What is gonadotropin

In the body, there are two types of gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), that are secreted from the anterior pituitary gland and that act on the gonads (i.e., the ovaries or testes). Gonadotrophs, cells that constitute about 10 percent of the pituitary gland, secrete two primary gonadotropins: luteinizing hormone (LH) and follicle-stimulating hormone (FSH). The amount and rate of secretion of these hormones vary widely at different ages and at different times during the menstrual cycle in women. Secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) is low in both males and females prior to puberty. Following puberty, more luteinizing hormone (LH) than follicle-stimulating hormone (FSH) is secreted. During the menstrual cycle there is a dramatic increase in the serum concentrations of both hormones at the time of ovulation, and the secretion of both hormones increases 10- to 15-fold in postmenopausal women. Another type of gonadotropin found in women is human chorionic gonadotropin (hCG), which is produced by the placenta during pregnancy. The detection of human chorionic gonadotropin (hCG) forms the basis of pregnancy tests.

Luteinizing hormone (LH) plays an important role in sexual development and is produced by the pea-sized pituitary gland in the brain.

In children, luteinizing hormone (LH) levels are high right after birth, but then fall, remaining low until puberty approaches (usually between ages 10 and 14). At this time the hypothalamus, an almond-sized area of the brain that links the nervous system with the hormone-producing endocrine system, releases gonadotropin-releasing hormone (GnRH) that starts the changes of puberty. Gonadotropin-releasing hormone (GnRH) signals the pituitary gland to release two puberty hormones into the bloodstream: luteinizing hormone (LH) and follicle-stimulating hormone (FSH).

Pulsatile secretion of gonadotropin-releasing hormone (GnRH) into the hypophyseal portal circulation represents the initial neuroendocrine step in the regulation of the hypothalamo-pituitary-gonadal axis in both sexes. Thus, this specialized gonadotropin-releasing hormone (GnRH) neuronal network plays a commanding role in this biologic hierarchy and controls episodic gonadotropin secretion, modulates gonadal steroid feedback, and ultimately determines the initiation or suppression of pubertal development and fertility across the life cycle ¹⁾.

Under normal conditions, the gonadotropin-releasing hormone (GnRH) neuronal network undergoes a series of dynamic changes from fetal life to adulthood. The initiation of gonadotropin-releasing hormone (GnRH) secretion is initiated in early fetal life and remains active until the first several months of infancy (representing the “mini-puberty”), and then becomes remarkably dampened during the years of the childhood “quiescence” ²⁾. At puberty, unknown biologic triggers re-ignite gonadotropin-releasing hormone (GnRH) secretion, resulting in full sexual maturation. Therefore, the controls of the reproductive axis are in dynamic flux, turning on and turning off in response to as-yet-unknown biologic signals at various points in the reproductive life cycle.

• In boys, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) work together to get the testes to begin producing testosterone, the hormone responsible for the physical changes of puberty and the production of sperm. Testosterone is the hormone that causes most of the changes in a boy’s body during puberty. Sperm cells must be produced for men to reproduce.
• In girls, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) prompt the ovaries to begin producing the hormone estrogen, which causes a girl’s body to mature and prepares her for menstruation. Estrogen, along with luteinizing hormone (LH) and follicle-stimulating hormone (FSH), causes a girl’s body to mature and prepares her for pregnancy.
• In men, follicle-stimulating hormone (FSH) stimulates the development of spermatozoa, in large part by acting on special cells in the testes called Sertoli cells. Luteinizing hormone (LH) stimulates the secretion of androgen (male) hormones by specialized cells in the testes called Leydig cells.
• In women, follicle-stimulating hormone (FSH) stimulates the synthesis of estrogens and the maturation of cells lining the spherical egg-containing structures known as Graafian follicles. In menstruating women, there is a preovulatory surge in serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) concentrations. The preovulatory surge of luteinizing hormone (LH) is essential for rupture of the Graafian follicle (ovulation), after which the egg enters the fallopian tube and travels to the uterus. The empty Graafian follicle becomes filled with progesterone-producing cells, transforming it into a corpus luteum. Luteinizing hormone (LH) stimulates the production of progesterone by the corpus luteum.
• Inhibin, a hormone secreted by the Graafian follicles of the ovaries and by the Sertoli cells of the testes, inhibits the secretion of follicle-stimulating hormone (FSH) from the pituitary gonadotrophs.

Patients with diseases involving the anterior pituitary gland often have gonadotropin deficiency. Thus, the disappearance of menstrual periods may be the first sign of a pituitary tumor or other pituitary disease in women. In men the most common symptoms of gonadotropin deficiency are loss of libido and erectile dysfunction. Isolated deficiencies of both luteinizing hormone (LH) and follicle-stimulating hormone (FSH) do occur but only rarely. In men isolated luteinizing hormone (LH) deficiency (“fertile eunuch”) is characterized by symptoms and signs of androgen deficiency; however, there is sufficient secretion of follicle-stimulating hormone (FSH) to permit the maturation of spermatozoa. Some pituitary tumors produce an excess of luteinizing hormone (LH) or follicle-stimulating hormone (FSH), whereas other pituitary tumors produce the hormonally inactive alpha chain subunit of the glycoprotein hormones.

Because luteinizing hormone (LH) and follicle-stimulating hormone (FSH) work so closely with each other, doctors often perform these tests together, as well as tests for testosterone (the major male sex hormone) and estradiol (a form of estrogen, the major female sex hormone). Taken together, the results can often provide a more complete picture of a person’s sexual maturation, and the well-being of the endocrine glands that produce these hormones.

Figure 1. Gonadotropins

Figure 2. The pituitary gland location

Figure 3. The hypothalamus and pituitary gland (anterior and posterior) endocrine pathways and target organs

What is human chorionic gonadotropin

Human chorionic gonadotropin (hCG) is a hormone produced by the placenta of a pregnant woman. Early in pregnancy, the level of human chorionic gonadotropin (hCG) increases in the blood and is eliminated in the urine. A pregnancy test detects human chorionic gonadotropin (hCG) in the blood or urine and confirms or rules out pregnancy.

