Lawrence C. Layman1
(1)
Section of Reproductive Endocrinology, Infertility, and Genetics, Department of Obstetrics and Gynecology, Developmental Neurobiology Program, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA 30912-3360, USA
Lawrence C. Layman
Email: llayman@mcg.edu
Abstarct
The patient is a 17-year-old white female who was seen for primary amenorrhea. Her mother states that she was healthy during her childhood years other than having several surgeries for repair of cleft lip and palate. Upon further questioning, she is not able to smell foods or other common smells. Her mother had normal puberty but she also has diminished sense of smell. Her mother’s brother reportedly had delayed puberty, cleft palate, and subsequent infertility.The patient is a 17-year-old white female who was seen for primary amenorrhea. Her mother states that she was healthy during her childhood years other than having several surgeries for repair of cleft lip and palate. Upon further questioning, she is not able to smell foods or other common smells. Her mother had normal puberty but she also has diminished sense of smell. Her mother’s brother reportedly had delayed puberty, cleft palate, and subsequent infertility.
Case Study
The patient is a 17-year-old white female who was seen for primary amenorrhea. Her mother states that she was healthy during her childhood years other than having several surgeries for repair of cleft lip and palate. Upon further questioning, she is not able to smell foods or other common smells. Her mother had normal puberty but she also has diminished sense of smell. Her mother’s brother reportedly had delayed puberty, cleft palate, and subsequent infertility.
On physical exam, she is 5′1′′ tall with Tanner 1 breasts and Tanner 3 pubic hair. By bimanual exam, the physician is able to place a finger into the vagina and palpate a cervix. Laboratory studies include a prolactin = 10ng/ml (3–20), TSH = 1.7 μU/ml (0.4–4), total T4 = 8.1 μg/dl (4.5–12.5), LH = 1 mIU/ml (1–11), and FSH = 1.5 mIU/ml (1–11). An MRI reveals no tumor in the brain, but the olfactory bulbs are not seen. Following these studies, additional laboratory studies are obtained: an 8:00 a.m. cortisol is 17ug/dl (8–20) and a growth hormone level rises from 1 to 8 ng/ml after 30 min of exercise.
Development of the Hypothalamic-Pituitary-Gonadal Axis
During embryological development, gonadotropin releasing hormone (GnRH) and olfactory neurons migrate from the olfactory placode region into the brain [1]. Once the olfactory neurons cross the cribriform plate into the brain, a dorsal branch migrates dorsally to reach the olfactory bulbs while a ventral branch migrates to the hypothalamus along with GnRH neurons. GnRH is secreted in a pulsatile fashion from the arcuate nucleus of the hypothalamus, where it is released into the hypophyseal-portal system to affect pituitary synthesis and secretion of gonadotropins luteinizing hormone (LH) and follicle stimulating hormone (FSH). Pituitary gonadotropins then stimulate the ovary to produce sex steroids and oocytes.
Without the use of green fluorescence protein labeling, it is very difficult to study the neurophysiology of individual GnRH neurons in the laboratory. The main reason that it is so difficult to study individual GnRH neurons is because they are widely dispersed throughout the hypothalamus and they are few in number – mice have only 800–1,000 and primates have only 1,000–2,000 [1]. Fortunately, there are two types of immortalized GnRH neurons commonly studied in the literature that are easily manipulated experimentally and maintain important characteristics of GnRH neurons (express GnRH mRNA and release peptide in a pulsatile pattern). GT1–7 cells represent one such group of immortalized GnRH neurons. These immortalized GnRH neurons were generated by cloning the rat GnRH promoter upstream to the T-antigen, which was then injected into single cell embryos [2]. This technique resulted in hypothalamic tumors which were harvested, cultured, and subsequently named GT1–7 cells. These cells are of neuronal origin (but not glia) and are postmigratory.
When the human GnRH promoter was cloned upstream to the T-antigen and injected into single cell embryos to create transgenic mice, tumors were observed along the migratory pathway. These GN11 and NLT cells not only express GnRH, albeit, at lower levels than GT1–7 cells, but they also display migratory properties in vitro [3].
