Collection of samples

The study was approved by the internal review board of the Instituto Nacional de Perinatología Isidro Espinosa de los Reyes in Mexico City (register 212250-02141). Patients provided written informed consent for the collection of samples and subsequent analysis. All patients included in this study lived in Mexico City, they were Hispanic and of medium socioeconomic status. Specific information from each patient is described in the Table . Patients with history of preterm delivery, hypertension, diabetes mellitus, thyroid, liver, or choric renal diseases were excluded.

Fetal membranes were collected from women who delivered by elective cesarean section, without evidence of active labor and with neither clinical nor microbiologic signs of chorioamnionitis or lower genital tract infection. General microbiologic cultures were conducted on the placenta and fetal membranes immediately after delivery, a sterile swab was rolled across randomly selected areas and only membranes with proven sterility were used for this study. The specimens were transported to the laboratory in sterile Dulbecco’s modified Eagle medium (DMEM; Gibco BRL, Bethesda, MD), and rinsed in sterile phosphate-buffered saline solution to remove adherent blood clots.

Fetal membrane explants

The membranes (including amnion and choriodecidua) were cut into disks (12-mm diameter) using a biopsy punch. We did not capture areas near to the placenta. One piece of membrane was placed in each well of a 24-well tissue culture plate with 1.0 mL of DMEM supplemented with 0.1% lactoalbumin hydrolysate, one mM sodium pyruvate, and antibiotic-antimycotic solution (penicillin 100 U/mL; streptomycin 100 μg/mL; amphotericin B 0.25 μg /mL). The explants were cultured in triplicate at 37°C under an atmosphere of 5% CO ₂ /95% air and saturated water, separate cultures were performed for each time point. All reagents for cell culture were purchased from Gibco BRL. All explants were routinely followed for cell viability using the XTT Assay (R&D Systems, Minneapolis, MN).

PRL’s effect on IL-1β, TNF-α, and MMP-9 secretion

To characterize the effects of PRL on the basal secretion and modulation of the different mediators, the explants were incubated alone or in the presence of 500 ng/mL human recombinant PRL (Sigma, St. Louis, MO), and in vitro secretion of IL-1β, TNF-α, and MMP-9 were plotted on a time-response curve at 4, 8, 24, 28, 32, and 48 hours.

In a different set of experiments, another set of explants from the same 15 women were incubated with 500 ng/mL PRL for the first 24 hours, and then conditioned media was eliminated and the explants were rinsed with phosphate-buffered saline solution Hanks solution and maintained for an additional incubation period of 24 in DMEM with 0.1% lactoalbumin hydrolysate (Gibco BRL) without PRL. Other explants were incubated during the first 24 hours in the absence of PRL and then, 500 ng/mL PRL was added, and the culture was maintained for an additional 24 hours.

A function-blocking anti-PRL antibody (MCA714 clone; Serotec, Oxford UK) was used as a control for the effect of PRL addition. The antibody was mixed (2.5 μg/mL) for 1 hour at 37°C with the media containing PRL and then added to selected independent experiments.

ELISA

IL-1β, TNF-α, MMP-2, and MMP-9 concentrations were measured in all conditioned media using specific DuoSet enzyme-linked immunosorbent sandwich assays (R & D Systems, Minneapolis, MN). Monoclonal antibodies against, IL-1β, TNF-α, MMP-2 and MMP-9 were used as the capture antibodies, and polyclonal biotinylated antibodies against both cytokines were used as the detection antibodies. For the IL-1β assay, a standard curve was developed from 4 to 260 pg/mL, and the sensitivity was 2 pg/mL. The TNF-α assay was linear from 15 to 960 pg/mL with a sensitivity of 5.0 pg/mL. The MMP-2 assay measured both active and proenzyme in a range from 12 to 2000 pg/mL. The MMP-9 assay measured the proenzyme, the active forms and MMP-9 bound to TIMP1. The assay was linear from 36 to 2000 pg/mL. The concentrations of IL-1β, TNF-α, MMP-2. and MMP-9 were normalized by μg of protein.

Total RNA isolation, reverse transcription, and real-time polymerase chain reaction for IL-1β, TNF-α, and MMP-9

We selected the 32-hour point in the experiments containing PRL, and we evaluated the effect of PRL on the respective gene expression. Total RNA was extracted with Trizol (Invitrogen, Carlsbad, CA). cDNA was synthesized with the Transcriptor First Strand cDNA Synthesis kit using random hexamers as directed by the manufacturer. Reverse transcription was carried out at 25°C for 10 minutes, followed by 30 minutes at 55°C and 85°C for 5 minutes.

Quantitative real-time polymerase chain reaction was performed in a Light Cycler 480 instrument (Perkin Elmer) using the Probes Master kit and TaqMan probes (hydrolysis probes labeled with fluorescein) according to the manufacturer’s recommended protocol (Roche Applied Science, Mannheim, Germany). The primers for IL-1β, TNF-α, and MMP-9 were described in a previous publication. The β-actin gene was used as a reference for normalization purposes. The relative gene expression was transformed to 2 ^ˆ ddCt using the control, 0 hour time point and β-actin as references.

Statistical analyses

For each variable, descriptive statistics (mean, standard deviation, standard error, median, and range) were obtained. A general linear model using variance analysis of repeated measurements between groups was used to compare the effect of PRL. Student’s t test was used to compare relative expression of IL-1β, TNF-α, and MMP-9 mRNA in the presence of PRL. All statistical analyses were performed using SPSS 20 (IBM Corp, Armonk, NY).