Primary Amenorrhea and Differences of Sex Development.

In order to achieve menstruation, normal hypothalamic, pituitary, and ovarian function, as well as hormone sensitivity and Mullerian anatomy, are necessary. Amenorrhea can be categorized into distinct categories: normal estrogen level and pelvic anatomy (anovulation) normal estrogen levels and abnormal pelvic anatomy Low estrogen status in the setting of hypothalamic or pituitary dysfunction (hypogonadotropic hypogonadism) Low estrogen status in the setting of gonadal dysfunction (hypergonadotropic hypogonadism) normal estrogen level and pelvic anatomy (anovulation) normal estrogen levels and abnormal pelvic anatomy Low estrogen status in the setting of hypothalamic or pituitary dysfunction (hypogonadotropic hypogonadism) Low estrogen status in the setting of gonadal dysfunction (hypergonadotropic hypogonadism) Individuals in the first category (with amenorrhea, normal estrogen, and normal anatomy) include those with anovulation. This can include hyperandrogenic anovulation as seen in polycystic ovary syndrome (PCOS), and congenital adrenal hyperplasia (CAH). Typically, these patients will present with secondary amenorrhea but may also present with primary amenorrhea. Those in the second category (amenorrhea, normal estrogen and abnormal uterovaginal anatomy) include those with congenital anatomic causes (imperforate hymen, transverse vaginal septum, cervical atresia, Mullerian agenesis) and iatrogenic causes (cervical stenosis and intrauterine adhesions). Obstructive anomalies in which there are uterine structures with endometrium, but the outflow tract is not patent (imperforate hymen, transverse vaginal septum, cervical atresia) will usually present with amenorrhea in the setting of cyclical pain and are not addressed in this chapter. Additionally, Mullerian agenesis can present in 46, XX (Mayer-Rokitansky-Küster-Hauser or MRKH) and 46, XY individuals [complete androgen insensitivity (CAIS)] Adolescents in the third category (amenorrhea, low estrogen due to central dysfunction) have pituitary disorders or hypothalamic amenorrhea (tumor of the pituitary or hypothalamus, an energy imbalance due to exercise or nutrition, or genetic conditions such as Kallman’s syndrome – a genetic cause of hypothalamic dysfunction and typically associated with anosmia). Finally, individuals in the fourth category (amenorrhea, low estrogen due to gonadal dysfunction) include those with abnormal gonadal development due to gonadal dysgenesis, which can be seen with sex chromosome DSD, 46, XY DSD and 46, XX DSD.

Adolescents with DSD presenting with primary amenorrhea and typical female genitalia fall into 2 general categories: gonadal dysgenesis (46, XY or 46, XX) and Mullerian agenesis (MRKH or CAIS). The former will present with lack of breast development, a patent vagina with mucosal atrophy, low estrogen levels and a high FSH with undetectable AMH. Those presenting with Mullerian agenesis will present with normal breast development and vaginal mucosa, but a short vagina on exam and absent uterus on imaging. Patients with Turner syndrome are often diagnosed prior to puberty due to evaluations of short stature 17 , however due to the variations in phenotype, the diagnoses should be considered in patients with typical female genitalia and primary amenorrhea due to premature ovarian insufficiency (POI) or hypergonadotropic hypogonadism. Management of patients with Turner syndrome should be directed by providers well versed in the treatment and long-term surveillance of the various associated conditions. In those with POI, exogenous estrogen (either transdermal or oral) is used to induce thelarche around the age of 11 or 12 (after a reasonable growth potential is obtained) and slowly titrated up over 2 to 3 years. 17 Because these patients have a uterus, endometrial protection is given with the addition of progestin therapy, started at the time of the first vaginal bleed or approximately 2 years after the initiation of estrogen. Hormone therapy should be continued until at least the normal age of menopause (around 50 in the United States). In a small subset of Turner Syndrome (approximately 10%), Y chromosome material is present. In these cases, there is a risk of germ cell tumor as in other DSD conditions discussed above. Current guidelines recommend prophylactic gonadectomy, but a thorough discussion remains imperative to discuss the potential for gonadal function against the potential risk of germ cell rumor. 