The pregnancy hormone, human chorionic gonadotropin (hCG), is crucially involved in processes such as implantation and placentation, two milestones of pregnancy whose successful progress is a prerequisite for adequate fetal growth. Moreover, hCG determines fetal fate by regulating maternal innate and adaptive immune responses allowing the acceptance of the foreign fetal antigens ³⁾. As one of the first signals provided by the embryo to its mother, human chorionic gonadotropin (hCG) has the potential to regulate very early pregnancy-driven immune responses, allowing the establishment and preservation of fetal tolerance.

During the early weeks of pregnancy, human chorionic gonadotropin (hCG) is important in maintaining function of the corpus luteum. Production of human chorionic gonadotropin (hCG) increases steadily during the first trimester (8-10 weeks) of a normal pregnancy, peaking around the 10th week after the last menstrual cycle. Levels then fall slowly during the remainder of the pregnancy. Human chorionic gonadotropin is no longer detectable within a few weeks after delivery.

When a pregnancy occurs outside of the uterus (ectopic pregnancy), the level of human chorionic gonadotropin (hCG) in the blood increases at a slower rate. When an ectopic pregnancy is suspected, measuring the level of human chorionic gonadotropin (hCG) in the blood (quantitative test) over time may be useful in helping to make a diagnosis of ectopic pregnancy.

Similarly, the human chorionic gonadotropin (hCG) blood level may be abnormal when the developing baby (fetus) has a chromosome defect such as Down syndrome. An human chorionic gonadotropin (hCG) test is used routinely in conjunction with a few other tests as part of screening for fetal abnormalities.

Human chorionic gonadotropin test

Human chorionic gonadotropin test is used for confirming pregnancy, the timing of testing depends on how accurate a woman is about the day she expects her menstrual period as well as the method used for testing. In general, blood tests are more sensitive than urine human chorionic gonadotropin tests and can be done two days before a woman would expect her period to start. A urine or blood human chorionic gonadotropin (hCG) test can be done reliably by 10 days after a missed menstrual period. Even using a urine human chorionic gonadotropin (hCG) test, a woman may be able to determine whether she is pregnant the day she misses her period, but the result could be falsely negative. Testing may be repeated at a later date if the first test is negative but pregnancy is still suspected.

Quantitative blood human chorionic gonadotropin (hCG) tests may be ordered over several days when a health practitioner wants to identify or rule out an ectopic pregnancy or to monitor a woman after a miscarriage. In these cases, a woman may experience the normal signs and symptoms of pregnancy at first but then may develop others that indicate that the pregnancy is not progressing as expected.

Some signs and symptoms of ectopic pregnancy include:

• Abnormal vaginal bleeding—because a woman is pregnant, she may not have a regular period but then may have light bleeding or spotting with an ectopic pregnancy
• Low back pain
• Pain or cramping in the lower abdomen or on one side of the pelvis

If untreated ectopic pregnancy, signs and symptoms may get worse and may include:

• Dizziness, weakness
• Feeling faint or fainting
• Low blood pressure
• Pain in the shoulder area
• Sudden, sharp pain in the pelvic area
• Fever, flu-like symptoms
• Vomiting

The area around an ectopic pregnancy may rupture and start to bleed, and, if undiagnosed, can lead to cardiac arrest and death.

An human chorionic gonadotropin (hCG) test may be ordered prior to a medical procedure or treatment that might be harmful during pregnancy.

How is human chorionic gonadotropin used?

Qualitative human chorionic gonadotropin (hCG) testing detects the presence of human chorionic gonadotropin (hCG) and is routinely used to screen for a pregnancy. This test may be performed by a laboratory, at a doctor’s office, or at home using a home pregnancy test kit. Methods will vary slightly but for most, a test strip is dipped into a collected cup of urine or exposed to a woman’s urine stream. A colored line (or other color change) appears within the time allotted per instructions, usually about 5 minutes. For accurate test results, it is important to carefully follow the test directions. (See the article on Home Testing: Avoiding Errors for more on this.) If the test is negative, it is often repeated several days later. Since human chorionic gonadotropin (hCG) rises rapidly, an initial negative test can turn positive within this time period.

Quantitative human chorionic gonadotropin (hCG) testing, often called beta human chorionic gonadotropin (β-hCG), measures the amount of human chorionic gonadotropin present in the blood. It may be used to confirm a pregnancy. It may also be used, along with a progesterone test, to help diagnose an ectopic pregnancy, to help diagnose and monitor a pregnancy that may be failing, and/or to monitor a woman after a miscarriage.

Human chorionic gonadotropin (hCG) blood measurements may also be used, along with a few other tests, as part of screening for fetal abnormalities.

Occasionally, an human chorionic gonadotropin test is used to screen for pregnancy if a woman is to undergo a medical treatment, be placed on certain drugs, or have other testing, such as x-rays, that might harm the developing baby. This is usually done to help confirm that the woman is not pregnant. It has become standard practice at most institutions to screen all female patients for pregnancy using a urine or blood human chorionic gonadotropin test before a medical intervention, such as an operation, that could potentially harm a fetus.

What does human chorionic gonadotropin test result mean?

