The most common conditions in which gonadal function tests are used are hypogonadism in males and menstrual disorders, fertility problems, and hirsutism or virilization in females. The hormones currently available for assistance include lutropin (luteinizing hormone; LH), follitropin (follicle-stimulating hormone; FSH), testosterone, human chorionic gonadotropin (hCG), and gonadotropin-releasing hormone (GnRH).

Gonadal function is regulated by hypothalamic secretion of a peptide hormone known as gonadotropin-releasing hormone (GnRH; also called luteinizing hormone-releasing hormone, LHRH). Secretion normally is in discontinuous bursts or pulses, occurring about every 1.5-2.0 hours (range, 0.7-2.5 hours). GnRH half-life normally is about 2-4 minutes (range, 2-8 minutes). There is little if any secretion until the onset of puberty (beginning at age 8-13 years in females and age 9-14 years in males). Then secretion begins predominately at night but later extends throughout the night into daytime, until by late puberty secretion takes place all 24 hours. GnRH is secreted directly into the circulatory channels between the hypothalamus and pituitary. GnRH causes the basophilic cells of the pituitary to secrete LH and FSH. In males LH acts on Leydig cells of the testis to produce testosterone, whereas FSH stimulates Sertoli cells of the testis seminiferous tubules into spermatogenesis, assisted by testosterone. In females, LH acts on ovarian thecal cells to produce testosterone, whereas FSH stimulates ovarian follicle growth and also causes follicular cells to convert testosterone to estrogen (estradiol). There is a feedback mechanism from target organs to the hypothalamus to control GnRH secretion. In males, testosterone and estradiol inhibit LH secretion at the level of the hypothalamus, and testosterone also inhibits LH production at the level of the pituitary. A hormone called inhibitin, produced by the Sertoli cells of the testis tubules, selectively inhibits FSH at both the hypothalamic and pituitary levels. There appears to be some inhibitory action of testosterone and estradiol on FSH production as well, in some circumstances. In females, there is some inhibitory feedback to the hypothalamus by estradiol.

Luteinizing hormone and follicle-stimulating hormone

Originally FSH and LH were measured together using bioassay methods, and so-called FSH measurements included LH. Immunoassay methods can separate the two hormones. The LH cross-reacts with hCG in some RIA systems. Although this ordinarily does not create a problem, there is difficulty in pregnancy and in patients with certain neoplasms that secrete hCG. Several of the pituitary and placental glycopeptides (TSH, FSH, hCG, LH) are composed of at least two immunologic units, alpha and beta. All of these hormones have the same immunologic response to their alpha fraction but a different response to each beta subunit. Antisera against the beta subunit of hCG eliminate interference by LH, and some investigators have reported success in producing antisera against the beta subunit of LH, which does not detect hCG. There is some cross-reaction between FSH and TSH, but as yet this has not seemed important enough clinically in TSH assay to necessitate substitution of TSH beta subunit antiserum for antisera already available. Assay of FSH in serum is thought to be more reliable than in urine due to characteristics of the antibody preparations available.

Serum luteinizing hormone

LH is secreted following intermittent stimulation by GnRH in pulses occurring at a rate of about 2-4 every 6 hours, ranging from 30% to nearly 300% over lowest values. Therefore, single isolated LH blood levels may be difficult to interpret and could be misleading. It has been suggested that multiple samples be obtained (e.g., four specimens, each specimen collected at a 20-minute interval from another). The serum specimens can be pooled to obtain an average value. FSH and testosterone have a relatively constant blood level in females.

Urine luteinizing hormone

In contrast to serum LH, urine LH is more difficult to obtain, since it requires a 24-hour specimen with the usual problem of complete specimen collection. It also has the disadvantage of artifact due to urine concentration or dilution. The major advantage is averaging of 24-hour secretion, which may prevent misleading results associated with serum assays due to LH pulsatile secretion. Another role for urine LH assay is detection of ovulation during infertility workups. LH is rapidly excreted from plasma into urine, so that a serum LH surge of sufficient magnitude is mirrored in single urine samples. An LH surge precedes ovulation by about 24-36 hours (range, 22-4 hours). Since daily urine specimens are obtained beginning 9-10 days after onset of menstruation (so as not to miss the LH surge preceding ovulation if ovulation should occur earlier than the middle of the patient cycle). It has been reported that the best time to obtain the urine specimen is during midday (11 A.M.-3 P.M.) because the LH surge in serum usually takes place in the morning (5 A.M.-9 A.M.). In one study, the LH surge was detected in 56% of morning urine specimens, 94% of midday specimens, and 88% of evening specimens. In contrast, basal body temperature, another method of predicting time of ovulation, is said to be accurate in only 40%-50% of cases. Several manufacturers have marketed simple immunologic tests for urine LH with a color change endpoint that can be used by many patients to test their own specimens. Possible problems include interference by hCG with some of the LH kits, so that early pregnancy could simulate a LH surge. Since the LH surge usually does not last more than 2 days, positive test results for more than 3 consecutive days suggest some interfering factor. Similar interference may appear in some patients with elevated serum LH levels due to ovarian failure (polycystic ovary [PCO] disease, early menopause, etc.).

Finally, an estimated 10% of patients have more than one urine LH spike, although the LH surge has the greatest magnitude. It may be necessary to obtain serum progesterone or urine pregnanediol glucuronide assays to confirm that elevation of LH values is actually the preovulation LH surge.