17 18 As accelerated follicle loss continues, any individual with Turner syndrome who achieves spontaneous menarche, fertility preservation with the option of egg harvesting should be discussed as an option of future family building. In 46, XY complete gonadal dysgenesis, gonadal tissue does not develop into testicular tissue, but rather are streak gonads. Neither testosterone nor anti-Mullerian hormone are made, leading to female typical internal and external genitalia. Mullerian structures are present. Aside from typical external female genitalia, patients will also have normal pubic hair and axillary hair, but breast development will not be noted. Upon diagnosis, exogenous estrogen (either transdermal or oral) is used to induce thelarche. Because these individuals have a uterus, endometrial protection is given with the addition of progestin therapy, started at the time of the first vaginal bleed or approximately 2 years after the initiation of estrogen. As in cases of premature ovarian insufficiency, hormone therapy should be continued until at least the normal age of menopause. Gonadectomies are performed due to the risk of developing germ cell tumors in the setting of gonads without fertility potential or function. Future fertility should be addressed and include discussion of utilizing an egg donor. Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome is a multifactorial agenesis of the Mullerian structures (uterus, fallopian tubes, cervix, and upper vagina). 1 These anomalies are usually classified into MRKH type 1 (isolated Mullerian agenesis) and MRKH type II which can be associated with MURCS - Mullerian ductal aplasia, Renal dysplasia, and Cervical Somite Anomalies. Because of normal ovarian development, individuals will have normal estrogen levels and progress through normal pubertal milestones at the appropriate tempo, however, never achieve menarche. Occasionally a Mullerian remnant which contains endometrial tissue is present and must be removed to prevent endometriosis and pain. A physical exam will demonstrate normal axillary hair, normal pubic hair, normal breast development, and a shortened vagina or a vaginal dimple. Upon diagnosis, it is important to rule out other possible associated anomalies. A renal ultrasound and spine imaging should be undertaken. Counseling and management include whether the patient desires to and timing of the creation of a functional vagina, and discussion about future fertility potential. A functional vagina can be created three ways, through sexual intercourse, self vaginal dilation, or surgically. The creation of a functional vagina (regardless of approach) should only be undertaken when a patient is able to partake in the discussion about vaginal creation. It is important to reiterate that there is no urgency or absolute need for vaginal creation and that the patient is an active part of the process. Self vaginal dilation achieves a functional vagina with a 90–96% success rate and should be considered first line, when compared to a surgically created neovagina, especially because regular dilation is also required following neovagina creation. 1 A discussion about future fertility potential is important covering available options for family building, such as egg harvesting and surrogacy (where legal) or adoption. In addition, births have been reported after uterus transplant in individuals with MRKH and there are several sites with active protocols for such an intervention around the world. 19 As patients with MRKH have functional ovaries, there is no need for sex hormone replacement. Patients with CAIS have a 46, XY karyotype and functional testicular tissue, but a mutation in the androgen receptor gene prevents androgen action. The fetal testes produce testosterone and AMH. AMH contributes to the regression of the Mullerian structures. Despite producing testosterone, the external fetal genitalia do not virilize, leading to typical female genitalia, without Mullerian structures. Persistence of the androgen receptor insensitivity into puberty usually leads to sparse pubic hair or axillary hair. Breast development is usually present due to the high levels of estrogen from aromatization of testosterone (which are often higher than a typical male level). Management includes discussion of gonadal management, given a risk of gonadal tumor, and timing of the creation of a functional vagina. As in cases of MRKH, vaginal lengthening can be created three ways, through sexual intercourse, self vaginal dilation, or surgically. The creation of a functional vagina (regardless of approach) should only be undertaken when a patient is able to partake in the discussion about her desire for vaginal creation. As in cases of MRKH, it is important to reiterate that there is no urgency for vaginal creation and that the patient is an active part of the process. Gonadectomies are delayed until pubertal development is complete, as the risk of malignancy is lower than other conditions (approximately 2%) with intraabdominal gonads with Y chromosomes. 20 More recently patients have advocated to retain their gonads given the relatively low risk of tumor as well as reports that gonadectomy results in significant distress due to symptoms of estrogen withdrawal. Thus, controversy currently exists regarding whether to offer gonadectomy, and there are no evidence based guidelines for tumor monitoring if gonads are left in situ. If a gonadectomy is performed, then exogenous sex hormones must be initiated for bone and cardiovascular health. The optimal dose of estrogen replacement for these individuals, however, has not been established.