• A negative human chorionic gonadotropin result means that it is unlikely that a woman is pregnant. However, tests performed too early in a pregnancy, before there is a significant human chorionic gonadotropin (hCG) level, may give false-negative results. The test may be repeated a few days later if there is a strong possibility of pregnancy.
• A positive human chorionic gonadotropin means that a woman is likely pregnant.
• However, blood or protein in the urine may cause false-positive pregnancy results. Urine human chorionic gonadotropin (hCG) tests may give a false-negative result if the urine is too diluted or if testing is done too soon in the pregnancy.
• Certain drugs such as diuretics and promethazine (an antihistamine) may cause false-negative urine results. Other drugs such as anti-convulsants, anti-parkinson drugs, hypnotics, and tranquilizers may cause false-positive results. The presence of protein in the urine (proteinuria), blood in the urine (hematuria), or excess pituitary gonadotropin may also cause a false positive.
• There are reports of false-positive blood human chorionic gonadotropin (hCG) results due to the presence of certain types of antibodies that some individuals produce or fragments of the human chorionic gonadotropin (hCG) molecule. Generally, if results are questionable, they may be confirmed by testing with a different method.

The blood level of human chorionic gonadotropin in a woman with an ectopic pregnancy usually rises at a slower rate than normal. Typically, human chorionic gonadotropin (hCG) levels double about every two days for the first four weeks of a normal pregnancy, then slow to every 31/2 days by six weeks. Those with failing pregnancies will also frequently have a longer doubling time early on or may even show falling human chorionic gonadotropin (hCG) concentrations during the doubling period. Human chorionic gonadotropin (hCG) concentrations will drop rapidly following a miscarriage. If human chorionic gonadotropin (hCG) does not fall to undetectable levels, it may indicate remaining human chorionic gonadotropin-producing tissue that will need to be removed (dilation and curettage – D&C).

How does the test that I do at home myself compare with the results of a test done in a lab?

Home pregnancy testing is very similar to qualitative urine human chorionic gonadotropin testing performed in the laboratory, but there are factors surrounding its use that are important to note.

• Home tests come with very specific directions that must be followed explicitly. If you are using a home test, follow the directions extremely carefully. There can be variability in sensitivity to detecting the presence of human chorionic gonadotropin with different brands of home pregnancy kits.
• Home tests are sometimes done too soon after the missed menstrual cycle to result in a positive test. It typically takes 10 days after a missed menstrual period before the presence of human chorionic gonadotropin can be detected by the urine test.
• All urine human chorionic gonadotropin tests should be done on a first morning urine sample, if possible. Urine becomes more dilute after ingestion of liquids (coffee, juice, water, etc.) and urine human chorionic gonadotropin concentrations may become too low to register as positive.

Generally, when used correctly, the home test should produce the same result as the urine human chorionic gonadotropin test done by your health practitioner. Blood testing for human chorionic gonadotropin is more sensitive than urine human chorionic gonadotropin testing, so sometimes a blood test will indicate pregnancy when the urine test is negative.

When is a blood human chorionic gonadotropin test ordered instead of a urine human chorionic gonadotropin?

Since human chorionic gonadotropin is not normally detected in the urine of a non-pregnant woman, a urine human chorionic gonadotropin is enough to confirm a pregnancy. This can also be done with a qualitative blood human chorionic gonadotropin test. Sometimes, however, it is important to know how much human chorionic gonadotropin is present to evaluate a suspected ectopic pregnancy or to monitor a woman following a miscarriage. In these circumstances, a health practitioner will order a quantitative blood human chorionic gonadotropin test.

How many days after a miscarriage would it take for a urine pregnancy test to show a negative result?

Urine human chorionic gonadotropin decreases at about the same rate as serum human chorionic gonadotropin, which can take anywhere from 9 to 35 days, with a median of 19 days. However, the timeframe for when an human chorionic gonadotropin result will be negative is dependent on what the human chorionic gonadotropin level was at the time of the miscarriage. Frequently, miscarriages are monitored with quantitative blood human chorionic gonadotropin testing. If the levels of human chorionic gonadotropin do not fall to undetectable levels, some human chorionic gonadotropin-producing tissue may remain and have to be removed.

What is an ectopic pregnancy?

An ectopic pregnancy occurs when the fertilized egg (ovum) implants somewhere other than in the uterus. This is a serious condition needing immediate treatment. Women with ectopic pregnancies often have abdominal pain and uterine bleeding. Usually, abnormally low levels of human chorionic gonadotropin are produced in ectopic pregnancies with slower-than-normal rates of increase.

Gonadotropin deficiency

Isolated gonadotropin-releasing hormone (GnRH) deficiency is characterized by inappropriately low serum concentrations of the gonadotropins – LH (luteinizing hormone) and FSH (follicle-stimulating hormone) in the presence of low circulating concentrations of sex steroids ⁴⁾. Isolated gonadotropin-releasing hormone (GnRH) deficiency is associated with a normal sense of smell (normosmic isolated gonadotropin-releasing hormone deficiency) in approximately 40% of affected individuals and an impaired sense of smell (Kallmann syndrome) in approximately 60% ⁵⁾. Isolated gonadotropin-releasing hormone (GnRH) deficiency can first become apparent in infancy, adolescence, or adulthood. Infant boys with congenital isolated gonadotropin-releasing hormone deficiency often have micropenis and cryptorchidism. Adolescents and adults with isolated gonadotropin-releasing hormone deficiency have clinical evidence of hypogonadism and incomplete sexual maturation on physical examination. Adult males with isolated gonadotropin-releasing hormone (GnRH) deficiency tend to have prepubertal testicular volume (i.e., <4 mL), absence of secondary sexual features (e.g., facial and axillary hair growth, deepening of the voice), decreased muscle mass, diminished libido, erectile dysfunction, and infertility. Adult females have little or no breast development and primary amenorrhea. Although skeletal maturation is delayed, the rate of linear growth is usually normal except for the absence of a distinct pubertal growth spurt.

Typically, a definitive diagnosis of isolated gonadotropin-releasing hormone (GnRH) deficiency is made around age 18 years. Occasionally, however, a high clinical suspicion of isolated gonadotropin-releasing hormone (GnRH) deficiency may be present in an adolescent presenting with anosmia and delayed puberty or in an infant with microphallus and cryptorchidism.

