman AJ, Finn JD, et al. Characteristics of secondary, primary, and compensated hypogonadism in ageing men: evidence from the European Male Ageing Study. J Clin Endocrinol Metab 2010; 95: 1810–8.

3. Rey A, Wayne J, Hellstroem G, Palmert MR, Corona G, Dohle GR, et al. Pediatric and adult-onset male hypogonadism. Nat Rev Dis Primers 2019; 5 (1). https://doi.org/10.1038/s41572-019-0087-y.

4. Nieschlag E. Hodenfunktion. In: Thomas L, ed. Labor und Diagnose. Frankfurt 2008; TH-Books: 1493–1502.

5. Nieschlag E, Swerdloff R, Behre HM, Gooren LJ, Kaufman JM, Legros JJ, et al. Investigation, treatment, and monitoring of late-onset hypogonadism in males: ISA, ISSAM, and EAU recommendations. J Androl 2006; 27: 135–6.

6. Borovickova I, Adelson N, Visvanath A, Rousseau G. Hypogonadism with normal serum testosterone Clin Chem 2017; 63: 1236–30.

7. Wu FCW, Tajar A, Beynon JM, Pye SR, Silman AJ, Finn JD, et al. Identification of late-onset hypogonadism in middle-aged and elderly men. N Engl J Med 2010; 363: 123–35.

8. Haring R, Völzke H, Felix SB, Schimpf S, Dörr M, Rosskopf D, et al. Prediction of metabolic syndrome by low serum testosterone levels in men. Diabetes 2009; 58: 2027–31.

9. Haring R, Völzke H, Steveling A, Krebs A, Felix SB, Schöfl C, et al. Low serum testosterone levels are associated with increased risk of mortality in a population-based cohort of men aged 20–79. Eur Heart J 2010; 31: 1494–1501.

10. Quassem A, Snow V, Denberg DT, Casey DE, Forciea MA, Owens DK, et al. Hormonal testing and pharmacologic treatment of erectile dysfunction: a clinical practice guideline from the American College of Physicians. Ann Intern Med 2009; 151: 639–49.

11. Giaguli VA, Kaufman JM, Vermeulen A. Pathogenesis of decreased androgen levels in obese men. J Clin Endocrinol Metab 1994; 79: 997–1000.

12. Ceccarelli C, Canale D, Battisti P, Caglieresi C, Moschini C, Fiore E, et al. Testicular function after 131I therapy for hyperthyroidism. Clin Endocrinol 2006; 65: 446–52.

13. Tajar A, Forti G, O’Neill TW, Lee DM, Silman AJ, Finn JD, et al. Characteristics of secondary, primary, and compensated hypogonadism in ageing men: evidence from the European Male Ageing Study. J Clin Endocrinol Metab 2010; 95: 1810–8.

14. Nieschlag E. Klinefelter syndrome: the commonest form of hypogonadism, but often overlooked or untreated. Dtsch Arztebl Int 2013; 110: 347–53. https://doi.org/10.3238/arztebl.2013.0347.

15. Mathers MJ, Sperling H, Rübben H, Roth S. Hodenhochstand: Diagnostik, Therapie und langfristige Konsequenzen. Dtsch Ärztebl 2009; 106: 527–32.

37.4 Gonadotropins

Follicle-stimulating hormone (FSH), luteinizing hormone (LH) and thyroid-stimulating hormone (TSH) are secreted by the anterior pituitary and have a similar structure to the placental hormone hCG. All four hormones have a molecular mass of about 30 kDa and a carbohydrate content of 15–30%. They are composed of two polypeptide chains, the α-subunit, which is identical in all the hormones, and the β-subunit, which varies between the hormones and determines the biological function (only after it combines with the α-subunit) /1/.

Gonadotropins regulate gonadal activity and the gonadal hormones estradiol, testosterone, and progesterone. These essential steroids are responsible for sexual development and for the differentiation and maintenance of the male and female phenotype.

The pattern of FSH, LH and of 17β-estradiol and progesterone during the menstrual cycle is shown in Fig. 37.1-2 – Serum levels of FSH, E2, LH, and progesterone synchronized to the day of the LH peak.

37.4.1 Follicle-stimulating hormone

In females, follicle stimulating hormone (FSH) regulates growth, sexual development and reproduction, including menstruation, follicular development and ovulation. In men FSH promotes spermatogenesis and androgen responsiveness in the testes.

37.4.1.1 Indication

In women

  • Assessment of menstrual cycle abnormalities
  • Diagnostic evaluation of infertility
  • Assessment of the need for hormone replacement therapy during the perimenopause.

In men

  • Etiological evaluation of impaired spermatogenesis in semen analysis
  • When basal testosterone levels are low, LH and FSH provide information about the type of hypogonadism that is present (primary or secondary).

37.4.1.2 Method of determination

Immunoassays, competitive and immunometric methods using various methods and labels /2/.

37.4.1.3 Specimen

Serum, plasma: 1 mL

37.4.1.4 Reference interval

Refer to Tab. 37.4-1 – Reference intervals for FSH.

37.4.1.5 Clinical assessment

Because FSH and LH determinations are requested together in most clinical situations, the clinical significance is discussed in Section 37.4.2.5 – Clinical assessment together with that of LH.

Functions in women

During the follicular phase, FSH stimulates the maturation of the Graafian follicle in the ovary and the secretion of 17β-estradiol by the theca cells of the follicle. When the follicle matures, 17β-estradiol also increases. The LH peak is triggered by high 17β-estradiol concentrations and ovulation occurs approximately one day after this peak. Because of the pulsatile GnRH secretion pattern, FSH is also secreted in a pulsatile fashion.

Functions in men

FSH stimulates spermatogenesis and is regulated by the protein inhibin, which is produced in the Sertoli cells and inhibits FSH secretion by the pituitary.

37.4.1.6 Comments and problems

Method of determination

Immunoassays from different manufacturers may provide different results in patient samples, regardless of whether polyclonal or monoclonal antibodies are used and despite the fact that these assays have been calibrated against the same standard: WHO International Standard Follicle Stimulating Hormone (FSH), human, recombinant, for immunoassay, NIBSC code 92/510.

Discrepancies in FSH determinations are the result of:

  • Varied specificity of the antibodies against different glycosylated forms of FSH, which, as genetic variants, include a wide population of variably glycosylated isoforms
  • Changes in epitopes in certain diseases such as renal insufficiency
  • Kinetic conditions of the immunoassay.

The immunoassays show less than 1% cross reactivity with LH, TSH, and hCG.

Reference interval in children

Refer also to the CALIPER cohort study; Clin Chem 2013; 59: 1215–27 and 1393–1405.

Stability

In serum and plasma at 4 °C for several days. Can be transported by mail, uncooled.

37.4.2 Luteinizing hormone (LH)

Luteinizing hormone (LH) is essential for sexual development and reproduction in both sexes. In women LH interacts with receptors of the ovarian follicles and promotes their maturation. In the middle of the menstrual cycle, a surge of LH triggers ovulation and production of progesterone by the corpus luteum. Progesterone is necessary for the maturation of the uterine endometrium for implantation of the fertilized egg. In men LH stimulates androgen synthesis in the testicular Leydig cells.

37.4.2.1 Indication

In women

  • Assessment of menstrual cycle abnormalities
  • Diagnostic evaluation of infertility
  • Assessment of the need for hormone replacement therapy during the perimenopause.

In men

  • When basal testosterone levels are low, LH provides information about the etiology of the hypogonadism (primary or secondary).

37.4.2.2 Method of determination

Immunoassays, competitive and immunometric assays /3/.

37.4.2.3 Specimen

Serum, plasma: 1 mL

37.4.2.4 Reference interval

Refer to Tab. 37.4-2 – Reference intervals for LH.

37.4.2.5 Clinical assessment

The assessment of FSH and LH in ovarian and testicular dysfunction are presented.

37.4.2.5.1 FSH in assisted reproductive technology (ART)

The Centers for disease Control and Prevention defined assisted reproductive technology (ART) as technology used to treat infertility in which the ovum and sperm are manipulated ex vivo. Included technologies are IVF, intracytoplasmic sperm injection and other related techniques.

Optimization of the treatment protocol and counselling of patients require evaluation of the ovarian reserve (OR) of follicles. Antral follicle count (AFC) on trans vaginal ultrasound is a reliable biophysical marker of OR. The AFC and the determination of FSH and estradiol are the traditional OR biomarkers of choice produced during the follicular development. FSH and estradiol are indirect markers and rely on the production of other hormones through a feedback loop. As women and their follicles age, FSH raises in reaction to decreased responsiveness of ovary /4/.FSH is affected by conditions other than ovarian causes even in the presence of optimum OR. Elevated levels are found in hormonal therapy (oral contraceptives), pituitary tumors, and Turner syndrome. Low levels of FSH are determined in non ovulatory polycystic ovary syndrome (PCOS) and non functioning pituitary tumors. In all such cases direct markers of OR that are produced during follicular stimulation (e.g.,anti-Muellarian hormone – Section 37.10 and inhibin – Section 37.11 reflect true OR consistently being independent of hypothalamic feedback loop /4/.

37.4.2.5.2 FSH, LH, and ovarian dysfunction

The World health organization classifies ovarian dysfunction on the bases of serum FSH and estradiol levels. FSH fluctuates with the menstrual cycle, therefore the samples are collected on day 3 of the menstrual cycle to reflect the basal level. The secretion of estradiol by the antral follicles is under the influence of FSH.

If FSH and LH are persistently elevated, primary ovarian failure is present /4/. If the FSH and LH levels are reduced or at the lower reference interval value in cases of amenorrhea, secondary ovarian failure is present. Most primary and secondary hypo gonadotropic or normo gonadotropic amenorrheas are the result of disturbed or absent hypothalamic pulsatile GnRH secretion. 17β-estradiol is always decreased in these cases.

Refer to Tab. 37.4-3 – Amenorrhea associated with low or low-normal FSH and LH levels.

A random gonadotropin peak in mid cycle may suggest primary ovarian failure. Simultaneous 17β-estradiol determination allows differentiation: high 17β-estradiol levels normally suggest a gonadotropin peak. However, the 17β-estradiol concentration may be transiently extremely low immediately after ovulation. During this time period, the distinction can be based on a slightly elevated progesterone (1–2 μg/L).

Conditions associated with persistent FSH and LH elevation are shown in Tab. 37.4-4 – Conditions associated with persistent elevations of FSH and LH in women.

Normal FSH and LH levels that are slightly elevated or within the upper reference interval, in conjunction with amenorrhea, oligomenorrhea, or other menstrual cycle abnormalities suggest the presence of hyperandrogenemic ovarian failure such as PCOS.

During the course of infertility treatment, serial LH determinations are important for predicting the timing of ovulation, which can be expected to occur about 30 hours after the rise in LH /5/.

37.4.2.5.3 LH, FSH, and testicular dysfunction

Luteinizing hormone (LH)

In the presence of low testosterone levels, serum LH concentration can be used to determine the etiology of hypogonadism /6/:

  • Elevated LH and low testosterone levels suggests testicular (primary) hypogonadism whereas low LH levels suggest a central (secondary) cause
  • Elevated LH and low normal testosterone levels suggests compensated hypogonadism
  • High LH levels in conjunction with high testosterone levels indicate the presence of an androgen receptor defect (androgen insensitivity).

Follicle stimulating hormone (FSH)

FSH serves as a biomarker for the diagnosis of abnormal spermatogenesis /7/:

  • Elevated FSH in association with small, firm testes (volume less than 6 mL) and azoospermia suggests the presence of Klinefelter syndrome
  • Elevated FSH in conjunction with azoospermia or very poor semen parameters and a testicular volume of greater than 6 mL suggests a primary disorder of spermatogenesis
  • Normal FSH levels in conjunction with azoospermia may indicate obstruction of the vas deferens. A simultaneous decrease in an epididymal marker that is secreted in the seminal plasma such as α-glucosidase, confirms the diagnosis.
  • Low FSH levels suggest the possibility of pituitary insufficiency (refer to Chapter 33 – Pituitary function).

The majority of men with fertility problems have oligospermia and normal FSH levels. The pathogenesis is often unclear in these cases and, when age and adiposity are taken into account, additional endocrine investigations are of limited use.

37.4.2.6 Comments and problems

Method of determination

Immunoassays from different manufacturers may provide different results in patient samples, regardless of whether polyclonal or monoclonal antibodies were used and despite the fact that these assays have been calibrated against the second International Standard for Human Pituitary LH (in ampoules coded 80/552; 2nd IS) and LH 81/535. The reasons for these discrepancies can be found in the section on FSH.

Reference interval in children

Refer also to the CALIPER cohort study; Clin Chem 2013; 59: 1215–27 and 1393–1405.

Stability

In serum and plasma at 4 °C for several days. Can be transported by mail, uncooled.

References

1. Bousfield GR, Dias JA. Synthesis and secretion of gonadotropins including structure-function correlate. Rev Endocr Metab discord 2011; 12: 289–302

2. Yin L, Tang Y, Chen X, Sun Y. Measurement differences between two immunoassay systems for LH and FSH: a comparison of Roche Cobas e601 vs. Abbott Arcitect i2000sr. Clin Lab 2018; 64: 295-301.

3. Mitchell R, Hollis S, Crowley V, McLoughlin J, Peers N, Robertson WR: Immunometric assays of luteinizing hormone (LH): differences in recognition of plasma LH by anti-intact and beta-subunit-specific antibodies in various physiological and pathophysiological situations. Clin Chem 1995; 41: 1139–45.

4. Jamil Z, Fatima SS, Ahmed K, Malik R. Anti-Mullarian hormone: above and beyond conventional ovarian reserve markers. Disease Markers 2016; Vol 2016: Article ID 5246217.

5. Plymate S. Hypogonadism. Endocrin Metab Clin North Am 1994; 23: 749–72.

6. Tajar A, Forti G, O’Neill TW, Lee DM, Silman AJ, Finn JD, et al. Characteristics of secondary, primary, and compensated hypogonadism in ageing men: evidence from the European Male Ageing Study. J Clin Endocrinol Metab 2010; 95: 1810–8.

7. Nieschlag E. Hodenfunktion. In: Thomas L, ed. Labor und Diagnose. Frankfurt 2008; TH-Books: 1493–1502.

8. Elmlinger MW, Kühnel W, Ranke MB. Reference ranges for serum concentrations of lutropin (LH), follitropin (FSH), estradiol (E2), prolactin, progesterone, sex hormone-binding globulin (SHBG), dehydroepiandrosterone sulfate (DHEAS), cortisol and ferritin in neonates, children and young adults. Clin Chem Lab Med 2002; 40: 1151–60.

9. Kratz A, Ferraro M, Sluss PM, Lewandrowski KB. Laboratory reference values. N Engl J Med 2004; 351: 1548–63.

37.5 17β-estradiol (E2)

17β-estradiol is the most potent of the naturally occurring estrogens. 17β-estradiol controls development and maintenance of female sex characteristic and is often referred to as the female hormone.

37.5.1 Indication

In women:

  • Monitoring the course of hormonal infertility treatment
  • Assessment of ovarian function.

