Manifestations of Polycystic Ovary Syndrome are Similar Regardless of the Degree of Menstrual Cycle Variation

Background: Menstrual irregularities may predict over adverse consequences in polycystic ovary syndrome (PCOS). Objective: To observe the relation of variants of menstrual cycles with clinical and biochemical features of PCOS. Methods: This cross-sectional study encompassed 200 PCOS women diagnosed by Rotterdam criteria and 120 age-matched healthy controls. Subgroups were classified  according  to  menstrual  cycle  length  as:  polymenorrheic  (<21  days),  eumenorrheic (21-35  days),  oligomenorrheic  (36  days-3 months)  and  amenorrheic  (>3 months). Glucose was measured by glucose oxidase, lipid by glycerol phosphate dehydrogenenase-peroxidase and all hormones by chemiluminescent immuno-assay method. Results: Around 86% of PCOS patients had menstrual irregularity, among which 75% had oligomenorrhea followed by amenorrhea (9%) and polymenorrhea (2%). All the subgroups of PCOS patients (polymenorrhea excluded from further analyses) had significantly poor metabolic manifestations than the control namely insulin resistance (IR), impaired glycaemic status, general and central obesity, metabolic syndrome and dyslipidaemia.  Acanthosis  nigricans  (AN),  hyperandrogenemia  (HA)  and  IR  had  significant predictive association with PCOS patients with both irregular [OR (95% CI)AN: 21.994 (6.427, 75.267), p<0.001; HA: 27.735 (8.672, 88.704), p<0.001; IR: 7.268 (2.647, 19.954), p<0.001] and regular cycle  [AN: 16.449  (3.830, 70.643), p<0.001; HA: 24.635  (6.349, 95.590), p<0.001;  IR: 6.071 (1.658, 22.234), p=0.006] in reference to control group.  None of the variables had significant predictive associations with irregular cycle in reference to regular cycle in patients with PCOS. Conclusion: Oligomenorrhea was the most common variant of menstrual irregularity in PCOS patients. All menstrual variants including eumenorrhea had similar manifestations in PCOS women, but poorer than controls.


Introduction
Polycystic ovary syndrome (PCOS) is an endocrine-metabolic disorder that implies various severe consequences to female health, including infertility, cutaneous complications as well as several metabolic complications of insulin resistance. PCOS manifests with a range of menstrual irregularities that frequently occur throughout adolescence or later in the reproductive years. 1 Regularity of menstrual cycle is regarded as a sign of women' reproductive health. Alterations in the menstrual cycle can occur for a variety of reasons and are frequently linked to ovaries-thyroid and pituitary axis dysfunctions. Menstrual irregularities constitute a prerequisite for the diagnosis of PCOS according to National Institute of Health and Androgen Excess-PCOS Society criteria, and one of the components of Rotterdam criteria. [2][3][4] Thus, regular cycle PCOS and other cyclical abnormalities like polymenorrhea remained underdiagnosed for a long period. In a large series of patients diagnosed with PCOS, approximately 80% had clinically evident menstrual dysfunction appearing as abnormal uterine bleeding like oligomenorrhoea. Polymenorrhoea was relatively rare, present in only 3.2% of untreated patient, whereas, prevalence of regular cycle was about 16%. 5 Previous research works have also found a link between severe cycle abnormalities and insulin resistance. When compared to controls, hyperinsulinemia was found to be substantially associated with amenorrhea in a retrospective, observational research. 6 In another study, Legro et al. found that hyperandrogenic sisters of PCOS patients with regular menses and sisters with PCOS and oligo/amenorrhea had similar levels of insulin resistance. 7 Thus this finding establishes the relation between IR and menstrual irregularities. If a relationship does exist between the degree of cycle irregularity and metabolic parameters in patients with PCOS, then menstrual dysfunction could be used as a simple clinical marker to identify patients at the greatest risk for metabolic abnormalities. The present study is designed to see the various pattern of menstrual disturbance and to clarify the significance of menstrual irregularities as predictor for adverse manifestations in PCOS.