The term “hypogonadism” refers to impaired sexual development based on findings from the individual’s clinical history (e.g., amenorrhea, hot flashes, erectile dysfunction) as well as physical examination (e.g., small testes, vaginal pallor).

With greater understanding of the hypothalamo-pituitary-gonadal axis (see Figures 1 to 3) and the introduction of urinary gonadotropin measurements, the term “hypergonadotropic” hypogonadism was used to identify those with a primary gonadal defect, while “hypogonadotropic” hypogonadism identified those with a central (i.e., pituitary or hypothalamic) defect.

When anatomic (and later functional) causes of central hypogonadism were identified, “idiopathic” or “isolated” hypogonadotropic hypogonadism came into use to indicate those individuals in whom secondary causes of hypogonadotropic hypogonadism had been excluded.

Subsequently the ability to measure the effect of exogenous gonadotropin-releasing hormone (GnRH) administration demonstrated that the vast majority of individuals with “idiopathic” hypogonadotropic hypogonadism had a functional deficiency of gonadotropin-releasing hormone (GnRH) resulting from a defect in GnRH biosynthesis, secretion, and/or action (hence “isolated GnRH deficiency” [IGD]). Aside from hypothalamic gonadotropin-releasing hormone (GnRH) deficiency, individuals with isolated gonadotropin-releasing hormone (GnRH) deficiency typically have normal pituitary function tests and their hypogonadism typically responds to a physiologic regimen of exogenous gonadotropin-releasing hormone (GnRH) ⁶⁾.

At this point, the term “isolated GnRH deficiency” (IGD) more properly reflects the current understanding of the clinical entity rather than the previous biochemical description of isolated hypogonadotropic hypogonadism and, thus, is the better term for what was previously called isolated or idiopathic hypogonadotropic hypogonadism.

Isolated gonadotropin-releasing hormone (GnRH) deficiency: Included Phenotypes

• Normosmic (with a normal sense of smell) gonadotropin-releasing hormone (GnRH) deficiency ~ 40 percent of cases
• Kallmann syndrome with impaired sense of smell ~ 60 percent of cases. The impaired olfactory function in Kallmann syndrome can be either hyposmia or complete anosmia) ⁷⁾. The difference between hyposmia and anosmia is quantitative and not qualitative (i.e., odorants can be variably affected in persons with hyposmia). Most individuals with impaired smell do not have any physical or social impairment and the finding often goes unnoticed until isolated gonadotropin-releasing hormone (GnRH) deficiency is diagnosed.

A recent epidemiologic study in Finland showed a minimal incidence of Kallmann syndrome of 1:30,000 in males and 1:125,000 in females ⁸⁾.

In the Seminara et al. ⁹⁾ cohort of 250 individuals with IGD, males predominate with a male-to-female ratio of nearly 4:1.

Kallmann syndrome accounts for nearly two thirds of individuals with isolated GnRH deficiency (IGD).

Gonadotropin deficiency diagnosis and testing

Isolated gonadotropin-releasing hormone (GnRH) deficiency is typically diagnosed in adolescents presenting with absent or partial puberty using biochemical testing that reveals low serum testosterone or estradiol (hypogonadism) that results from complete or partial absence of gonadotropin-releasing hormone (GnRH)-mediated release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) (hypogonadotropic hypogonadism) in the setting of otherwise normal anterior pituitary anatomy and function and in the absence of secondary causes of hypogonadotropic hypogonadism. Pathogenic variants in more than 25 genes account for about half of all isolated gonadotropin-releasing hormone (GnRH) deficiency; the genetic cause for the remaining cases of isolated gonadotropin-releasing hormone (GnRH) deficiency is unknown.

In individuals with isolated gonadotropin-releasing hormone (GnRH) deficiency, analyses of the pulsatile pattern of gonadotropins have demonstrated a rather broad spectrum of abnormal developmental patterns varying from completely absent gonadotropin-releasing hormone (GnRH)-induced luteinizing hormone (LH) pulses to sleep-entrained gonadotropin-releasing hormone (GnRH) release that is indistinguishable from that of early puberty ¹⁰⁾. This broad spectrum of neuroendocrine activity accounts for the variable reproductive phenotypes observed in persons with isolated gonadotropin-releasing hormone (GnRH) deficiency (IGD).

Suggestive findings

Isolated gonadotropin-releasing hormone (GnRH) deficiency should be suspected in individuals with the following:

• Absent or partial puberty at presentation in adolescents; low serum testosterone or estradiol on biochemical testing
• Findings of incomplete sexual maturation on physical examination as determined by Tanner staging (see Table 1):
  • Men with isolated gonadotropin-releasing hormone (GnRH) deficiency typically have Tanner stage I-II genitalia (prepubertal testicular volumes; i.e., <4 mL); however, some males show evidence of partial pubertal maturation ¹¹⁾.
  • Women with isolated gonadotropin-releasing hormone (GnRH) deficiency typically have Tanner stage I breast development and amenorrhea; however, some have spontaneous breast development and occasional menses ¹²⁾.
  • Both men and women with isolated gonadotropin-releasing hormone (GnRH) deficiency typically have Tanner stage II-III pubic hair, since pubic hair is controlled in part by adrenal androgens.
• In rare males, isolated gonadotropin-releasing hormone (GnRH) deficiency may present later in adulthood (i.e., adult-onset isolated gonadotropin-releasing hormone deficiency). However, in these patients, as puberty was not disrupted, sexual maturation is complete and secondary sexual characteristics may be fully developed. Diagnosis of adult-onset isolated gonadotropin-releasing hormone (GnRH) deficiency relies on documentation of hypogonadotropic hypogonadism and absence of other secondary causes of hypogonadotropic hypogonadism.

