Utility of Serum Anti-Müllerian Hormone Measurement as Part of Polycystic Ovary Syndrome Diagnosis

The 2023 international evidence-based guideline update for the assessment and management of polycystic ovary syndrome (PCOS) recommends using the Rotterdam criteria for the diagnosis of PCOS. The updated guideline has evidence-based recommendation for the diagnosis, and it now also includes serum anti-Müllerian hormone (AMH) measurement as an alternative tool for gynecological ultrasound to diagnose polycystic ovary morphology (PCOM). The aim of this new recommendation was to facilitate PCOS diagnostic workup in primary care and other disciplines, as currently most diagnosing is done in gynecology and infertility clinics. Here, we review factors affecting AMH levels as well as the utility of AMH in PCOS diagnosis. We identified relevant studies that report different cut-offs for AMH to diagnose PCOM as part of PCOS diagnosis. There are, however, some limitations when using AMH that should be acknowledged. These include physiological aspects like age, ethnicity, and obesity and iatrogenic causes like hormonal medication and ovarian surgery. Also reference ranges are different depending on AMH assay used. As a summary, we conclude that AMH is a usable tool in PCOM diagnostics, but it does not have a single cut-off. Therefore, further studies are needed to establish age and assay-based reference ranges.

increases pregnancy-related risks, including miscarriages, gestational diabetes, pregnancy-induced hypertension, preeclampsia, and premature delivery. 1,6Given the high risk for several comorbidities, women with PCOS and young individuals with or at risk of PCOS should be identified from the population early on to enable early prevention and support, including during pregnancy.Moreover, by educating healthcare professionals and patients, some of the medical, psychosocial, and economic burdens associated with PCOS could be prevented including its related comorbidities. 7However, there is a delicate balance between under-and overdiagnosis.Underdiagnosis and delayed diagnosis is common and contributes to patient distress and distrust, while simultaneously limiting opportunities for prevention and intervention. 8Even though some data show more depression and anxiety among women who are aware of their condition, 4 it seems as if the PCOS diagnosis itself is not a cause of distress for women with PCOS, but rather the related comorbidities. 9Overdiagnosis or misdiagnosis, on the other hand, may generate anxiety regarding potential risks for infertility, diabetes, cardiovascular disease, and obesity. 8

Polycystic Ovary Syndrome Diagnosis
The 2023 updated international evidence-based guideline for the assessment and management of PCOS recommends using the Rotterdam criteria for the diagnosis of PCOS, but in this updated guideline these criteria are now better specified and evidence-based. 1For diagnosis in adults, two out of the following three criteria are required: (1) ovulatory dysfunction (OD); (2) clinical or biochemical hyperandrogenism (HA); and (3) polycystic ovary morphology (PCOM).In addition, other causes, for example, hypothyroidism and hyperprolactinemia, should be excluded.In adolescents, both OD and HA are required, whereas the PCOM criterion should not be used due to low specificity in this age group.The flow chart for diagnosis is shown in ►Fig. 1.

Ovarian Dysfunction
The OD criterion indicates oligo-or anovulation, with irregular menstrual cycles as the most common symptom.In the updated 2023 PCOS guideline, criteria for OD are defined as a menstrual cycle < 21 or > 35 days, 3 years after menarche until perimenopause.Adolescents within 3 years after menarche need special consideration, as reviewed in the guideline. 1

Hyperandrogenism
HA can be assessed either clinically or biochemically.Regarding clinical HA, hirsutism is highly correlated with biochemical HA, whereas acne and alopecia are less specific.For objective assessment of hirsutism, the use of the modified Ferriman-Gallwey score is recommended, recognizing that self-treatment is frequently employed. 10In these cases, self-reported scores can be used.If clinical HA is not present, biochemical HA can be determined.Biochemical HA is best assessed using total testosterone or free androgen index analyzed with highly accurate tandem mass spectrometry (liquid chromatography with tandem mass spectrometry) assays.

