Introduction

Oligomenorrhoea is defined as having fewer than 9 menstrual cycles per year or a cycle length greater than 35 days, and it affects approximately 13% of the fertile female population. The most common causes of oligomenorrhoea are polycystic ovary syndrome, hyperprolactinaemia, and functional hypothalamic anovulation. In addition, thyroid dysfunction, non-classical congenital adrenal hyperplasia (NCAH), premature ovarian failure, androgen producing tumours, and Cushing’s syndrome (CS) cause oligomenorrhoea [1].

Polycystic ovary syndrome (PCOS) is characterized by hyperandrogenism (clinical and/or biochemical), oligo-amenorrhoea and polycystic ovaries detected by pelvic ultrasound. Two out of these 3 criteria are sufficient for diagnosis according to the most widely used Rotterdam criteria (2003) [2]. The Androgen Excess Society (2006) defined PCOS by the presence of hyperandrogenism (clinical and/or biochemical), ovarian dysfunction (oligo-anovulation and/or polycystic ovaries), and the exclusion of related disorders such as CS, congenital adrenal hyperplasia, and/or androgen-secreting tumours [3]. PCOS is associated with important reproductive comorbidities including infertility, irregular uterine bleeding, and increased pregnancy loss during the reproductive years. PCOS is also associated with increased metabolic and cardiovascular risk factors such as type 2 diabetes, dyslipidaemia, and obesity related to insulin resistance [1, 4, 5].

Hyperprolactinaemia may lead to hypogonadism, infertility, and galactorrhoea in addition to oligomenorrhoea [6]. It might result from lactotroph adenomas of the pituitary, conditions associated with decreased dopaminergic inhibition of prolactin secretion, such as drugs that block dopamine receptors or disorders of hypothalamus, or pituitary, such as tumours, infiltrative diseases [7]. Hypothalamic anovulation is associated with low-weight eating disorders, intensive exercise, and stress. The diagnoses should be made after the exclusion of other organic causes that lead to oligomenorrhoea [8].

CS may lead to important clinical morbidities and mortality. It may cause menstrual changes, obesity, diabetes, hypertension, hypercoagulability, decreased bone mineral density, psychopathologies, and cognitive impairment. These morbidities may resolve after treatment, but in adrenocorticotropic hormone (ACTH)-dependent CS, hypercortisolaemia may recur. Increased cardiovascular and metabolic risk necessitate early diagnosis of CS. However, the prevalence of CS was very low in previous studies that evaluated patients with chronic anovulation and hirsutism. Hence, the authors recommended that screening of CS in those groups should only be performed in patients with typical signs of the disease [9, 10].

There are various reports that evaluate prevalence of disorders that cause hirsutism [11–13], but there is only one retrospective study that evaluated the prevalence of conditions leading to chronic anovulation [14]. Therefore, in this study, we aimed to thoroughly evaluate premenopausal women who were admitted to our clinic for oligomenorrhoea, and we also intended to screen for CS in this population.

Material and methods

Patients study design

This was a single-centre, prospective, clinical study that included 304 premenopausal women (aged 18–44 years) referred to our clinic for oligomenorrhoea. The study was carried out between January 2022 and February 2023 at the Department of Endocrinology at Tepecik Research and Training Hospital. Women with chronic diseases such as overt hypothyroidism or hyperthyroidism, kidney or liver failure, galactorrhoea, psychiatric disorders, or having a history of intense low-calorie diet or exercise or losing more than 10% of body weight within 6 months were excluded from the study. Additionally, women who had been receiving hormonal therapy, including oral contraceptive pills or steroids (glucocorticoids) or dopamine antagonists, within 6 months were excluded. PCOS was defined in accordance with the Rotterdam criteria [2]. The study was approved by the medical ethics committee of the Tepecik Research and Training Hospital, University of Health Sciences (Date: 15 November 2021; Meeting Number: 11; Decision: 7) and in accordance with the Declaration of Helsinki, and all participants provided written informed consent.

Body mass index (BMI) and waist circumference were measured in all study subjects. BMI was calculated by the ratio between weight and height squared in kg/m2. Waist circumference was measured on bare skin between the tenth rib and the iliac crest in centimetres. Hirsutism was evaluated based on the Ferriman-Gallwey scoring (FGS) index over 9 body areas [15].