In men:

  • Gynecomastia.

37.5.2 Method of determination

In serum and plasma, 17β-estradiol is bound to proteins, including SHBG and albumin. Prior to the determination, estradiol must be released from the proteins. This is performed by specific reagents contained in commercially available immunoassays.

37.5.3 Specimen

Serum, heparin plasma: 1 mL

37.5.4 Reference interval

Refer to Tab. 37.5-1 – Reference intervals for 17β-estradiol.

37.5.5 Clinical assessment

37.5.5.1 17β-estradiol (E2) in women

E2 determination is considered useful:

  • In monitoring follicular growth during assisted reproductive technology (ART). Poor ovarian response is suggested with basal E2 levels of < 20 or > 80 pg/mL.
  • In detecting mid-cycle gonadotropin peaks in cases of progesterone negative amenorrhea. A positive progesterone withdrawal test indicates adequate endogenous synthesis of E2.

Diseases and conditions associated with changes in E2 concentrations are shown in Tab. 37.5-2– Diseases and conditions associated with changes in E2 levels.

37.5.5.2 17β-estradiol (E2) in men

More than 80% of the circulating E2 is produced by aromatization of testosterone. E2 deficiency is a consequence of severe testosterone deficiency and can be corrected by the administration of testosterone. Like testosterone, E2 has a role in normal libido, erectile function, and bone metabolism. The subcutaneous and intra abdominal fat mass depends on E2 while sexual function depends on both testosterone and E2.

17β-estradiol deficiency

E2 deficiency underlies a number of important consequences in concern to hypogonadism. For example, men with testosterone levels of 2–4 μg/L (6.9–13.9 nmol/L) and E2 levels of ≥ 10 ng/L (36.7 pmol/L) show a reduction in the sexual desire score of 13%, while those with an E2 level < 10 ng/L show a decrease of 31% /1/.

17β-estradiol excess

Elevated E2 levels can be associated with gynecomastia. The causes of gynecomastia are: idiopathic 25%, puberty 25%, medication 10–20%, liver cirrhosis and malnutrition 8%, primary hypogonadism 8%, testicular tumors 3%, secondary hypogonadism 2%, hyperthyroidism 2%, renal disease 1%, and other causes 8%.

E2 producing tumors can occur in the testes, adrenal glands, and elsewhere. For example, hCG is synthesized by germ cell tumors and activates testicular steroid synthesis. hCG is also synthesized para neoplastically by tumors of the lungs and gastrointestinal tract. It stimulates aromatase activity, thereby leading to the production of E2 from testosterone.

Tumors of the adrenal cortex lead to feminization by producing large quantities of androgens (DHEA, androstenedione), which are then converted peripherally to E2 by aromatization.

37.5.6 Comments and problems

Method of determination

The comparability of commercial immunoassays for the determination of E2 is limited /1/. The immunoassay used must cover a wide measuring range, including, for example:

  • 50–2,000 pmol/L (14–545 ng/L) for infertility testing
  • 200–15,000 pmol/L (54–4,086 ng/L) for monitoring of in vitro fertilization.

Stability

Samples can be transported by mail, uncooled, within a 24-h time period.

37.5.7 Pathophysiology

17β-estradiol (E2; C18H24O2) the primary female sex hormone, is a C18 steroid and responsible for the key physiological processes related to growth, differentiation and physiology of the reproductive system. Aromatase catalyses the final rate limiting step of 17β-estradiol biosynthesis (Fig. 37.1-1 – Biosynthesis of important sex steroids). It is localized in the endoplasmic reticulum of estrogen producing cells (ovarian granulosa cells, placental syncytiotrophoblast, bone, brain, skin fibroblasts, adipose tissue, mesenchymal cells) and encoded by the CYP19A1 gene, located on chromosome 15, band q21 of the human genome.

E2 is the primary estrogen of ovarian hormone and formed by a aromatization of androgens. The formation is a complex process that involves three hydroxylation steps. The aromatase enzyme complex is comprised of the aromatase cytochrome P450 (P450arom), and the flavoprotein NADPH-cytochrome P450 reductase. The biosynthesis of 17β-estradiol occurs through binding of the aromatase enzyme to C19 androgenic steroid substrates and catalyzing the aromatase reaction leading to the formation of the phenolic A ring which is characteristic of estrogens /2/. E2 is readily oxidized to estrone (E1) in the liver. Estrone can be further hydrated to estriol (E3). 17β-estradiol is bound to sex hormone binding globulin (SHBG) and transported through plasma.

E2 is synthesized in both women and men. In women of reproductive age, it is mainly synthesized in the ovary; in men, 20% is synthesized in the testes and the remaining 80% is produced in the adrenal gland. However, the ovary and adrenal cortex also synthesize a range of androgens (androstenedione, dehydroepiandrosterone, testosterone) that are converted to E2 by aromatization in peripheral tissues such as brain, bone, and adipose tissue.

Levels of E2 are low in male and female children (less than 100 pmol/L).

In women, the level of E2 rises at puberty and is a sign of ovarian activity. During the menstrual cycle, the highest levels of E2 are reached in the preovulatory phase (approximately 1,000 pmol/L) and in the luteal phase, when it is secreted by the corpus luteum. Postmenopausal levels of E2 are low (less than 100 pmol/L). Estrone (E1) is the predominant estrogen in postmenopausal women; it is produced by the conversion of androstenedione in the adipose tissue.

References

1. Finkelstein JS, Lee H, Burnett-Bowie SAM, Pallais JC, Yu EW, Borges LF, et al. Gonadal steroids and body composition, strength, and sexual function in men. N Engl J Med 2013; 369: 1011–22.

2. To SQ, Knower KC, Cheung V, Simpson ER, Clyne CD. Transcriptional control of local estrogen formation by aromatase in the breast. J Steroid Biochem and Molecular Biology 2015; 145: 179–86.

3. Elmlinger MW, Kühnel W, Ranke MB. Reference ranges for serum concentrations of lutropin (LH), follitropin (FSH), estradiol (E2), prolactin, progesterone, sex hormone-binding globulin (SHBG), dehydroepiandrosterone sulfate (DHEAS), cortisol and ferritin in neonates, children and young adults. Clin Chem Lab Med 2002; 40: 1151–60.

4. Kratz A, Ferraro M, Sluss PM, Lewandrowski KB. Laboratory reference values. N Engl J Med 2004; 351: 1548–63.

5. Hinney B, Wuttke W. Ovarialfunktion. In: Thomas L, ed. Labor und Diagnose. Frankfurt 2008; TH-Books: 1478–92.

37.6 Progesterone

Progesterone is the most important of the corpus luteum hormones, whose main function is to prepare for and maintain pregnancy. During the follicular phase, it is detectable in the serum only in small amounts. Along with the LH peak, a slight rise in progesterone occurs shortly prior to ovulation; subsequently, the corpus luteum produces significant amounts of progesterone.

Progesterone is synthesized from pregnenolone by action of 3β-HSD in the corpus luteum, by the placenta during pregnancy; as well as by the adrenals, as a step in androgen and mineralocorticoid synthesis (Fig. 37.1-1 – Biosynthesis of sex steroids).

Progesterone causes secretory transformation of the endometrium and, if pregnancy occurs, it serves to maintain the decidual transformation of the endometrium. The corpus luteum is necessary for progesterone secretion up to the 8th gestational week, at which point the placenta takes over this function /1/.

37.6.1 Indication

  • Confirmation of ovulation
  • Assessment of corpus luteum function
  • Assessment of early pregnancy.

37.6.2 Method of determination

More than 90% of progesterone in serum and plasma is bound to proteins. When commercially available immunoassays are used, progesterone is released from the proteins by means of reagents contained in the kits. Heterogeneous immunoassays are employed.

37.6.3 Specimen

Serum, heparin plasma, saliva: 1 mL

37.6.4 Reference interval

Refer to Tab. 37.6-1 – Reference intervals for progesterone.

37.6.5 Clinical assessment

Progesterone reaches its maximum levels 6–8 days after ovulation and then declines steeply approximately 3 days prior to menstruation. Due to its thermogenic effect, progesterone causes a rise in basal temperature. The assessment of progesterone levels is facilitated by the use of basal temperature charts. During pregnancy, there is a continuous rise in progesterone between the 5th and 40th gestational week, with a ten to forty-fold increase in the concentration.

Diseases and conditions associated with changes in progesterone levels are shown in Tab. 37.6-2 – Diseases and conditions associated with changes in progesterone levels.

37.6.6 Comments and problems

Stability

Samples are stable at 4–8 °C for one week; can be transported by mail, uncooled, within 24 h

37.6.7 Pathophysiology

Refer to Ref. /5/.

References

1. Taraborreli S. Physiology, production and action of progesterone. Acta Obstet Gynecol Scand 2015; 94 Suppl.: 8–16

2. Elmlinger MW, Kühnel W, Ranke MB. Reference ranges for serum concentrations of lutropin (LH), follitropin (FSH), estradiol (E2), prolactin, progesterone, sex hormone-binding globulin (SHBG), dehydroepiandrosterone sulfate (DHEAS), cortisol and ferritin in neonates, children and young adults. Clin Chem Lab Med 2002; 40: 1151–60.

3. Kratz A, Ferraro M, Sluss PM, Lewandrowski KB. Laboratory reference values. N Engl J Med 2004; 351: 1548–63.

4. Dart R, Ramanujam P, Dart L: Progesterone as a predictor of ectopic pregnancy when the ultrasound is indeterminate. Am J Emerg Med 2002; 20: 575–9.

5. Mizrachi D, Wang Z, Sharma KK, Gupta MK, Xu K, Dwyer CR, et al. Why human cytochrome P450c21 is a progesterone 21-hydroxylase. Biochemistry 2011; 50 (19): 3968-74

37.7 Sex hormone binding globulin (SHBG)

SHBG is a glycoprotein possessing high affinity binding for 17β-hydroxy steroid hormones such as testosterone and 17β-estradiol. SHBG delivers hormones to target tissues. Measurement of SHBG is useful in the evaluation of disorders of androgen and 17β-estradiol metabolism and enables identification of patients with excessive hormone activity from normal individuals /1/.

37.7.1 Indication

Determination of SHBG becomes significant in conjunction with the determination of testosterone if the level of testosterone is not consistent with the clinical picture, for example:

  • Clinically evident androgenization with testosterone levels within the reference interval
  • Elevated testosterone in the absence of clinical signs of androgenization
  • Hypogonadism with a normal testosterone level.

37.7.2 Method of determination

Immunoassays, usually using the sandwich principle /2/. The commercially available assays are calibrated against the WHO Standard code 08/266.

37.7.3 Specimen

Serum, heparin plasma, saliva: 1 mL

37.7.4 Reference interval

Refer to Tab. 37.7-1 – Reference intervals for SHBG.

37.7.5 Clinical significance

SHBG concentrations exhibit a high degree of inter individual variability /3/:

  • Age, exogenous estrogen use, physical activity, regular coffee intake and post menopause are positively associated with SHBG concentration
  • Increased body mass index (BMI) is inversely associated with SHBG level.

37.7.5.1 Relationship between SHBG and testosterone

There is a strong positive correlation between the SHBG concentration and total testosterone but only a weak correlation between SHBG and free (bioavailable) testosterone

Elevated SHBG concentrations are associated with higher total testosterone levels in the setting of unchanged testosterone production.The biologically active free testostrone in blood is decreased.

Decreased SHBG concentrations are associated with decreased total testosterone levels in the setting of unchanged testosterone production.The biologically active free testostrone in blood is increased.

The SHBG concentration affects testosterone production indirectly. Only the concentration of free testosterone exerts feedback inhibition on the release of LH. As the SHBG concentration increases, the free testosterone concentration decreases. The resulting increase in LH release leads to increased production of bioavailable testosterone and SHBG-bound testosterone. SHBG, however, has very little direct influence on the concentration of bioavailable testosterone /4/.

Slightly elevated SHBG concentrations are observed in older men and are thought to be one cause of hypogonadism due to a decrease in the concentration of free testosterone. Some investigators, however, think that the cause is more likely to be a disturbance in the feedback inhibition of LH secretion by free testosterone /4/. In patients with hypogonadism and infertile males determination of total testosterone and SHBG was used to calculate free testosterone. Using calculated free testosterone as a standard in the measurement of definitive biochemical hypogonadism in males (less than 1.56 ug/L, 5.4 nmol/L) revealed 81% sensitivity and 83% specificity if SHBG related total testosterone was determined /5/.

37.7.5.2 Causes of elevated SHBG concentrations

Hormonal contraceptives, anti-epileptics, hormone replacement therapy, liver cirrhosis, hyperthyroidism, hypogonadism, gynecomastia (men) /6/.

37.7.5.3 Decreased SHBG concentrations

A decreased SHBG concentration is likely in women who present with symptoms of androgenization but in whom no elevation of testosterone is found in at least two blood samples.

37.7.5.4 Free androgen index (FAI)

The FAI can provide further information in patients with symptoms of andogenization. The FAI is calculated as follows:

FAI (%) = (Total testosterone/SHBG) × 100

Concentrations are measured in nmol/L. The median FAI value (%) is around 45 in men and 1.5 in women.

The FAI is a measure of the biologically active testosterone in the blood. By calculating the FAI, one can get a measurement of physiologically active testosterone in the blood.

An increased FAI is likely in women who present with symptoms of androgenization but in whom no elevation of testosterone is found in at least two blood samples. Obesity, glucocorticoid therapy, growth hormone therapy, androgenization (women), polycystic ovary syndrome, Cushing’s syndrome, hypothyroidism, acromegaly, hyperprolactinemia are pathologies of increased FAI /6/.

37.7.6 Comments and problems

Specimen

Concentrations measured in EDTA plasma are up to 50% lower than those measured in serum.

Stability

4 hours at 20 °C, 3 days at 2–8 °C.

37.7.7 Pathophysiology

Androgens and estrogens circulate in blood either unbound as a free fraction or bound to binding proteins such as SHBG and albumin. Whereas the binding with albumin rapidly dissociates during tissue transit, the binding of these hormones with SHBG is strong, high affinity binding, and therefore hormones bound to SHBG are considered not readily available for biological action. The two other fractions, free and bound to albumin, have direct access to target cells, and together they are often referred to as bioavailable fraction.

SHBG is a large glycoprotein with a molecular mass of 95 kDa and exists as a homodimer composed of two identical subunits. Each subunit contains two disulfide bonds. SHBG is synthesized in the liver and has a plasma half life of 7 days. It has binding affinities for C18 and C19 steroids with a 17α-hydroxyl group. Characteristics of SHBG are:

  • High binding affinity to dihydrotestosterone
  • Medium binding affinity to testosterone and estradiol
  • Low binding affinity to estrone, DHEA, androstenedione, and estriol.