Methods
This study was conducted in the Department of Endocrinology of BSMMU, Dhaka, Bangladesh. Sample size was estimated according to a previous study. 8 3 Control group included 120 age-matched healthy women having regular menstrual cycle without clinical and biochemical hyperandrogenism and any known endocrine disease. Patients were not included if they had: primary amenorrhea, hyperprolactinemia (serum prolactin >25 ng/ ml), hypothyroidism (serum thyroid stimulating hormone, TSH >10 μIU/ml) and non-classical congenital adrenal hyperplasia diagnosed in case of basal or adrenocorticotrophic hormone (ACTH) stimulated 17-hydroxyprogesterone greater than 10 ng/ml. 9 Women on medication for <6 months prior to the study (including oral contraceptives, glucocorticoids, metformin, ovulation induction agents, and estrogenic or anti-androgenic drugs or any medication for dyslipidemia or anti-obesity drugs) or suffering from other systemic diseases (e.g. chronic kidney disease, liver diseases etc.) were also excluded from the study. The study was conducted according to the ethical standards of Helsinki's declaration. Prior to commencement, the research protocol was approved by the Institutional Review Board (IRB) of the university. Informed written consent was taken from all participants.
Anthropometric measurements were done by the same investigator and hirsutism was assessed using a modified Ferriman-Gallwey (mFG) score. 10 Blood sample for total testosterone (TT), folliclestimulating hormone (FSH), luteinising hormone (LH), TSH and prolactin were collected on any day between 2 nd -5 th of a spontaneous bleeding episode or randomly in the case of amenorrhea. Trans-abdominal (in unmarried) or trans-vaginal ultrasonography was performed in early follicular phase. Samples for glucose (fasting and 2 hours after 75gm glucose load) and lipid profile were taken to assay on the same day of collection whereas for fasting insulin (FI) blood was centrifuged, serum separated and preserved at -20 0 C until assay. Plasma glucose was assayed by glucose oxidase method, insulin by chemiluminescent microparticle immunoassay (CMIA, Architect Plus ci4100) and lipid profile by glycerol phosphate dehydrogenenase-peroxidase method with automated analyzer (Architect Plus ci8200). Assay were verified by use of internal quality control (QC) method of the respective laboratory. Any missing or unusual data were verified immediately and there were no missing data.
Women with PCOS were grouped according to the interval between menstrual cycles and classified as polymenorrheic (<21 days), eumenorrheic (21 -35 days), oligomenorrheic (36 days -3 months) and amenorrheic (>3 months). 11 Clinical hyperandrogenism (mFG score of >8) or biochemical hyperandrogenism (TT >46 ng/ dl), polycystic ovarian morphology on USG (any ovarian volume ≥10 cm 3 ) were taken from a previous study. 12 Surrogate markers were used to define IR; fasting plasma insulin (FI) and HOMA-IR. IR was diagnosed by homeostasis model assessment of insulin resistance, HOMA-IR >2.6; HOMA-IR was calculated using the formula= {fasting plasma glucose, FPG (mmol/L) × FI (µU/ml)}÷22.5. 13 Metabolic syndrome (MetS) was defined by the harmonizing criteria for metabolic syndrome. 14 Prediabetes was defined as follows: impaired fasting glucose (IFG) when fasting plasma glucose (FPG) was between 5.6-6.9 mmol/L and impaired glucose tolerance (IGT) when the 2-h plasma glucose (2-h PG) value during a 75 g oral glucose tolerance test (OGTT) was between 7.8-11.0 mmol/L. Diabetes mellitus was confirmed by FPG ≥7.0 mmol/L and/or a 2-h PG value during a 75 g OGTT of ≥11.1 mmol/L. Comparison between groups was done by Chisquare/ Fisher's exact test with post-hoc analysis from adjusted residuals for qualitative variables. Quantitative variables with normal distribution were analyzed by independent-samples t-test or one-way ANOVA with post hoc Tukey test and skewed variables were analyzed by Mann-Whitney U test or Kruskal-Wallis one-way ANOVA with pairwise comparison by Dunn's test. Considering eumenorrhea as reference category, multinomial multivariate logistic regression analysis was done to see the predictive association of irregular and regular menstrual cycle of PCOS with different manifestations. P values <0.05 were considered as statistically significant.  Figure-1 shows the pattern of menstrual cycle in patients with PCOS. Most of the patients had menstrual irregularity (86%). Among them 75% had oligomenorrhea followed by amenorrhea (9%) and only 2% had polymenorrhea.

This
The baseline characteristics of the study population with menstrual pattern are shown in Table 1 and     Table 3). None of the variables had significant predictive associations with irregular cycle in reference to regular cycle in patients with PCOS.

