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Purpose:
To compare uterine ultrasound measurements in women with normogonadotropic anovulation, specifically those with polycystic ovary syndrome (PCOS) and hypothalamic-pituitary-ovarian dysfunction (HPOD), with those of regularly menstruating women, and to assess the influence of clinical and biochemical parameters on these measurements.
Methods:
Uterine length, width, height, and volume, along with endometrial thickness and volume, measured using two- and three-dimensional transvaginal ultrasonography, were prospectively compared in women aged 18 to 45 from the aforementioned groups. Correlations between clinical parameters and uterine measurements, as well as between biochemical parameters and these measurements in anovulatory women, were analyzed.
Results:
Women with normogonadotropic anovulation had significantly reduced uterine and endometrial measurements compared to healthy women (all p ≤0.001). Women with PCOS showed significantly lower uterine length (p=0.045), height (p=0.004), and volume (p=0.009) than those with HPOD, with no significant endometrial differences. Among women with PCOS, those with hyperandrogenemia had thicker endometrium (p=0.036), with no significant differences in other measurements. All myometrial measurements significantly negatively correlated with anti-Müllerian hormone (AMH) and follicle-stimulating hormone (FSH), while positively correlating with estradiol and prolactin. Endometrial measurements negatively correlated with AMH and FSH concentrations, and positively with estradiol, prolactin, 17-hydroxyprogesterone, fasting insulin, and insulin resistance.
Conclusions:
A significant association was identified between menstrual cycle regularity and uterine morphology, influenced by hormonal, metabolic, and clinical factors. Estrogenic stimulation and metabolic status significantly affected uterine and endometrial dimensions, indicating that uterine morphology reflects the cumulative impact of reproductive and endocrine-metabolic influences, with important clinical implications for women with normogonadotropic anovulation.
1 IntroductionUltrasonography, due to its widespread accessibility, reliability, and non-invasive nature, is the preferred imaging modality for evaluating pelvic structures. The assessment of uterine morphology, encompassing the measurement of its size and endometrial thickness, is crucial for determining its functional status and requires a comprehensive understanding of the anatomy and physiology of the reproductive system to accurately acquire and interpret ultrasonographic images (1, 2). Although a body of research has addressed the assessment of uterine normograms in specific populations and the analysis of measurement variations throughout the menstrual cycle (3) and over the lifespan (4), a substantial deficiency of data persists regarding the variability of uterine morphology in the context of normogonadotropic anovulation. The uterus serves as a target organ for steroid hormones and understanding uterine and endometrial morphology is essential for the diagnosis and treatment of various reproductive health conditions. The ultrasonographic assessment of the morphology of the uterine corpus is essential for the diagnosis of abnormal uterine bleeding, dysmenorrhea, pregnancy complications, and infertility. Furthermore, the visual evaluation of the endometrium is particularly relevant for diagnosing menstrual irregularities and for identifying structural alterations that may underlie abnormal uterine bleeding. The dimensions and volume of the myometrium and endometrium can influence clinical pregnancy rates in women undergoing assisted reproductive techniques, with both excessively small and large measurements potentially decreasing this rate. Inadequate dimensions may result from hypoestrogenism, while enlarged dimensions can arise from estrogen-progesterone imbalances associated with polycystic ovary syndrome (PCOS), increased uterine vascularization, or estrogen-dependent myometrial conditions such as adenomyosis. Additionally, both excessively thin and thick cystic endometrium can compromise endometrial receptivity and adversely affect embryo implantation. Despite established ultrasonographic criteria for ovarian assessment in PCOS (5), there is limited data regarding the myometrium and endometrium in women with PCOS and hypothalamic-pituitary-ovarian axis dysfunction (HPOD) (6). Moreover, considerable attention has been devoted to investigating complications specific to PCOS, such as insulin resistance, hyperinsulinemia, hyperandrogenemia, and systemic inflammation, which are known to act synergistically to impair endometrial function, leading to menstrual irregularities, infertility, and obstetric failure (7). However, the impact of these complications on the ultrasonographic morphology of the myometrium and endometrium in the context of normogonadotropic anovulation, as well as the differences between PCOS and HPOD, have not yet been studied. This study aimed to conduct a comparative ultrasonographic assessment of myometrial and endometrial measurements among women with PCOS and HPOD, while also evaluating the influence of selected clinical and biochemical variables on these measurements.