The Diagnosis of Delayed Puberty
Following the childhood period and for reasons not completely understood, central inhibition of GnRH pulses is reduced, and GnRH pulses stimulate first nocturnal LH pulses, which then later occur throughout the 24-h period. These LH pulses stimulate ovarian estrogen production and initiate the onset of the pubertal transition. Normal puberty is usually a progression of events. Once the pubertal process is initiated, it is generally completed within 3–4 years. Thelarche is typically the first sign of estrogen production, and occurs at about age 10.5 years, while pubarche (pubic hair growth) generally occurs about 6 months later. In 10–20% of girls, pubarche precedes thelarche. The peak height velocity occurs at about age 12, and on the descending arm of the growth spurt, menarche occurs at age 12–13 [4].
In girls, delayed puberty is defined as either the absence of thelarche by age 13 or menarche by age 15, which represents 2.5 standard deviations above the mean for North American children. The differential diagnosis is complex and may include hypogonadism, anatomic abnormalities, such as the absence or obstruction of the outflow tract, and certain disorders with ongoing estrogen production – most commonly polycystic ovary syndrome (PCOS). An overview is shown in Fig. 3.1. Although not discussed here, the evaluation and diagnostic categories are similar for males.
Fig. 3.1
An overview of the diagnostic steps in girls with delayed puberty. The two most common causes of outflow obstruction causing primary amenorrhea are complete androgen insensitivity syndrome and mullerian aplasia (Mayer–Rokitansky–Kuster–Hauser syndrome). PCOS polycystic ovary syndrome; T4thyroxin; TSH thyroid stimulating hormone; PRL prolactin
All patients who present with delayed puberty should have a TSH, total T4 (more robust assay than the problematic free T4 – unless it is done by dialysis and subsequent immunoassay), serum prolactin, and detailed psychosocial history. Hypothyroidism (central, rather than primary), hyperprolactinemia, and hypothalamic disorders (eating disorders/stress/exercise) may occur in patients who are eugonadal or hypogonadal depending upon how long the process has been ongoing.
A careful history and physical exam of the female with delayed puberty are extremely important. It is necessary to ascertain any initial signs of puberty and estrogen production. If there are no signs of breast development, the patient clearly has hypogonadism. If she has breast development, she has evidence of having estrogen exposure at some time in her life, but her estrogen status must be determined at the time of presentation (as she could be now hypogonadal).
Several different methods could be considered to determine estradiol exposure, but it should be remembered that estradiol levels are usually not very helpful because discrimination between low and low-normal levels is not easy. A vaginal maturation index can be determined with a vaginal smear performed at the first visit by inserting a Q-tip and rolling it in the fornices of the vagina. Collected cells are then gently rolled on to a slide and set with urine Sedi-Stain or another quick prep stain (liquid prep pap smear fixative may also be used) and the ratio of parabasal, intermediate, and superficial (P/I/S) cells counted in ∼100 cells. A predominance of parabasal cells (such as 80:20:0 ratio) indicates that the patient is hypoestrogenic, and therefore, hypogonadal. It is important to remember that this does not tell anything about the etiology of the hypogonadism – just that hypogonadism is present. If superficial cells are present (as in 0:20:80), the patient is probably making estrogen and is probably eugonadal.
Another method used to assess gonadal status is the progestin challenge test. It is usually performed by administering medroxyprogesterone acetate 10 mg for 5–10 days (after a negative pregnancy test if the patient has normal breast development). Progesterone could also be used, but norethindrone should probably not be used since it may be contaminated with and/or converted to estrogen (which could cause the hypogonadal patient to bleed) [5]. If the patient is hypogonadal, she may not bleed at all or have only minimal spotting. A normal menstrual bleed after the medication is completed indicates that sufficient endogenous estrogen is being produced to prime the endometrium. This test does not need to be performed in patients with no breast development, or who have a vaginal smear inconsistent with estrogen exposure (predominant parabasal cells).
Hypogonadism
If hypogonadism is suspected on the basis of physical exam (Tanner 1 breasts), vaginal maturation index, or negative progestin withdrawal, then serum gonadotropins should be obtained. If elevated, they should be repeated in several weeks for confirmation because of the pulsatile secretion of gonadotropins. If gonadotropins are elevated on several occasions, gonadal failure, also known as hypergonadotropic hypogonadism, is present. A karyotype should be performed in patients (boys and girls) with elevated gonadotropins to rule out a chromosomal abnormality (most commonly a 45,X with or without mosaicism or a pure 46,XY cell line in a girl; or a 47,XXY or 46,XX in a male) [6, 7]. If gonadotropins are low or normal in the face of hypogonadism, then the patient has hypogonadotropic hypogonadism (HH) due to hypothalamic or pituitary dysfunction or disease. It is usually taught that chromosome abnormalities are uncommon and hence unnecessary to evaluate in patients with HH. However, this presumption may be challenged by studies from our lab which found that as many as 2–3% of patients with HH have chromosomal rearrangements [8].