Polycystic Ovary Morphology
The assessment of PCOM has until recently been done by ultrasonography but in the 2023 international PCOS guideline, measurement of serum anti-Müllerian hormone (AMH) has been added as an alternative.Neither method is recommended to be used within 8 years after menarche, due to low specificity.
Ultrasound is at present still the primary method for assessing PCOM in most clinical settings; however, it is expensive, and the availability is often limited or even absent.According to the diagnostic criteria as defined in the evidence-based guideline, the cut-off for PCOM is !20 follicles with a diameter of 2 to 9 mm in at least one ovary on transvaginal ultrasonography. 1 As alternatives, the ovarian volume or follicle number per cross-section can be used, with a cut-off of !10 mL for ovarian volume and !10 follicles per cross-section.If abdominal ultrasound is used, ovarian volume should be the assessment method of choice.
As AMH strongly correlates with antral follicle count (AFC) on ultrasound, the 2023 guideline has now incorporated this as an alternative method to estimate PCOM. 1

Phenotypes, Other Diagnostic Criteria
The Rotterdam criteria/ international evidence-based diagnostic criteria results in four different possible phenotypes in adults: A: HA þ OD þ PCOM; B: HA þ OD; C: HA þ PCOM; and D: OD þ PCOM, each encompassing different hormonal and metabolic profiles. 11The differences in consequences depending on phenotypes are not yet fully understood, especially regarding longterm outcomes.According to a Finnish multicenter study, AMH levels were highest in PCOS patients with phenotype A. 12 Over time, other diagnostic criteria have been used; two of these are still applied to some extent.The National Institutes of Health (NIH) criteria require HA and oligo-amenorrhea and corresponds to phenotypes A and B but does not include Fig. 1 Diagnostic workup of polycystic ovary syndrome (PCOS) according to 2023 international evidence-based guideline for the assessment and management of polycystic ovary syndrome, including anti-Müllerian hormone (AMH) as an alternative diagnostic method for polycystic ovary (PCO).PCOM, polycystic ovary morphology.
PCOM. 13 The Androgen Excess PCOS Society (AE-PCOS) diagnostic criteria, on the other hand, involve essential androgen excess and, additionally, either oligo/amenorrhea or PCOM, thus corresponding to phenotypes A, B, and C but not D. 14 Since the diagnostic criteria differ and include different phenotypes, clinical presentation differs and contributes to significant heterogeneity.This is further influenced by variation across the life course, symptoms influenced by excess weight, and ethnic diversity.As a result, both diagnosis and treatment of PCOS are challenging, and can lead to delayed diagnoses, poor diagnostic experiences, and dissatisfaction with care. 11

AMH as Marker of Ovarian Reserve
AMH is a glycoprotein that belongs to the transforming growth factor-β family, and in women it is secreted from the granulosa cells of preantral and small antral follicles in the ovary.AMH is absent in primordial as well as larger (>8 mm) antral follicles. 15In the ovary, AMH acts as a gatekeeper, inhibiting the recruitment of primordial follicles from the follicle pool also regulating ovarian folliculogenesis by inhibiting FSH action on the follicles. 16Recently, some animal and human data have suggested AMH having a role in GnRH-neuron signaling. 17,18MH has been shown to correlate well with the ovarian preantral and small antral follicles and can thus serve as a surrogate measurement of ovarian reserve as well as for the AFC to assess PCOM. 16AMH levels are two-to threefold higher in women with PCOS. 19AMH levels have also been used to predict menopause, as menopause occurs when the ovarian pool reaches a critically low level.Together with age, AMH levels can be adjusted for the prediction of menopausal age, and it has been shown that women with low age-specific AMH levels reach menopause earlier than those with high age-specific levels. 20,21ssessment of AMH is currently used as part of the evaluation before in vitro fertilization (IVF) and can predict ovarian response to gonadotrophin stimulation.While AMH is a predictor of oocyte yield after controlled ovarian hyperstimulation in an IVF treatment, no convincing evidence exist of AMH being a valid predictor of oocyte quality or the chance of achieving natural pregnancy. 22

Factors Affecting AMH Levels
The 2023 updated PCOS guideline has included measurement of AMH as an alternative to ultrasound when assessing PCOM.This enables general practitioners, endocrinologists, and other health personnel who do not have access to ultrasound, to assess PCOM and will hopefully lead to more effective and continuous overall care, while also saving resources for specialized care if the diagnostic workup could be mainly concentrated on primary care.Given the complexity of PCOS, the guideline does not support using serum AMH as a sole marker for PCOS. 23ealthcare professionals also need to be aware that there are several factors that might influence AMH in the general population, including laboratory assays, age, body mass index (BMI), ethnicity, menstrual cycle stage, pregnancy, use of oral contraceptive pill, and ovarian surgery. 2,24The effects of these factors on the AMH level are described in ►Table 1.

