Study Design And Subjects
The study has been designed according to the CONSORT methodology [20]. The randomized superiority double blinded placebo controlled trial involved two Italian ART clinics (IRCCS Fondazione Ca’ Granda, Ospedale Maggiore Policlinico, Milan and IRCCS San Raffaele Scientific Institute, Milan). The study has been conducted in accordance to the ethical principles of the Helsinki Declaration guidelines. The study was approved by the Ethical Committees of the two participating centers (Comitato Etico Area B, Milan, protocol n° 602 05/04/2016 and Comitato Etico Istituto di Ricovero e Cura a Carattere Scientifico - Ospedale San Raffaele, protocol n° 189/2016). The study was approved by the Italian Medicines Agency (AIFA) (Protocol AIFA/RSCP/P/65768) and registered (EUDRACT 2015-004233-27).
Selected population were infertile patients undergoing ART cycles with insufficient serum levels of Vitamin D (25-hydroxyvitamin D serum level < 30 ng/ml) according to the most recent International Guidelines [21]. Inclusion criteria were female age between 18–39 years, with body mass index (BMI) between 18 and 25 Kg/m2 undergoing autologous ART cycles with less than three previous cycles. Exclusion criteria included contraindications/side effects for consumption of Vitamin D, anti-mullerian hormone serum level < 0.5 ng/ml and ART cycles with surgical retrieval of the spermatozoa or frozen gametes.
Written informed consents were obtained from eligible patients. The first 50 eligible women that agree to participate to both the randomized double-blinded clinical and the uterine fluid collection were enrolled. In one patient, the uterine fluid collection did not result in an adequate sampling. Researchers were unaware of which treatment was being provided to enrolled women. At the time of randomization, from 2 to 12 weeks before oocyte retrieval, women received a single dose of 600,000 IU of 25-hydroxyvitamin D or placebo. Randomization was performed centrally by Fondazione IRCCS Ospedale Maggiore Policlinico. The allocation sequence were computer-generated and hidden from the participants as well as from the physicians and biologists involved in the clinical management of the patients.
The two centers followed their own standard regarding ovarian stimulation, ART laboratory procedures and endometrial preparation as described elsewhere [22–24]. Serum 25-hydroxyvitamin D was assessed at the time of hCG administration. All Vitamin D levels measurements was performed with a commercially available kit (DiaSorin). Clinical pregnancy was defined after the ultrasound presence of at least one intrauterine gestational sac with viable fetus.
Endometrial Secretion Aspiration
Uterine fluid samples were collected during the secretory phase of the menstrual cycle that proceeded the oocyte retrieval. Dating was estimated according to the previous cycles and to the presence of a corpus luteum cyst at ultrasound. After insertion of a sterilized speculum, vaginal secretions were cleaned by cotton buds. The uterine flushing was performed by using a disposable catheter for sonohysterography with a balloon opening the cavity when it is inserted in the uterus to minimize vaginal contamination (Wallace® Trial Transfer Catheter, CooperSurgical Fertility & Genomic Solutions, Denmark). To get representative sampling of uterine secretion, 1.5 mL of physiologic solution was injected and gently suctioned. Samples were immediately centrifuged at 1200xr.p.m. for 15 min in order to separate cell debris, mucus and minimal blood contamination from liquid fraction. The liquid fractions were stored at − 80°C. After thawing, total protein concentration was measured by Bradford assay (Quick Start™ Bradford, Bio-Rad) for normalization purposes.
Determination of endometrial Secretion cytokine concentrations with bead-based multiplex immunoassays
After thawing, the quantitative determination of IL-1beta, IL-1Ralpha, IL-2, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17A, IFN-alpha, TNF-alpha, G-CSF, GM-CSF, VEGF, PDGF, FGF, IP-10, MCP-1, RANTES, eotaxin, MIP-1-alpha, and MIP-1-beta in endometrial secretion samples was performed by using a bead-based multiplex immunoassay (Biorad Laboratories, Hercules, CA, USA) and the Bioplex 200 system (Biorad Laboratories, Hercules, CA, USA), as we have previously described elsewhere [24]. In brief, in a 96-well filter plate (Bio-Rad) 50µl of each serum sample were added to 50 µl of antibody-conjugated beads directed against the cytokines listed above (Bio-Rad). After a 30-min incubation, the plate was washed and 25 µl of biotinylated anti-cytokine antibody solution were added to each well before another 30-min incubation. The plate was then washed and 50 µl of streptavidin-conjugated phycoerithrin were added to each well. After a final wash, each well was resuspended with 125 µl of assay buffer (Bio-Rad) and analyzed by Bioplex 200 (Biorad Laboratories, Hercules, CA). Standard curves were derived from various concentrations of the cytokine standards and followed the same protocol as the endometrial secretion samples. The concentration of the 27 cytokines (pg/ml) in each endometrial secretion sample was calculated thanks to the Bioplex 200 software.
The lower detection limits of the multiplex immunoassay kit were as follows: Interleukin (IL)-1β (0.39 pg/ml), IL-1RA (42.01 pg/ml), IL-2 (0.33 pg/ml), IL-4 (0.1 pg/ml), IL-5 (0.86 pg/ml), IL-6 (0.22 pg/ml), IL-7 (29.25 pg/ml), IL-8 (1.09 pg/ml), IL-9 (2.28 pg/ml), IL-10 (0.98 pg/ml), IL-12 (0.23 pg/ml), IL-13 (0.09 pg/ml), IL-15 (8.96 pg/ml), IL-17 (1.7 pg/ml), Eotaxin (0.13 pg/ml), Fibroblast Growth Factor (FGF) (2.62 pg/ml), Granulocyte-colony stimulating factor (G-CSF) (2.33 pg/ml), Granulocyte-macrophage colony-stimulating factor (GM-CSF) (0.43 pg/ml), Interferon (IFN)-γ (0.2 pg/ml), IFN-γ-inducible 10 kDa protein (IP-10) (5.64 pg/ml), Monocyte chemo-attractant protein-1 (MCP-1) (0.45 pg/ml), Macrophage Inflammatory Protein(MIP)-1α/CCL3 (0.05 pg/ml), MIP-1β/CCL4 (0.46 pg/ml), Platelet-Derived Growth Factor-BB (PDGF-BB) (4.56 pg/ml), (4.56 pg/ml), Chemokine ligand 5 also known as regulated on activation, normal T cell expressed and secreted (RANTES) (3.76 pg/ml), Tumor Necrosis Factor-α (TNF-α) (3.28 pg/ml), Vascular Endothelial Growth Factor (VEGF) (10.38 pg/ml). Relative concentration of uterine mediators was performed based on total protein concentration of each sample measured by Bradford assay (Quick Start™ Bradford, Bio-Rad).
Statistical Analysis
All data were initially examined for normality using the Kolmogorov Smirnov test: the normal distributed data was analyzed with the Student’s t test, while the not normal data was analyzed with the Mann-Whitney test. The frequency of patients’ characteristics was analyzed with the chi-square test. Data are presented as Number (%), Mean ± SD, Median [IQR] as described in each legend. Significance was set at p < 0.05. Soluble mediators in endometrial secretion aspiration in which more than 50% of the samples were detectable were analyzed as continuous variables (Median [IQR]) and the difference between the two groups was tested by Mann-Whitney test. For the remaining molecules, a dichotomous analysis (presence or absence in the sample) was carried out and they were tested by chi-square test.