SHBG regulates the availability of these hormones for the target tissues. Other binding and transport proteins for sex steroids include corticosteroid binding protein (CBG) and albumin. In men, 44–65% of circulating testosterone is bound to SHBG, 33–50% to albumin, and 3.5% to CBG; only 2–3% exists as free testosterone /3/. In women, 66–78% of circulating testosterone is bound to SHBG and 20–30% to albumin. SHBG binding reduces the renal clearance of androgens and estrogens and prevents the conversion of testosterone to androstenedione in the tissues (Fig. 37.1-1 – Biosynthesis of important sex steroids). SHBG also reduces the amount of testosterone that can bind to intracellular androgen receptors and exerts its biological effects.

Free (bioavailable) testosterone

Testosterone circulates in the blood either in free or protein-bound form. Albumin-bound testosterone dissociates readily as it passes through the tissues, releasing free testosterone; this is not the case for testosterone that is bound to SHBG. Free testosterone and albumin-bound testosterone are therefore referred to as the bioactive fraction. They can bind directly to intracellular receptors and exert their biological actions.

References

1. Selby C. Sex hormone binding globulin: origin, function and clinical significance. Ann Clin Biochem 1990; 27: 532–41.

2. Evaluation of a sex hormone-binding globulin automated chemiluminescent assay. scand J Clin Lab Invest 2013; 73 (6): 480–4.

3. Goto A, Chen BH, Song Y, Cauley J, Cummings SR, Farhat GN, et al. Age, body mass index, usage of exogenous estrogen, and lifestyle factors in relation to circulating sex hormone-binding globulin concentrations in postmenopausal women. Clin Chem 2014; 60: 174–85.

4. De Ronde W, van der Schouw Y, Pierik FH, Pols HAP, Muller M, Grobbee DE, et al. Serum levels of sex hormone binding globulin (SHBG) are not associated with lower levels of non-SHBG-bound testosterone in male newborns and healthy adult men. Clin Endocrinol 2005; 62: 498–503.

5. Ring J, Welliver C, Parenteau M, Markwell S, Branningan RE, Köhler TS. The utility of sex hormone-binding globulin in hypogonadism and infertile males. J Urol 2017; 197 (5): 1326–31.

6. Thaler MA, Seifert-Klauss V, Luppa PB. The biomarker sex hormone-binding globulin – from established applications to emerging trends in clinical medicine. Best Pract Res Clin Endocrinol Metab 2015; 29 (5): 749–60.

7. Vanbillemont G, Lapauw B, Bogaert V, Goemaere S, Zmierczak HG, Taes Y, Kaufman JM. Sex hormone-binding globulin as an independent determinant of cortical bone status in men at the age of peak bone mass. J Clin Endocrinol Metab 2010; 95: 1579–86.

8. Elmlinger MW, Kühnel W, Ranke MB. Reference ranges for serum concentrations of lutropin (LH), follitropin (FSH), estradiol (E2), prolactin, progesterone, sex hormone-binding globulin (SHBG), dehydroepiandrosterone sulfate (DHEAS), cortisol and ferritin in neonates, children and young adults. Clin Chem Lab Med 2002; 40: 1151–60.

9. Kratz A, Ferraro M, Sluss PM, Lewandrowski KB. Laboratory reference values. N Engl J Med 2004; 351: 1548–63.

37.8 Testosterone

The failure of the testes to produce physiologic levels of testosterone and a normal number of spermatozoa are clinical symptoms of hypogonadism.

37.8.1 Indication

In men:

  • Suspected primary or secondary hypogonadism
  • Suspected late onset hypogonadism.

In women:

  • Clinical signs of androgenization
  • Suspected androgen induced ovarian insufficiency
  • Suspected polycystic ovary syndrome.

37.8.2 Method of determination

Total testosterone

Immunoassays; the candidate reference method is liquid chromatography tandem mass spectrometry (LC-MS/MS) /1/.

Free (bioactive) testosterone

Immunoassay for determination of total testosterone and SHBG and use of the free testosterone index or the Sodergard formula (www.issam.ch/freetesto.htm). Other methods are the prior separation of free testosterone by equilibrium dialysis or ultrafiltration /2/.

37.8.3 Specimen

Serum, morning blood sample: 1 mL

37.8.4 Reference interval

Refer to Tab. 37.8-1 – Reference intervals for testosterone.

37.8.5 Clinical assessment

The assessment of a testosterone level found to be low on a sample taken at 9.00 requires a serum prolactin, LH and FSH measurement in order to rule out secondary hypogonadism. Also, sex hormone binding globulin (SHBG) measurement will help in investigation of male hypogonadism /3/.

37.8.5.1 Testosterone in males

Two phases in a man’s life are characterized by a particularly high level of activity in the hypothalamic-pituitary-gonadal axis are the neonatal period and the period before and after puberty. In the first 6 months of life, LH and testosterone are elevated to levels within the lower adult reference interval. They then decline sharply and do not rise again until the start of puberty /4/. During adulthood, levels of both hormones are high and remain high. In older men, there can be a slight decline in testosterone and a slight rise in SHBG. If this is associated with hypogonadism, this is known as late- onset hypogonadism (LOH).

For more about LOH and about primary and secondary hypogonadism in males, refer to:

Testosterone deficiency leads to a decrease in lean body mass, muscle mass, and strength and 17β-estradiol deficiency leads to increased body fat; deficiency of both hormones causes reduced sexual function. Testosterone levels are correlated with hypogonadism in type 2 diabetics /5/.

In a study /6/, 198 men aged 20–50 years were provided with goserelin acetate (to suppress endogenous testosterone and estradiol) and randomly assigned to receive a placebo gel or 1.25 g, 2.5 g, 5 g, or 10 g of testosterone gel daily for 16 weeks. Another 202 healthy men received goserelin acetate, placebo gel or testosterone gel, and anastrozole (to suppress the conversion of testosterone to 17β-estradiol). The results found the following:

  • Erectile function, lean body mass, and thigh muscle area were reduced at a testosterone dose (1.25 g per day) that elicited serum testosterone levels of approximately 2 μg/L (7 nmol/L), justifying testosterone supplementation
  • At testosterone levels of 2–4 μg/L (7–15 nmol/L) and 17β-estradiol levels of ≥ 10 ng/L (36.7 pmol/L), the sexual desire decreased by 13%, however, if estradiol levels were < 10 ng/L the sexual desire decreased by 31%.

The total testosterone was measured by means of an immunoassay and using LC-MS/MS.

37.8.5.2 Testosterone in females

Oligomenorrhea with selective LH hypersecretion is observed in 6–8% of women, often in conjunction with the symptoms of polycystic ovary syndrome (PCOS). A significant proportion of these patients have symptoms of hyper androgenism and elevated testosterone levels /7/.

For hyper androgenism in women, refer to:

37.8.6 Comments and problems

Method of determination

Compared to Isotope dilution/GC-MS, immunoassays overestimate testosterone levels in women and underestimate testosterone levels in men. Ten different assays overestimated testosterone levels in women by a mean of 46% and underestimated testosterone levels in men by a mean of 12% /8/.

DHEAS interferes in several testosterone immunoassays; testosterone concentrations in two assays correlated positively with the DHEAS concentration /9/. In women with testosterone levels of 5–7 nmol/L, interference by DHEAS led to a mean increase of 1.4 nmol/L in the measured level /10/.

Determination of free testosterone using equations

When four different equations were used to calculate free testosterone in male serum, the mean bias ranged from 5.8–56% /11/.

Stability

4 hours at 20 °C, 3 days at 2–8 °C.

Accuracy

Immunoassays can have differences in testosterone concentration ranging from 200% to 500% compared with LC-MS/MS. The desirable limits for imprecision, bias, and total error are 4.7%, 5.4%, and 13.1%, respectively /12/.

37.8.7 Pathophysiology

The androgen testosterone (17β-hydroxy androstenone) is a steroid hormone with a molecular mass of 288 Da. It is secreted by the testicular Leydig cells and, in small amounts, by the ovaries. The enzyme 17β-hydroxy steroid dehydrogenase type 3 (17β-HSD3) catalyzes the conversion of androstenedione to testosterone in the testes (Fig. 37.1-1 – Biosynthesis of sex steroids), the secretion is regulated by LH.

Testosterone exists in the plasma in different forms with different names /12/:

  • Total testosterone includes SHBG-bound and albumin-bound testosterone as well as free testosterone
  • Free testosterone (2–3% of total testosterone)
  • Bioavailable testosterone (35–52% of total testosterone) includes albumin bound testosterone, which dissociates readily, and free testosterone. Refer also to Section 37.7 – Sex hormone binding globulin (SHBG).

Testosterone in males

Secreted by the testes, testosterone acts as an anabolic hormone promoting secondary sexual characteristics such as hair growth, muscle mass, penile enlargement and libido, as well as sexual differentiation and spermatogenesis. The amount of testosterone required to maintain lean body mass, fat mass, and muscular strength varies widely in men. The diurnal variation in the testosterone concentration corresponds to that of LH. Highest levels are measured in the morning and lowest concentrations between 9.00 p.m. and midnight. In older men with suspected hypogonadism, in whom the total testosterone may be normal, free testosterone is more meaningful than total testosterone.

Testosterone in females

The physiological effect of testosterone in females is the growth of pubic and axillary hair. It also has an influence on the libido. At physiological concentrations, testosterone has no specific effects in women. It is synthesized in the ovaries and adrenal glands. Dehydroepiandrosterone sulfate (DHEAS) is an indicator of androgen production by the adrenals.

In women, the total plasma testosterone level is low and the determination of elevated concentrations is less reliable than in men. For this reason, DHEAS is also determined in initial investigations in order to verify and distinguish the origin of the testosterone.

References

1. Botelho JC, Shacklady C, Cooper S, Tai SSC, van Uytfranghe K, Thienpoint L, Vesper HW. Isotope-dilution liquid chromatography-tandem mass spectrometry candidate reference method for total testosterone. Clin Chem 2013; 59: 372–80.

2. Giton F, Fiet J, Guechot J, Ibrahim F, Bronsard F, Chopin D, Raynaud JP. Serum bioavailable testosterone: assayed or calculated? Clin Chem 2006; 474–81.

3. Livingston M, Kalansooriya A, Hartland AJ, Ramachadran S, Heald A. Serum testosterone levels in male hypogonadism: why and when to check – a review. Int J Clin Pract 2017; 71: e12995

4. Goto A, Chen BH, Song Y, Cauley J, Cummings SR, Farhat GN, et al. Age, body mass index, usage of exogenous estrogen, and lifestyle factors in relation to circulating sex hormone-binding globulin concentrations in postmenopausal women. Clin Chem 2014; 60: 174–85.

5. Herrero A, Marcos M, Galindo P, Miralles JM, Corrales JJ. Clinical and biochemical correlates of male hypogonadism in type 2 diabetes. Andrology 2018; 6: 58–63.

6. Finkelstein JS, Lee H, Burnett-Bowie SAM, Pallais JC, Yu EW, Borges LF, et al. Gonadal steroids and body composition, strength, and sexual function in men. N Engl J Med 2013; 369: 1011–22.

7. Santoro N. Update in hyper- and hypogonadotropic amenorrhea. J Clin Endocrinol Metab 2011; 96: 3281–8.

8. Taieb J, Mathian B, Millot F, Patricot MC, Mathieu E, Queyrel N, et al. Testosterone measured by 10 immunoassays and isotope-dilution gas chromatography-mass spectrometry in sera from 116 men, women and children. Clin Chem 2003; 49: 1381–95.

9. Benton SC, Nuttall M, Nardo L, Laing I. Measured dehydroepiandrosterone sulfate positively influences testosterone measurement in unextracted female serum: comparison of 2 immunoassays with testosterone measured by LCMS. Clin Chem 2011; 57: 1074–5.

10. Heald AH, Butterworth A, Kane JW, Borzomato J, Taylor NF, Kilpatrick ES, et al. Investigation into possible causes of interference in serum testosterone measurement in women. Ann Clin Biochem 2006; 43: 189–95.

11. Ho CKM, Stoddart M, Walton M, Anderson RA, Beckett GJ. Calculated free testosterone in men: comparison of four equations and with free androgen index. Ann Clin Biochem 2006; 43: 389–97.

12. Yun Y-M, Cook-Botelho J, Chandler DW, Katayev A, Roberts WL, Stanczyk FZ, et al. Performance criteria for testosterone measurement based on biological variation in adult males: recommendations from the Partnership for the Accurate Testing of Hormones. Clin Chem 2012; 58: 1703–10.

13. Goldman AL, Bhasin S, Wu FCW, Krishna M, Matsumoto AM, Jasuja R. A reappraisal of testosteron´s binding in circulation: physiological and clinical implications. Endocrine Reviews 2017; 38 (4): 302–24.

14. Pesant MH, Desmarais GD, Baillargeon JP. Reference ranges for total and calculated free bioavailable testosterone in a young healthy women population with normal menstrual cycles or using oral contraception. Clin Biochem 2012; 45: 148–50.

15. Kratz A, Ferraro M, Sluss PM, Lewandrowski KB. Laboratory reference values. N Engl J Med 2004; 351: 1548–63.

37.9 Inhibin

Inhibin is a major hormone in reproductive biology, secreted primarily by ovarian granulosa cells and testicular Sertoli cells. Inhibin antagonizes the hormone activin. Activin A and activin B are pleiotropic factors that affect proliferation, differentiation, and apoptosis in a variety of cell types. As one of the hormones that regulate activin induced FSH (not LH) formation and folliculogenesis, inhibin is a diagnostic marker in the assessment and management of infertility- and pregnancy-related conditions. Activin and inhibin share the type II activin receptor (ActRII) and inhibin blocks the activin induced signalling of the receptor and formation of FSH. Beta glycan a membrane-anchored proteoglycan acts as an inhibin coreceptor and increases the binding affinity of inhibin to the receptor /1/.

37.9.1 Indication

Assessment and management of infertility- and pregnancy-related conditions, especially in the assessment of ovarian reserve.

Prenatal screening of Down syndrome as part of the quadruple test.

37.9.2 Method of Determination

Inhibins are heterodimers of a common α-subunit and a βA- or βB-subunit. The inhibins are produced as precursor molecules that undergo further processing into major subunits that assemble into active inhibin dimers /1/.

The inhibin A and B ELISAs are sandwich assays. Two monoclonal antibodies (anti-βA-subunit and anti-βB-subunit) are utilized as capture antibodies and a detection antibody directed against a peptide of the inhibin α-subunit conjugated with alkaline phosphatase are used /2/.

A commercially available ELISA measures inhibin B using a biotinylated detection antibody directed against the α-subunit of inhibin /3/.

37.9.3 Specimen

Serum, heparinized plasma: 1 mL

37.9.4 Reference interval

The reference intervals of a commercial inhibin B test are:

Females: ≤ 341 pg/mL

Females 3. cycle day: ≤ 273 pg/mL

Females post menopause: ≤ 4 pg/mL

Males: 25–325 pg/mL

37.9.5 Clinical assessment

Inhibins are glycoprotein hormones that belong to the TGFβ super family and are composed of two subunits, an alpha-subunit (20 kDa) and a beta-subunit (13 kDa) linked by a disulfide bridge. Heterodimers of the inhibin α-subunit and β-subunit are the mature inhibin forms. There are two main isoforms of the β-subunit, βA and βB, resulting in two isoforms of the mature 32 kDa inhibin protein, inhibin A (αβA) and inhibin B (αβB) /4/. The inhibitory activity of inhibin B for activin is higher in comparison to inhibin A. Inhibins antagonize the activin signal transduction pathway and are negative regulators of FSH release from the anterior pituitary.