Assay
The updated PCOS guideline recommends that laboratories involved in AMH measurements in females should use population and assay-specific cut-offs. 1Commercial assays for the measurements of AMH have been available since the late 1990s, the AMH Gen II ELISA (marketed by Beckman-Coulter, Inc.) being the most widely used assay kit for many years.In recent years, however, new AMH measurement kits have become available, including the Elecsys AMH Immunoassay (Roche Diagnostics International Ltd, Indiana), Ultra-Sensitive AMH/MIS ELISA kit (Ansh Labs, Texas), and the automated Access AMH assay (Beckman-Coulter Diagnostics, California).
A study by Li et al evaluated the three newer AMH assay methods, namely, the Access AMH assay, Elecsys AMH Immunoassay, and Ultra-Sensitive AMH/MIS ELISA, and compared them to the older AMH Gen II ELISA.Results showed that values obtained from the Elecsys AMH Immunoassay were lower than the Gen II and Access AMH assays (0.88-fold and 0.86-fold, respectively). 25These findings were also confirmed by Moolhuijsen et al. 26 AMH values obtained with the Ultra-Sensitive AMH/MIS ELISA were higher than those obtained with the Gen II and Access AMH assays (1.77-fold and 1.65-fold, respectively). 25It is important to acknowledge that clinical cut-off for AMH in PCOS would acquire assay-specific cut-offs. 27

Age
The concentration of serum AMH is dependent on the number of remaining oocytes in the ovaries. 2 Follicle development varies across the lifespan and is increased in adolescence, after which the number falls subsequently until menopause, when oocytes are depleted.According to previous studies, age-based decline in AMH is also known to be much less pronounced in PCOS women compared with controls. 28,29There is thus a need for age-specific cut-offs for both PCOM and AMH.According to a systematic review, the sensitivity and specificity suggests greater accuracy of AMH in PCOS diagnosis in adults than in adolescents.Thus, the updated PCOS guideline recommends that PCOM should not be used in adolescent or young adults 8 years or less from menarche. 1

Body Mass Index
Obesity is more common in women with PCOS compared with women without PCOS and a recent study showed that women with PCOS gain more weight annually compared with women without PCOS. 30Previous studies have found a strong correlation between decreasing AMH levels and increasing BMIs in patients both with and without PCOS, suggesting different AMH cut-offs for different BMI groups. 31,32Indeed, a recent study looking at different BMI subgroups and correlation between oligo-anovulation and AMH suggested progressively lower AMH cut-offs for women with increasing BMI to diagnose PCOS. 33

Ethnicity
Previous studies have suggested a variation in AMH according to ethnicity.One study comparing Chinese and European women from the Netherlands, Belgium, Germany, France, and Turkey found that from the age of 25 years onward, Chinese women had significantly lower AMH than women of European origin. 34This was confirmed in another study, where Chinese women had a lower AMH cut-off value for diagnosing PCOS compared with non-Asian women. 35When comparing Caucasian women to Afro-Americans and Hispanic women, AMH levels were highest in Caucasian women.The clinical impact of these differences may be substantial and one of the studies found the natural menopause being 1 to 2 years earlier in Chinese women compared with European. 34

Menstrual Cycle Stage
Some fluctuations of serum AMH levels according to menstrual cycle stage have been demonstrated in previous studies, but generally these changes are considered clinically irrelevant for the estimation of the ovarian reserve in individual woman. 36ne recent study measured AMH levels in 47 women every second day during two menstrual cycles. 37All participants had a regular menstrual cycle, a BMI between 19 and 26 kg/m 2 , and were 18 to 40 years old.The study showed that inter-participant and intra-cycle variability of serum AMH levels were larger than inter-cycle variability and hence were in line with previous findings.It is, however, important to remember that these studies were performed in the general population, not specifically on women with PCOS. 37

Pregnancy
9][40][41] There have been conflicting results on AMH kinetics in pregnancy, with some studies finding that AMH remained stable, 42 whereas others found a decrease in AMH as the pregnancy progressed. 43A recent systematic review, consisting of eight studies and 1,719 participants, found an association between reduced maternal AMH and advancing gestational age. 44These results were confirmed in a prospective, longitudinal cohort study where the median difference of AMH was -39.8% between the first and second trimester of pregnancy. 45However, in postpartum, increased AMH levels were found when comparing with AMH levels during pregnancy.These findings should be taken into consideration when assessing PCOS in pregnant women, especially during last two terms.

