Patients and samples
This case–control study was performed between March 2013 and February 2017 at the Recurrent Pregnancy Loss Outpatient Clinic of the Gemelli Hospital of Rome, Italy. The study population included 70 women with history of idiopathic RPL with three or more spontaneous consecutive pregnancy losses (≤ 12 weeks of gestation) clinically documented by ultrasonography [1] at least 6 months before the study and 30 healthy women with two or more previous uncomplicated term pregnancies (control group).
The inclusion criteria for both groups were as follows: caucasian, age ≤ 39 years, healthy, regular ovulatory cycles (28–32 days), normal serum levels of follicle-stimulating hormone (FSH < 10 mIU/ml), luteinizing hormone (LH < 10 mIU/ml) and anti-mullerian hormone (AMH > 2 ng/ml) on day 3 of the menstrual cycle, absence of abnormal ovarian and endometrial ultrasonographic features, no use of any contraceptive drugs or intrauterine device in the last 6 months. Exclusion criteria were: smoking, alcohol consumption, presence of abnormalities at the screening for RPL (anatomical abnormalities, endometrial cavity anomalies assessed by hysteroscopy, luteal phase deficiency, hyperprolactinemia, hyperinsulinemia or insulin resistance, endocrine disorders, vaginal infections, karyotype anomalies, thrombophilic disorders, autoimmune diseases), obesity, endometriosis, refusal to undergo intestinal permeability test, any pathological conditions potentially interfering with intestinal permeability, like celiac disease or gluten sensitivity, history of viral hepatitis, diarrhea, diverticulosis, irritable bowel syndrome, inflammatory bowel diseases, or bariatric or other abdominal surgery, immunoglobulin A or immunoglobulins deficiency, impaired renal function, use of non-steroidal anti-inflammatory drugs, antibiotics, probiotics, or anti-secretory drugs within the 3 months preceding enrollment.
Samples collection
Peripheral blood samples (5 ml) from all RPL (n = 70) and healthy control women (n = 30) enrolled were collected by vein puncture. Samples were spun at 3000 g at 4 °C for 20 min, then the supernatant were aliquoted and stored at − 80 °C until use.
Thirty-five of seventy RPL women and twenty of thirty controls accepted a diagnostic mini-hysteroscopy during the putative window of implantation (days 19th to 24th), with endometrial biopsies. The biopsies timing was chosen according to the last menstrual period, monitoring the follicle size by transvaginal ultrasound and then confirmed by histologic assessment [15]. All women were advised to avoid sexual intercourses from menses to planned hysteroscopic procedure. Serum progesterone and β-hCG levels were determined just before hysteroscopy. Endometrial biopsy were performed using a 3-mm Novak curette, avoiding any contact between the curette and the vaginal walls, and then immediately washed in normal saline solution and stored at − 80 °C.
Biopsies were also analysed for Chlamydia trachomatis, Mycoplasma, Ureaplasma urealyticum, Neisseria gonorrheae or yeast colonization. Patients with endometrial samples positive for pathogens colonization as well as those with evidence of chronic endometritis at hysteroscopy were not included in the study.
Anxiety and depression state evaluation
Acute or stable anxiety of enrolled women was assessed by administration of the State-Trait Anxiety Inventory (STAI) Y test [16, 17], divided in two parts: the first evaluating the anxiety state by inquiring current emotional state asking twenty questions with responses ranging from ‘‘not at all’’ (score 1) to ‘‘extremely’’ (score 4) on a 1–4 Likert scale. Total scores ranged from 20 to 80, with higher scores indicating higher degrees of state anxiety. The second part of STAI Y test assesses trait anxiety by asking to subjects to describe how they usually feel. Twenty questions were asked to enrolled patients with responses ranging from ‘‘almost never’’ (score 1) to ‘‘almost always’’ (score 4) on a Likert 1–4 scale. Higher scores reflected greater symptomatology and a suggested cutoff of 39 was established for clinically significant anxiety (score: 40–50 mild; 50–60 moderate; > 60 severe [17]. STAI -Y questionnaires were completed in approximately 20 min.
Depressive symptomatology was investigated with the use of the Zung Self-Rating Depression Scale (Z-SDS) [18], a short self-administered survey. The Z-SDS scale consists of 20 items rating the four common characteristics of depression: (1) the pervasive effect, (2) the physiological equivalents, (3) other disturbances and (4) psychomotor activities. Each question is scored on a scale of 1–4 [corresponding to (1) a little of the time, (2) some of the time, (3) good part of the time and (4) most of the time]. The scores range from 25 to 100 to be set on a depression scale with higher scores reflecting greater symptomatology: 25–49 Normal Range; 50–59 Mildly Depressed; 60–69 Moderately Depressed; 70 and above Severely Depressed.