Inhibin is a gonadal hormone that down regulates FSH production and release by the anterior gonadotropes and a paracrine factor that regulates ovarian folliculogenesis /5/and steroidogenesis /6/. Inhibins modulate FSH synthesis by antagonizing the actions of activin to induce FSH formation and folliculogenesis. Gonadal inhibins and locally produced activin B directly regulate the biosynthesis of FSH. Activin stimulates and inhibin decreases the level of FSH.

Serum levels of inhibin are undetectable after gonadectomy in both males and females. In addition to reproductive organs, inhibin is present in the eye, lung, kidney, bone marrow, brain, pituitary and the adrenal glands. The role of inhibins in the reproduction is shown in Tab. 37.9-1 – Clinical applications of inhibin determination.

References

1. Makanji Y, Zhu J, Mishra R, Holmquist C, Wong WPS, Schwartz NB, Mayo KE, Woodruff TK. Inhibin at 90: from discovery to clinical application, a historical review. Endocr Rev 2014; 35 (5): 747–94.

2. Groome NP, Illingworth PJ, O’Brien M, Pai R, Rodger FE, Mather JP, McNeilly AS. Measurement of dimeric inhibin B throughout the human menstrual cycle. Clin Endocrinol (Oxf) 1994; 40: 717–23.

3. Groome NP, Lawrence M. Preparation of monoclonal antibodies to the βA subunit of ovarian inhibin using a synthetic peptide immunogen. Hybridoma 1991; 10: 309–16.

4. Ling N, Ying SY, Ueno N, Esch F, Denoroy L, Guillemin R. Isolation and partial characterization of a Mr 32,000 protein with inhibin activity from porcine follicular fluid. Proc Natl Acad Sci USA 1985; 82: 7217–21.

5. Woodruff TK, Lyon RJ, Hansen SE, Rice GC, Mather JP. Inhibin and activin locally regulate rat ovarian folliculogenesis. Endocrinology 1990; 127: 196–205..

6. Hsueh AJ, Dahl KD, Vaugham J,Tucker E, Rivier J, Bardin CW, Vale W. Heterodimers and homodimers of inhibin subunits have different paracrine action in the modulation of luteinizing hormone-stimulated androgen biosynthesis. Proc Natl Acad Sci USA 1987; 84: 5082–6.

7. Groome NP, Illingsworth PJ, O’Brien M, Rodger FE, Mather JP, McNeilly AS. Measurement of dimeric inhibin B throughout the human menstrual cycle. J Clin Endocrinol Metab 1996; 81 (4): 1401–5.

8. Muttukrishna S, North RA, Morris J, Schellenberg JC, Taylor RS, Asselin J, et al. Serum inhibin A and activin A are elevated prior to the onset of pre-eclampsia. Hum Reprod 2000; 15 (7): 1640–5.

9. Wright VC, Chang J, Jeng G, Macaluso M. Assisted reproductive technology surveillance – United States ,2005. MMWR Surveill Summ 2008; 57: 1–23.

10. Coccia ME, Rizzello F. Ovarian reserve. Ann NY Acad Sci 2008; 1127: 27–30.

11. Ocal P, Aydin S, Cepni I, Idil S, Idil M, Uzzun H, Benian A. Follicular fluid concentrations of vascular endothelial growth factor, inhibin A and inhibin B in IVF cycles: are they markers for ovarian response and pregnancy outcome? Eur J Obstet Gynecol Reprod Biol 2004; 115: 194–9.

12. Seifer DB, Lambert-Messerlian G, Hogan JW, Gardiner AC, Blazar AS, Berk CA. Day 3 serum inhibin B predictive of assisted reproductive technologies outcome. Ferti Steril 1997; 67: 110–4.

13. Welt CK Hall JE, Adams JM, Taylor AE. Relationship of estradiol and inhibin to the follicle stimulating hormone variability in hypergonadotropic hypogonadism or premature ovarian failure. J Clin Endocrinol Metab 2005; 90: 826–30.

14. Toulis KA, Iliadou PK, Venetis CA, Tsametis C, Tarlatzis BC, Papadimas I, Goulis DG. Inhibin B and anti-Mullerian hormone as markers of persistent spermatogenesis in men with non-obstructive azoospermia: a metaanalysis of diagnostic accuracy studies. Hum Reprod Update 2010; 16: 713–24.

15. Pierik FH, Vreeburg JT, Stijnen T, De Jong FH, Weber RF. Serum inhibin B as a marker of spermatogenesis. J Clin Endocrinol Metab 1998; 83: 3110–4.

37.10 Anti-Muellarian hormone

Anti-Muellarian hormone (AMH) is a dimeric glycoprotein and a member of the transforming growth factor β (TGF-β) family of growth differentiation factors. AMH has been predominantly known for its role in male sexual differentiation. However, AMH has developed into a factor with a wide array of clinical applications, mainly based on its ability to represent the number of antral and pre-antral follicles present in the ovaries /1/.

37.10.1 Indication

AMH has been suggested to predict /1/:

  • Ovarian response to hyper stimulation of the ovaries for assisted reproductive technology (ART)
  • Timing of menopause
  • Iatrogenic damage of the ovarian follicle reserve.

AMH is proposed as a surrogate for antral follicle count in the diagnosis of polycystic ovary syndrome /1/.

37.10.2 Method of determination

Immunoassay

Principle: AMH of the sample forms a sandwich complex with a biotinylated monoclonal antibody and a ruthenium labeled monoclonal antibody complex. Both antibodies are raised against recombinant human AMH and recognize epitopes in the pro region of AMH. After addition of streptavidin-biotin coated micro particles the sandwich complex is bound to a solid phase. Substances not bound to the micro particles are removed. The chemiluminescence of the micro particle bound sandwich complexes is measured.

37.10.3 Specimen

Serum, heparinized plasma: 1 mL

37.10.4 Reference interval

Males /2/

0.77–14.5 ng/mL (5.5–103 pmol/L)

Females /2/

20–24 years: 1.22–11.7 ng/mL (8.71–83.6 pmol/L)

25–29 years: 0.89–9.85 ng/mL (6.35–70.3 pmol/L)

30–34 years: 0.58–8.13 ng/mL (4.11–58.0 pmol/L)

35–39 years: 0.15–7.49 ng/mL (1.05–53.5 pmol/L)

40–44 years: 0.03–5.47 ng/mL (0.19–39.1 pmol/L)

45–50 years: 0.01–2.71 ng/mL (0.07–19.3 pmol/L)

Values are 2.5th and 97.5th percentiles of the Elecsys AMH Plus assay.

Conversion factors: ng/ml × 7.14 = pmol/l; pmol/l × 0.14 = ng/mL

37.10.5 Clinical assessment

The determination of AMH is used in various ways in clinical diagnostics to evaluate problems in reproduction.

37.10.5.1 AMH in females

AMH is specifically expressed in granulosa cells of small growing follicles. AMH has a potential role in dominant follicle selection in the follicular phase of the menstrual cycle. AMH acts as a follicular gatekeeper and ensures that each antral follicle produces little estradiol prior to selection (i.e., up to a follicular diameter of about 8 mm) allowing direct ovarian/pituitary dialogue regulating the development of the selected follicle that will undergo ovulation /3/.

During menstrual cycle AMH exhibits mild fluctuation due to the very high variability in the number of antral follicules within women of similar age. However the inter cyclic variation is much lower (13%) than the inter-individual variability (72%) amongst women of the same age /4/. The concentration of AMH is relative stable through the menstrual cycle since the dominant follicle and corpus luteum do not secrete AMH. Because of relative stability through the menstrual cycle blood collection is not limited to special days.

AMH is detectable in girls of all ages, unlike other reproductive hormones, and rises steadily through childhood thus may be of value in the assessment of ovarian function in prepubertal girls.

In pregnancy a decline in serum AMH concentration is measured with advancing gestational age. In the post-partum period AMH concentrations are increased /5/.

AMH is negatively related to body mass index but the relationship is age dependent. AMH serum levels are approximately 30% lower in hormonal oral conception users than controls. Serum AMH is not a predictor of ovarian reserve in women using long-term hormonal contraception.

AMH is an important marker in the assessment of ovarian reserve in:

  • Assisted reproductive technology (ART)
  • Ovarian damage from chemotherapy, radiotherapy and surgery
  • Polycystic ovary syndrome.

Refer to Tab. 37.10-1 – Clinical applications of AMH determination.

37.10.5.2 AMH in pediatrics and in adolescents

In boys, AMH determination is useful in the clinical setting as a marker of Sertoli cell function. Serum AMH is low in infants with hypo gonadotropic hypogonadism (and increases with FSH treatment), in patients with primary hypogonadism from early postnatal life and in Klinefelter syndrome from mid puberty. In boys with non palpable gonads, AMH determination is useful to distinguish between cryptorchism and anorchism, as well as differentiating the dysgenetic causes of disorders of sexual development from those due to defective androgen synthesis or action /6/.

37.10.6 Comments and problems

Specimen

EDTA plasma should not be used for the determination of AMH.

Method of determination

Because commercial assays use different antibody pairs and different AMH calibrators, the values of serum AMH differ significantly between the assays.

Stability

Three days at 20–25 °C, 5 days at 2–8 °C

37.10.7 Pathophysiology

AMH is a homodimeric glycoprotein, a member of the transforming growth factor β (TGF-β) family and its gene is located on chromosome 19 p 13.3, containing 5 exons. The hormone binds to its receptor (AMRH) that is expressed on target organs such Muellerian ducts, Sertoli- and Leydig cells of testes and granulosa cells of the ovary /1/. The structure of AMH is shown in Fig. 37.10-1 – Structure of anti-Muellarian hormone.

AMH is also recognized as Muellarian inhibiting hormone. Wolfian as well as Muellarian ducts consist in the sexually undifferentiated embryo.

In the male embryo Sertoli cells secrete AMH that induce the regression of Muellarian duct as early as 7th week of gestation. The Wolfian duct gives rise to epididymis and the seminal vesicles under the influence of testosterone. Since then AMH is continuously secreted until puberty when it rapidly declines in response to testosterone synthesis /78/.

In the female embryo absence of AMH allows the Muellarian duct to give rise to uterus, fallopian tubes, and part of vagina. The earliest production of AMH is the 36th week of gestation. Female newborns have about 35 times lower AMH serum levels than males of the same age /78/. Females are born with a fixed number of primordial follicles, resting in a dormant state of meiosis II until they enter different states starting at puberty. The quantity and quality of primordial follicles constitute the ovarian reserve. Dormant primordial follicles do not produce AMH. However, as soon as they are recruited for development AMH is specifically expressed in granulosa cells of small and large pre antral follicles and small antral follicles. AMH inhibits follicle recruitment and selection of antral follicles to the preovulatory follicle. As soon as the follicles enter FSH dependent stages of development (large antral stage > 8–10 mm diameter) and are selected for dominance the secretion of AMH ist lost. This supports the role of AMH as a major regulator of initial as well as cyclic recruitment of follicles by maintaining their threshold for FSH sensitivity /9/. Refer to Fig. 37.10-2 – AMH actions in the ovary.

In summary: AMH inhibits FSH induced pre-antral follicle growth and functions as a gatekeeper of follicular estrogen production and is involved in the fine-tuned and delicate balance between estradiol secretion by the preovulatory follicle and gonadotropin secretion by the pituitary to ensure that ovulation is triggered exactly at the right time. It has been suggested that AMH may exert a physiological role in down regulating the aromatizing capacity of granulosa cells until the time of follicular selection.

In the absence of AMH, primordial follicles are recruited at a faster rare, resulting in an exhausted primordial follicle pool at a younger age. AMH serum levels decline with increasing chronological age. In women from 21 years of age and onwards the annual decline has been calculated to be 5.6% /10/.

References

1. Dewailly D, Andersen CY, Balen A, Broekmans F, Dilaver N, Fanchin R, et al. The physiology and clinical utility of anti-Müllarian hormone in women. Human Repoduction Update 2014; 20 (3): 370–85.

2. Elecsys AMH Plus Test.

3. Van Houten EL, Themmen EP, Visser JA. Anti-Müllarian hormone (AMH): regulator and marker of ovarian function. Ann Endorcrinol (Paris) 2010; 71: 191–7.

4. Van Disseldorp J, Lambalk CB, Kwee J, Looman CW, Eikjmans MJ, Fauser BC, et al. Comparison of inter- and intra-cyclic variability of anti-Mullarian hormone and follicle counts. Human Reprod 2010; 25: 221–7.

5. McCredie S, Ledger W, Venetis CA. Anti-Müllerian hormone kinetics in pregnamcy and post-partum. Reprod Biomed Online 2017; 34 (5) 522–33.

6. Weintraub A, Eldar-Geva T. Anti-Mullarian hormone (AMH) determinations in the pediatric and adolescent endocrine practice. Pediatr Endocrinol Rev 2017; 14 (4): 364–70.

7. Lindhardt Johansen M, Hagen CP, Johannsen TH, Main KM, Picard JY, Jorgensen A, et al. Anti-Müllarian hormone and its clinical use in pediatrics with special emphasis on disorders of sex development Int J Endocrinol 2013; Vol 2013: Article ID 198698.

8. Jamil Z, Fatima SS, Ahmed K, Malik R. Anti-Mullarian hormone: above and beyond conventional ovarian reserve markers. Disease Markers 2016; Vol 2016: Article ID 5246217.

9. Jeppesen JV, Anderson RA, Kelsey TW, Christiansen LW, Kristensen SG, Yayaprakasan K, et al. Which follicles make the most anti-Müllarian hormone in humans? Evidence for an abrupt decline in AMH production at the time of follicle selection. Mol Hum Reprod 2013; 19: 519–27.

10. Broer SL, Broekmans FJM, Laven JSE, Fauser BCJM. Anti-Müllarian hormone: ovarian reserve testing and its potential clinical implications. Human Reproduction Update 2014; 20 (5): 688–701.

11. Van Rooij A, Broekmans FJ, te Velde ER, Fauser BC, Bancsi LF, Jong FH, Themmen AP. Serum anti-Müllarian hormone levels: a novel measure of ovarian reserve. Hum Reprod 2002; 17: 3065–71.

12. Stracquadanio M, Ciotta L, Palumbo MA. Relationship between serum anti-Müllarian hormone and intrafollicular AMH levels in PCOS women. Gynecol Endocrinol 2018; 34 (3): 223–8.

13. Depmann M, Eijkemans MJC, Broer SL, Scheffer GJ, van Rooij IAJ, Laven JSE, Broekmams FJM. Does anti-Müllarian hormone predict menopause in the general population? Results of a prospective ongoing cohort. Human Reproduction 2016; 31 (7): 1579–87.

