In this retrospective study, a consecutive series of 277 female patients attending our Andrology, Women’s Endocrinology and Gender Incongruence Unit at Careggi University Hospital, Florence, Italy for sexual symptoms, between January 2016 and January 2021, were analyzed. The inclusion criteria were: being sexually active in the previous 4 weeks, having a partner and being able to give an informed consent. Two-hundred sixty four of these patients constituted the normo-PRL Female Sexual Dysfunction (FSD) women group, while 13 were diagnosed with hyperprolactinemia and formed the hyper-PRL FSD group. The diagnostic workflow for these pathological conditions was performed according to the current Endocrine Society guidelines and the reasons for hyper-PRL were identified as follows: pharmacological treatment (78%), macroprolactinemia (11%) and undefined cause due to loss at follow-up (11%). A third group (n = 42) was identified as that composed by healthy controls, namely women consulting the same Unit for other endocrinological symptoms, contraceptive counseling or menopausal check-up, in which non-compensated endocrine disorders had been excluded, sexually active in the previous 4 weeks, and having a partner. For these women, the Female Sexual Function Index (FSFI) and the Female Sexual Distress Scale-Revised had been used as screening tools and were retrospectively available, and the FSFI total score was ≥ 26.55, excluding FSD (see Clinical Assessment).

All procedures were in accordance with the ethical standards and approved by the institutional research committee. All participants provided an informed consent before any diagnostic procedure (protocol 37.589/SPE.13.034, Careggi Hospital, Florence, Italy).

For all patients, a demographic, clinical and anthropometric [weight, body mass index (BMI), waist circumference (WC) and systolic (SBP) and diastolic blood pressure (DBP)] evaluations were performed. Current use of medications, in particular those potentially interfering with PRL levels (psychiatric medications, hormonal contraception, hormonal replacement therapy), was investigated.

All participants were asked to complete the FSFI [25], the most common psychometric instrument for the screening of FSD. The validated Italian version was used [26]. This self-reported questionnaire explores different domains of the female sexual response: desire, arousal, orgasm, satisfaction, and pain. For each question, the possible score ranges from 0 or 1 to 5, with the minimum score representing the most pathological condition. By adding individual questions’ scores and multiplying these by a specific factor, a score for each domain is obtained. The sum of the 6 domain scores provides a Total score, with a threshold of <  = 26.55 classifying the woman as at risk for FSD. The FSFI does not evaluate the presence of distress, which plays a key role in the diagnosis of FSDs, including HSDD. Therefore, we included in our questionnaires also the Female Sexual Distress Scale-Revised (FSDS-R) [27], a 13-items self-reported questionnaire which quantifies sexually related personal distress in women with HSDD. The cut-off score for discriminating affected from non-affected patients is >  = 11.

Psychopathologic parameters were evaluated by the Middlesex Hospital Questionnaire (MHQ), a self-administered measure of psychoneurotic pathology in non-psychiatric settings [28]. The MHQ provides scores for free-floating anxiety (MHQ-A), phobic anxiety (MHQ-P), obsessive–compulsive traits and symptoms (MHQ-O), somatization (MHQ-S), depressive symptoms (MHQ-D), and histrionic or hysterical symptoms and traits (MHQ-H) and a total score. Body image concerns, which represent a crucial aspect in women’s sexuality, were explored by the Body Uneasiness Test (BUT), a validated self-reported questionnaire [29]. This measure includes two separate sheets, one for the evaluation of body experiences (BUT-A), i.e., weight phobia or compulsive self-monitoring, and one regarding dissatisfaction with individual body parts (BUT-B); the answers are scored on a 6-point Likert scale, with higher scores identifying greater concerns or dislike. Both sub-scales provide total indexes.

Finally, FSD patients completed the Sexual Inhibition and Sexual Excitation Scales (SIS/SES) [30, 31]. The SIS/SES is composed of 3 factors, the first (SES) describing sexual arousal related to sexual or non-sexual situations, the second (SIS1) describing inhibition due to threat of performance failure (e.g., arousal difficulties, concern for the partner’s pleasure, etc.), and the third (SIS2) describing inhibition due to threat of performance consequences (e.g., the risk of being caught, sexually transmitted diseases, pain, unwanted pregnancies) [30].

Biochemical assessment was performed in the morning, in the early follicular phase of the cycle for pre-menopausal women—in any day of the month in post-menopausal women- in fasting conditions to measure glucose (esokinase method; Dimension Vista 1500 Medical Solutions by Siemens Healthcare, Newark, USA), total cholesterol, high-density lipoprotein (HDL) and triglycerides (automatic enzymatic colorimetric method; Dimension Vista 1500 Medical Solutions by Siemens Healthcare, Newark, USA); insulin (electrochemiluminescence immunoassay, “ECLIA”; Roche Diagnostics, Mannheim, Germania) and glycated hemoglobin (HbA1c) (high prestation liquid chromatography, HPLC, Variant II method, Biorad Laboratories, Hercules, CA, USA), prolactin (PRL), thyroid-stimulating hormone (TSH), follicle-stimulating hormone (FSH), luteinizing hormone (LH), 17β-estradiol (using the chemiluminescence method; DIMENSION VISTA ® System, Siemens), cortisol (electro-chemiluminescence immunoassay; COBAS 600, Roche Diagnostics, Basel, Switzerland); sex hormone binding globulin (SHBG) (using the electrochemiluminescence immunoassay; COBAS, ROCHE, Germany). Androgens, including D-4-androstenedione, were measured by liquid chromatography-mass spectrometry (Agilent Technologies, Santa Clara, CA). To estimate low-density lipoprotein (LDL) cholesterol the Friedewald equation was used: LDL cholesterol = total cholesterol-(HDL cholesterol + triglycerides/5).

Metabolic syndrome (MetS) was diagnosed according to the National Cholesterol Education Program—Third Adult Treatment Panel (NCEP-ATPIII; Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults, 2001), based on the presence of ≥ 3 of the following factors: central obesity (waist circumference ≥ 88 cm), hypertriglyceridemia (≥ 150 mg/dL or specific therapy), arterial hypertension (systolic blood pressure ≥ 130 mmHg and/or diastolic blood pressure ≥ 85 mmHg or specific therapy), impaired fasting glycemia (≥ 110 mg/dL or specific therapy) and low HDL cholesterol serum levels.

Data were reported as mean ± SD when normally distributed, as median (quartiles) when non-normally distributed and as percentage and number when categorical. Linear analyses were performed to assess the association of continuous variables (PRL) using the Pearson’s method. Significant correlations at univariate analysis were tested at multivariate analysis after adjusting for confounding factors (i.e., years since menopause). One-way ANOVA was used to test differences in means among groups (i.e., PRL quintiles), followed by post hoc analyses, applying a Bonferroni correction. The unpaired 2-sided Student t-test and the Mann–Whitney U test were applied for assessing differences between 2 groups, whenever appropriate. Receiver operating characteristic (ROC) analysis was carried out to determine the PRL cut-off value that yielded optimal sensitivity and specificity in identifying the presence of HSDD.

All analyses were carried out with SPSS 26.0 statistical package and a p < 0.05 was considered statistically significant.