Urine pregnanediol

The ovarian corpus luteum formed shortly after ovulation secretes increasing amounts of progesterone. Progesterone or its metabolite pregnanediol glucuronide begins to appear in detectable quantities about 2-3 days after ovulation (3-5 days after the LH surge) and persists until about the middle of the luteal phase that ends in menstruation. Conception is followed by progesterone secretion by the placenta. A negative baseline urine pregnanediol assay before the LH surge followed by a positive pregnanediol test result on at least 1 day of early morning urine specimens obtained 7, 8, and 9 days after the LH surge confirm that the LH surge did occur and was followed by ovulation and corpus luteum formation. Urine specimens are collected in the morning rather than at the LH collection time of midday. At least one manufacturer markets a simple kit for urine pregnanediol assay.

Problems with interpretation of a positive urine pregnanediol glucuronide assay include the possibility that early pregnancy may be present. Also, 5%-10% of patients throughout their menstrual cycle nonovulatory phase have slightly or mildly increased pregnanediol levels compared to the reference range. A urine sample collected before the LH surge can be tested to exclude or demonstrate this phenomenon.

Serum androgens

The most important androgens are dehydroepiandrosterone (DHEA), a metabolite of DHEA called DHEA-sulfate (DHEA-S), androstenedione, and testosterone. DHEA is produced in the adrenal cortex, ovary, and testis (in the adrenal cortex, the precursors of DHEA are also precursors of cortisol and aldosterone, which is not the case in the ovary or testis). DHEA is the precursor of androstenedione, and androstenedione is a precursor both of testosterone and of estrogens (see Fig. 30-2). About 50%-60% of testosterone in normal females is derived from androstenedione conversion in peripheral tissues, about 30% is produced directly by the adrenal, and about 20% is produced by the ovary. DHEA blood levels in females are 3 times androstenedione blood levels and about 10 times testosterone blood levels. In normal males after onset of puberty, testosterone blood levels are twice as high as all androgens combined in females. Androstenedione blood levels in males are about 60% of those in females and about 10%-15% of testosterone blood levels.

Serum testosterone

About 60%-75% of circulating serum testosterone is bound to a beta globulin variously known as sex hormone-binding globulin (SHBG) or as testosterone-binding globulin (TBG, a misleading abbreviation because of its similarity to the more widely used abbreviation representing thyroxine-binding globulin). About 20%-40% of testosterone is bound to serum albumin and 1%-2% is unbound (“free”). The unbound fraction is the only biologically active portion. Serum assays for testosterone measure total testosterone (bound plus unbound) values. Special techniques to assay free testosterone are available in some reference laboratories and in larger medical centers. Circulating androstenedione and DHEA are bound to albumin only.

Several conditions can affect total serum testosterone levels by altering the quantity of SHBG without affecting free testosterone. Factors that elevate SHBG levels include estrogens (estrogen-producing tumor, oral contraceptives, or medication), cirrhosis, hyperthyroidism, and (in males) decreased testis function or testicular feminization syndrome. Conditions that decrease SHBG levels include androgens and hypothyroidism.

There is a relatively small diurnal variation of serum testosterone in men, with early morning levels about 20% higher than evening levels. There is little diurnal change in women. Serum levels of androstenedione in men or women are about 50% higher in the early morning than in the evening.

In males, testosterone production is regulated by a feedback mechanism with the hypothalamus and pituitary. The hypothalamus produces gonadotropin-releasing hormone (GnRH; also called LHRH), which induces the pituitary to secrete LH (and FSH). LH, in turn, stimulates the Leydig cells of the testis to secrete testosterone.

Serum estrogens

There are three major estrogens: estrone ((E1), estradiol (estradiol-17b; E2), and estriol (E3). All of the estrogens are ultimately derived from androgenic precursors (DHEA and androstenedione), which are synthesized by the adrenal cortex, ovaries, or testis. The adrenal is unable to convert androgens to estrogens, so estrogens are directly produced in the ovary or testis (from precursors synthesized directly in those organs) or are produced in nonendocrine peripheral tissues such as the liver, adipose tissue, or skin by conversion of androgenic precursors brought by the bloodstream from one of the organs of androgen synthesis. Peripheral tissue estrogens are derived mainly from adrenal androgens. Estradiol is the predominant ovarian estrogen. The ovary also secretes a much smaller amount of estrone. The primary pathway of estradiol synthesis is from androstenedione to estrone and then from estrone to estradiol by a reversible reaction. This takes place in the ovary and to a much lesser extent in peripheral conversion of testosterone to estradiol. Estrone is produced directly from androstenedione (mostly in peripheral tissues) and to a lesser extent in the ovary from already formed estradiol by the reversible reaction. Estriol in nonpregnant women is formed as a metabolite of estradiol or estrone. In pregnancy, estriol is synthesized by the placenta from DHEA (actually from DHEA-S) derived from the fetal adrenals. This is a different pathway from the one in nonpregnant women. Estriol is the major estrogen of the placenta. The placenta also produces some estradiol, derived from both fetal and maternal precursors. Nonendocrine peripheral tissues (liver, skin, fat) synthesize a small amount of estrone and estradiol in pregnancy, mainly from adrenal precursors.

In females, there is a feedback mechanism for estradiol production involving the ovaries, hypothalamus, and pituitary. As already mentioned, the hypothalamus produces GnRH. The GnRH is secreted in a pulsatile manner about every 70-90 minutes and is excreted by glomerular filtration. The GnRH stimulates FSH and LH secretion by the pituitary. Some investigators believe there may be a separate (undiscovered) factor that regulates FSH secretion. The FSH acts on the ovarian follicles to stimulate follicle growth and development of receptors to the action of LH. The LH stimulates the follicles to produce estradiol.

Estradiol values can be measured in serum, and estriol values can be measured in serum or urine by immunoassay. Total estrogen values are measured in urine by a relatively old but still useful procedure based on the Kober biochemical reaction. All of the estrogens can be assayed by gas chromatography. The DHEA and androstenedione values can also be measured by special techniques, but these are available only in large reference laboratories.