14. Promberger R, Ott J. Anti-Müllarian hormone as a parameter for endometrial trauma in Asherman syndrome: a retrospective data analysis. Reprod Biol 2017; 17 (2): 151–3.

37.11 Kisspeptin

Kisspeptines are a family of neuropeptides and act upstream of gonadotropin releasing hormone (GnRH) as high-level mediators of the reproductive axis. Kisspeptins stimulate the release of GnRH from hypothalamic neurons and are involved in the control of human reproduction bridging the gap between sex steroid levels and feedback mechanisms that control the GnRH secretion /1/. Exogene kisspeptin or kisspeptin receptor agonists can stimulate physiological GnRH responses in both healthy subjects and those with disorders of reproduction. Kisspeptin administration stimulates the reproductive endocrine cascade in both men and women /2/.

37.11.1 Indication

In combination with kisspeptin agonists to localize lesions in the hypothalamic-pituitary-gonadal axis dysfunction to evaluate the gonadotrophic potential of infertile individuals /3/.

37.11.2 Method of determination

Enzyme immunoassay /4/: several enzyme-linked immunosorbent assays are commercially available.

37.11.3 Specimen

Serum, heparinized plasma: 1 mL

37.11.4 Reference interval

Refer to the recommendation of the manufacturer; some manufacturers recommend intervals of approximately 0.2–2 ng/mL, some 10 to 100 fold higher levels.

37.11.5 Clinical assessment

Kisspeptins are critical for initiating puberty and regulating ovulation in sexually mature females via the central control of the hypothalamic-pituitary gonadal axis (HPO axis) /5/. The pulsatile secretion of GnRH and therefore the gonadotropins FSH and LH regulate the HPO axis at puberty and maintain cyclic function in adulthood. The GnRH secretion is modulated by a negative feedback of serum estrogen secreted from the ovarian follicle. However, there are several functional limitations of the GnRH network. The most important issue is that GnRH neurons do not express estrogen receptor-α that mediates both positive and negative estrogen feedback actions in tissues /6/. The upstream regulator that exerts positive and negative feed back actions in response to estradiol is kisspeptin (KISS1) that functions through the kisspeptin receptor (KISS1R) to stimulate and release GnRH. Inactivating mutations in the kisspeptin molecule or its receptor can lead to hypo gonadotropic hypogonadism /5/.

Kisspeptin administration stimulates GnRH induced LH release in healthy men /7/. In postmenopausal women continuous administration of kisspeptin demonstrated a significant increase in LH pulse amplitude in direct proportion to the circulating estradiol concentration /2/.

HCG is the most used trigger for oocyte maturation, but is associated with increased risk of ovarian hyper stimulation syndrome (OHSS). Kisspeptin-54 can be used as an oocyte maturation trigger /8/ and augments the expression of genes involved in ovarian steroidogenesis in granulosa lutein cells including, FSH receptor, LH/hCG receptor, steroid acute regulatory protein, aromatase, estrogen receptors, 3β-hydroxy steroid dehydrogenase and inhibin A, when compared to traditional maturation triggers, but does not alter markers of OHSS. In a study /8/ women undergoing IVF treatment for infertility received either hCG or kisspeptin-54 to trigger oocyte maturation. Granulosa lutein cells from women who had received kisspeptin-54 had a 8–14 fold higher expression of FSH receptor and a 2–2.5 fold higher expression of LH/hCG receptors than granulosa cells who had received hCG or GnRH agonist, respectively. Kisspeptin-54 had no direct effects on either OHSS genes or steroidogenic genes.

After intracytoplasmic sperm injection (ICSI) kisspeptin measured on hCG day (clinical pregnancy with β-hCG > 25 IU/L) can be used as a marker for success of treatment. Kisspeptin values are higher than in non-pregnant with β-hCG < 25 IU/L /9/.

Women with PCOS have increased kisspeptin levels. In a study /10/ kisspeptin concentrations were negatively correlated with serum FSH and positively correlated with serum total testosterone and DHEAS levels.

37.11.6 Comments and problems

The gene KISS1 encodes the kisspeptin precursor, a peptide comprising 145 amino acids. This is proteolysed to fragments of various lengths. Kisspeptin-54 is the major fragment, while other fragments include kisspeptin-10, kisspeptin-13, and kisspeptin-14. The commercially available kisspeptin assays measure different forms of kisspeptins a reason for different results.

37.11.7 Pathophysiology

Kisspeptin describes a family of polypeptide hormones of varying amino acid length cleaved from the product of the KISS1 gene. The kisspeptin peptides share a common carboxy-terminal sequence necessary for their action on kisspeptin receptors. Secreted by neurons within the hypothalamus kisspeptin activates kisspeptin receptors resulting in GnRH release. GnRH arriving the gonadotrophs of the anterior pituitary stimulates the release of FSH and LH that stimulate the gonads to release estradiol as well as progesterone in females and testosterone in males. Kisspeptin also occurs in other extra hypothalamic brain regions and kisspeptin signalling plays a role in sexual and emotional brain processing /11/.

Kisspeptin is expressed abundantly in the arcuate nucleus (ARC) and the anteroventral periventricular nucleus (AVPV) of the forebrain. Both estradiol and testosterone regulate KISS1 gene expression in ARC and AVPV; however while estradiol and testosterone down regulate KISS1 mRNA in the ARC, they up regulate KISS1 expression in the AVPV. Thus, kisspeptin neurons in the ARC may participate in the negative feedback regulation of gonadotropin secretion, whereas kisspeptin neurons in the AVPV may contribute to generating the preovulatory gonadotropin surge in the female /12/.

At puberty hypothalamic levels of kisspeptin and its receptor increase dramatically, suggesting that kisspeptin signaling mediate the neurocrine events that trigger the onset of puberty /12/.

References

1. Trevisan CM, Montagna E, de Oliveira R, Christofolini DM, Barbosa CP, Crandall KA, Bianco B. Kisspeptin/GPR54 system: what do we know about its role in human reproduction? Cell Physiol Biochem 2018; 49: 12597–76.

2. Lippincott MF, Chan YM, Rivera Morales D, Seminara SB. Continuous kisspeptin administration in postmenopausal women: impact of estradiol on luteinizing hormone secretion. J Clin Endocrinol Metab 2017; 102 (6): 2091–9.

3. Whitlock BK, Daniel JA, Amelese LL, Tanco VM, Chameroy KA, Schrick FN. Kisspeptin receptor agonist (FTM080) increased plasma concentrations of luteinizing hormone in anestrous ewes. Peer J 2015; https://doi.org/10.7717/peerj.1382.

4. Mondal M, Baruah KK, Prakash BS. Determination of plasma kisspeptin concentrations during reproducrtive cycle and different phases of pregnancy in crossbred cows using bovine specific enzyme immunoassay. General and Comparative Endocrinology 2015; 224: 168–75.

5. Hu KL, Zhao H, Chang H-M, Yu Y, Qiao J. Kisspeptin/kisspeptin receptor system in the ovary. Frontiers in Endocrinology 2017; https://doi.org/10.3389/fendo.2017.00365.

6. Herbisonn AE, Pape JR. New evidence for estrogen receptors in gonadotropin releasing hormone neurons. Front Neuroendocrinol 2001; 22: 292–308.

7. George JT, Veldhuis JD, Roseweir AK, Newton CL, Faccenda E, Millar RP, et al. Kisspeptin 10 is a potent stimulator of LH and increases pulse frequency in men. J Clin Endocrinol Metab 2011; 96: E1228-E1236.

8. Owens LA, Abbara A, Lerner A, O’floinn S, Christopoulos G, Khanjani S, et al. The direct and indirect effects of kisspeptin-54 on granulosa lutein cell function. Human Reprod 2018; 33 (2): 292–302.

9. Jamil Z, Fatima SS, Arif S, Alam F, Rehman R. Kisspeptin and embryo implantation after ICSI. Reprod Biomed online 2017; 34 (2): 147–53.

10. Gorkem U, Togrul C, Arslan E, Sargin Oruc A, Buyukayaci Duman N. Is there a role for kisspeptin in pathogenesis of polycystic ovary syndrome? Gynecol Endocrinol 2018; 34 (2): 157–60.

11. Comnios AN, Dhillo WS. Emerging roles of kisspeptin in sexual and emotional brain processing. Neuroendocrinology 2018; 106: 195–202.

12. Hussain MA, Song WJ, Wolfe A. There is kisspeptin – and then there is kisspeptin. Trends Endocrinol Metab 2015; 26 (10): 564–72.

Table 37.1-1 Production rates of sex steroids in women /6/

Hormone

Reproductive
age

Post
menopausal

Ovarian
ablation

Androstene-
dione

2–3

0.5–1.5

0.4–1.2

DHEA

6–8

1.5–4

1.5–4

DHEAS

8–16

4–9

4–9

Testosterone

0.2–0.25

0.05–0.18

0.02–0.12

Estrogens

0.350

0.045

0.045

Data expressed in mg/24 h. DHEA, dehydroepiandrosterone; DHEAS, dehydroepiandrosterone sulfate.

Table 37.1-2 Serum concentrations of sex steroids in women /6/

Hormone

Premenopause

Postmenopause

Estradiol (E2)

40–400

10–20

Estrone (E1)

30–200

30–70

Testosterone

200–800

150–700

Androstenedione

600–3,000

300–1,500

Data expressed in ng/L

Table 37.2-1 Laboratory tests for the diagnosis of ovarian dysfunction

Hormonal investigations

Functional tests

Prolactin*

Follicle-stimulating hormone (FSH)*

Luteinizing hormone (LH)

Estradiol (E2)

Progesterone, 17α-hydroxyprogesterone

Testosterone*, androstenedione

Dehydroepiandrosterone sulfate (DHEAS)

TSH*

Progesterone withdrawal test

Estrogen-gestagen test

GnRH test

ACTH test

* Initial investigations in amenorrhea

Table 37.2-2 Functional tests in ovarian dysfunction

Functional test

Progesterone withdrawal test (progestogen test, progestin challenge)

Indication: after the exclusion of pregnancy and anatomical causes, the progesterone withdrawal test is the first step in the diagnostic evaluation of amenorrhea.

Principle: progestin intake for 10–12 days causes secretory transformation of the endometrium so that a withdrawal bleed occurs when the progestin is discontinued. Progestins are synthetic progestogens that have progestogenic effects similar to those of progesterone.

Test protocol: progestin intake for a period of 10–12 days at a dose sufficient to induce secretory transformation.

Interpretation: if so-called withdrawal bleeding occurs a few days after the cessation of medication, the test is said to be positive. This indicates that the endometrium adequately stimulated by basal estrogen production underwent secretory transformation in response to the progestin administration. A positive progesterone withdrawal test therefore indicates adequate estrogen synthesis by the ovaries. Estrogen supplementation is not indicated in the presence of a positive progesterone withdrawal test.

Estrogen test or estrogen-progestogen test

Indication: if the progestogen test is negative, the estrogen test may be done to aid in further differentiation.

Principle: in ovarian failure, the administration of estrogen stimulates secretory transformation of the endometrium.

Test protocol: intake of an estrogen (e.g., in the form of a natural estrogen or combined with a progestogen or a two-phase oral contraceptive for a period of 3 weeks).

Interpretation: normally, discontinuance of estrogens will evoke withdrawal bleeding (positive result). A positive result verifies the presence of a responsive endometrium, while a negative result indicates amenorrhea of uterine failure, which can be classified into primary or secondary ovarian failure based on the determination of FSH. It is important to note that a false negative test result may occur in pregnancy, in the absence of an uterus or if there is an impediment to the outflow of blood due to an anatomical anomaly.

GnRH test

Indication: this test is used to assess the severity of progestin-negative amenorrhea and in suspected hypothalamic-pituitary dysfunction.

Principle: GnRH stimulates the release of FSH and LH by the pituitary. If this does not occur, this indicates hypothalamic dysfunction.

Test protocol: basal blood sampling with immediate i.v. bolus administration of 25 μg GnRH followed by repeat blood sampling 30 minutes later. FSH and LH are determined in both blood samples.

Interpretation: in adults, LH increases to 2–8 times the basal value while FSH increases to 2–3 times the basal value. In pubertal children, an increase of twice the baseline level of each hormone is considered normal. An absent or inadequate increase suggests hypothalamic dysfunction.

The test is well suited:

  • To differentiate forms of ovarian failure due to hypothalamic causes from those due to pituitary causes. If a negative test is repeated after one week of pulsatile GnRH administration, this can distinguish definitively between a hypothalamic etiology (rise in LH/FSH is detectable), pituitary etiology (no rise in LH/FSH is detectable), and ovarian failure.
  • To differentiate between constitutional delays in sexual development (associated with a detectable rise in LH and FSH) and hypo gonadotropic hypogonadism (no detectable rise in LH and FSH)
  • To diagnose hyperandrogenemic ovarian failure, excessive LH rises are noted in conjunction with levels already elevated at baseline
  • To investigate children with pre cox puberty (i.e., in the appearance of secondary sexual characteristics prior to the age of 8 years in girls and 9 years in boys). In girls with centrally induced precocious puberty, both the basal and stimulated LH levels are higher than normal whereas only the basal levels of FSH are elevated; the stimulated FSH levels, in contrast, show the same pattern as in healthy pubertal girls /4/.

ACTH test

Indication: exclusion of adrenal enzymatic defects in the case of hirsutism.

Test protocol: following a fasting blood sample taken at 8:00 a.m. during the first half of the cycle to determine 17α-hydroxy progesterone, 250 μg of ACTH is administered by i.v. injection; a second blood sample is obtained 60 min. later.

Interpretation: normal test result, if the difference in 17α-hydroxy progesterone between the first and second sample amounts to less than 2.5 μg/L. Otherwise, classical or nonclassical congenital adrenal hyperplasia is likely if 17α-hydroxy progesterone increases to ≥ 10 μg/L. It is important to perform this test during the first half of the cycle because of the cross reactivity between 17α-hydroxy progesterone and progesterone.

Table 37.2-3 Clinical and laboratory findings in menstrual disorders

Clinical and laboratory findings

Oligomenorrhea

Menses occurring at intervals of 33–90 days are referred to as oligomenorrhea (fewer than 9 menses per year). It is frequently associated with abnormal follicular maturation and subsequent anovulatory cycles. Polycystic ovarian changes on ultrasound and suspected polycystic ovary syndrome (PCOS) are not uncommon. Hyper androgenemia and hyperprolactinemia are frequent causes of oligomenorrhea, each accounting for approximately 25% of cases. In addition to hyperprolactinemia, thyroid dysfunction must also be excluded. In the presence of pronounced virilization, determination of 17α-hydroxy progesterone is recommended to rule out a diagnosis of 21-hydroxylase deficiency (Section 34.5 – 17α-hydroxy progesterone).

Laboratory findings: FSH > 12 IU/L, LH < 2 IU/L; other possible findings include: prolactin > 600 mIU/L (30 μg/L), testosterone > 0.6 μg/L, DHEAS > 3500 μg/L, TSH < 0.4 mIU/L or > 4.5 mIU/L. If polycystic ovary syndrome is suspected, determination of sex hormone binding globulin (SHBG) is recommended. Levels of less than 15 nmol/L are typically observed in PCOS.