Sex steroids are necessary for growth therefore performing a bone age should be considered in hypogonadal patients. These patients can have delayed bone age compared to chronological age. They do not usually have a bone age beyond 11–12 years; however, if bone age is markedly delayed, growth hormone deficiency and/or hypothyroidism should strongly be considered.
Idiopathic Hypogonadotropic Hypogonadism
When the patient is hypogonadal with low serum gonadotropins (or “normal,” an inappropriate finding on a background of hypogonadism), the diagnosis is HH. An MRI with and without gadolinium of the brain is necessary to exclude a CNS tumor, most commonly a prolactinoma or craniopharygioma. It is also reasonable to visualize the olfactory tracts and bulb – if they are absent or hypoplastic, this suggests Kallmann syndrome (KS). If a tumor is not present, the cause is usually considered to be hypothalamic by exclusion. Although it is possible to perform a triple test (insulin induced hypoglycemia, GnRH, and TRH stimulation and check baseline and hormone levels every 15 min for 1–2 h – TSH, prolactin, cortisol, LH, FSH, and GH), the cost is great and the yield is very low except in patients who have extreme short stature, which could suggest pituitary failure. However, cryptic adrenal failure is very serious and could indicate pituitary insufficiency with reduced ACTH, therefore, it is reasonable to obtain an 8:00 AM cortisol. If the patient has a height below the 5th percentile, particularly with a family history of pituitary failure, then combined pituitary hormone deficiency (CPHD) should be considered. Genetic counseling and testing for mutations in genes such as PROP1, HESX1, SOX2, SOX3, LHX3, or LHX4 could be considered in these patients (Fig. 3.1) [9]. In the absence of a tumor, strong consideration must be given to the history and physical exam with particular attention to body mass index, eating and exercise patterns, and stress.
For patients with HH who have no pituitary tumor and are of normal weight, two diagnoses must be entertained. The first is constitutional delay of puberty (CDP), which really can only be documented in retrospect if girls subsequently initiate spontaneous puberty before age 17 (CDP is more common in boys). If girls remain hypogonadal by age 17, then the diagnosis is idiopathic or isolated hypogonadotropic hypogonadism (IHH). A history should be sought for anosmia/hyposmia, midline facial defects, associated neurologic deficits such as synkinesia (on exam she raises both arms when she is asked to raise one), hearing loss, or visual abnormalities. When IHH is combined with anosmia/hyposmia, she has KS. Certainly, a history for eating disorders (anorexia or bulimia), extreme stress, or strenuous exercise must be excluded (discussed in Chap. 8).
The Genetic Basis of IHH/KS
Chromosome Analysis
Although they do not occur as commonly as in women with hypergonadotropic hypogonadism, karyotype abnormalities may occur in several percent of IHH/KS patients [8]. Although their immediate significance is less obvious than a 45,X cell line in a patient with gonadal dysgenesis, a balanced chromosomal translocation could provide an indication of a new IHH/KS gene. These types of studies are being actively pursued in our molecular reproductive endocrinology laboratory. For example, it is possible that a chromosomal translocation could disrupt a gene at the breakpoint, which could cause IHH/KS in that patient. Then, this gene becomes a candidate gene to test in other patients with IHH/KS who do not have chromosome abnormalities. It is also very reasonable to karyotype patients with multiple anomalies and IHH/KS, as this could indicate an unbalanced chromosomal rearrangement with the loss of multiple genes. Prader-Willi syndrome, due to a 15q deletion by FISH, should be considered if associated anomalies are present. Comparative genomic hybridization (CGH) could also be very helpful in ascertaining if deletions or duplications occur.
