GnRH Agonist-Induced Ovarian Suppression and Ovarian Steroids in PMDD and Controls

In this study, lymphoblastoid cell line cultures (LCLs) from women with premenstrual dysphoric disorder (PMDD) and asymptomatic controls were compared via whole-transcriptome sequencing (RNA-seq) during untreated (ovarian steroid-free) conditions and after estradiol and progesterone treatment. In untreated LCLs, pathway analysis of the LCL transcriptome revealed, among others, over-expression of ESC/E(Z) complex genes (an ovarian steroid-regulated gene silencing complex) with significant overexpression of MTF2, PHF19, and SIRT1 (P<0.05) in cells from women with PMDD compared with controls. Finally, mRNA expression of several ESC/E(Z) complex genes were increased by progesterone in controls only, and decreased by estradiol in PMDD LCLs. ]]> Participant Selection: Women between the ages of 18 and 48 years who were medication-free, with regular menstrual cycles (range, 21 to 35 days), not medically ill and not pregnant were included in the clinical study. Women with PMDD were self-referred in response to newspaper advertisements or were referred by their physician. The diagnosis of PMDD was confirmed prospectively prior to enter into this study by self-administered symptom ratings (a 100mm visual analog scale) completed daily over three consecutive menstrual cycles. In addition to meeting DSM IV criteria for PMDD, each woman had an increase of at least 30 percent (relative to the range of the scale she used) in average self-ratings of negative moods (irritability, depression, anxiety, and mood stability) in the seven days before menses compared with the ratings for the seven days after the end of menses, in at least two of the three baseline cycles. These are more stringent criteria than those of DSM-IV or V. Women without PMDD (control women) were recruited, and studied, in parallel. Absence of premenstrual mood symptoms was confirmed using the same daily rating scales during a two-month baseline period. Women with PMDD had no other current Axis I psychiatric diagnosis within the past two years per Structured Clinical Interview for DSM-IV (SCID), while control women had neither current nor past Axis I diagnosis as confirmed by SCID. All women received remuneration according to guidelines from the NIH Healthy Volunteer Office. The study protocol was reviewed and approved by the National Institute of Mental Health Institutional Review Board, and all women gave written informed consent. Clinical Hormone Manipulation Protocol: The hormone manipulation protocol has been previously described (Schmidt et al, NEJM, 1998) and is briefly presented here. All participants received six monthly injections of the GnRH agonist leuprolide acetate, Lupron (3.75 mg IM q monthly). After three months of leuprolide alone, all participants (while continuing to receive monthly leuprolide injections for an additional three months) were randomly assigned in double-blind, crossover fashion to receive either estradiol or progesterone replacement lasting for five weeks each (with a two-week washout between hormone administration periods). We employed this GnRH agonist, ovarian suppression protocol to first confirm the ovarian steroid phenotypes that were the focus of this study; i.e., women with GnRH agonist-induced remission and ovarian steroid-triggered recurrence of PMDD symptoms and those with no mood symptoms during the same hormone manipulation (i.e. control women). Symptom ratings and criteria for response: Symptom-rating forms were completed daily by all women prior to the study and during the six-month Lupron protocol. Significant recurrence of PMDD symptoms was defined by a weekly average score of greater than three (moderate severity) in irritability, anxiety, or sadness. Controls were defined by absence of affective symptoms throughout the 6 month hormone manipulation protocol (i.e., no weekly average score >2). Selection of participants for generation of lymphoblastoid cell lines (LCLs): Two main sets of lymphoblastoid cell lines were created for this study: a focus set and a larger replication set. For the focus set, we selected from participants who completed participation in the GnRH agonist-induced hormone manipulation protocol. Group 1 (n=10) consisted of women with PMDD based on suppression of PMDD symptoms during leuprolide treatment and recurrence of symptoms during either the E or P add-back conditions. Group 2 (n=9) consisted of women without PMDD (controls) based on absence of symptoms throughout the entire protocol. Group 1 and Group 2 were matched for age and ethnicity and compared against each other within experimental protocols (no significant differences in Age (t17 =1.3, p=0.03) or BMI (t17=0.2, p=0.8). For the larger replication set, we selected 29 PMDD women (Group 3) and 30 control women (Group 4) who did not necessarily participate in the hormone manipulation protocol. Some women in groups 3 and 4 had participated in the GnRH agonist study, but their participation was not an inclusion criterion. These women underwent the same screening as those in Groups 1 and 2, and completed the DRF to confirm the diagnosis or absence of PMDD according to the DSM V. Thus, Group 3 represents an expanded PMDD phenotype, not necessarily confirmed as hormone-triggered. Nonetheless, we predict based on clinical studies, and observation, that approximately 70% of the women ascertained as PMDD will experience symptom remission with GnRH agonist treatment, and 90% of control women would not develop clinically significant mood symptoms on leuprolide (Lupron). They were also matched for ethnicity, although there was a significant difference in age (t57=4.4, p<0.0001), but not BMI (t57=1.8, p=0.08). ]]> Participants in this study were enrolled in NIMH Protocols NCT00001259 and NCT00001322 within the Intramural Research Program of the NIMH and NIAAA, NIH. NIMH Project # MH002865; NIAAA Project # AA000301. Samples from women with PMDD and controls were collected from 1997-2010 (mean 2003) and year of collection and subsequent storage did not significantly differ between the two groups. Nor did year of collection predict levels of gene expression in the ESC/E(Z) complex genes. ]]>