Disease condition and impact on patients Estrogens are steroid hormones that are responsible for the development and regulation of the female reproductive system and secondary sex characteristics. In women, they are synthesized primarily in the ovaries (in response to follicle stimulating hormone (FSH) stimulation) and in the placenta during pregnancy. Lesser amounts of estrogens are produced in other tissues, such as liver, adipose, adrenal, skin and brain. In men, estrogens are synthesized in response to FSH in the testes but are produced in much lower amounts than in women. There are three main naturally occurring estrogens: estrone (E1), estradiol (E2) and estriol (E3) (1). In terms of estrogenic activity, E2 is the predominant estrogen during reproductive years and thus is the most clinically relevant. After menopause when E2 levels fall, E1 becomes the predominant circulating estrogen. E3 is the main estrogen during pregnancy; it does not have a significant role in non-pregnant women or men (1). Given its predominant role and importance in clinical utility, this application focuses on the measurement of E2. Measuring serum E2 levels has an integral role in the assessment of reproductive function in women, specifically infertility, menstrual irregularities and menopausal status. E2 levels are also commonly measured to monitor ovulation induction, and during preparation for in vitro fertilization (IVF). Infertility. Infertility is recognized as an essential component of reproductive health by the UN Programme of Action of the International Conference on Population and Development (2). Paradoxically, nations with the highest overall fertility are also the ones with the greatest prevalence of infertility and these often include LMICs. Given the economic, resource, cultural and religious constraints in these countries, infertility services among them will vary significantly. Assays that measure E2 levels are, however, relatively non-invasive (requiring only a blood draw or finger stick), inexpensive and accessible from laboratories throughout the world. Measuring E2 levels can be used in infertility management to assess ovarian reserve (3) and predict IVF success (4) in women, and as an aid in determining the etiology of azoospermiaError! Bookmark not defined. in men (5). Menstrual irregularities. E2 levels can also be useful in evaluating irregular menstrual cycles, particularly suspected anovulation and amenorrhoea, which generally indicate a defect at some point in the hypothalamic-pituitary-ovarian-uterine axis. Anovulation. In addition to impacting fertility, ovulation disorders can also cause menstrual irregularities ranging from amenorrhoea to dysfunctional uterine bleeding (DUB). Primary amenorrhoea is defined as no menses by age 14 in the absence of development of secondary sexual characteristics, or no menses by age 16 regardless of sexual development (6). Most cases are caused by gonadal dysgenesis, most commonly Turner syndrome or (HH) as well as Kallmann syndrome. Secondary amenorrhoea is defined in women who have menstruated before as no menses for 6 months or the equivalent of at least three cycles (6). It is most commonly caused by primary ovarian insufficiency and menopause. Hypothalamic amenorrhoea, another category of secondary amenorrhoea, is essentially a diagnosis of exclusion in the face of low estrogen levels and low or normal FSH. It is often associated with the excessive exercise and/or weight loss that can be seen in elite athletes (7). But it can also be precipitated by weight loss due to malnutrition or extreme psychological stress. Although there are no known studies that have documented this, hypothalamic amenorrhoea is likely underdiagnosed in LMICs. Because these women are estrogen deficient, they are at risk for its natural clinical outcomes, and failure to identify their deficiency may also lead to conditions such as osteoporosis and genital atrophy. Anovulation can also lead to DUB (8), which causes anaemia and can negatively impact psychological well-being, personal economics and social interactions, particularly in LMICs. Studies report that women in LMICs face increased physical and psychological obstacles coping with menstrual flow compared to women in HICs. Menstruating women in LMICs often face challenges in accessing menstrual materials, as well as religious or cultural shunning, lack of privacy, absences from school or work due to stained clothing and overall poor self-esteem (9). General reproductive function. Primary