Use of Hormonal Contraceptive
Results on the effect of hormonal contraceptive use on AMH levels have been conflicting, with some studies finding up to 55% reduction of AMH levels in contraceptive users compared with controls, whereas other studies have found no difference in AMH between the two groups. 46A large American study, comprising of 27,125 participants, aged 20 to 46 years, found that women using oral contraceptive pills, implants, or vaginal ring had the largest reduction in AMH levels compared with those not using contraceptives (À24%, À23%, and À22%, respectively).In a Finnish prospective spin-off study, it was also observed that continuous use of all combined contraceptives decreased AMH levels significantly during 9 weeks of treatment and decease was of same magnitude for contraceptive patch, pill, and vaginal ring. 47omen using progesterone only pills or hormonal intrauterine devices had smaller reductions in AMH compared with those not using contraceptives (À15% and À7%, respectively).Among those using oral contraceptive pills, duration of contraceptive use (ranging from 1 month to 20 years) was not associated with a further decrease of AMH levels. 46

Ovarian Surgery
In fertile aged women, a conservative ovarian cyst enucleation, with as little damage as possible on the normal ovarian tissue, is the preferred surgical intervention for the treatment of benign ovarian cysts.Even though cyst enucleation techniques aim at being fertility-sparing, conservative surgery will affect to some extent AMH and fertility, due to unavoidable removal of normal ovarian tissue or surgical damage to the remaining normal ovarian tissue. 48A recent Swedish study on 75 fertileaged women showed that type of cyst might also play a role.In this study, AMH decreased more in women with endometriomas than in women with dermoid cysts. 49

Utility of AMH in PCOS Diagnosis
The utility of AMH in PCOS diagnosis has been an area of interest for a long time.Some of the relevant studies are summarized in ►Table 2. 35,[50][51][52][53][54][55][56][57][58][59][60][61][62][63] Given that serum AMH levels have been shown to be two to three times higher in women with PCOS, it has been proposed as an alternative tool for the diagnosis of PCOS. 19,28,60,64However, use of AMH as a single marker for PCOS has poor sensitivity and specificity 58,61,62 and therefore AMH as a single marker for PCOS is not recommended.Studies conducted over the past two decades have indicated that serum AMH provides a practical and costeffective biomarker for detecting PCOM, with AMH levels correlating with the number of antral follicles on ultrasound.In the first international PCOS guideline in 2018, evidence was not enough to recommend AMH as a diagnostic marker for PCOM. 23,65In the recent 2023 guideline, 1 evidence had emerged and AMH is now an alternative tool to assess PCOM and is thus part of the diagnostic criteria (►Fig.1).
As evident in ►Table 2, there is currently not one single AMH cut-off to assess PCOM.As laboratory methods vary in different parts of the world, the updated PCOS guideline has recommended the use of population and assay-specific cutoffs.However, the traditional way of defining the "normal" range as a cut-off of within 2 standard deviations is not appropriate for defining diagnostic cut-offs for a clinical condition like PCOS.Here, more important considerations include clustering with other clinical features such as clinical HA, oligo-anovulation, or prediction of long-term health outcomes such as fertility.Unfortunately, large studies on PCOS with cluster analyses are lacking.A recent epidemiological study was, however, able to analyze different cut-offs for AMH and the relation to clinical outcomes showing that as low as 3.2 ng/mL suggested by Dietz de Loos et al 54 was able to detect women with PCOS with typical hormonal and metabolic profile. 59s evident in ►Table 2, different cut-offs have indeed been used to identify PCOM, emphasizing that the cut-off is dependent on the population and the assay used.Consequently, the sensitivity and specificity for PCOM also differ depending on the cut-off used, 50,[52][53][54] and the cut-off will also affect the prevalence of PCOS. 55,56,59Thus, in future studies, it is important to define how the AMH cut-off has been established. 23,59efining in which population AMH is assessed is also important; women with obesity have lower AMH levels also in PCOS 35 ; and younger women might need higher cut-offs than older women to define PCOM. 60o summarize, AMH assessment is now an evidencebased alternative to assess PCOM in the diagnosis of PCOS.However, it is relevant only when using the Rotterdam criteria, which is also now evidence-based, or when using AE PCOS criteria, but not when using the NIH criteria where PCOM is not part of the diagnostic criteria.

Conclusion
AMH serves as a new tool to diagnose PCOM.It will enable PCOS diagnosis in primary care and could facilitate early diagnosis, prevention, and support.There are, however, some limitations in the usage that should be acknowledged.These include physiological aspects such as age, ethnicity, and obesity and iatrogenic causes such as hormonal medication and ovarian surgery.Age and platform-related reference ranges are warranted to optimize the usage of AMH as part of the PCOS diagnosis workup.

Table 2
Studies assessing AMH cut-off values for PCOM and/or PCOS Utility of Serum AMH Measurement as Part of PCOS Diagnosis Piltonen et al. 53 Seminars in Reproductive Medicine Vol.42No.1/2024 © 2024.The Author(s).

Table 2 (
Continued) Utility of Serum AMH Measurement as Part of PCOS Diagnosis Piltonen et al.