Primary amenorrhea can be a diagnostic puzzle; however, a strategic approach can evaluate embryologic, anatomic, or endocrinologic causes. Careful evaluation of an individual’s estrogen status, hypothalamic status, and presence of Mullerian structures can lead to a diagnosis. A multi-disciplinary team approach is needed during the workup of primary amenorrhea to focus on both the medical and psychological aspects of a possible DSD diagnosis. Care should be taken to address concerns about pubertal development and timing, anatomic considerations, and future fertility.

As stated above, the ability to achieve menses will depend on whether an individual has estrogen, a functional endometrium and patent genital tract. Additionally, the hormonal fluctuations and feedback during the menstrual cycle allow for endometrial proliferation and ovulation followed by menstruation approximately 2 weeks later. Thus, both anatomy and endocrine physiology impact menstruation. There are three main steps in sex development, including establishment of chromosomal sex, gonadal sex, and phenotypic sex (internal and external genital anatomy). Chromosomal sex, or the X and/or Y complement, is established at the time of fertilization. Gonadal sex refers to the development of bipotential gonadal tissue into testis or ovary (around 5–8 weeks of gestation) 5 6 Initial testis development occurs by expression of the SRY gene (on Y chromosome) and by subsequent expression of many other genes. Initial ovarian development is also dependent on specific genetic expression (ex. WNT4) 7 . The gonads (ovary and testis) are responsible for the production of hormones which can affect anatomy, pubertal development and in conjunction with the pituitary and hypothalamus the fluctuations in hormones which allow for ovulation and menstruation. 8 Just as fetuses have bipotential gonadal tissue, they also initially possess internal and external genitalia which can develop along a male or female-typical pathway. Sex-specific development occurs as a result of hormone action. The developing testis produces AMH from Sertoli cells, causing regression of Mullerian structures, and androgens from Leydig cells, causing stabilization of Wolffian structures and further development of external anatomy. In the absence of AMH and with relatively low androgen production, Mullerian structures develop further, Wolffian structures regress and external anatomy is typical for a female. Once Mullerian structures develop, they remain quiescent until puberty, at which point pulses of luteinizing hormone activate the hypothalamic-pituitary-gonadal axis. Estrogen causes breast development – either from ovarian origin or from peripheral conversion of testosterone (as seen in CAIS). A work-up for delayed puberty should occur if breast development is not present by 13 years of age or if menses is absent at 15 years of age or 3 years after thelarche. 2 Presence of estrogen in the setting of a uterus with a functional endometrium will cause endometrial proliferation. Gonadotropins (luteinizing hormone and follicle stimulating hormone) cause the recruitment of ovarian follicles, eventually leading to ovulation and subsequent progesterone secretion. Progesterone causes a change in the endometrial lining from secretory to proliferative. Subsequent physiologic decrease of progesterone causes the lining to shed, thus achieving menarche. 6 Once an individual starts to menstruate, it can be interrupted by a variety of factors, leading to secondary amenorrhea. Typical causes of secondary amenorrhea include androgen excess, hyper or hypothyroidism, or functional hypothalamic deficiencies. These can be present even during puberty and could be a cause of primary amenorrhea. As illustrated above, a multitude of factors work together synchronously to allow menstruation. When establishing a diagnosis of primary amenorrhea, a systematic consideration of the steps leading to menarche is important and will lead to a diagnosis.