Polymenorrhea

The cycle length is less than 25 days and it often occurs in combination with hyper menorrhea or hypomenorrhea in the perimenopausal phase. Chronic anovulation with follicle persistence is present in many cases. The main priority is to exclude ovarian failure due to the onset of menopause.

Laboratory findings: blood sample taken during the second phase of the menstrual cycle: FSH > 12 IU/L, estradiol < 40 ng/L if perimenopausal. Determination of prolactin is important for the differential diagnosis.

Amenorrhea

Amenorrhea of at least 3 month duration is present in 3–5% of adult women while approximately 11% report oligomenorrhea of lesser duration. Among hyper gonadotropic conditions early menopause (cessation of menses before age 45) occurs in about 5% of women. Hypo gonadotropic hypogonadism, or hypothalamic ovarian failure, is observed in around 2–5% of adult women. Oligomenorrhea with selective LH hypersecretion is present in women with PCOS and occurs in about 6–8% of adult female population /4/.

Primary amenorrhea: by definition, primary amenorrhea refers to menarche not occurring by 16 years of age.

Secondary amenorrhea: by definition secondary amenorrhea is the absence of menses for 4 months or more. Amenorrhea is rare in patients with fertility problems or in those who are trying to conceive. Pregnancy must be excluded prior to any extensive diagnostic evaluation. Secondary amenorrhea can be due to a number of different causes, such as poorly controlled diabetes mellitus, celiac disease, anorexia nervosa, emotional stress, or previous chemotherapy or radiotherapy.

However, the four main etiologies are: primary ovarian insufficiency, hypothalamic amenorrhea, hyperprolactinemia, and polycystic ovary syndrome.

Laboratory findings: ovarian (primary) disorders are characterized by significant FSH elevation whereas central (secondary) amenorrhea is characterized by low to normal FSH and LH and low estradiol. Important investigations in the differential diagnosis include the determination of prolactin, testosterone, DHEAS, and TSH.

Primary ovarian insufficiency

Primary ovarian insufficiency is considered to be present when a woman who is less than 40 years old has had amenorrhea for 4 months or more with elevated serum FSH levels in the menopausal range (> 20 IU/L) /3/. The condition differs from menopause in that there is intermittent and unpredictable ovarian function in 50% of cases and about 5–10% of women conceive and deliver a child after they have received the diagnosis. Primary ovarian insufficiency occurs through two major mechanisms: follicle depletion and follicle dysfunction. In follicle dysfunction, a pathological process (e.g., an FSH receptor mutation) prevents normal follicle development. In most cases, the condition develops after a normal puberty and established regular menses, with the abrupt cessation of menses. In some women, menses fail to resume after pregnancy or after they have stopped taking hormonal contraceptives. In 90% of cases, the cause remains unknown. Spontaneous 46,XX primary ovarian insufficiency can occur as part of a syndrome (for a list, see Ref. /3/) or single gene mutations (FMR1 pre mutation) account for some of the causes. Several single genes like bone morphogenetic protein 15 (BMP15), diaphanous homolog 2 (DIAPH2), and inhibin alpha subunit (INHA) are associated with non syndromic primary ovarian insufficiency. There is also a positive family history with an affected first degree relative in 10–15% of cases and there may also be a history of autoimmune disease (hypothyroidism, adrenal insufficiency, or hypoparathyroidism).

Laboratory findings: after pregnancy is ruled out by hCG determination, serum FSH and prolactin should be measured. If the FSH level is in the menopausal range, the test should be repeated in 1 month along with a serum estradiol measurement. A progesterone withdrawal test is not recommended since nearly 50% of women with primary ovarian insufficiency have withdrawal bleeding in response to the test, despite the presence of menopausal FSH levels.

Hypothalamic-pituitary ovarian failure

Hypo gonadotropic ovarian failure is present. This is due to a disorder of the hypothalamic pulse generator, which leads to irregular gonadotropin release as a result of the loss of the normal pulsatile secretion of GnRH. Some patients have psychological disorders, some are competitive athletes, others have experienced major weight gain, and some have an intracranial space occupying lesion. Because of their low estradiol levels, patients are at risk of osteoporosis and hyperlipidemia /67/.

Laboratory findings: FSH < 20 IU/L, LH < 1 IU/L, estradiol < 30 ng/L.

Hyperprolactinemia

Serum prolactin levels of ≥ 1,000 mIU/L (50 μg/L) inhibit the hypothalamic GnRH pulse generator. Increasing prolactin levels result in a disorder of the ovarian cycle, which starts as luteal insufficiency and progresses to hypo gonadotropic ovarian failure. For more information, refer to Chapter 36 – Prolactin (PRL).

Polycystic ovary syndrome (PCOS)

PCOS is a common endocrine disorder with a prevalence of 5–10% in the adult female general population. PCOS is present if there is ultrasound evidence of polycystic ovaries in addition to at least one of the following /8/:

  • Oligomenorrhea or amenorrhea
  • Hyper androgenism (hirsutism, acne, alopecia) or hyper androgenemia (elevated free or total testosterone).

Other causes such as hyperprolactinemia, nonclassical CAH, Cushing’s syndrome, androgen secreting tumors, and acromegaly must be ruled out.

Biochemistry and physiology: LH regulates androgen synthesis in the theca cells and FSH is responsible for aromatase activity in the granulosa cells. Increased GnRH pulsatility is thought to promote the synthesis of LH, with a subsequent increase in androgen synthesis and reduction in estradiol synthesis (Fig. 37.1-1 – Biosynthesis of important sex steroids). It is not yet clear whether the increased GnRH pulse frequency is due to an intrinsic abnormality of the pulse generator or simply to reduced levels of pro gestogens, which have an inhibitory effect on pulsatile LH secretion.

Clinical findings: patients are oligomenorrheic or amenorrheic and may have dysfunctional uterine bleeding and reduced fertility as a result of anovulatory cycles. External symptoms include: hirsutism, acne, and androgenic alopecia. A proportion of patients are overweight, and obesity in turn is thought to act as an initiator for the development of the syndrome since it can cause metabolic and reproductive disturbances regardless of the diagnosis of PCOS. Initial symptoms can be present as early as menarche but can also arise after puberty in response to environmental triggers such as obesity. Apart from its effects on the gonadotropic system, other consequences of PCOS include obesity, reduced glucose tolerance, type 2 diabetes, hypertension, coronary heart disease, thrombosis, and sleep apnea, and it has also been associated with an increased predisposition to certain types of cancer.

Laboratory findings: LH and testosterone are elevated, estradiol is decreased. Because the concentration of LH fluctuates throughout the menstrual cycle, some investigators state that LH elevation is not required for a diagnosis of PCOS. In a study /9/, laboratory findings in patients with hypothalamic ovarian failure (HOF) were compared with those of patients who had both HOF and PCOS (HOF + PCOS). Although the concentrations of FSH (4–5 IU/L) and E2 (31 ± 14 ng/L) were almost identical in both groups, LH and testosterone concentrations differed significantly (HOF: LH 3.1 ± 2.6 IU/L, testosterone 322 ± 142 ng/L; HOF + PCOS: LH 7.7 ± 7.5 IU/L, testosterone 539 ± 307 ng/L).

Androgenization

The clinical signs of androgen excess may appear before menopause or after menopause as a consequence of normal aging. Androgen excess can only be confirmed by hyper androgenemia in laboratory investigations. The following causes must be considered in the differential diagnosis /10/:

  • Obesity: not all obese women have PCOS; most have a history of normal menarche and regular periods, often followed by a normal pregnancy, after which they became obese. They failed to lose the weight gained during pregnancy or continued to gain weight, resulting in disturbances in the gonadotropic system. Increased 5α-reductase and aromatase activity in adipose tissue is thought to be responsible for increased local production of androgens and estrogens, which in turn leads to irregular menses, hirsutism, and acne.
  • Iatrogenic causes: medications such as androgens, anabolic steroids, pro gestogens, danazol, glucocorticoids, diuretics, and anti rheumatics are the main causes of iatrogenic androgen excess.
  • PCOS: this is not always diagnosed before menopause, so it must also be considered in postmenopausal women. However, it is difficult to distinguish in this age group.
  • Hyperthecosis: this is a severe form of PCOS and results from an overproduction of androgens in the ovarian stromal cells. Premenopausal and postmenopausal women may present with this disorder. Hyperthecosis is typically bilateral and characterized by a hyperplastic ovarian stroma with cellular luteinization. The increased ovarian steroid synthesis is thought to be due to ovarian hyper stimulation.
  • Ovarian or adrenal tumors (Leydig cell tumors, Sertoli cell tumors, ovarian thecomas): the ovary tumors may present with hyper androgenism; adrenal tumors produce and secrete androgenic pro hormones (DHEA), glucocorticoids and/or estrogens.
  • Congenital adrenal hyperplasia (CAH). A distinction is made between classical CAH and late-onset CAH: Tab. 34.6-2 – Enzyme deficiencies in congenital adrenal hyperplasia).

Laboratory findings: primary investigations include measurement of testosterone and DHEAS; 17α-hydroxy progesterone may be determined in addition. DHEA is a pulsatile hormone whose determination is of limited use in the presence of stress. Testosterone levels are only reliable if they lie within the male range. In the event of borderline 17α-hydroxy progesterone levels, the ACTH test is recommended. Prolactin should also be determined since hyperprolactinemia can be associated with hirsutism and urinary cortisol excretion should be measured if Cushing’s syndrome is suspected. According to a study /3/, the likelihood ratio for an ovarian or adrenal tumor in postmenopausal hyper androgenism is approximately 10 times higher if the testosterone level is ≥ 1.4 μg/L (4.9 nmol/L) and the FSH level is ≤ 35 IU/L.

Thyroid dysfunction

Hypothyroidism is associated with infertility, spontaneous abortion, stillbirth, and congenital anomalies. This is thought to be caused by a reduction in the release of GnRH. It may also be due to a decrease in thyroid hormone binding proteins, which leads to elevated intrafollicular androgen concentrations and disturbed follicle development. Furthermore, the increased release of TRH by the hypothalamus is also thought to increase prolactin secretion. In the case of hyperthyroidism, infertility mainly occurs in those patients who have severe autoimmune hyperthyroidism.

Table 37.3-1 Functional tests in male gonadal dysfunction /4/

Functional test

hCG stimulation test

Indication: to confirm the presence of functional testicular tissue (e.g., in cases of bilateral cryptorchidism). To determine the testicular reserve capacity for testosterone secretion.

Principle: hCG has LH activity and stimulates testosterone production by the Leydig cells.

Test protocol: collection of a blood sample between 8:00 a.m. and 10:00 a.m. for determination of basal testosterone, followed by a single intramuscular injection of 5,000 IU hGC (in children, 5,000 IU/m2 body surface area, up to a maximum of 5,000 IU). Further blood samples are obtained for testosterone determination after 48 and/or 72 hours.

Interpretation: if functional testicular tissue is present, testosterone should increase by at least a mean factor of 2 (1.5–2.5).

GnRH test

Indication: this test is used in the event of borderline LH and FSH concentrations to differentiate between hypothalamic and pituitary hypogonadism. It can also differentiate between delayed puberty and hypo gonadotropic eunuchoidism. The test should not be performed if gonadotropin levels are elevated.

Principle: in the case of hypothalamic dysfunction, the pituitary responds to GnRH stimulation by secreting FSH and LH.

Test protocol: baseline blood sample followed by repeat sampling at 25 and 45 minutes after i.v. injection of 100 μg GnRH. In children, 60 μg/m2 body surface area is administered (minimum 25 μg, maximum 100 μg). FSH and LH are determined in the samples.

Interpretation: at least a 3-fold increase in LH and at least a 1.5-fold increase in FSH with respect to the baseline concentration suggest the presence of hypothalamic dysfunction. In the absence of the signs of puberty, a rise in LH and FSH in response to GnRH indicates the presence of constitutional delayed puberty.

Table 37.3-2 Primary hypogonadism /13/

Clinical and laboratory findings

Klinefelter syndrome

Klinefelter syndrome is the most common form of congenital hypogonadism, with a prevalence of 1–2/1,000 male life births /14/. In the Klinefelter man all of the cells carry an XXY karyotype, however, many men are mosaic, and only a portion of their cells have the XXY constitution. Clinical features include small, firm testes (volume 2–10 mL, normal 30–60 mL), symptoms of androgen deficiency (sparse body hair, tall stature, erectile dysfunction), gynecomastia, and azoospermia. Co morbid conditions include osteoporosis, metabolic syndrome, type 2 diabetes mellitus, varicosis, thrombosis, and epilepsy. Prior to puberty the only consistent finding is a testicular volume below 1.5 mL.

Laboratory findings /14/: low testosterone (7–15 nmol/L, normal 10–18 nmol/L), elevated sex hormone binding globulin (SHBG), elevated FSH (25–42 IU/L, normal 10–30 IU/L), elevated LH (15–21 IU/L, normal 5–11 IU/L). Testosterone substitution is indicated for levels below 12 nmol/L.

XX male syndrome

Patients with this chromosomal disorder may appear as a normal women, a women with female gonadal dysgenesis, a hermaphrodite, or male with gonadal dysgenesis. XX males tend to be shorter than normal and may have eunuchoid body proportions and hypospadia. Frequency: 1 in 10,000 live births.

Laboratory findings: same as in Klinefelter syndrome.

XX/X0 mixed gonadal dysgenesis

Patients with a 45X0 or 46XY karyotype may have a male or female phenotype. The gonads are dysgenetic and usually located intraabdominally. These patients are invariably infertile. A dysgerminoma, gonadoblastoma, or embryonal cell tumor is present in up to 20% of cases.

XYY syndrome

These men are taller than their peers but do not have a “super male” physique. Spermatogenesis is significantly reduced due to hyalinization of the seminiferous tubules.

Laboratory findings: testosterone normal or low, FSH and LH elevated.

Del Castillo’s syndrome

Men with this syndrome are infertile, with small testes and azoospermia. Testicular biopsy is required for diagnosis. Because of azoospermia, the syndrome is also known as Sertoli-cell-only syndrome.

Laboratory findings: testosterone levels are usually normal, but the response to the hCG stimulation is decreased, LH concentrations are in upper reference interval.

Functional prepubertal castrate (vanishing testis syndrome)

Patients have complete testicular failure but a normal XY karyotype. It is thought that the testes are destroyed in utero as a result of trauma.

Laboratory findings: elevated LH and FSH, low testosterone, lack of response or slight response only to hCG stimulation.

-reductase deficiency

Congenital deficiencies of individual enzymes involved in steroid biosynthesis can occur. These deficiencies may affect testosterone biosynthesis only or may occur in combination with a disorder of cortisol synthesis.

