Introduction
According to DMS-5 criteria, anorexia nervosa (AN) is characterized by persistent restriction in caloric intake, intense fear of gaining weight or behavior that interferes with weight gain, bodily disrespect that affects self-esteem levels or non-recognition of the severity of the underweight condition [
1].
One of the most frequent clinical complications of AN is functional hypothalamic amenorrhea (FHA), a form of chronic anovulation without organic causes but due to multiple interacting factors [
2‐
4].
Amenorrhea and anorexia are so closely related that in DSM-IV, this complication had been included among the diagnostic criteria of AN; in DSM-V, however, it has been excluded because it cannot be evaluated in some categories of patients such as pre-pubertal patients or patients with delayed puberty, menopausal women, those taking oral contraceptives and male patients [
5].
In AN, FHA is the result of an energy deficit due to malnutrition, physical hyperactivity or both; these conditions can induce alterations in metabolic and reproductive hormones [
6]. During a chronic caloric restriction, adaptive mechanisms act to diminish basal metabolic rate and preserve calories for vital functions. In such condition, GnRH pulsatility is decreased and LH secretion becomes similar to that of pre-pubertal females with a diminsihed pulse amplitude and frequency, mainly during the day hours [
7,
8].
The leptin decrease is considered one of the major signal dysfunctions leading to FHA. A positive correlation between leptin and LH secretion was indeed demonstrated and the increase in circulating leptin concentrations > 1.85 ng/mL was reported to be associated with menstrual recovery [
9,
10]. However, the persistence of amenorrhea is not uncommon in women who have regained weight, regardless of the normalization of leptin levels [
11]. Nevertheless, the restoration of a normal body weight was reported to be the most important clinical parameter for the functional recovery of the hypothalamic–pituitary–gonadal (HPG) axis [
12‐
14], even if the recovery of menses can frequently be delayed by several months [
15,
16]. Generally, the resumption of menses is associated with a 90% recovery of the ideal body weight, but a considerable number of patients (about 1/5) was reported to have a prolongation of amenorrhea even after an adequate weight gain [
17‐
21]. High urinary cortisol, physical hyperactivity and a low body fat mass have been found as risk factors for persistent FHA [
18‐
22].
Furthermore, a subgroup of AN patients do not present FHA, but maintain regular menstrual cycles despite a manifest underweight. One previous study described significant differences in fat mass, leptin levels and in the severity of psychological disorder when eumenorrheic or amenorrheic patients were compared [
23].
Despite the efforts of several groups, the relation underlying FHA in AN is still largely unexplained. The aim of our study is to investigate the anthropometric, hormonal and psychological parameters in patients with restrictive-type anorexia nervosa to obtain insights into the factors associated with the onset, resolution or persistence of FHA.
Materials and methods
Patients
We revised data from 297 patients affected with eating disorders who were part of a hospital rehabilitation program in the Division of Endocrine and Metabolic Diseases of the IRCCS Istituto Auxologico, since 2011 up to 2018. This is a semi-resident rehabilitation program generally proposed for AN patients with a disease history shorter than 2 years. The program is multidisciplinary and includes either medical, psychological or nutritional interventions.
We selected 226 female patients with AN diagnosis based on DMS-5 criteria, who had reached a BMI > 17.5 kg/m2 after a rehabilitative intervention of 6–12 months and did not worsen their BMI during a follow-up period of 12 months. Criteria of exclusion were (i) primary amenorrhea, (ii) previous diagnosis of polycystic ovary syndrome (PCOS), (iii) use of all the drugs potentially interfering with the HPG axis (e.g., anti-dopaminergic agents or estroprogestins), and (iv) intense exercise for >5 h a week.
After informed consent, 114 patients were divided into three groups, according to the variable menstrual history: Group 0 (n = 74) included AN patients who experienced FHA during the critical illness and then recovered menstrual cyclicity during rehabilitation; Group 1 included 26 AN patients having persistent amenorrhea despite significant weight gain during rehabilitation; Group 2 (n = 14) included all eumenorrheic AN patients, who persistently had regular menses throughout the illness period despite a severe weight loss and prominent underweight.
Detection of anthropometric parameters
Body weight was measured using the WUNDER Rb300 electronic scale. The body height was measured with a HARPENDEN stadiometer and body mass index (BMI) was calculated as weight (kg)/height (m)2.