Individuals presenting with amenorrhea can either have a known diagnosis of a DSD or this could be the first time a diagnosis of a DSD is considered. Patients with a known diagnosis will be typically counseled about expected menstruation at the time of diagnosis, but it is important to readdress the issue and any concerns as they proceed through puberty. Accurate assessments about timing of pubertal milestones (thelarche, adrenarche, pubarche, growth spurts, etc.) along with identification of similar issues in family members is needed. A detailed history should include evaluation for short stature, hypertension, learning issues and other endocrinopathies as well as inquiries about family history of atypical genitalia, abnormal pubertal development, or infertility. In review of systems, it is important to assess for effects of estrogen or excess androgens. Breast development is the primary effect of estrogen. Exposure to exogenous testosterone can cause amenorrhea and genital changes so it is important to inquire about individual testosterone use or testosterone use within the family which can lead to accidental transdermal exposure. Excess androgens can present as hirsutism, increased muscle mass, voice deepening or growth of the clitoris which can be elicited on history or physical exam. On physical exam, a thorough evaluation of estrogen status is needed by examining breast development and genital estrogenization. The external genitalia can be visualized by gently spreading the labia after thorough counseling and permission from the patient. Estrogen status can be determined by visualizing the hymenal and introital mucosa which will become thicker and a light pink hue with estrogen exposure. In a virginal teen with normal external genitalia and primary amenorrhea the vagina can be evaluated by introducing a moist cotton swab after assuring visualization of a separate urethra. The external genitalia should be further evaluated for vaginal rugae and/or pigmentation of the labia, posterior labial fusion or presence of a single urogenital sinus, or enlargement of the clitoris. Evaluation should also include Tanner staging, along with evidence of axillary or pubic hair. The skin should be evaluated for signs of hirsutism, acne, and acanthosis nigricans. Initial laboratory testing in an adolescent presenting with primary amenorrhea aims to assess reproductive hormone and androgen levels. Initial endocrine evaluation should usually include, LH, FSH, TSH and prolactin. An estradiol level and progesterone withdrawal test (giving a short course of progesterone to evaluate for withdrawal bleeding) will elucidate the estrogen status as well as patency of the genital tract. If there are signs of anovulation (positive progesterone withdrawal test) and/or severe acne or hirsutism, an evaluation for causes of androgen excess should be undertaken, including testosterone, 17-hydroxyprogesterone, 17-hydroxypregnenolone, DHEAS, androstenedione and dihydrotestosterone. Pelvic ultrasound, to assess for Mullerian structures and appearance of gonads, is usually informative and sufficient although in rare cases magnetic resonance imaging may provide further useful information. A karyotype is performed if there is a high FSH consistent with gonadal dysgenesis and in cases of uterovaginal agenesis. As the differential diagnosis is narrowed, further genetic evaluation via single gene analysis or next generation sequencing (targeted panel or whole exome sequencing) may be indicated. Even the initiation of a workup for primary amenorrhea can be fraught with many feelings for an individual. Puberty and menarche are viewed as important landmarks in the progression from childhood to adulthood. Particularly in adolescence, the need to feel conventional is often important. Individuals that are concerned about pubertal development or lack of menarche often feel like they are not like their peers. Careful consideration must be given to assure individuals that they are healthy and while their amenorrhea might be unexpected, it is not unnatural. Thoughtful discussions must occur at each step of the workup to allow for adequate time for questions and education about chromosomal sex, gonadal sex, and anatomic sex. Mental health providers (particularly those with expertise in sex development) are useful in a multidisciplinary approach to the care of these individuals to support them as they adapt to new information about their body development. Prior to initiating treatment for any condition, it is imperative that the individual understands the condition and feels ready to undergo any therapy required. Mental health providers may also be helpful at this stage to assess the individuals’ understanding and readiness. Individuals with atypical genitalia at birth may have DSD that leads to primary amenorrhea Some infants with genitalia most typical for a female may have unappreciated, mild signs of androgen exposure at birth or may not develop androgen effects until later in childhood or adolescence thus some DSD conditions with primary amenorrhea may not be previously diagnosed. 