After an overnight fast of 12 hours, venous blood was collected from the antecubital veins of all study subjects to evaluate biochemical parameters including plasma glucose and lipid profile (total cholesterol, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and triglycerides) as well as hormones including oestradiol, progesterone, total testosterone, 17-hydroxyprogesterone (17-OHP), prolactin, insulin, dehydroepiandrosterone sulphate (DHEA-S), follicle-stimulating hormone (FSH), luteinizing hormone (LH), free triiodothyronine (fT3), free thyroxine (fT4), and thyroid-stimulating hormone (TSH). If basal 17-OHP was > 2 ng/mL, we evaluated ACTH-stimulated 17-OHP levels, and if the value was > 10 ng/mL, it was determined as NCAH due to 21-hydroxylase deficiency. Also, screening for CS was done by evaluating cortisol levels after 1 mg dexamethasone suppression test. If the value was > 1.8 µg/dL, we performed additional confirmatory tests and measured ACTH levels according to the proposed algorithm recommended by the Endocrine Society Clinical Practice Guideline [9]. The blood samples were obtained during the third to ninth days of the menstrual cycle or 60 days after the last menstrual period.

Pelvic ultrasonography was performed for all participants.

Laboratory assessments

Glucose, triglycerides, total cholesterol, and HDL-C levels were measured by enzymatic methods using an AU5800 autoanalyser (Beckman Coulter Inc., CA, United States). LDL-C was calculated by the Friedewald ’s equation method. FSH, LH, total testosterone, prolactin, DHEA-S, and cortisol levels, were analysed by chemiluminescence assay method using a DxI immunoanalyser (Beckman Coulter Inc., CA, United States). fT3, fT4, TSH, and anti-thyroid paroxidase antibody (anti-TPO) levels were measured by chemiluminescent method by Immulite 2000 autoanalyser (Immulite XPi, Siemens, Germany). ACTH was measured by chemiluminescence assay method using an IMMULITE-2000 autoanalyser (Siemens Healthcare Diagnostic Products, USA). 17-OHP levels were measured by radioimmunoassay by using a diagnostic system laboratories (TX, United States).

Pelvic ultrasonography

Transabdominal pelvic ultrasonography was performed by using a Logiq 5 Pro unit (GE Medical Systems, WI, United States) and a C1-5-RS (2–5 MHz) transducer. The ovaries were imaged in the sagittal and transverse planes. The presence of polycystic ovaries were defined as the existence of 12 or more follicles throughout the ovary measuring 2–9 mm in diameter [3]. All ultrasonographic evaluations were performed by the same radiologist.

Pituitary imaging and inferior petrosal sinus sampling (IPSS)

For patients in whom we established ACTH-dependent CS, we evaluated pituitary magnetic resonance imaging (MRI) and referred them to interventional radiology for pituitary imaging and inferior petrosal sinus sampling (IPSS).

Statistical analysis

The results are expressed as means ± standard deviation (SD). Comparisons between the groups were performed using Student’s t-test. p < 0.05 was considered statistically significant. Statistical analysis was performed with SPSS 20 statistical software.

Results

A total of 304 premenopausal women with oligomenorrhoea were evaluated prospectively. The mean age of the patients was 26.34 ± 5.98 years. In addition to oligomenorrhoea, hirsutism was present in 164 patients (53.94 %), and polycystic ovarian morphology in ultrasound imaging was detected in 199 (65.46%) patients. The most common cause of oligomenorrhoea was PCOS, as shown in Table 1, and its prevalence was 81.57% (n = 248) in the study population. Among the PCOS patients, 100 had phenotype A, which is characterised by hyperandrogenism (HA), ovulatory dysfunction (OD), and polycystic ovarian morphology (PCOM), 52 had phenotype B (HA + OD), and 93 had phenotype D (OD + PCOM). Because our study included patients with oligomenorrhoea, there were no PCOS patients with phenotype C (HA + PCOM).

Hyperprolactinaemia was present in 22 (7.23%) patients, and 18 had pituitary microadenoma. Functional hypothalamic anovulation was detected in 16 (5.26%) patients, and these patients had a history of intense psychological stress. Although we excluded patients with psychiatric disorders or with a history of very intense low-calorie diet or exercise, we still detected hypothalamic anovulation in our study population.