Biochemical parameters
Blood samples were periodically taken at 8 am after overnight fasting of at least 8 h. Hormone parameters, such as estradiol, gonadotropins and thyroid function tests, were assessed by routine immunoassays (Roche); leptin was assessed by ELISA (DRG Leptin Sandwich ELISA).
EDI-2 questionnaires
The severity of psychiatric symptoms was evaluated by administering the EDI-2, a 64-item, self-report questionnaire with eight subscales [
24] recognized as valid in the identification and characterization of patients with eating disorders. EDI-2 evaluates behavior and attitude related to nutrition, weight and body shape (drive for thinness, bulimia, body dissatisfaction, impulsiveness) and psychological characteristics (such as inadequacy, perfectionism, self-confidence, fear of maturity, asceticism, social insecurity and enteroceptive capacity).
The EDI-Symptom Checklist is used to measure the frequency of manifestations such as binge eating, abuse of laxatives or diet pills, physical hyperactivity and amenorrhea [
25].
Statistical analysis
Quantitative variables were reported as mean and standard deviation (or as median and interquartile range if the variable was not normally distributed). Shapiro–Wilk test was used to evaluate the normal distribution of data. The three groups were compared with one-way ANOVA test, or non-parametric Kruskal–Wallis test in case of non-normality. Comparisons between two groups were explored with Student’s t test, or non-parametric Wilcoxon test in case of non-normality. Predictors of menstrual cycle relapse were analyzed with a logistic regression. For this analysis, we considered only two groups: patients who experienced FHA and recovered their menses (Group 0) and patients remaining in persistent amenorrhea (Group 1). Repeated measures model was used to explore differences of estradiol, fT3 and leptin levels in the three groups and between admission and discharge. The strength and direction of the association between continuous variables were evaluated with Spearman correlation coefficient. Comparisons of EDI-2 scores at admission and at discharge for each group were explored with the Wilcoxon signed rank test. Statistical significance was defined as a two-sided p value < 0.05. Statistical analysis was performed using R software version 4.3.0.
Discussion
In this study, we evaluated the gonadal function of patients suffering from AN by analyzing anthropometric, endocrinological, metabolic and psychological data at the onset of illness, during rehabilitation and after recovery. To our knowledge, this is the first study analyzing clinical and biochemical parameters in the three categories of AN patients with distinct menstrual histories: (i) those with FHA resolving during a stable improvement of the disease conditions, (ii) those with protracted amenorrhea despite BMI and psychological recovery or (iii) persistence of regular menses throughout the course of the disease.
The inclusion and exclusion criteria allowed the selection of patients in whom FHA was apparently related to the critical phase of the AN. It is known that weight and BMI are relevant factors influencing the function of the gonadal axis [
13,
25,
26]. However, in our cohort, premorbid BMI, age of disease onset or amount of weight loss was similar among the three groups of patients with variable menstrual history, and in contrast with a previous study which defined premorbid BMI as the main predictor for longer duration of amenorrhea [
27]. In addition, despite the patients with persistent amenorrhea experienced the lowest BMI values when compared with the other two groups, they were also the ones to enter FHA at higher BMI values. Indeed, also the logistic regression studying together the factors associated with persistence of FHA found the BMI at which patients experience amenorrhea to be the strongest predictors of the recurrence of menses. This finding, together with the lack of significance of the overall weight loss, points to a higher contribution of the individual predisposition to HPG axis disruption than the effect of the severity of the disease. Accordingly, the last weight at follow-up was statistically higher in patients with persistent amenorrhea even when compared with the weight at which the patients of Group 0 resolved the amenorrhea, thus supporting the thesis that weight is not an independent parameter in determining FHA. Even when we compared Group 2 of the eumenorrheic patients with the other two groups, we found that the weight was not a determining factor for the functionality of the HPG axis. In fact, the lowest BMI at which these patients maintained the menstrual cycle is statistically lower compared to the others.