Discussion
Menstrual irregularity is one of the key factors in diagnosing PCOS. Pattern of menstrual abnormalities in PCOS and its relationship with various manifestations of PCOS was evaluated in present study. It was revealed that all menstrual cycle variants had significantly adverse metabolic indices such as BMI, WC, fasting insulin, and HOMA-IR than control group.
Majority of patients (86%) had menstrual irregularity with a predominance of oligomenorrhea (75%), followed by amenorrhea (9%) and only 2% had polymenorrhea. On the other hand, 14% PCOS women had regular cycle. These findings were almost similar with the study done by others. 5 All the subgroups of PCOS patients had poor metabolic profile than the control namely IR, impaired glycemic status, general and central obesity, Mets, clinical and biochemical hyperandrogenemia and dyslipidemia, supported by similar findings found in a study done in Bangladeshi by our PCOS Study group. 16 Normocyclic women with PCOS as defined by the Rotterdam criteria have a decreased risk of insulin resistance than women with various degrees of cycle abnormalities and PCOS. 17 In the present study, age group 21-30 years in comparison to age group 16-20 years had significant predictive association with irregular cycle in PCOS patients. This may indicate some effects of advancing age or the fact that pathophysiologic aberration of PCOS started in early age which was noticed and identified at some time after advancement of age thereby permitting elapse of time for menstrual abnormality be manifested. Hyperandrogenemia and IR had predictive association with PCOS patients with both irregular and regular cycle in reference to control. However, none of the variables had predictive associations with irregular cycle in reference to regular cycle in PCOS patients. 17 Therefore it is pertinent from this study to assume that despite menstrual abnormality is a key manifestation of PCOS, it cannot apprehend over hyperandrogenemia and IR. Among metabolic and clinical manifestations in PCOS subgroups, general and central obesity, significant hirsutism, acanthosis nigricans, prediabetes, hypertriglyceridemia, IR as well as MetS were all present significantly with higher percentages in patients with oligomenorrhea than control. This would virtually indicate that PCOS is associated with menstrual irregularity which is considered as a key element in diagnosis is applicable in distinguishing between PCOS and control; but cannot be extrapolated for the discrimination among subtypes of PCOS. Percentages of T2DM was significantly higher in patients with amenorrhea than control along with history of subfertility and family history of PCOS. Likewise, hyperandrogenemia was present at a higher percentages in patients with amenorrhea and oligomenorrhea than control.
Several studies have found the overall prevalence of IR ranges from 44% to 70%; our study revealed around 78% in eumenorrheic, 72% in oligomenorrheic and 63% in amenorrheic group were insulin resistant. 18,19 In the present study, the prevalence of MetS ranged from 30-40% in PCOS subgroups. This frequency is similar to that reported in the United States (43%-46%) but higher than that reported in Italy (8%-16%) and China (2.3%-12.2%). 20,21 The variations in these metabolic findings could possibly be due to the influence of ethnic origin, genetic and environment, and dietary habits on the prevalence of PCOS and its comorbidities.
The study questionnaire did not use standard criteria to define regular menstruation. According to recommendations from International Federation of Gynecology and Obstetrics (FIGO), normal menstrual cycle should be defined according to the: [i] frequency of menses (24-38 days), [ii] regularity of menses (variation±2-20 days), [iii] duration of menstrual flow (4.5-8 days) and [iv] volume of monthly blood loss (5-80 ml). 22 Thus, subjects might have experienced confusion regarding their menstrual pattern which may have led to inaccurate data. In the current study, after adjusting for BMI, age and race, women with PCOS were grouped according to the interval between menstrual cycles and classified as polymenorrheic, eumenorrheic, oligomenorrheic and amenorrheic respectively. Besides, the number of study participants in amenorrhea group was small, so the associations might not significant with other menstrual variants in PCOS group.

Conclusion
Our data suggest that one out of ten PCOS patients might present with regular menstrual cycle and oligomenorrhea was the predominant variant of irregular menstrual cycle in PCOS. Among different manifestations, acanthosis nigricans, insulin resistance and hyperandrogenemia were significantly associated with both irregular and regular menstrual cycle of PCOS patients in comparison to control. However, the manifestations were not different across different menstrual patterns in patients with PCOS.