ovarian insufficiency (POI), previously called “premature menopause” or “premature ovarian failure”, is a heterogeneous disorder resulting in the depletion or dysfunction of ovarian follicles with menses stopping before 40 years of age. Follicle depletion can be caused by chromosomal abnormalities (e.g. fragile X syndrome), chemotherapy or radiation therapy, autoimmune disease or infiltrative or infective processes. POI affects approximately 1% of women. Does the test meet a medical need? See “How the test is used”. How the test is used Infertility. Since E2 is secreted by developing ovarian follicles, it can be used to evaluate ovarian reserve. The number of ovarian follicles present in the ovaries (oocytes) is established at birth and declines with time throughout a woman’s lifetime without regeneration. It can therefore become necessary to confirm the presence of viable oocytes and the potential to ovulate in infertile women by measuring E2 and FSH levels (10). Although testing for anti-Müllerian hormone (AMH) has gained favour in predicting IVF success (10), measurement of E2 (with FSH) levels has also been shown to be a significant prognostic marker and may be more accessible in low-resource settings (11). Menstrual irregularities. An E2 measurement provides an accessible and relatively inexpensive objective assessment of ovarian function. Once a diagnosis of anovulation is confirmed, measuring E2 levels can also help clarify the etiology, according to the classification adopted by WHO (12): • Group I (hypogonadotropic hypogonadal anovulation) accounts for around 5–10% of anovulatory women and is characterized by low E2 in the presence of low FSH. • Group II (normogonadotropic normestrogenic anovulation) accounts for around 75–85% of anovulatory women and is characterized by normal E2 with normal FSH. • Group III (hypergonadotropic anovulation) accounts for 10–20% of anovulatory women and is characterized by suppressed E2 in the presence of elevated FSH. • Group IV (hyperprolactinaemic anovulation) accounts for 5–10% of anovulatory women and is characterized by low E2 levels with generally low FSH. Amenorrhoea and hypogonadism. There are many causes of primary and secondary amenorrhoea, and multiple laboratory measurements are used in their evaluation and diagnosis. E2 levels are primarily used in conjunction with FSH and luteinizing hormone (LH). Decreased E2 levels indicate hypogonadism, and subsequent measurement of gonadotropins is essential to distinguish whether the abnormality is gonadal or of pituitary/hypothalamic origin (13). Physical manifestations of gonadal dysgenesis may be subtle, so measuring reproductive hormone levels can facilitate diagnosis, particularly in LMICs, where laboratory menus and diagnostic resources may be limited. Early diagnosis of gonadal dysgenesis is essential, as these syndromes often involve congenital heart defects and other organ anomalies. Timely diagnosis also allows for initiation of growth hormone treatment in early childhood to address short stature and estrogen in adolescence to promote pubertal development and prevent osteoporosis (14). E2 levels can be used to monitor this treatment. In males, increased E2 levels may help confirm a diagnosis of primary hypogonadism. General reproductive function. When POI is suspected on the basis of history and physical exam, measuring E2 and FSH can confirm the diagnosis. If E2 levels indicate hypogonadism and basal FSH levels are elevated into the menopausal range, a repeat measurement 1 month later is indicated for final confirmation (15). In precocious puberty (PP), E2, LH and FSH tend to be above the prepubertal range (16). E2 measurement in children suspected of having PP is used to support the diagnosis and to determine the etiology. Increased E2 levels can be caused by exogenous estrogens or an ovarian cyst that has produced transient estrogens. Early diagnosis of central PP allows for treatment with gonadotropin-releasing hormone (GnRH) agonists that can delay the onset of puberty until a more appropriate age. The psychosocial and physical burdens of undiagnosed and untreated delayed or precocious puberty are significant, and it is important that all tools available to aid in a timely diagnosis be available. The combination of low E2 and high FSH levels may be used to confirm menopausal status, particularly in women who have had hysterectomies (with ovaries intact) and in whom the absence of menses cannot be used as a reliable indicator of ovarian function (17).