Molecular Analysis
Mutations have been identified in approximately 30% of IHH/KS families, including KAL1, GNRHR, NR0B1, FGFR1, KISS1R, PROK2, PROKR2, CHD7, FGF8, TAC3, TAC3R, NELF, and GNRH1(reviewed in Kim et al. [9] and summarized in Table 3.1). In this section, the more common genes, particularly those that have consequences for genetic counseling will be briefly reviewed. Mutations in the first identified gene – KAL1 [10, 11] – on the X chromosome account for about 5–10% of the causes of KS in males (not females since it is X-linked recessive) [12]. If a clear X-linked recessive family history is present, 30–60% of these patients will have KAL1 mutations [12]. Males with mutations in the KAL1 gene may also have unilateral renal agenesis (50% in one series), which should be tested. To date KAL1 mutations have only been reported in males with either anosmia or hyposmia – not normosmia. Mutations in the FGFR1 (KAL2) gene occur in about 10% of patients with either KS or normosmic IHH [13, 14]. This autosomal dominant disorder can lead to individuals with mutations who are not affected (reduced penetrance) or to patients with a mild phenotype, such as anosmia only (variable expressivity), which can complicate the diagnosis. Midline facial defects and dental agenesis may occur in patients with FGFR1mutations.
Table 3.1
IHH/KS genes. The gene symbol and its chromosomal location are shown in addition to the phenotype, inheritance pattern, and frequency
|
Gene |
Localization |
Phenotype |
Inheritance |
Frequency |
|
GnRH and olfactory neuron migration |
||||
|
KAL1 |
Xp22.3 |
KS |
XLR |
5–10%; 30–70% familial |
|
FGFR1 |
8p12 |
KS, IHH |
AD |
−10% |
|
CHD7 |
8q12.2 |
KS, IHH, CHARGE syndrome |
AD, sporadic |
6% |
|
FGF8 |
10q24 |
KS, IHH |
AD |
1.3% |
|
NELF |
9q34.3 |
IHH/KS |
Digenic and monogenic |
1–2% |
|
PROK2 |
3p21.1 |
KS |
Unknown |
1–2% |
|
PROKR2 |
20p13 |
KS |
Unknown |
5% |
|
Hypothalamus |
||||
|
KISS1R |
19p13.3 |
IHH |
AR |
Low |
|
LEP |
7q31.3 |
Obesity, IHH |
AR |
Low |
|
LEPR |
1p31 |
Obesity, IHH |
AR |
Low |
|
NROB1 a |
Xp21 |
Adrenal hypoplasia congenita and IHH |
XLR |
Low |
|
PCSK1 |
5q15-q23 |
Obesity, IHH |
AR |
Rare |
|
TAC3 |
12q13-q21 |
IHH |
AR |
Unknown |
|
TACR3 |
4q25 |
IHH |
AR |
Unknown |
|
GNRH1 |
8p21-p11.2 |
IHH |
AR |
0.3–0.8% |
|
Pituitary |
||||
|
GNRHR |
4q21.2 |
IHH |
AR |
3–5% |
|
FSHB |
11p13 |
Isolated FSH deficiency |
AR |
Rare |
|
LHB |
19q13.3 |
Isolated LH deficiency |
AR |
Rare |
|
HESX1 |
3p21.1–21.2 |
Septo-optic dysplasia, CPHD |
AR, AD |
Low |
|
PROP1 |
5q |
Short stature, hypothyroid, CPHD |
AR |
Low |
|
LHX3 |
9q34.3 |
CPHD |
AR |
Low |
|
LHX4 |
1q25 |
CPHD |
AD |
Low |
|
SOX2 |
3q26.3-q27 |
Microphthalmia/midline CNS defects, CPHD |
AD, sporadic |
2–3% |
|
SOX3 |
Xq26.3 |
Midline CNS anomalies, CPHD |
XLR |
Low |
FGFR1, CHD7, FGF8, PROKR2, and PROK2 mutations cause both normosmic IHH and KS. XLR X-linked recessive; AD autosomal dominant; AR autosomal recessive; CPHD combined pituitary hormone deficiency
aNROB1 has hypothalamic and pituitary effects
Mutations in the CHD7 gene cause CHARGE syndrome (Coloboma of the eye, Heart defects, choanal Atresia, Retardation in growth and development, Genito-urinary anomalies, and Ear – both vestibular and auditory) [15]. This disorder can be autosomal dominant or sporadic. Recently, CHD7 mutations were found to be present in 6% of KS patients and 6% of those with normosmic IHH [16]. Mutations in CHD7 and FGFR1 are particularly important because they cause autosomal dominant disease with an attendant 50% risk to each pregnancy, indicating that the patient should be properly counseled prior to pregnancy.