9 10 Therefore, for the purposes of this article, we have addressed all these conditions. MGD is a sex chromosome DSD (karyotype 45, X/46, XY) with asymmetric anatomic findings. A streak gonad is present on one side and a dysgenetic testicle (descended or undescended) on the other leading to asymmetric internal and external development. The side with the streak gonad retains some Mullerian structures due to the lack of AMH production during development while Mullerian structures are usually absent on the side with the dysgenetic testicle. External genital development is also often asymmetric, with more testosterone effects on the side with the dysgenetic testicle. Most MGD presents in infancy, but if the dysgenetic testicle produces only a small amount of testosterone, it can present in adolescence with abnormal pubertal development and primary amenorrhea. Laboratory work-up in these individuals will reveal low estrogen with high gonadotropins (LH/FSH) with or without elevated testosterone. As they may harbor Mullerian structures, they can achieve menses with estrogen replacement. Karyotype, imaging, and gonadal biopsy will confirm findings of streak gonad and dysgenetic testicle. When a streak gonad or dysgenetic gonadal tissue is present in the presence of a Y chromosome, a discussion about gonadal management is necessary, to address the increased risk of germ cell tumor, potentially undesirable testosterone production, and fertility potential. Risk of germ cell tumor, with potential for malignant transformation, increases in adolescence. Monitoring of undescended testicular tissue is not fully reliable due to inadequacies of current imaging technology. 11 Ongoing testicular function will cause further androgen effect (hirsutism, voice deepening, clitoral enlargement) which may be undesirable to the patient. It is possible, although relatively unlikely, for dysgenetic testicular tissue to produce viable sperm, thus this should be considered in a discussion about biological fertility potential, but eggs will not be produced. 10 Given these considerations, the option for gonadectomy should be discussed and shared decision making should be undertaken with the patient and family. Investigational methods for fertility preservation, like gonadal tissue cryopreservation, may be considered at the time of gonadectomy. 12 Ovotesticular DSD can occur with any karyotype (45, X/46, XY, 46, XX, 46, XY) and is characterized by the presence of both ovarian and testicular tissue. Both tissues may be present in one gonad, an ovotestis, or separately as an ovary on one side and a testis on the other side. Mullerian structures may be present, depending on the amount of AMH produced during development. Either the ovarian or testicular component may continue to function throughout puberty and adulthood, although it is more common for the ovarian component to continue to function while the testicle, which is often dysgenetic, develops insufficiency over time. 13 Diagnosis is made with karyotype, imaging, laboratory testing and gonadal biopsy to confirm the presence of ovarian and testicular tissue. Evaluation of gonadotropins and sex steroids can be complicated by the dual tissue components. Sometimes a genetic cause for OVT can be found in gene sequencing. In the case of OVT, again there is a risk of germ cell tumor, in both the ovarian or testicular tissue 14 , 15 , and discussion about gonadal management is indicated. Hormone evaluation will allow better understanding of how the current tissue is functioning, and how much androgen is being produced. Fertility via eggs and sperm has been reported in individuals with OVT, although infertility is common. As in other cases, shared decision making regarding gonadectomy in light of these facts should be carefully undertaken. 15 Partial gonadal dysgenesis is a 46, XY DSD in which testicles are present bilaterally, but the testicular tissue is dysgenetic. If relatively little AMH was present, Mullerian structures may remain. Again, gonadal biopsy is helpful for diagnosis, but laboratory evaluation will also likely show elevated gonadotropin levels with testosterone levels higher than expected for the female range, but lower than expected for the male range. Many genetic causes of PGD are known. Some are heritable and next generation sequencing may detect a cause. Again, gonadal management must be discussed as there is a risk of germ cell tumor and potential for unwanted androgen production. While infertility is common, sperm production is possible, but eggs will not be produced. Partial Androgen Insensitivity Syndrome is an X-linked condition which occurs in individuals with 46, XY karyotype and normal testicles, but with a mutation in the androgen receptor which prevents androgens from acting as effectively on the receptor. The degree to which the receptor is functioning dictates the amount of androgen effect seen in genital development. If there is relatively little androgen response, this may not present until puberty. Aromatization of the testosterone may lead to relatively robust