Prevalence and socioeconomic impact Infertility. Estimating the prevalence of global infertility is challenging due to differences in definitions and methodology and the lack of population-based studies (18). One analysis based on health surveys estimates that more than 40 million couples are affected by infertility (18). And data on global prevalence of childlessness suggest that as many as 70 million couples would benefit from medical intervention to achieve pregnancy (19). The socioeconomic impact of infertility is significant, particularly for women, who often suffer from social isolation, discrimination, disinheritance, depression, abuse, divorce and possible abandonment in old age. Infertility can also have broader negative impacts on families, particularly in LMICs, where children contribute to family incomes and older parents depend on their children for support. Menstrual irregularities. The prevalence of menstrual disorders is estimated to range from 5% to 36% (20); occurrence depends on age, nutritional status and country of residence. Anovulation. Estimates of the incidence of anovulation are imprecise due to variables such as age, general health, nutritional status and country of residence. Up to 25% of infertile women are estimated to be anovulatory (12). Turner syndrome has a reported incidence of 1 per 2500 live births (21); Kallmann syndrome has a reported incidence of 1 in 120 000 women (22).

A 2016 Endocrine Society clinical practice guideline on hormonal replacement in hypopituitarism in adults (13) recommends measuring E2, FSH and LH in females with oligomenorrhoea or amenorrhoea. In 2014, the American College of Obstetricians and Gynecologists (ACOG) (15) listed measuring FSH and E2 (two random tests at least 1 month apart) when there has been menstrual irregularity for at least three consecutive months as a way of diagnosing and initially evaluating POI. The American Society for Reproductive Medicine (ASRM) Practice Committee (11) states that ovarian reserve tests should include both biochemical tests and ultrasound imaging of the ovaries, and that biochemical tests should include both basal measurements (of FSH, E2, inhibin B and AMH) and provocative tests such as the clomiphene citrate challenge test. The committee go on to say that basal E2 alone should not be used to screen for DOR, explaining that the test is only of value as an aid to correctly interpreting a ‘‘normal’’ basal FSH value. An early rise in serum E2 is a classic characteristic of reproductive ageing and can lower an otherwise elevated basal FSH level into the normal range, thereby causing a misinterpretation of the test. When the basal FSH concentration is ‘‘normal’’ but the E2 level is elevated (> 60–80 pg/mL) in the early follicular phase, there is limited evidence for an association with poor response, increased cancellation rates or lower pregnancy rates.

Because E2 testing is a quantitative measurement that is not used in screening for diseases, and it is generally done in conjunction with other testing to support a diagnosis of disease or to monitor a treatment, sensitivity and specificity have not been determined for many indications.

A 2019 review by Mikhael et al. (23) suggests that, in general, basic evaluation of hypothalamic-pituitary failure includes measurement of E2 (with FSH and LH). Overall management of hypothalamic-pituitary failure will depend on patient needs. For example, in young women of reproductive age where E2 levels (with FSH) are used to diagnose ovarian insufficiency, management can include reassurance and education about alternate reproductive options. This may also apply to patients with mosaic Turner syndrome who become aware of impending follicular atresia and who wish to preserve fertility through oocyte or embryo cryopreservation. In cases where E2 is used to confirm ovarian failure, either premature or due to natural menopause, the diagnosis allows for monitoring the effects of estrogen deficiency and consideration of hormonal replacement to prevent or delay the associated morbidity. Infertility. A 2003 WHO study (24) looked at high singleton live birth rate following classical ovulation induction in normogonadotrophic anovulatory infertility. All patients started with clomiphene for ovulation induction: those who did not ovulate within three treatment cycles of incremental daily doses up to 150 mg for five consecutive days or ovulatory clomiphene citrate (CC) patients who did not conceive within six cycles underwent gonadotrophin induction of ovulation applying a step-down dose regimen. Of 240 consecutive women, there were 134 pregnancies ending in a singleton live birth (56% of women). The cumulative pregnancy rate after 12 and 24 months of follow-up was 50% and 71%, respectively. The authors concluded that classical ovulation induction produces very good results in normogonadotrophic anovulatory infertility. A 2014 study in India by Prasad et al. (25) looked at the E2 level on downregulated day 2 and on the day of human chorionic gonadotropin (hCG) trigger as a predictor of IVF success. The authors found that for women with E2 levels > 31.2 pg/mL on downregulated day 2, 61.8% will achieve pregnancy; 59% of women with E2 levels greater than 1400 pg/mL on the day of hCG trigger will achieve pregnancy. Menstrual irregularities. The clinical community generally accepts that hormonal therapy is highly successful in treating DUB. A 2004 study by Hurskainen et al. (26) followed more than 200 women with DUB who were treated with a triphasic preparation of norgestimate and ethinyl E2. More than 80% were documented to have improvement in bleeding, and this was significantly increased over the placebo group. The authors concluded that the triphasic combination of norgestimate and ethinyl E2 is an effective treatment for metrorrhagic, menometrorrhagic, oligomenorrhoeic and polymenorrheic dysfunctional uterine bleeding. General reproductive function. In 2012, Lee et al. (27) studied 76 girls with PP who were treated with leuprolide acetate, a GnRH agonist, every 3 months. At 6 months, 98% exhibited LH suppression and 100% E2 suppression. The authors concluded that treatment with leuprolide acetate 3-month depot formulations (11.25 and 30 mg) effectively suppressed the GnRH axis.

The cost (in US dollars) of an E2 test in the USA is around US$ 43, compared with US$ 4.50–7 in India and US$ 34 in Australia. By contrast, an AMH test is around US$ 100 in the USA, US$ 17–28 in India and US$ 64 in Australia. It is several times more expensive to predict IVF success through ultrasound determination of follicle number and size. An E2 measurement that prompts patients to abandon a cycle of assisted reproductive technology could also result in significant savings (thousands of dollars in the USA). It is also relatively inexpensive to determine whether anovulation has occurred using an E2 measurement, particularly in cases of amenorrhoea. Given the significant health repercussions of anovulation in terms of disorders of sexual development (DSDs) and also the sequelae of estrogen deficiency, there should be no question that identifying these risks and reducing morbidity would be cost–effective.

No information provided.