5α-reductase is responsible for the reduction of the double bond in the A ring of testosterone that results in the lack of conversion of testosterone to dihydrotestosterone (DHT). In 5α-reductase deficiency, testosterone is not converted to dihydrotestosterone (Fig. 37.1-1 – Biosynthesis of important sex steroids). DHT is responsible for the development of the scrotum, penis, and prostate. Patients with this syndrome therefore appear as girls until puberty, despite having an XY karyotype. At puberty testes begin to produce testosterone and the men take on a normal male appearance, they experience an increase in muscular development and loss of subcutaneous fat. It is DHT that is responsible for the development of the scrotum, prostate, and testes. As a result of DHT deficiency the scrotum does not develop and the prostate remains rudimentary.

Laboratory findings: FSH and LH are slightly elevated. Testosterone is normal or slightly elevated. Measurement of the testosterone/DHT ratio is indicated. After puberty, the normal ratio is ≤ 20, but in 5α-reductase deficiency, it is greater than 35. This ratio can also be used to diagnose the condition prepubertally. In normal boys, the ratio is ≤ 20, while in boys with 5α-reductase deficiency, it is greater than 50.

LH-resistant testes

Absence or reduced function of Leydig cell LH receptors due to inactivating mutations of the LH receptor gene. Patients have an XY karyotype with a female phenotype.

Laboratory findings: reduced testosterone, elevated LH.

Pseudo hermaphrodism

Pseudo hermaphrodism can be caused by inactivating mutations of the nuclear androgen receptor gene.

Testicular feminization

Patients with testicular feminization have no functional tissue receptors for testosterone and dihydrotestosterone, which results in a complete loss of androgenic activity. However, testosterone is still be converted to estrogen (Fig. 37.1-1 – Biosynthesis of important sex steroids). At puberty, boys with this syndrome develop breast tissue and have female subcutaneous fat distribution due to increased conversion of testosterone to estrogen.

Laboratory findings: testosterone is in the reference interval or slightly elevated; FSH and LH are elevated because of the lack of androgen feedback on the pituitary and hypothalamus.

Reifenstein syndrome

Like in testicular feminization, there is a lack of androgen binding to the corresponding receptors but this deficiency is often only partial. Phenotypically, men with this condition display varying degrees of pseudo hermaphrodism. As adults, they have decreased androgen initiated hair growth and reduced muscle mass.

Laboratory findings: elevated FSH, LH, and testosterone. Because of the partial nature of the defect, the increases are not as great as seen in testicular feminization.

Postpubertal orchitis

Postpubertal mumps is associated with orchitis in up to 25% of cases.

Laboratory findings: reduction in spermatogenesis and mono tropic elevation of FSH. If the orchitis persists, testosterone decreases and LH increases.

Cryptorchidism

Up to 10% of men will be cryptorchid at birth, however following puberty, the incidence of undescended testes is 0.3 to 0.4%. Undescended testes result in hypogonadism and are associated with infertility in 70% of cases. Unilateral cryptorchism is also associated with infertility but to a lesser extent than bilateral cases. In phenotypically male newborns with bilateral non palpable testes, a female karyotype must be ruled out and the presence of testosterone producing tissue confirmed /315/.

Laboratory findings: LH and FSH elevation in combination with undetectable anti-Muellerian hormone and a negative testosterone response to hCG stimulation suggest the presence of anorchia.

Table 37.3-3 Secondary and mixed acquired hypogonadism /34/

Clinical and laboratory findings

Kallmann syndrome

Classic hypo gonadotropic hypogonadism (Kallmann syndrome) is a congenital disorder characterized by absent secretion of FSH and LH, hyposmia or anosmia due to a defective development of the olfactory bulbs. The incidence of the syndrome is 1 in 10,000 male births. Patients present as pubertal eunuchs. The testes tend to be prepubertal, in some patients there is limited growth. FSH and LH secretion can be stimulated by repeated administration of GnRH, which indicates that the primary defect is at the level of the hypothalamus rather than the pituitary /3/.

Laboratory findings: low concentrations of FSH, LH, and testosterone.

Non secretory pituitary adenoma

Hypogonadism is an early feature of non functioning adenoma and frequently develops shortly after growth hormone deficiency.

Laboratory findings: reduced levels of FSH and LH; LH is often more significantly affected than FSH. Testosterone concentration is decreased. It is important to test for hyperprolactinemia. Approximately 80% of men with hyperprolactinemia have a macro adenoma. In young patients, the GnRH test can be used to differentiate between a constitutional delay in sexual development and secondary hypogonadism. A positive GnRH test suggests the presence of secondary hypogonadism (e.g., adenoma).

Isolated LH deficiency,isolated FSH deficiency

Isolated LH deficiency in men has been termed the “fertile eunuch” syndrome /3/. Although men with this condition have an androgen deficiency, they still have enough FSH to support spermatogenesis and are therefore fertile. Men with an isolated FSH deficiency, on the other hand, have normal male androgen levels but do not produce spermatozoa. They are therefore infertile.

Critically ill patients

Severe stress acutely reduces FSH and LH release and testosterone secretion. This also occurs in patients admitted for acute severe illness or in critically ill patients in intensive care units.

Pituitary stalk transection

Pituitary stalk transection as the result of severe accidental head injury presents with diabetes insipidus in the acute phase, followed by secondary hypogonadism.

Increased SHBG production

Increased production of sex hormone binding globulin (SHBG) in men with alcoholic liver cirrhosis and a decreased testosterone reserve can result in a reduction in free testosterone. Although the total testosterone level is normal, the decrease in free testosterone causes hypogonadism. Elevated SHBG and total testosterone can also occur in hyperthyroidism.

Hemochromatosis

A mixed clinical picture with features of primary and secondary hypogonadism can occur due to increased iron storage in the testes and pituitary gonadotropic cells. This also occurs in cases of secondary iron overload (e.g., following a large number of blood transfusions in thalassemia major or myelodysplastic syndrome).

Chronic kidney disease

Patients with chronic renal disease (especially those undergoing chronic hemodialysis) experience dysfunction at all levels of the gonadotropic system as well as reduced spermatogenesis. Hyperprolactinemia and secondary hyperparathyroidism also intensify the development of impotence.

Metabolic syndrome, diabetes mellitus

Men with metabolic syndrome or type 2 diabetes mellitus not only have an increased cardiovascular risk but can also experience reduced testosterone and erectile dysfunction. Men with a BMI of 30–35 kg/m2 have decreased total testosterone due to a reduction in SHBG while men who are morbidly obese (BMI greater than 40 kg/m2) experience decreased testosterone as a result of reduced LH secretion /11/. Erectile dysfunction occurs frequently in type 1 diabetes as a result of microvascular and macro vascular damage and neuropathy.

Drugs

Alkylating agents such as cyclophosphamide, busulfan, and chlorambucil destroy the seminiferous tubules, leading to azoospermia and, less frequently, reduced testosterone secretion. Amino glutethimide, etomidate, and ketoconazole interfere with androgen synthesis. Spironolactone, cyproterone acetate, and flutamide inhibit androgen receptors.

Radioiodine therapy

Radioiodine-131 is commonly used for the treatment of differentiated thyroid cancer and hyperthyroidism. According to a study /12/ the 131J treatment of hyperthyroidism may account for a small amount of damage both to the germinal epithelium and Leydig cells. The results are unlike that observed in cancer patients, in whom testicular damage after 131J treatment is well documented.

Laboratory findings /12/: testosterone was significantly reduced 45 days after treatment of hyperthyroidism, levels returned to basal values after 12 months. FSH and LH were not significantly affected.

Table 37.4-1 Reference intervals for FSH

 

(IU/L)

(IU/L)

Children /8/

1–7 days

0.10–3.43

0.11–2.97

8–15 days

0.13–1.04

0.17–1.43

16 days – 3 years

0.25–3.20

0.12–2.50

4–6 years

 0.19–3.28

0.10–6.68

7–8 years

0.17–11.05

0.13–4.10

9–10 years

0.36–6.91

0.20–4.52

11 years

0.44–8.97

0.41–8.87

12 years

0.95–17.15

0.51–10.46

13 years

1.84–9.94

0.69–10.75

14 years

0.91–11.79

0.45–10.46

15 years

1.19–12.43

0.43–18.45

16 years

1.09–12.38

0.16–9.65

17 years

1.17–9.63

2.22–12.93

18–19 years

< 0.10–9.50

1.95–15.41

Men /9/

 

1–12

Women /9/

Follicular phase

3–20

Ovulation

9–26

Luteal phase

1–12

Postmenopause

18–153

Values expressed as 2.5th and 97.5th percentiles. Reference preparation 2nd IRP 78/549.

Table 37.4-2 Reference intervals for LH

 

(IU/L)

(IU/L)

Children /8/

16 days – 3 years

0.25–2.48

0.20–2.95

4–6 yrs

 0.23–1.85

0.22–2.99

7–8 yrs

0.21–2.97

0.22–2.67

9–10 yrs

< 0.20–3.96

0.37–2.64

11 yrs

< 0.20–6.46

0.30–1.82

12 yrs

0.41–9.92

0.25–4.04

13 yrs

0.34–5.36

0.33–5.97

14 yrs

0.49–31.18

0.48–7.93

15 yrs

0.50–20.68

0.50–10.73

16 yrs

0.40–29.35

0.48–9.65

17 yrs

1.56–12.43

0.86–10.83

18–19 yrs

1.82–11.17

1.51–5.92

Men /9/

2–12

Women /9/

Follicular phase

2–15

Ovulation

22–105

Luteal phase

0.6–19

Post menopause

16–64

Values expressed as 2.5th and 97.5th percentiles. Reference preparation 2nd IRP 80/552.

Table 37.4-3 Amenorrhea associated with low or low-normal FSH and LH levels /5/

Disease/condition

Assessment

Negative gestagen test

Secondary amenorrhea, depending on the degree of severity.

Hyperprolactinemia

Refer to Chapter 36 – Prolactin (PRL).

Anorexia nervosa

Psychogenic amenorrhea related to weight loss.

Kallmann syndrome (olfacto-genital syndrome)

Primary amenorrhea (often in conjunction with anosmia). This condition is extremely rare in women, with an incidence of 1 : 50,000. If patients wish to conceive, therapy with pulsatile GnRH administration or gonadotropins can be used.

Trauma, adenoma

Traumatic or adenoma related damage to the hypothalamic-pituitary-gonadal axis may result in primary amenorrhea.

Empty sella in the case of β-thalassemia

This leads to delayed puberty and amenorrhea. The condition can be treated using hMG/hCG and growth hormone.

Table 37.4-4 Conditions associated with persistent elevations of FSH and LH in women /5/

Clinical and laboratory findings

Gonadal dysgenesis in conjunction with 45X0 (Turner syndrome)

Primary amenorrhea, stunted growth, infantile genitalia, characteristic stigmata such as broad shield-like chest and webbing of the neck (pterygium colli). Numerous mosaic forms are possible, mainly 46XX, 45X0. In the presence of mosaic forms, pregnancy is rare but possible.

Gonadal dysgenesis in conjunction with 45X0, 46XY mosaic

Primitive testicular gonad present on one side with gonadal aplasia present on the other side. Because of the risk of malignant degeneration, surgical removal of the gonads is recommended, especially if Y chromosome portions are detected.

Gonadal dysgenesis in conjunction with 46XY (Sywer syndrome)

Primary amenorrhea, normal growth, infantile genitalia. Because of the risk of malignant gonadal degeneration, surgical removal of the gonads is recommended.

Premature menopause

Secondary amenorrhea and elevated gonadotropins in women prior to 40 years of age. Autoimmune disease with genetic predisposition.

Resistent ovary syndrome (intermittent ovarian failure)

Symptoms as in premature menopause; however, the condition is reversible and subsequent pregnancy is possible. It can be differentiated from premature menopause by histological examination of ovarian biopsy material. Elevated FSH levels in conjunction with regular menstrual cycles indicate perimenopause.

Post chemotherapy or post radiation

Chemotherapy may lead to the development of primary ovarian failure. The same is true of ovarian irradiation. Not infrequently, however, the condition is reversible.

Premature ovarian failure

Premature ovarian failure is defined as the cessation of menstrual periods before the age of 40. It occurs in 1 in 1,000 women between the ages of 15 and 29 and 1 in 100 women between the ages of 30 and 39. In most cases no cause is ever identified. Premature ovarian failure may occur abruptly over one to two months or gradually over several years. Some women may experience symptoms of menopause such as hot flashes, no menses, and vaginal dryness.

Laboratory findings: usually, the cycle day 3 FSH or estrogen levels may be elevated.

Table 37.5-1 Reference intervals for 17β-estradiol

 

(pmol/L)

(pmol/L)

Children /3/

1–7 days

25–116

< 20–229

8–15 days

42–134

31–126

16 days – 3 yrs

21–113

< 20–65

4–6 yrs

< 20–81

29–121

7–8 yrs

23–88

20–83

9–10 yrs

< 20–176

< 20–81

11 yrs

33–188

28–110

12 yrs

< 20–221

26–131

13 yrs

< 20–157

< 20–232

14 yrs

42–541

22–273

15 yrs

25–909

< 20–302

16 yrs

76–849

40–137

17 yrs

49–507

40–103

18–19 yrs

53–688

28–129

Men /4/

< 184

Women /4/

Follicular phase

184–532

Ovulation

411–1626

Luteal phase

184–885

Postmenopause

< 217

Values are expressed as 2.5th and 97.5th percentiles. Conversion formula: ng/L × 3.671 = pmol/L

Table 37.5-2 Diseases and conditions associated with changes in 17β-estradiol (E2) levels /5/

Clinical and laboratory findings

Ovarian failure

E2 concentrations are below 37 pmol/L (10 ng/L).

Anovulatory cycle

Subnormal E2 concentrations during the follicular phase; the dominant follicle does not reach the preovulatory stage and becomes atretic. Subsequently, estrogen withdrawal bleeding occurs.

Corpus luteum insufficiency

Preovulatory E2 levels are often reduced; the second maximum during the luteal phase is not detectable.

Monitoring of infertility treatment, especially in the case of clomiphene or gonadotropin therapy

Regular E2 determinations in conjunction with sonographic investigations of follicular growth allow optimal dosing of medication. Once the dominant follicle has reached a satisfactory size and the corresponding E2 level is adequate (roughly 1,000 pmol/L per follicle greater than 14 mm), ovulation is induced by hCG administration. Very high E2 concentrations (i.e. > 7,350 pmol/L; 2,000 ng/L) or an increase in values on consecutive days that is too steep indicate impending hyper stimulation syndrome. However, hyper stimulation syndromes have been observed even at concentrations of around 1,000 ng/L (3,670 pmol/L). As far as the occurrence of multiple pregnancies is concerned, E2 can only provide preliminary indications; more definitive evaluation by sonographic examination is required.

Estradiol-producing tumor

Elevated E2, low gonadotropin levels. A rare occurrence (e.g., as seen in granulosa cell tumors).

