Dissertation committee:
Δημήτριος Λουτράδης, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Σταυρούλα Μιχαλά, Επίκουρος Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Αλεξάνδρα Σολδάτου, Επίκουρος Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Νικόλαος Βλάχος, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Πέτρος Δρακάκης, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Καλλιόπη Παππά, Επίκουρος Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Μαριάννα Θεοδωρά, Επίκουρος Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Summary:
Although the proximate cause of functional hypothalamic amenorrhoea (FHA) is the abnormal gonadotropic-releasing hormone (GnRH) secretion, it is seemingly more than an isolated desynchronisation of the GnRH axis. There is fair evidence to support the association of FHA with behavioural, emotional, cognitive and psychological factors. Previous studies underscored the psychological FHA correlates; notwithstanding, the pivotal role of sleep disorders (SleD) has not been elucidated thus far. Moreover, it remains uncertain, whether this variability is also attributable to a genetic predisposition. The Prokineticin 2 (PROK2) and Prokineticin receptor 2 (PROKR2) singling pathway is implicated in the cause of idiopathic hypogonadotropic hypogonadism (IHH). Two heterozygous PROKR2 mutations (g. 254 G>A and g. 518 T>G) have also been identified amongst patients with FHA. The latter were found to be loss-of-function mutations.
We hypothesised that FHA women suffer from SleD. We aimed to ascertain the anxiety levels (AL) role on FHA pathophysiology and to identify plausible associations with other known FHA predisposing factors. We also aimed to identify any possible mutations in PROKR2 gene in subjects with FHA.
We conducted a prospective case-control study spanning the period January 2016 to Οctober 2019. We recruited forty-one FHA women and 86 healthy controls. We assessed SleD and other FHA predisposing factors via self-reported questionnaires. The Spearman’s correlation coefficient (rho) was used to examine possible correlations among the different variables. Multivariate logistic regression analysis was conducted to identify independent factors.
We also analysed the coding sequence of PROKR2 in 84 women: 41 with FHA, 23 with IHH, and 20 healthy controls respectively. Genomic DNA was extracted from peripheral blood samples using PureLink Genomic DNA Kits for purification of genomic DNA. PCR was performed using primers designed to amplify the two coding exons and the exon-intron boundaries. The amplified PCR products were sequenced using the ΑΒΙ Prism 3130 Genetic Analyzer, Applied Biosystems. Variants were validated using the sequencing Analysis v 5.2 software.
Women with FHA reported having higher SleD (p=0.004). There was a significant positive correlation between SleD and AL (rho=0.879, p<0.0001). Significant correlation was also found between AL and several AIS-8 constituents, including sleep induction (rho=0.529, p=0.0004), awakenings during the night (rho=0.603, p<0.0001), final awakening (rho=0.421, p=0.0061), total sleep duration (rho=0.638, p<0.0001), quality of sleep (rho=0.629, p<0.0001), well-being during the day (rho=0.34, p=0.029) and sleepiness during the day (rho=0.51, p=0.007). High AL were correlated with 2.83-fold increased SleD risk (p=0.04).
Mean scores on Eating Attitudes Test-26 (EAT-26) were significantly higher in females with FHA (p<0.0001). Women with FHA were characterised by significantly higher scores at the sub-scale items of dieting (p=0.03) and bulimia & food preoccupation (p<0.001) compared to healthy controls. Significant difference was also observed between the mean scores of the two groups in all other questionnaires: State-Trait-Anxiety-Inventory (STAI) (p<0.0001), Multidimensional Body-Self-Relations Questionnaire (MBSRQ) (p<0.0001) and International Physical Activity Questionnaire (IPAQ) (p=0.004). EAT-26 scores were positively correlated with scores on STAI (rho=0.26, p=0.04), MBSRQ (rho=0.79, p<0.0001) and IPAQ (rho= 0.35, p=0.03). High scores on IPAQ and STAI were correlated with a 12.2-fold (p=0.008) and 4.3-fold (p=0.04) increased risk for high scores on EAT-26, respectively.
Eight novel PROKR2 mutations were identified in five of the 41 women with FHA, either in heterozygous state (two cases) or in homozygous or compound heterozygous state (three cases) : g. 241 G>C, g. 296 G>T, g. 375 A>G, g. 404 A>G, g. 404 C>G, g. 410 C>A, g. 421 G>A, and g. 475 G>T. Furthermore, three PROKR2 heterozygous mutations were identified in three of 23 women with IHH: g. 246 C>T, g. 252 C>T and g. 259 G>C. No mutations were found in the cohort of 20 controls with normal menstrual cycles.
Disordered eating behaviours (DEBs) may occur in FHA populations at a higher frequency compared to the general population. Anxiety and overweight preoccupation may underlie and independently contribute to development and maintenance of both DEBs and FHA. Furthermore, FHA women are seemingly more prone to SleD and those with SleD suffer from higher AL. In view of this evidence, the potential rationale of adding psychological and SleD evaluation to their clinical care is highlighted. Finally, novel mutations in PROKR2 gene were found in women with FHA, suggesting that these variants may contribute to an extent to the variable vulnerability of women to functional changes in GnRH secretion. Identification of gene mutations underlying FHA may lead to new pathophysiology insights, improved diagnostics, and novel treatment approaches. Further research to ascertain the functional impact of these variants is warranted. Differences in the genetic backgrounds of various ethnic populations should also be considered.
Keywords:
Functional hypothalamic amenorrhoea, Prokineticin receptor 2, Mutations, Psycho-physiological factors, pathophysiology