Premature ovarian failure (POF) was present in 5.26 (1.6%) patients, and only one of them had autoimmune polyglandular syndrome 1 with additional disorders including primary adrenal failure and primary hypothyroidism.

NCAH due to 21-hydroxylase deficiency was found in 6 (1.97%) patients. Basal 17-OHP level was > 2 ng/mL in 12 patients, but ACTH-stimulated 17-OHP level was > 10 ng/mL in 6 patients. Unfortunately, genetic evaluation was not carried out in these patients.

Unexpectedly, Cushing’s syndrome was detected in 7 (2.30%) patients. All the patients with CS were found to have Cushing’s disease (CD) due to pituitary adenoma confirmed by pituitary MRI and IPSS. Although 3 patients with CD had classical signs and symptoms such as abdominal obesity, plethora, ecchymoses, hirsutism, and hypertension, 4 patients with CD had no classical signs and symptoms except oligomenorrhoea, and they even had BMI < 30 kg/m2.

Clinical characteristics and hormonal parameters of the patients according to aetiologies of oligomenorrhoea are described in Table 2. No significant differences were observed between the groups according to age, BMI, and waist circumference. FGS was similar in non-hyperandrogenaemic aetiological groups including hyperprolactinaemia, hypothalamic anovulation, and POF, but significantly lower compared to hyperandrogenaemic aetiological groups that included PCOS, NCAH, and CD (p = 0.002). Metabolic parameters and thyroid hormone levels were similar in all aetiology groups. As expected, serum gonadotrophin levels were higher with respect to all groups in the POF group (p = 0.001). Serum total testosterone and DHEA-S values were similar in non-hyperandrogenaemic aetiological groups and were significantly lower than values measured in hyperandrogenaemic aetiological groups (p-test = 0.001, p-DHEAS = 0.01). Although patients with CD had similar total testosterone values to the PCOS group and patients with NCAH, they had significantly higher DHEA-S compared to the PCOS and NCAH groups (CD vs. PCOS, p = 0.001 and CD vs. NCAH, p = 0.030) (Fig. 1).

Figure 1. Serum testosterone and dehydroepiandrosterone sulphate (DHEAS) values in hyperandrogenaemic groups. CS — Cushing’s syndrome; PCOS — polycystic ovary syndrome; NCAH — non-classical congenital adrenal hyperplasia

All the patients with CD underwent TSS, and hypercortisolaemia and hyperandrogenaemia was normalized in 5 of them. Two of them also had biochemical remission after the second surgery, performed by a neurosurgeon in a specialized centre.

Discussion

PCOS is the most common cause of oligomenorrhoea and anovulation in premenopausal women. Apart from reproductive morbidities, it is also frequently associated with metabolic dysfunction (including type 2 diabetes) and cardiovascular disease [3, 16]. The diagnosis of PCOS relies on the exclusion of other disorders that might cause hyperandrogenism and/or ovulatory dysfunction, such as NCAH, and ovarian and adrenal androgen secreting tumours [3].

NCAH due to 21-hydroxylase deficiency is one of the most common autosomal recessive disorders that affects 1–10% of women with hyperandrogenaemia. Clinical features are not very different from PCOS. Therefore, routine screening for 21-hydroxylase deficiency by measuring basal 17 (OH) P is recommended especially in high-risk populations in the diagnosis of PCOS for the exclusion of NCAH [3, 17]. Likewise, it is suggested that all patients presenting with hyperandrogenic symptoms should be screened for androgen secreting neoplasms [3, 18].

CS comprises a large group of signs and symptoms, such as reddish-purple stria, plethora, easy bruising, proximal muscle weakness, and unexplained osteoporosis. Also, pituitary CD may lead to hyperandrogenaemia and menstrual irregularities that are major components of PCOS. Although CS is associated with cardiovascular and infectious complications, the mortality rate is significantly decreased after successful normalization of hypercortisolaemia with the advent of surgical techniques and treatment strategies [8]. In patients who have persistent, moderate hypercortisolism, mortality was found to be increased 3–5-fold compared to general population [9, 19–22]. Treatment of patients with moderate to severe Cushing’s syndrome clearly reduces mortality and morbidity. Because Cushing’s syndrome tends to progress, and severe hypercortisolism is probably associated with a worse outcome, it is likely that early recognition and treatment of mild disease would reduce the risk of residual morbidity [9, 10, 23–26].