Table 1. Tanner Staging

Stage	Normal Findings
	Pubic Hair	Male Genitalia	Female Breast Development
I	None	Childhood appearance of testes, scrotum, and penis (testicular volume <4 mL)	No breast bud, small areola, slight elevation of papilla
II	Sparse hair that is long and slightly pigmented	Enlargement of testes; reddish discoloration of scrotum	Formation of the breast bud; areolar enlargement
III	Darker, coarser, curly hair	Continued growth of testes and elongation of penis	Continued growth of the breast bud and areola; areola confluent with breast
IV	Adult hair covering pubis	Continued growth of testes, widening of the penis with growth of the glans penis; scrotal darkening	Continued growth; areola and papilla form secondary mound projecting above breast contour
V	Laterally distributed adult-type hair	Mature adult genitalia (testicular volume >15 mL)	Mature (areola again confluent with breast contour; only papilla projects)

[Source ¹³⁾]

Laboratory findings of isolated gonadotropin-releasing hormone (GnRH) deficiency (see Figure 4 and Figure 5 for algorithm)

• Total testosterone (T) <100 ng/dL in males and estradiol (E2) <50 pg/mL in females
• Inappropriately low or normal serum concentration of LH (luteinizing hormone) and FSH (follicle stimulating hormone) in the presence of low circulating concentrations of sex steroids. Levels of other anterior pituitary hormones are typically normal.

Figure 4. Testing algorithm to establish the diagnosis of isolated GnRH deficiency (IGD) in males

[Source ¹⁴⁾]

Figure 5. Testing algorithm to establish the diagnosis of isolated GnRH deficiency (IGD) in females

[Source ¹⁵⁾]

Imaging findings of isolated gonadotropin-releasing hormone (GnRH) deficiency

• In persons with isolated gonadotropin-releasing hormone (GnRH) deficiency: typically, normal-appearing hypothalamus and pituitary on MRI exam
• In persons with Kallmann Syndrome: typically, aplasia or hypoplasia of the olfactory bulbs/sulci/tracts.

Olfactory findings

Olfactory function is evaluated by history and by formal diagnostic smell tests, such as the University of Pennsylvania smell identification test (UPSIT), a “scratch and sniff” test that evaluates an individual’s ability to identify 40 microencapsulated odorants and can be easily performed in most clinical settings ¹⁶⁾. Anosmia, hyposmia, or normosmia is identified using the University of Pennsylvania smell identification test (UPSIT) manual normogram, which incorporates an individual’s score, age at testing, and gender.

Individuals with isolated gonadotropin-releasing hormone (GnRH) deficiency with either self-reported complete anosmia or a score of hyposmia/anosmia on University of Pennsylvania smell identification test (UPSIT) are diagnosed with Kallmann Syndrome, while those with normal olfactory function are diagnosed with normosmic IGD (nIGD) ¹⁷⁾.

Establishing the Diagnosis

The diagnosis of isolated gonadotropin-releasing hormone (GnRH) deficiency is established in a proband based on clinical and biochemical investigations above; a genetic diagnosis can be made with identification of pathogenic variant(s) in one of the genes listed in Table 2 and Table 3.

See Table 2 for the most common genetic causes (i.e., pathogenic variants of any one of the genes included in this table account for >2% of isolated gonadotropin-releasing hormone (GnRH) deficiency) and Table 3 for less common genetic causes (i.e., pathogenic variants of any one of the genes included in this table are reported in only a few families).

Molecular testing approaches can include serial single-gene testing, use of a multi-gene panel, and more comprehensive genomic testing.

Serial single-gene testing can be considered based on mode of inheritance and clinical findings, especially non-reproductive phenotypic features that indicate that pathogenic variation of a particular gene is most likely. Sequence analysis of the gene of interest is performed first, followed by gene-targeted deletion/duplication analysis if no pathogenic variant is found.

To help prioritize the order of serial single-gene testing, the following can be considered (see Figure 6 and Figure 7):

Sense of smell

• Pathogenic variants in CHD7, FGF8, FGF17, FGFR1, HS6ST1, NSMF (NELF), PROK2, PROKR2, and WDR11 cause both Kallmann syndrome (KS) and normosmic IGD (nIGD).
• Pathogenic variants in ANOS1 (KAL1), CCDC141, FEZF1, IL17RD, SEMA3A, SEMA3E, and SOX10 cause Kallmann syndrome (KS).
• Pathogenic variants in GNRH1, GNRHR, KISS1, KISS1R (GPR54), TAC3, and TACR3 cause nIGD.

Mode of inheritance

• X-linked. Sequence analysis of ANOS1 (KAL1) is the highest-yield molecular genetic test.
• Autosomal dominant. In families with clear autosomal dominant inheritance, testing of CHD7, FGFR1, FGF8 and SOX10 can be considered.
• Autosomal recessive. Testing of GNRH1, GNRHR, KISS1, KISS1R, TAC3, and TACR3 can be considered in families with autosomal recessive normosmic isolated gonadotropin-releasing hormone (GnRH) deficiency; testing of FEZF1, PROK2 and PROKR2 can be considered in families with autosomal recessive Kallmann syndrome (KS).

Associated phenotypic features

• The presence of some associated clinical phenotypic features may also help prioritize genetic testing in isolated gonadotropin-releasing hormone (GnRH) deficiency ¹⁸⁾. See Figure 7.

Figure 6. Genes associated with isolated GnRH deficiency (IGD) by sense of smell and mode of inheritance 

[Source ¹⁹⁾]

Figure 7. Suggested guidelines for prioritization of genetic testing for persons with isolated GnRH deficiency (IGD) based on phenotype 

[Source ²⁰⁾]

Table 2. Summary of Molecular Genetic Testing Used in Isolated Gonadotropin-Releasing Hormone (GnRH) Deficiency: Most Common Genetic Causes

Gene 1, 2	% of IGD Attributed to Pathogenic Variants in This Gene 3	Proportion of Pathogenic Variants 4 Detected by Test Method
		Sequence analysis 5	Gene-targeted deletion/duplication analysis 6
ANOS1 (KAL1)	5%-10% (KS)	~88%-99%	≤12% in one study (4/33 persons w/KS) 7
CHD7	5%-10% (KS or nIGD)	~100%	Unknown 8
FGFR1	~10% (KS or nIGD)	~99%	Rare 9
GNRHR	5%-10% (nIGD)	~100%	Unknown 8
IL17RD	2%-5% (KS or nIGD)	~100%	Unknown 8
PROKR2	~5% (KS or nIGD)	~100%	Unknown8
SOX10	2%-5% (KS)	~100%	Unknown 8
TACR3	~5% (nIGD)	~100%	Unknown 8

Footnotes: Pathogenic variants of any one of the genes included in this table account for >2% of isolated gonadotropin-releasing hormone (GnRH) deficiency (IGD).