2 Materials and methodsA prospective cohort study was conducted among women diagnosed with menstrual irregularities from January 5, 2024, to December 31, 2024, in units affiliated with the Clinical Department of Gynecological Endocrinology and Gynecological Oncology at the University Hospital in Krakow. The study was approved by the Bioethics Committee of Jagiellonian University (no. 118.6120.53.2023) and conducted in accordance with the Helsinki Declaration, with informed written consent obtained from all participants. The study was registered in the ClinicalTrials.gov Protocol Registration and Results System (no. NCT06211608). The inclusion criteria were as follows: i) age 18 to 45 years, ii) no prior diagnosis and treatment for menstrual disorders or infertility. The exclusion criteria included: i) history of ovarian surgery, ii) use of medications that disrupt the hypothalamic-pituitary-ovarian axis, iii) presence of uterine tumors or congenital malformations. The control group consisted of regularly menstruating women of reproductive age, with non-pregnant and non-pathological uterus, reporting for preventive health care. All women participating in the study underwent a comprehensive physical examination, which included a medical interview and a gynecological evaluation featuring a vaginal speculum examination, bimanual examination, and pelvic ultrasound. Body Mass Index (BMI) was calculated using the formula: BMI= body weight [kg]/height [m]². Excess body hair was assessed using the modified Ferriman-Gallwey scale (mFG) (5). Based on the obtained data, a characterization of the population was conducted regarding demographic information (age, body weight, BMI), gynecological aspects (cycle length, bleeding pattern, menstrual-related pain, and infertility), obstetric history (number of pregnancies, deliveries, and miscarriages), ultrasonographic measurements, and, for women with irregular menstruation, biochemical parameters, along with an assessment of the correlations between these variables and the measured outcomes.
2.1 Ultrasonography of the reproductive organsTwo-dimensional and three-dimensional ultrasound imaging of the female reproductive organs was performed using the Samsung HERA W9 ultrasound device, employing transvaginal (EV2-10A) volume transducer (Samsung Electronics, Republic of Korea). The transvaginal examination was conducted with an emptied bladder, in a dorsal lithotomy position. The examination protocol included the evaluation of the cervix, uterine body, adnexa, and Douglas pouch. Uterine morphology was assessed in three planes: sagittal, transverse and coronal, achieved through HDVI™ volume rendering technology (Samsung Electronics, Republic of Korea). Volume acquisition was conducted using a standardized method (8), maintaining a maximum sweep angle of 180° after obtaining a sagittal section of the uterus, with the ultrasound beam oriented at approximately a 90° angle to the uterine axis. The ultrasound transducer was held stationary, and the examiner instructed the participant to remain still. The length of the uterine body was measured from the apex of the fundus to the internal cervical os, while the height was assessed in the anteroposterior dimension at the level of the fundus, both on the sagittal section. The width was measured at the fundal level using a coronal section obtained through 3D reconstruction. The volume was calculated using an automatic function based on the formula for an ellipsoid, accounting for three dimensions: length, width, and height of the organ, employing the simplified formula: V = (π/6) × length × width × height (in cm and reported in ml). The detection of at least 20 follicles ranging from 2 to 9 mm in diameter, along with the absence of a corpus luteum, dominant follicle, or functional cyst, was indicative of polycystic ovarian morphology (PCOM) (5). Ultrasonographic representations of the female reproductive organs in women with regular menstrual cycles (healthy controls), polycystic ovary syndrome, and hypothalamic-pituitary-ovarian axis dysfunction, along with an illustration of the measurement techniques, were presented in Figure 1. Uterine malformations identified through ultrasound were classified according to the European Society for Gynaecological Endoscopy (ESGE) consensus (9), with women presenting such anomalies excluded from the study. Ultrasound examinations were performed exclusively by two designated investigators (I.G., K.Z.) collaborating closely to ensure consistency in conducting and reporting measurements, thereby minimizing variability and enhancing the reliability of the collected data.
Ultrasonographic images of the female reproductive organs representative of women with regular menstrual cycles (healthy controls) (1.1), polycystic ovary syndrome (1.2), and hypothalamic-pituitary-ovarian axis dysfunction (1.3), accompanied by an illustration of the measurement techniques employed.
2.2 Biochemical analysis of blood plasmaConcentrations of specific biochemical parameters were determined using an automated Roche Cobas PRO/e801 analyzer (Roche Diagnostics, Basel, Switzerland) on a 10 ml venous blood sample collected after a minimum eight-hour overnight fast. The concentrations of serum follicle-stimulating hormone (FSH) [mIU/ml], luteinizing hormone (LH) [mIU/ml], prolactin (PRL) [μIU/ml], Anti-Müllerian hormone (AMH) [pmol/l], estradiol [pmol/l], testosterone [nmol/l], sex hormone-binding globulin (SHBG) [nmol/l], dehydroepiandrosterone sulfate (DHEA-S) [μmol/l], fasting insulin (FI) [μU/ml], and those measured at the 120-minute mark during the 75 g glucose tolerance test (120’75OGTTI) [μU/ml], along with thyroid-stimulating hormone (TSH) [μIU/ml], free triiodothyronine (fT3) [pmol/l], free thyroxine (fT4) [pmol/l], thyroid peroxidase antibodies (TPOAb) [IU/ml], thyroglobulin antibodies (TGAb) [IU/ml], and 17-hydroxyprogesterone (17-OHP) [ng/ml] were quantified utilizing electrochemiluminescence immunoassay (ECLIA). Fasting glucose (FG) [mmol/l] and glucose levels at 120 minutes during the 75 g glucose tolerance test (120’75OGTTG) [mmol/l], triglycerides (TG) [mmol/l], and total cholesterol (TC) [mmol/l] were assessed using enzymatic methods, while high-density lipoprotein cholesterol (HDL) [mmol/l] was evaluated spectrophotometrically. C-reactive protein (CRP) [mg/l] was analyzed using an immunoturbidimetric method. Low-density lipoprotein (LDL) cholesterol [mmol/l] was calculated using the Friedewald formula (10). The Homeostasis Model Assessment for insulin resistance (HOMA-IR) was determined by multiplying FI by FG and dividing by 22.5. The free androgen index (FAI) was calculated by dividing total testosterone concentration by SHBG concentration. Hyperandrogenemia was defined as a testosterone concentration greater than 1.67 nmol/L or a FAI greater than 5.
