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:

• Tab. 37.1-1 – Production rates of sex steroids in women
• Tab. 37.1-2 – Serum concentrations of sex steroids in women.

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:

• Tab. 37.2-1 – Laboratory tests for the diagnosis of ovarian dysfunction
• Tab. 37.2-2 – Functional tests in ovarian dysfunction.

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.

11. Stuenkel CA, Gompel A. Primary ovarian insufficiency. N Engl J Med 2023; 388 (2): 154–63.

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:

• Tab. 37.3-1 – Functional tests in gonadal dysfunction
• Tab. 37.3-2 – Primary hypogonadism.

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 /5, 6/.

37.3.3.0.1 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.

37.3.3.0.2 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).

37.3.3.0.3 Diagnosis of LOH

Diagnosis of LOH requires /1, 7/:

• 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

37.3.3.1 Testosteron body composition and sexual function in men

Testosteron body composition and sexual function in men.

37.3.3.1.1 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/.

37.3.3.2 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.

37.3.3.2.1 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/.

37.3.3.2.2 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).

37.3.3.2.3 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/.

37.3.3.2.4 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). doi: 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. doi: 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.

Controlled ovarian hyperstimulation is critical for follicle development and oocyte retrieval for in vitro fertilization. The serum indicators 17β-estradiol (E2), anti müllarian hormone (AMH) and inhibin B have been suggested as predictors of the ovarian response during controlled ovarian hyperstimulation /10/.

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.

10. Zhai J, Li S, Zhu Y, Chen Zi J. Sex hormone binding globulin concentration as a predictor of ovarian response during controlled ovarian hyperstimulation. Frontiers in Medicine 2021; doi: 10.3389/fmed.2021.719818

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, a glycated homodimeric plasma transport protein, has 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 Androgens (testosterone)

Androgens are synthesized from cholesterol through sequential conversions by enzymes in the adrenal glands and gonads. Refer to Fig. 34.1-2 – Biosynthesis of adrenal steroids.

Serum concentrations of androgens change during the different phases of life. Androgens are testosterone, dihydrotestosterone, androstenedione, 17OH progesterone, and dehydroepiandrosterone. Androgen synthesis happens as follows:

• male embryos: from week 8 on Leydig cells of the testes start to produce androgens
• in the second half of pregnancy stimulated by the gonadotropins LH and FSH testosterone stabilizes the Wolffian ducts, which form the male internal genitalia. Testosterone is converted into dihydrotestosterone by the enzyme 5-alpha reductase type 2 in the external genitalia.
• during childhood testosterone is not detectable
• in male puberty testosterone concentrations rise, resulting in male secondary sex characteristics
• in female puberty testosterone in serum also rises, but levels in adulthood are 10–15 fold lower than in males
• large amounts of dehydroepiandrosterone-sulfate (DHEAS) are synthesized by the fetal adrenals and serve as substrate for the production of estrogens.
• The adrenals also synthesize high concentrations of another group of steroids, the 11-oxygenated androgens.

The failure of the testes to produce physiologic levels of androgens 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 (POCS).

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

37.8.5.1 Testosterone in males

The assessment of a testosterone concentration 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/.

Testosterone deficiency leads to a decrease in lean body mass, muscle mass, and strength.

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/.

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. A significant proportion of these patients have symptoms of hyperandrogenism and elevated testosterone levels /7/.

For hyperandrogenism in women, refer to:

• Section 37.2.1 – Female infertility
• Tab. 37.2-3 – Clinical and laboratory findings in menstrual disorders.

37.8.5.3 Androgen excess and deficiency /16/

Differences of sex development present at birth, in newborns, or at later age, e.g. premature pubarche in children, delayed puberty, hirsutism in adolescence, or frank virilization of girls will be discussed.

37.8.5.3.1 Atypical genitalia

Atypical genitalia can be caused by:

• various differences in sex development including androgen resistance or androgen deficiency in individuals with an XY genotype
• androgen excess in individuals with an XX genotype, or by variations of the sex chromosomes such as mosaic, e.g. X/XY.
• In the case of XX genotypes causing congenital the adrenogital hyperandrogenism (CAH) is the main reason. 17OH progesterone is determined in the first 72 hours after birth.
• In the case of XY genotype testosterone concentration may be low and for evaluation of testicular function anti-Müllarian hormone and Inhibin A are measured.

37.8.5.3.2 Premature pubarche

Premature pubarche (defined as the appearance of pubic or axillary hair before the age of 8 years. Premature pubarche is 5 to 9 times more often in girls and is characterized by:

• elevated DHEAS, slightly increased androstenedione, total or free testosterone and decreased sex hormone binding globulin (SHBG)
• in the absence of DHEAS or other androgens and without signs of puberty pubarche is idiopathic.

37.8.5.3.3 Delayed onset of puberty or pubertal progression

• Constitutional delay is the most common cause. No laboratory test can differentiate constitutional delay from hypogonadotropic hypogonadism, both have low concentrations of FSH, LH and testosterone.
• Hypergonadotropic hypgonadism is indicated by increased concentrations of FSH and LH
• Klinefelter syndrome: Increase in LH, low concentration of testosterone.

37.8.5.3.4 Hirsutism/virilization in females

In pubertal and postpubertal females hirsutism, acne, and irregular menses can be the reason of androgen excess. The most cause of androgen excess is polycystic ovary syndrome (POCS).

37.8.5.3.5 Androgen excess or androgen deficiency in men

Two phases in a man’s life are characterized by a particularly high level of androgen activity, 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:

• Section 37.3.1 – Primary hypogonadism in males
• Section 37.3.2 – Secondary hypogonadism in males
• Section 37.3.3 – Late-onset hypogonadism in males.

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.

37.8.5.4 Androgen excess in females

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

For hyperandrogenism in women, refer to:

• Section 37.2.1 – Female infertility
• Tab. 37.2-3 – Clinical and laboratory findings in menstrual disorders.

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 below 5–7 ng/dL (2 nmol/L), interference by DHEAS leads 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/.

Testosterone, androstenedione, 17OHP, DHES in men show a diurnal rhythm with the highest serum concentrations in the early morning between 5.30 and 8.00 AM and the lowest concentration in the afternoon. For women it is less clear whether they show a circadian rhythm.

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.

16. Heijbor AC, Hannema SE. Androgen excess and deficiency: Analytical and diagnostic approaches. Clin Chem 2023; 69 (12): 1361–73.

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

Variability

In women inter-participant and intra-cycle variability of AMH levels were greater than inter-cycle variability using the Elecsys AMH Plus immunoassay /15/.

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 /7, 8/.

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 /7, 8/. 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.

15. Biniasch M, Laubender RP, Hund M, Buck K, de Geyter C. Intra- and inter-cycle variability of anti Müllarian hormone (AMH) levels in healthy women during non-consecutive menstrual cycles; the Bicycle Study. Clin Chem Lab Med 2022; 60 (4): 597–605.

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; doi: 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; doi: 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.