KS = Kallmann syndrome

nIGD = normosmic isolated gonadotropin-releasing hormone deficiency

1. Genes are listed in alphabetic order.
2. See Table A. Genes and Databases for chromosome locus and protein (https://www.ncbi.nlm.nih.gov/books/NBK1334/#kms.molgen.TA).
3. Proportion of IGD attributed to these genes is determined from the author’s cohort of 950 probands with IGD who were screened for rare sequence variants (<1% of control cohort).
4. See Molecular Genetics for information on pathogenic allelic variants detected.
5. Sequence analysis detects variants that are benign, likely benign, of uncertain significance, likely pathogenic, or pathogenic. Pathogenic variants may include small intragenic deletions/insertions and missense, nonsense, and splice site variants; typically, exon or whole-gene deletions/duplications are not detected. For issues to consider in interpretation of sequence analysis results, click here.
6. Gene-targeted deletion/duplication analysis detects intragenic deletions or duplications. Methods that may be used include: quantitative PCR, long-range PCR, multiplex ligation-dependent probe amplification (MLPA), and a gene-targeted microarray designed to detect single-exon deletions or duplications.
7. 12% of persons with KS harbored intragenic deletions in ANOS1 ²¹⁾.
8. No data on detection rate of gene-targeted deletion/duplication analysis are available.
9. FGFR1 deletions are rare ²²⁾.

[Source ²³⁾]

Table 3. Molecular Genetics of Isolated Gonadotropin-Releasing Hormone (GnRH) Deficiency (IGD): Less Common Genetic Causes

Gene 1, 2, 3	Comments
AXL	Described in 1 report: 4/104 persons w/KS or nIGD
CCDC141	Described in 1 report: 1/20 persons w/KS
DUSP6	Described in 1 report: 5/386 persons w/KS or nIGD
FEZF1	Described in 1 report: 2/30 persons w/KS
FGF8	<2% 4 of persons w/KS or nIGD
FGF17	Described in 1 report: 3/386 persons w/KS or nIGD
FLRT3	Described in 1 report: 3/386 persons w/KS or nIGD
GNRH1	Typically AR; <2% 4 of persons w/nIGD
HS6ST1	<2% of persons w/KS or nIGD 4, 5
KISS1	Typically AR; <2% of persons w/nIGD 4
KISS1R	Typically AR; <2% of persons w/nIGD 4
POLR3B	Described in 1 report: 3/565 persons w/KS or nIGD
PROK2	Typically AR; <2% 4 of persons w/KS or nIGD 4
SEMA3A	<2% of persons w/KS or nIGD 4, 5
SEMA3E	Described in 1 report: 1/121 persons w/KS or nIGD
SPRY4	Described in 1 report: 14/386 persons w/KS or nIGD
SRA1	Described in 1 report: 3/136 persons w/nIGD
TAC3	Typically AR; <2% of persons w/nIGD 4
WDR11	Described in 1 report: 1 person w/balanced translocation; 6/201 persons w/KS or nIGD

Footnotes: Pathogenic variants of any one of the genes listed in this table are reported in only a few families [i.e., <2% of isolated gonadotropin-releasing hormone (GnRH) deficiency (IGD)]

KS = Kallmann syndrome

AR = autosomal recessive

nIGD = normosmic isolated gonadotropin-releasing hormone deficiency

1. Genes are listed in alphabetic order.
2. See Table A. Genes and Databases for chromosome locus and protein (https://www.ncbi.nlm.nih.gov/books/NBK1334/#kms.molgen.TA)
3. Genes are not described in detail in Molecular Genetics but may be included here (https://www.ncbi.nlm.nih.gov/books/NBK1334/bin/kms-less_common_genes.pdf).
4. Proportion of isolated gonadotropin-releasing hormone (GnRH) deficiency attributed to these genes is determined from the author’s cohort of 950 probands with isolated gonadotropin-releasing hormone (GnRH) deficiency who were screened for rare sequence variants (<1% of control cohort).
5. Pathogenic variants in this gene are not thought to cause isolated gonadotropin-releasing hormone (GnRH) deficiency without contributions from other isolated gonadotropin-releasing hormone (GnRH) deficiency-related genes; thus, the proportion of isolated gonadotropin-releasing hormone (GnRH) deficiency caused by pathogenic variants in this gene is unknown.

[Source ²⁴⁾]

Genetic counseling

Isolated gonadotropin-releasing hormone (GnRH) deficiency can be inherited in an X-linked, autosomal dominant, or autosomal recessive manner. Almost all isolated gonadotropin-releasing hormone (GnRH) deficiency-related genes have also been associated with indeterminate or oligogenic inheritance. Recurrence risk counseling is based on family history and the results of molecular genetic testing when available. Carrier testing for at-risk relatives in families with X-linked isolated gonadotropin-releasing hormone (GnRH) deficiency or autosomal recessive isolated gonadotropin-releasing hormone (GnRH) deficiency is possible if the pathogenic variant(s) in the family are known. Prenatal testing for pregnancies at increased risk is possible if the pathogenic variant(s) in the family are known.