As far as hormonal parameters are concerned, it is known that leptin correlates with patient's weight and nutritional status and with the amount of fat mass [
28‐
32]. Some authors have questioned such correlations: Miller and colleagues [
33] reported hypoleptinemia as independent of fat mass in women with FHA. In our study, we found similar leptin levels at admission, with average values below the lower limit of the healthy population (< 3.6 μg/mL). Leptin concentrations statistically improved at the last follow-up visit only in patients who were menstruating at discharge (Groups 0 and 2). In contrast, the increase of leptin levels was limited and not significant in subjects with persistent amenorrhea (Group 1). Low leptin can account for the decreased pulsatility of LH [
30], and the lack of its significant increase justifies the persistence of FHA in Group 1; in this group, low leptin concentrations despite weight recovery are indicative of a different body composition, perhaps due to an unbalanced diet with a low fat content, as previously suggested [
33].
Interestingly, the average fT3 values at admission were above the lower reference limit (> 3.7 pmol/L) in eumenorrheic patients (Group 2) and statistically higher (
p value = 0.0249) than in the group of patients recovering from amenorrhea (Group 0), despite similar clinical characteristics at admission (Table
1), and tended to be higher than in the group of patients with persistent amenorrhea (Group 1). At the end of rehabilitation, there was a general improvement in fT3 concentration, but they remained below or at the lower limit of the normal range in a consistent number of patients with permanent amenorrhea (Group 1), thus indicating the persistence of a hypothalamic dysfunction despite the increase of body weight which might result from the lack of a significant leptin rise.
Reinehr et al. [
34] hypothesized that leptin could be the link between BMI and fT3; this observation appears partially in disagreement with the results of our study because patients maintaining the gonadal function during the critical phase of the disease (Group 2) had low levels of leptin and normal fT3 at admission; moreover, patients with persistent amenorrhea had an improvement in fT3, but not in leptin levels after rehabilitation. Swenne et al. [
35] concluded that fT3 can be used as a nutritional indicator and that the severity and the velocity of weight loss are the strongest predictors of fT3 concentrations. Consistently, we found an inverse correlation of fT3 with the amount of weight loss and a positive correlation between fT3 and leptin levels, as previously described [
36]. But in addition, here we observed a highly significant positive correlation between fT3 and estradiol, with fT3 levels tending to an earlier normalization before resumption of menses and rise of circulating estradiol in Group 0, thus indicating fT3 rise as an early marker of the resumption of hypothalamic activity.This adds to the evidence already available for IGF1 [
37] as opposed to cortisol levels which are reported to be a predictor of hypothalamic inhibition [
38]. Still, the usefulness of fT3 levels in this setting should always be put in context of clinical parameters. In eumenorrheic patients of Group 2 at admission, fT3 levels were significantly higher in association with the conserved gonadal function; this combination may indicate the portion of AN patients with a hypothalamic function that is less sensitive to the effects of caloric restriction and weight loss.
The main limitation of our study is represented by the lack of body composition determinations to support and correlate with the leptin measurements, leaving open the possibility for a relevant role of the psychopathological profile of the patients [
39]. Finally, only some endocrine/metabolic parameters have been evaluated, but many others (GH/IGF-1, hypothalamus–pituitary–adrenal axis, other adipokines and gastro-intestinal hormones) may contribute to the HPG axis dysfunction. Eating disorder manifestations, quantified by the EDI-2 questionnaire, were similar among the three groups, both at admission and discharge. However, when we evaluated the improvement of the score within the three groups, we found that only the patients recovering gonadal function (Group 0) showed a significant general improvement of the score involving almost all the categories. The patients with persistent amenorrhea (Group 1) had an intermediate improvement. These results are in partial agreement with those of Miller et al. [
40].
In conclusion, to our knowledge this study is the first comparing three different categories of AN patients defined on the basis of menstrual activity. We found that the premorbid weight and the weight loss during AN are not the main determinants of the GnRH neuron activity, but a more important role is played by the minimal BMI reached during the critical phase of the disease. Nevertheless, a significant portion of patients maintain menstrual cycles even at BMI significantly lower than the other two groups, indicating the significant role played by individual factors in the definition of the set-point of hypothalamic dysfunction. This view is reinforced by the findings in patients with persistent amenorrhea (Group 1) showing a body weight higher than in patients resuming their menses, both at the amenorrhea onset and during the follow-up, further supporting the hypothesis that persistent FHA may be a consequence of an intrinsic fragility of the HPG axis, thus supporting the multifactorial origin of the FHA in the AN patients [
41,
42].
At admission, fT3 levels are statistically higher in eumenorrheic patients and fT3 variations during rehabilitation often precede the restoration of gonadal function in Group 0, thus indicating fT3 rise as an early indicator of FHA recovery.
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