Table 37.6-1 Reference intervals for progesterone

 

(nmol/L)

(nmol/L)

Children /2/

1–7 days

0.8–9.6

1.0–12.5

8–15 days

1.0–4.7

1.0–8.2

16 days – 3 yrs

0.3–3.2

0.3–3.6

4–6 yrs

0.3–3.5

0.4–8.7

7–8 yrs

0.8–3.6

0.7–3.5

9–10 yrs

0.4–3.5

0.4–3.9

11 yrs

1.1–3.0

0.7–3.6

12 yrs

1.5–5.9

1.0–5.1

13 yrs

1.2–4.8

1.2–4.8

14 yrs

1.5–41.7

1.1–4.1

15 yrs

1.50–45.7

2.0–9.6

16 yrs

1.8–46.9

2.2–14.5

17 yrs

2.3–41.2

2.2–6.9

18–19 yrs

3.8–43.2

3.7–9.6

Men /3/

< 0.6–4.45

Women /3/

Follicular phase

< 0.6

Mid luteal

9.5–63.6

Post menopause

Up to 3

Values expressed as 2.5th and 97.5th percentiles. Conversion formula: μg/L × 3.18 = nmol/L

Table 37.6-2 Diseases and conditions associated with changes in progesterone levels

Condition

Clinical and laboratory findings

Confirmation of ovulatory cycle

Progesterone levels in the second half of a cycle indicate the prior occurrence of ovulation.

Detection of corpus luteum insufficiency

2–3 blood samples should be collected at around 5 and 11 days after ovulation. Both values should be > 32 nmol/L (10 μg/L) in normal corpus luteum function.

Detection of early pregnancy

Progesterone levels of < 32 nmol/L (10 μg/L) in early pregnancy indicate an abnormal pregnancy. It is particularly important to consider the possibility of ectopic pregnancy /4/.

Table 37.7-1 Reference intervals for SHBG

 

(nmol/L)

(nmol/L)

Children /8/

1–7 days

7.4–34.8

8.8–50.7

8–15 days

10.1–51.2

13.7–68.7

16 days – 3 yrs

12.9–96.6

19.8–114.4

4–6 yrs

 42.5–130.8

34.4–141.1

7–8 yrs

41.8–149.4

42.9–120.3

9–10 yrs

30.4–178.1

30.3–169.0

11 yrs

34.9–158.0

46.9–153.5

12 yrs

30.6–144.1

30.8–173.6

13 yrs

25.2–160.0

22.9–159.0

14 yrs

13.4–134.3

14.6–100.6

15 yrs

25.1–154.8

17.8–142.7

16 yrs

28.0–164.4

17.9–113.1

17 yrs

28.3–129.1

19.6– 77.4

18–19 yrs

25.8–103.4

19.7–60.4

Men /9/

 

13–71

Women /9/

18–114

Post menopause

15–70

Values expressed as 2.5th and 97.5th percentiles. Conversion formula: nmol/L × 0.095 = mg/L; mg/L × 10.53 = nmol/L

Table 37.8-1 Reference intervals for testosterone

Test

Women /14/

Men /15/

Total
testosterone

0.54–2.72 nmol/L

9.4–37.1 nmol/L

Free
testosterone

3–39 pmol/L

0.42–1.39 nmol/L

Conversion formula: μg/L × 3.467= nmol/L; nmol/L = μg/L × 0.288

Table 37.9-1 Clinical applications of Inhibin determination /1/

Clinical and laboratory findings

Female reproductive axis

In females, the granulosa cells of the ovary produce inhibin, and inhibin production by each follicle increases with the number of granulosa cells during normal follicle growth and maturation. Inhibin and activin also act as intraovarian paracrine signaling molecules that regulate follicular dominance during the preovulatory phase of the menstrual cycle /1/. In a study /7/ measurement of inhibin B through the human menstrual cycle showed the following results: the plasma concentration of inhibin B increased rapidly in the early follicular phase to a peak of 85.2 ± 9.9 pg /mL on the day after the inter cycle FSH rise, then fell progressively during the remainder follicular phase. Two days after the mid cycle LH peak, there was a short lived peak in the inhibin B concentration (133.6 ± 31.2 pg/mL), which then fell to a low concentration (< 20 pg/mL) for the remainder in of the luteal phase. The concentration of inhibin B in individual follicular fluid samples was 20–200 fold higher than the level of inhibin A.

Pregnancy

Maternal inhibin level increases over the course of pregnancy. The levels are low during the first and second trimesters and rise dramatically during the third trimester /1/.

Pre-eclampsia

Compared to women with normal pregnancy, when matched for duration of gestation, parity and maternal age, elevated maternal inhibin A correlates with both onset and severity of pre-eclampsia /8/.

Ovarian reserve

The Centers for disease Control and Prevention defined assisted reproductive technology (ART) as technology used to treat infertility in which the ovum and sperm are manipulated ex vivo /9/. Included technologies are IVF, intracytoplasmic sperm injection and other related techniques.

Tests used for the assessment of ovarian reserve in women undergoing ART are /10/:

  • Hormones e.g., FSH, estradiol, progesterone, inhibin, anti-Muelleranian hormone
  • Ultrasound e.g., antral follicle count, ovarian volume
  • Dynamic testing e.g., clomiphen citrate test, exogenous FSH reserve test, GnRH test.

Normal inhibin concentration provides a good measure of follicle health and viability. The granulosa cells of growing ovarian follicles release inhibins; as follicles progress to the antral stage (secondary follicles), the population of inhibin producing cells and the release of inhibins increases /1/. Women with declining ovarian reserve, show reduced inhibin B and low FSH levels because of low granulosa cell formation at day 3 of the cycle /11/. High levels of follicular fluid inhibin levels correlate with increased pregnancy rate and better ovarian response in women undergoing hormonal stimulation for oocyte retrieval in IVF /12/.

Female reproductive function

Approximately 1% of women under the age of 40 years have premature ovarian failure. The disorder is associated with amenorrhea, decreased estrogen concentration and elevated FSH level. The levels of inhibin A and B are decreased, which explains the elevation of FSH /13/.

Male reproductive function

Inhibin B is the biologically active form of inhibin in men and circulating concentrations of inhibin B and FSH are inversely correlated in healthy and sub fertile men. Serum inhibin B level is a direct marker of Sertoli cell function and an indirect indicator of spermatogenesis. Inhibin B demonstrated a diagnostic sensitivity of 65% and a specificity of 83% /14/. Significantly lower concentrations of serum inhibin B were measured in men with impaired spermatogenesis i.e., severe oligozoospermia, idioapathic azoospermia, cryptorchism, and Klinefelter syndrome in comparison to men with normospermia /15/.

Table 37.10-1 Clinical applications of anti-Muellarian hormone (AMH) determination

Clinical and laboratory findings

Ovarian reserve in infertility and assisted reproductive technology (ART)

Diminished ovarian reserve (OR) is one of the major causes of infertility. AMH is regarded as an excellent marker of the OR, a measure of the biological age of the ovaries and a diagnostic marker of ovarian dysfunction. Serum AMH levels are found to be reduced earlier before any increase in baseline FSH levels. The conventional tests for the determination of OR include day 3 FSH level and antral follicle count (AFC). Levels of AMH strongly correlate with the size of the early growing follicle pool. The ovary secretes AMH directly into the blood and is easy to measure in serum. In the progress of folliculogenesis AMH inhibits the recruitment of primordial follicle cell and the selection of follicles that are under the dominance of FSH. AMH expression is high in primary, secondary, pre-antral and early antral follicles (< 4 mm diameter) and disappears in follicles between 4 and 8 mm diameter /110/.

A strong correlation between the AMH and the AFC exists and a similar association between AMH and age, AFC, and FSH was described in women undergoing IVF /11/.

AFC is considered the most reliable method to evaluate the OR. Normal OR of AFC is 10 ± 4 follicles. In the context of assisted reproductive technology (ART), AFC is used in routine to monitor the OR. AMH concentration has a clinical value in providing useful information regarding OR, was well as responsiveness to treatment. Using the Elecsys AMH Plus assay threshold values of AMH have been correlated to AFC distribution (Tab. 37.10-2 – Threshold values of AMH correlated to AFC distribution).

Normal ovarian FSH stimulation in ART is associated with an AFC ≤ 15 follicles and significant decline in serum AMH concentration. This is not the case in FSH hyper stimulation that can result in follicle diameters of ≥ 12 mm, AFC of > 15 with diameter > 12 mm, and estradiol levels > 11.700 pmol/L. Using the Elecsys AMH Plus assay and the cutoff of ≥ 2.10 ng/mL (15 pmol/L) as indicator of ovarian hyper stimulation the diagnostic sensitivity was 81.3%, specificity 64.7%, positive predictive value 21.7%, negative predictive value 96.6%.

Prevalence of infertility is higher in obese women due to decreased ovarian reserve as well as follicle dysfunction. It is hypothesized that low levels of adiponectin stimulate aromatase activity in the ovary. As a result, AMH production declines, reflecting dysfunctional folliculogenesis /8/. Refer to Fig. 37.1-1 – Biosynthesis of sexual steroids.

Polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is a heterogenous condition, affecting 5–10% of the female population. Clinical symptoms are acne, hirsutism, menstrual irregularities and increased risk of type 2 diabetes. PCOS is a genetic condition. Polycystic ovaries are characterized by an increase in the number of follicles at all growing stages. AMH levels are 2 to 4 fold higher than in women with PCOS, reflecting the increased number of small antral follicles in which AMH production is highest. AMH decreases FSH- and LH induced aromatase expression in granulosa cells as well as reducing the activity of the ovary-specific aromatase promoter II. This results in reduction of estradiol and overproduction of androgens /1/. AMH values above 5 ng/mL (35 pmol/L) might be considered as a diagnostic criterion for PCOS /12/.

Ovarian damage from chemotherapy, radiotherapy and surgery

Women who had childhood cancer but who still have regular menses can have a decrease in serum AMH because of reduced ovarian reserve. Alkylating based therapeutic protocols, radiotherapy (including abdominal pelvic therapy in children or total body irradiation) can be associated with very low or undetectable AMH level. In endometrioma surgery a decline of AMH indicates the removal of a significant part of the ovarian reserve. On the basis of AMH monitoring timely referral to the reproductive endocrinologist can assist childhood cancer survivors in their puberty progression /8/. For further information refer to Ref. /1/.

Time to menopause

Menopause occurs at a mean age of 51 years. However, a considerable variation in age exists; some women reach menopause at 40 years of age, while others at age 60. Menopause results from a decline in the number of primordial follicles below a critical threshold. Since the number of antral follicles is related to the size of the primordial follicle pool a marker like AMH correctly reflecting the number of antral follicles is potentially capable of predicting timing of menopause. A study /13/ has shown that age-specific AMH is capable of predicting time to menopause making this marker a possible candidate in the preventive management of age-specific fertility. However, a reduced predictive effect of AMH was observed with increasing age of the women. AMH should therefore not be used to make individual forecasts of menopause, or of fertile lifespan, in the day-to-day clinical practice based on the current research.

Asherman’s syndrome

Asherman’s syndrome is an acquired condition that refers to the existence of scar tissue in the uterus. Symptoms include having light or no menstrual cycles and problems getting pregnant. FSH, LH and estradiol concentrations did not differ between with and without Asherman’s syndrome, whereas significantly lower AMH levels were found in patients [median 0.50 pg/mL (3.5 pmol/L)] than in controls [median 1.14 pg/mL (8.1 pmol/L)] /14/.

Table 37.10-2 Threshold values of anti-Muellarian hormone (AMH) correlated to antral follicle count (AFC) /11/

AMH cutoff
ng/mL (pmol/L)

Proportion (%) of women with antral follicle count in relation to the AMH level

AFC 0–7

AFC 8–15

AFC > 15

≤ 0.681 (4.68)

63.2%

32.4%

4.4%

0.681–2.27
(4.68 – ≤ 16.2)

12%

56.9%

31.1%

> 2.27 (16.2)

1.4%

24.1%

74.5%

Figure 37.1-1 Biosynthesis of important sex steroids. 1) 20–22 desmolase; 2) 3β-hydroxy steroid dehydrogenase; 3) 17α-hydroxylase; 4) 17–20 desmolase; 5) 17β-hydroxysteroid dehydrogenase; 6) 5α-reductase

C CH 3 O HO C CH 3 O HO OH O HO OH HO C CH 3 O O C CH 3 O O OH O O OH O O HO OH HO Cholesterol Δ5-Pregnenolone 17α-Hydroxy-pregnenolone Dehydroepian-drosterone Androstenediol Progesterone 17α-Hydroxy-progesterone Androstenedione Testosterone Aromatases Estradiol-17β 1 3 4 5 5 5 4 3 2 2 2 2 Estrone Aromatases OH O 6 Dihydrotestosterone

Figure 37.1-2 Serum levels of FSH, E2, LH, and progesterone synchronized to the day of the LH peak /1/.

LH (IU/L) 60 50 40 30 20 10 Progesterone (μg/L) 15 10 5 0 14 28 Days FSH (IU/L) 15 10 5 E 2 (ng/L) 200 100

Figure 37.2-1 Example of cycle monitoring in a 28-day cycle. In this case, ultrasound and endocrinological investigations were performed on two occasions and blood samples to check luteal function were collected on two occasions /1/.

Cycle- Day Bleeding • Ultrasound of ovaries • E 2 , progesterone and LH in serum Progesterone and E 2 in serum Follicular phase Luteal phase Ovulation 1 3 5 7 9 11 13 15 17 19 21 23 25 27

Figure 37.2-2 Diagnostic approach to female androgenization. Before extensive diagnostic procedures are performed, hormone levels should be checked multiple times and iatrogenic causes excluded /1/.

Testosterone > 0.6 μg/L or DHEAS > 3,000 μg/L Repeat of the investigation of three collected samples at intervals of 20 min. Tumor search in the area of ovaries and adrenals(Imaging or angiographic investigation of organs) Possible causes: PCOS, especially if LH/FSH quotient > 2, Hyperthecosis ovarii, NNR hyperplasia.Therapy: If no wish for child antiandrogen effective ovulation inhibitor. On wish for child low metered corticoids. ACTH test Pathological Molecular genetic testing CYP21 defect etc. Corticosteroid therapy, possiblyhuman genetic counseling Normal > 1.5 mg/L > 7000 mg/L > 3000 and< 7000 mg/L > 0.6 and< 1.5 mg/L DHEAS Testosterone

Figure 37.10-1 Full length homodimer AMH molecule consisting of proregion and mature peptide (top) cleaved by proprotein convertase. The regions that are recognized by monoclonal antibodies used in some immunoassays are indicated (bottom). With kind allowance of Ref. /1/.

Full lengthhomodimer Proregion55 kD Mature peptide12.5 kD Cleavage by proproteinconvertases Furin, PC5 RAQR Cleavednoncovalentcomplex

Figure 37.10-2 AMH actions in the ovary. AMH, secreted by the granulosa cells of small growing follicles, inhibits initial follicle recruitment and FSH dependent growth and selection of pre antral and small antral follicles The inset shows the inhibitory effect of AMH on FSH-induced of Cyp19a1 expression leading to decreased estradiol levels. T, testosterone; Cyp19a1, aromatase. With kind allowance of Ref. /13/.

Primordialfollicle pool Small Preantral Large Preantral Small Antral Large Antral Preovulatory Theca cell FSH AMH T T E2 Cyp19a1 Granulosa cell Initial recruitment Cyclic recruitment FSH AMH
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