estrogen levels and potential breast development during puberty. No Mullerian structures are present, as AMH was produced during development. Karyotype, imaging, endocrine evaluation, and genetic testing are helpful in diagnosis. During puberty, gonadotropins are pubertal, testosterone is high and AMH is higher than expected for elevated testosterone. Androgen receptor gene sequencing may detect a pathologic variant. In discussion of gonadal management, there is a risk of germ cell tumor. Androgens will continue to be produced by the testicles and, depending on the degree of androgen receptor function, may cause undesirable effects. The testosterone will also, however, continue to be aromatized to estrogen. Fertility via sperm is reported in PAIS, but azoospermia is common and eggs will not be produced. 5-alpha reductase deficiency is an autosomal recessive condition which occurs in individuals with 46, XY karyotype and normal testicles, but who have a mutation in the gene coding for 5-alpha reductase, the enzyme responsible for converting testosterone to dihydrotestosterone. Severe enzyme deficiency leads to external genitalia typical for a female at birth and not diagnosed until puberty fails to progress. No Mullerian structures are present. The testosterone to dihydrotestosterone ratio is elevated and a mutation may be found in the 5ARD2 gene. There is low risk of germ cell tumor, likely consistent with cryptorchidism, in these undescended gonads, but if testosterone production is undesirable, gonadectomy may be preferred. Sperm production is possible although again, azoospermia and infertility are common. Cholesterol is synthesized to aldosterone, cortisol, and testosterone via enzymatic action by multiple necessary enzymes. Autosomal recessively inherited steroidogenic defects in these enzymes may lead to a 46, XX or 46, XY DSD. Many severe forms will present in infancy with atypical genitalia as well as potentially life-threatening features of salt wasting and cortisol insufficiency. More mild forms may present later with more subtle atypical genitalia and abnormalities in pubertal development which may include primary amenorrhea. Conditions which would be most likely to present with primary amenorrhea and mild virilization are 46, XY 17α-hydroxylase/17,20-lyase deficiency, 46, XX 11β-hydroxylase deficiency, and 46, XY 17β-hydroxysteroid dehydrogenase-3 deficiency. The former two conditions also typically have hypertension due to excess mineralocorticoid activity. Non-classical CAH due to 21-OH deficiency may also lead to menstrual irregularities and amenorrhea, typically secondary, but could present as primary amenorrhea. Diagnostic evaluation includes measurement of blood pressure, karyotype, and serum steroid biosynthesis intermediates. Genetic evaluation of associated genes may confirm a diagnosis. 16

Menarche represents an important milestone in adolescence. It is the concluding event of a pubertal pathway that includes multiple hormonal, metabolic, genetic, and anatomic factors working in concert. In the United States, median age of menarche is 12 years old. 11 Primary amenorrhea (the absence of the menstrual cycles) requires an investigation if not present by age 15 or approximately 2 to 3 years after breast development. 2 Careful tracking of pubertal milestones is important to identify patients with primary amenorrhea early. It is important to distinguish between primary amenorrhea (never menstruated) and secondary amenorrhea (previously menstruated but absent menstruation greater than 6 months 1 , 3 ) – for the purposes of this chapter, we will focus on primary amenorrhea. Since 2006, differences in sex development (abbreviated DSD, and also referred to as disorders of sex development and intersex conditions) are separated into the following categories: sex chromosome DSDs, 46,XX DSDs and 46,XY DSDs. 4 Although controversial with some affected individuals, in an attempt to include all differences in sex development, congenital anomalies of the genital tract are included in this classification and useful for us to consider in this chapter. These include 46, XX individuals with Mullerian agenesis (Mayer-Rokitansky-Kuster-Hauser or MRKH) or 46, XY individuals with cloacal anomalies. When a DSD is suspected, a thorough workup is warranted as this informs associated health parameters, future reproductive potential, and psychological development. A multi-disciplinary team approach is valuable as patients often have multiple questions and concerns that are best addressed by specialists with varying expertise who collaborate to guide evaluation and management. In this chapter, we will discuss amenorrhea in the setting of an adolescent assigned female sex, with a known or suspected DSD. Patients with a DSD and typical female genitalia often present at the time of puberty, while patients with atypical genitalia at birth are often identified and diagnosed prior to puberty. Thus, the approach to the patient will differ depending on whether they present with atypical genitalia at birth or present with typical female genitalia.