However, routine screening for CS is not recommended in the diagnoses of PCOS for the exclusion of hyperandrogenaemic disorders. Screening is only recommended for patients with specific and suggestive symptomatology by the measurement of 24-hour urine free cortisol level [3].

There is only one study that evaluated the prevalence of conditions causing chronic anovulation, and there is no clinical study that specifically evaluated the prevalence of disorders that cause oligomenorrhoea [14]. In that retrospective study, 32% of the study population had oligomenorrhoea and 39% had secondary amenorrhoea. It was found that the most common cause of anovulation was PCOS, followed by prolactin disorders, idiopathic chronic anovulation, and thyroid disorders. Among the 293 women involved, 4 had NCAH and one had CS. Therefore, the author recommended that serum androstenedione, testosterone, TSH, and prolactin, and that pelvic ultrasonography should be performed initially for the evaluation of anovulation. 17 (OH) P and DHEA-S examinations could be secondary investigations for anovulation [14]. Routine evaluation of CS was not suggested if there was no clinical sign of the syndrome. In our study, we prospectively evaluated 304 premenopausal women with oligomenorrhoea. Likewise, in our study, the most common cause of oligomenorrhoea was PCOS, followed by hyperprolactinaemia and functional hypothalamic anovulation. NCAH due to 21-hydroxylase deficiency was found in 1.97% and POF in 1.64% of the patients. However, in contrast to the previous study, we found higher prevalence of CS, at 2.30% (n = 7), and 4 of the CS patients did not have clinical signs of the disease. We attributed this finding to the fact that we evaluated every patient with oligomenorrhoea for CS whether or not she had clinical signs.

In contrast to anovulation and oligomenorrhoea, hirsutism, which is the major clinical sign of hyperandrogenaemia, was evaluated previously in premenopausal women in detail [11–13]. In those studies, approximately half of the study population were women with oligomenorrhoea. As expected, the most common cause of hirsutism was PCOS. Idiopathic hirsutism, idiopathic hyperandrogenaemia, and NCAH were other causes of hirsutism. CS was detected only in one patient in 2 of the studies and in no patient in the other study. CS was due to CD because of pituitary adenoma. Hence, the authors recommended that screening for CS should only be performed in women with hirsutism, who had clinical stigmata of the disease [11–13]. In discordance with our study, half of the women of the study population had oligomenorrhoea in the aforementioned studies. Also, in our study we detected CD due to pituitary adenoma in all patients with CS.

In our study, we found slightly higher but non-significant total testosterone values in patients with CD with respect to PCOS or NCAH. Also, DHEA-S levels were significantly higher in patients with CD compared to PCOS or NCAH patients.

In conclusion, common causes of oligomenorrhoea are PCOS, followed by hyperprolactinaemia and functional hypothalamic anovulation. In contrast to previous findings, we found a higher prevalence of CS in patients with oligomenorrhoea even in patients without clinical signs of the disease. Screening for CS only in patients with obvious clinical signs might preclude the diagnosis of unrecognized cases without specific clinical findings. Therefore, we suggest routine screening for CS even in the absence of clinical stigmata during the evaluation of patients with oligomenorrhoea and/or PCOS. The likelihood of CS is greater in patients with elevated androgen levels, especially DHEA-S levels.

Conflict of interest

Nothing to declare.

Data availability statement

This manuscript has not been previously published and is not under consideration in the same or substantially similar form in any other peer-reviewed media.

Ethics statement

The study was approved by the medical ethics committee of the Tepecik Research and Training Hospital, University of Health Sciences, (Date: 15 November 2021; Meeting Number: 11; Decision: 7) and in accordance with the Declaration of Helsinki, and all participants provided written informed consent.

Author contributions

All the authors (H.Y.Y., M.D., U.B., I.O., A.C., S.C., M.S., E.Y.) were involved in: 1) substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; 2) drafting the article or revising it critically for important intellectual content; and 3) final approval of the version to be published.

Funding

None.

Acknowledgments

None.