For patients with normosmia, FGFR1 and CHD7 are the most common. GNRHR mutations [17, 18] comprise approximately 3–5% of the cases and are inherited in an autosomal recessive fashion [19]. They have only been reported in normosmic IHH – not KS. Although mutations in other genes have been reported, they are currently thought to be rare or the role of genetic counseling is marginally understood. There have also been some cases in which mutations in two different genes – digenic inheritance – has been reported [20, 21].
Currently, the molecular basis of IHH/KS usually relies upon research labs, including our own. Blood from the affected patient, parents, and siblings (both affected and unaffected) are obtained so that segregation studies can be performed. In this way, if a mutation is identified, other family members can be tested to see if the putative mutation is found in those affected and absent in those who are unaffected. Molecular analysis should be offered to any patient who desires to understand more about her disease, as well as for the risk of recurrence to her child if she elects to attempt conception.
Treatment
Treatment for patients with IHH involves administration of estrogen for girls [22]. Usually, estrogen-only preparations are started at a low dose and increased gradually until normal breast development is reached (or she begins bleeding). This could be 0.3 mg of conjugated equine estrogens that are increased every 3–6 months to 1.25 mg/day. Alternatively, 0.5 mg of estradiol could be given and increased by 0.5 mg increments until the desired result is observed. This could take a year or longer. Then, a progestin is added, or for ease of administration, a combined estrogen–progestogen preparation could then be prescribed.
When pregnancy is desired the estrogen–progestogen is discontinued. Since clomiphene acts principally in the hypothalamus, and these patients have hypothalamic dysfunction, it usually will not work. In contrast to patients with gonadal failure, patients with IHH or KS can be treated with subcutaneous FSH and LH (usually 150 IU/day with monitoring of ovarian follicular development) for ovulation induction in females. These patients are at high risk for ovarian hyperstimulation syndrome and high-order multiple pregnancies. Therefore, it is prudent to be cautious and not aggressive with exogenous stimulation of the ovaries. Previously, this risk was circumvented with the use of a GnRH pump which supported monofollicular ovarian development. Unfortunately, this method of ovulation induction is not available at this time in the United States. Nonetheless, if tubal patency and normal semen parameters exist, ovulation induction as a therapeutic approach yields cycle fecundity that is similar with age-matched fertile women [22].
Case Discussion
This patient has no evidence of puberty – she has Tanner 1 breasts and primary amenorrhea. The fact that she does not demonstrate any stigmata of puberty and there is an absence of breast development strongly indicates hypoestrogenism. There is no reason to obtain a serum estradiol level in this case. In fact, most estradiol assays are not designed to distinguish between low (<30 pg/ml) and early follicular phase levels (30–60 pg/ml). Low or normal serum levels of gonadotropins in the presence of clinical evidence consistent with hypoestrogenism indicate that the patient has HH. An MRI excludes the presence of a tumor, which could be either a prolactinoma or a craniopharygioma. In this patient, as in many others, the cause is usually unknown; and it is often termed idiopathic hypogonadotropic hypogonadism (IHH). She has anosmia with absent olfactory bulbs, findings that strongly suggest that she has KS. If she were tested with a smell identification test, it is likely she would be anosmic or hyposmic. Midline facial defects may also be present in patients with IHH.
The findings of: (1) KS in the patient, (2) her mother who has anosmia only, and (3) an uncle who had delayed puberty and infertility suggest autosomal dominant inheritance. Mutations in either FGFR1or CHD7 genes cause autosomal dominant KS and could be present in this family. DNA sequencing of first the FGFR1 gene, which is more common (10%), and if negative, the CHD7 gene (6%) could be considered in this patient. Autosomal dominant diseases may manifest either reduced penetrance (meaning that not all patients with a mutation manifest the disease), or as in this family, variable expressivity (indicating variable severity). The patient has KS; the mother has only anosmia; and an uncle probably has IHH without anosmia, but who had a more serious defect – cleft palate.
This patient can be treated with estrogen to induce breast development. Once breast development is adequate or she has menses, then adding a progestin or switching to a combined estrogen–progesterone combination preparation can be done. When she considers pregnancy, treatment with gonadotropins (both FSH and LH will be necessary) can be used for ovulation induction. It must be remembered that she should be counseled that there could be up to a 50% chance of having an affected child with the same disorder.
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