Gonadotropin deficiency treatment

Treatment of manifestations: To induce and maintain secondary sex characteristics, gradually increasing doses of testosterone or human chorionic gonadotropin (hCG) injections in males or estrogen and progestin in females; to stimulate spermatogenesis or folliculogenesis, either combined gonadotropin therapy (hCG and human menopausal gonadotropins [hMG] or recombinant FSH) or pulsatile gonadotropin-releasing hormone (GnRH) therapy. If conception fails despite spermatogenesis in a male or ovulation induction in a female, in vitro fertilization may be an option.

Males with isolated gonadotropin-releasing hormone deficiency Age ≥18 Years

Treatment options include sex steroids, gonadotropins, and pulsatile gonadotropin-releasing hormone (GnRH) administration. Choice of therapy in adults is determined by the goal(s) of treatment (i.e., to induce and maintain secondary sex characteristics and/or to induce and maintain fertility). The selection of hormone replacement therapy is also based on the preference of the individual being treated; however, when fertility is not immediately desired, replacement with testosterone therapy is the most practical option. As the majority of individuals with isolated gonadotropin-releasing hormone (GnRH) deficiency have not progressed through puberty at the time of diagnosis, initial therapy should be started at low doses and gradually increased to adult doses once the development of secondary sexual characteristics is achieved.

Hormone replacement therapy for males not desiring fertility

Testosterone therapy

Testosterone therapy in the form of injectable and transdermal routes of testosterone administration is typically used to both induce puberty and maintain adult levels of testosterone. Recently nasal testosterone has become available but use has not been reported in patients with isolated gonadotropin-releasing hormone (GnRH) deficiency.

The injectable testosterone preparations have a “roller-coaster” pharmacokinetic effect, with peak and trough levels that can go to extraphysiologic levels; thus, the transdermal preparations have the added benefit of offering a more favorable pharmacokinetic profile. A typical adult dose of testosterone replacement is 200 mg of testosterone ester injection every two weeks or 5 g of a 1% testosterone gel every day. Doses do vary with newer testosterone preparations; manufacturer’s instructions should be followed for individual testosterone preparations.

Men using topical androgen replacement must take care to avoid exposing other individuals to treated skin. Anecdotal reports suggest that the transmission of clinically effective levels of testosterone from the patient to other family members (including women and children) is possible with undesirable side effects.

Once puberty is initiated, testosterone replacement therapy is usually required indefinitely to ensure normal sexual function and maintenance of proper muscle, bone, and red blood cell mass. However, in approximately 10% of males, reversal of isolated gonadotropin-releasing hormone (GnRH) deficiency may occur; thus, if clinical evidence shows endogenous activity of the hypothalamo-pituitary-axis (e.g., testicular growth on testosterone, maintained testosterone levels despite missing/withholding therapy), a brief washout of testosterone therapy should be done with monitoring of testosterone levels. If testosterone levels fall, therapy should be reinitiated. If levels are normal, no further testosterone therapy will be required; serial monitoring of levels should be undertaken, as some individuals may require reinitiation of therapy.

Human chorionic gonadotropin injections

Human chorionic gonadotropin injections is an alternative to testosterone therapy, human chorionic gonadotropin (hCG) injections promote testicular growth, normalize serum concentration of testosterone, and induce development of secondary sexual characteristics.

In adults, treatment with human chorionic gonadotropin (hCG) is usually initiated at 1,500 IU intramuscularly or subcutaneously every other day to normalize serum testosterone concentrations. Dose should be increased by increments of 250 IU if serum testosterone levels remain low.

Treatment with human chorionic gonadotropin (hCG) must be weighed against the increased risk of developing gynecomastia (resulting from the estrogen produced by stimulation of the testes with human chorionic gonadotropin). To some extent the risk of gynecomastia can be minimized by gradually reducing the dose of human chorionic gonadotropin (hCG) to the minimum required to sustain a serum testosterone concentration in the mid-normal range (~500 ng/dL).

Male Infants/Adolescents with Suspicion of isolated gonadotropin-releasing hormone deficiency

If isolated gonadotropin-releasing hormone (GnRH) deficiency is clinically suspected (e.g., low testosterone levels with low/normal gonadotropins) low-dose testosterone or hCG therapy can be given in early infancy to boys with microphallus to increase penile length ²⁵⁾.

Since a definitive diagnosis of isolated gonadotropin-releasing hormone (GnRH) deficiency may not be possible until age 18 years, after infancy these boys do not generally need to be treated until around the time of puberty. At this time, if a high suspicion of isolated gonadotropin-releasing hormone (GnRH) deficiency remains (e.g., associated anosmia and delay in onset puberty), these subjects may benefit from early initiation of hormonal replacement therapy with either testosterone or human chorionic gonadotropin (hCG) treatment early in the pubertal period. A suggestive puberty induction regimen in adolescents is to start a long-acting testosterone ester at a dose of 25-50 mg, given intramuscularly every two weeks. The doses can be gradually increased by 25-50 mg every two to three months until full virilization is achieved. Once adult doses (~200 mg/2 weeks) are reached, further adjustments are based on serum testosterone levels.

Hormone replacement therapy for males desiring fertility (fertility induction in males)

As testosterone replacement therapy suppresses spermatogenesis in the testes, gonadotropins or pulsatile gonadotropin-releasing hormone (GnRH) therapy is usually required to realize the fertility potential in males.

Gonadotropin therapy

In most males with isolated gonadotropin-releasing hormone (GnRH) deficiency, a combination of gonadotropins (hCG along with either human menopausal gonadotropins [hMG] or recombinant FSH) is used to stimulate spermatogenesis. In males with very low testicular volumes (≤~8 mL) the initiating dose of hCG is usually 1,500 IU intramuscularly or subcutaneously every other day; follicle-stimulating hormone (FSH) is added at doses ranging from 37.5 to 75 IU as either human menopausal gonadotropins [hMG] or recombinant formulation. Trough serum testosterone concentrations (target: mid-normal range [~500 ng/dL]), trough serum FSH levels (target: mid-normal reference range), and sperm count are monitored to assess response. Recent trials show that in those with lower testicular volumes, priming with FSH prior to combination therapy may improve spermatogenic outcomes ²⁶⁾.