2.3 Statistical analysisQuantitative variables were analyzed using descriptive statistics, which included the mean, standard deviation, median, quartiles, and range. Qualitative variables were evaluated by calculating the absolute and percentage frequencies of all possible values. The distributions of the variables were examined using the Kolmogorov-Smirnov test. The assessment of quantitative variable values between two groups was conducted using the Mann-Whitney U test. To examine quantitative variable values among three or more groups, the Kruskal-Wallis test was employed, followed by Dunn’s post-hoc test where statistically significant differences among groups were identified. Comparative analysis of qualitative variables across different groups was performed using the chi-square test (with Yates’ continuity correction for 2x2 contingency tables) or Fisher’s exact test in cases where the assumptions for the chi-square test regarding expected frequencies were not met. Correlations among quantitative variables were analyzed using Spearman’s rank correlation coefficient. A significance threshold of 0.05 was established, indicating that all p-values below 0.05 were considered indicative of statistically significant associations. The analyses were conducted using R software, version 4.5.1 (11). Assuming a 95% confidence level, a 5% margin of error, and a 50% population proportion, the normogonadotropic anovulation arm required a minimum sample size of 285 participants from a cohort of 1100 women undergoing diagnostic evaluation of menstrual irregularities. The healthy control group, based on 200 women undergoing routine examinations, required a minimum sample size of 132 participants.
3 ResultsThe study consecutively enrolled 648 women, including 452 with irregular menstrual cycles in the study arm and 196 with regular cycles in the control arm. The developed database has been made publicly available https://doi.org/10.7910/DVN/HZNRLK.
A comparative analysis of selected clinical parameters and ultrasonographic measurements of the uterus and ovarian characteristics in both study arms was presented in Table 1. Women with irregular cycles were significantly younger (p<0.001), scored higher on the mFG scale (p=0.008), exhibited a higher BMI (p=0.001), had a longer average cycle length (p<0.001), and lower percentage of pregnancies (p=0.025) and miscarriages (p=0.001). No significant intergroup differences were identified in the prevalence of dysmenorrhea, AUB, or history of childbirth (all p>0.05). All myometrial measurements in women with irregular menstrual cycles were significantly reduced, including length (p=0.001), height (p<0.001), width (p<0.001), and consequently the volume of the uterine corpus (p<0.001), as well as thickness (p<0.001) and volume of the endometrium (p<0.001). Ultrasonographic ovarian volume (p=0.016) and the prevalence of PCOM (p<0.001) were greater among women with irregular menstrual cycles. The frequencies of other ovarian findings, such as dominant follicle, simple cyst, and ovarian lesion, were not significantly different (all p-values >0.05).
VariableIrregular cycles (N = 452)Regular cycles (N = 196)Total (N = 648)pAge [years]Mean (SD)26.82 (5.14)28.73 (5.72)27.4 (5.39)p<0.001 *Median (quartiles)26 (23-30)28 (24.75-33)27 (24-31)Range18-4418-4518-45mFerriman -Comparative analysis of selected clinical variables between the study arms of women with regular and irregular menstrual cycles.
p, Qualitative variables, chi-squared or Fisher’s exact test; Quantitative variables: Mann-Whitney test, * statistically significant (p<0.05); SD, standard deviation; quartiles, lower quartile (Q1), upper quartile (Q3); BMI, body mass index.
The comparative analysis of uterine measurements and concentrations of selected biochemical parameters in women with irregular menstrual cycles diagnosed with PCOS, compared to those with HPOD, was presented in Table 2. Women with PCOS demonstrated significantly reduced uterine length (p=0.045), height (0.004), and volume (p=0.009) compared to women with HPOD. Endometrial measurements, however, did not reveal significant differences (all p-values >0.05). Significant differences in concentrations were observed for AMH, LH, testosterone, DHEA-S, fT3, FI, TG, and 17-OHP, which were higher in individuals with PCOS, while estradiol, SHBG, and vitamin D were lower (all p-values <0.05).
ParameterPCOS
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