In males with higher baseline testicular volumes, treatment with human chorionic gonadotropin alone may be sufficient to achieve spermatogenesis and conception ²⁷⁾. However, if after six to nine months, semen analysis reveals persistent azoospermia or marked oligospermia, follicle-stimulating hormone (FSH) is added to the regimen at doses ranging from 37.5 to 75 IU as either human menopausal gonadotropins [hMG] or a recombinant formulation.

In either treatment, testicular volume must be tracked, as this is one of the primary determinants of successful spermatogenesis. In fact, sperm are rarely seen in the semen analysis until testicular volume reaches 8 mL ²⁸⁾. In most males without a history of cryptorchidism, sperm function is usually normal and conception can occur even with relatively low sperm counts.

Note: Liu et al ²⁹⁾ have noted that previous treatment with gonadotropins may reduce the period of subsequent gonadotropin treatment required for initiation of spermatogenesis.

If a pituitary defect exists, gonadotropin therapy becomes the treatment of choice.

Pulsatile GnRH stimulation vs. gonadotropin therapy

While either gonadotropin therapy or pulsatile GnRH stimulation can induce spermatogenesis in approximately 90%-95% of men with isolated gonadotropin-releasing hormone (GnRH) deficiency, some men have a better response to pulsatile GnRH stimulation than to gonadotropin therapy.

Subcutaneous administration of gonadotropin-releasing hormone (GnRH) in a pulsatile manner through a portable pump that delivers a GnRH bolus every two hours is an efficient way of inducing testicular growth and spermatogenesis ³⁰⁾. As the primary defect of isolated gonadotropin-releasing hormone (GnRH) deficiency is typically localized to the hypothalamus, the pituitary responds appropriately to physiologic doses of GnRH.

Note: In the US, pulsatile GnRH therapy is not currently approved by the Food and Drug Administration for the treatment of infertility in men and, thus, is available for such treatment only at specialized research centers.

Females with isolated gonadotropin-releasing hormone deficiency

Hormone replacement therapy for females not desiring fertility. Although a definitive diagnosis of isolated gonadotropin-releasing hormone (GnRH) deficiency in females is usually made around age 18 years, occasionally a high clinical suspicion of isolated gonadotropin-releasing hormone (GnRH) deficiency may be present in an adolescent presenting with anosmia and delayed puberty, and therapy may need to be initiated earlier (age ~14 years)

• To allow optimal breast development, initial treatment should consist of unopposed estrogen replacement via oral or topical preparations. Many formulations of estrogens are available; a suggested oral regimen is using premarin 0.3 mg daily to be increased gradually to an adult replacement dose of 1-1.25 mg daily.
• Once breast development is optimal, a progestin should be added for endometrial protection (e.g., cyclical Prometrium® 200 mg daily for 10-12 days).
• Although preference of the individual plays an important role in choice of treatment plan, low-estrogen formulations should be considered in women with clotting abnormalities (e.g., Factor V Leiden Thrombophilia and Prothrombin Thrombophilia).

Hormone replacement therapy for females desiring fertility (fertility induction in females). Pulsatile GnRH stimulation and exogenous gonadotropins are FDA approved for folliculogenesis in women with isolated gonadotropin-releasing hormone (GnRH) deficiency. Either therapy should be administered with close supervision by physicians specializing in ovulation induction. Intravenous administration of GnRH at various frequencies throughout the menstrual cycle closely mimics the dynamics of normal menstrual cycles resulting in ovulation of a single follicle ³¹⁾. This therapy offers a clear advantage over the traditional treatment with exogenous gonadotropins, which results in higher rates of both multiple gestation and ovarian hyperstimulation syndrome. For either approach, however, the rate of conception is approximately 30% per ovulatory cycle ³²⁾.

Fertility Options in Patients with isolated gonadotropin-releasing hormone (GnRH) deficiency if Fertility Induction is Unsuccessful

In vitro fertilization (IVF). Although successful spermatogenesis can be obtained in most males with isolated gonadotropin-releasing hormone (GnRH) deficiency through pulsatile GnRH therapy or combined gonadotropin therapy, some men with Kallmann syndrome caused by an ANOS1 (KAL1) pathogenic variant may have an atypical response to therapy ³³⁾. In those who respond to therapy, low sperm numbers can often result in conception; however, if infertility continues despite successful spermatogenesis or if spermatogenesis fails to occur, in vitro fertilization (IVF) is an option.

Similarly, if spontaneous conception fails to occur in women with isolated gonadotropin-releasing hormone (GnRH) deficiency who have undergone ovulation induction, IVF may be an option.

Prevention of secondary complications

Optimal calcium and vitamin D intake should be encouraged and specific treatment for decreased bone mass as needed.

Surveillance

For children of both sexes with findings suggestive of isolated gonadotropin-releasing hormone (GnRH) deficiency, monitor at regular intervals after age 11 years:

• sexual maturation (by Tanner staging on physical examination);
• gonadotropin and sex hormone levels (measurement of serum concentrations of LH, FSH, and total testosterone (T) in males and estradiol (E2) in females);
• bone age.

In individuals with confirmed isolated gonadotropin-releasing hormone (GnRH) deficiency, monitor at regular intervals: serum sex steroid levels (to guide optimal hormone replacement); bone mineral density.

Evaluation of relatives at risk

If the pathogenic variant(s) in a family are known, genetic testing of prepubertal at-risk relatives may be indicated to clarify their genetic status. Because of variable expressivity, a prepubertal child with a known pathogenic variant may progress through puberty in a normal or delayed fashion, or not at all; therefore, clinical reevaluation over time is necessary.