2.3 Gingival crevicular fluid sample collection and elution protocol:
Oral examination and the collection of GCF samples were performed between 11 0/7 and 13 6/7 weeks of gestation. After a tooth was isolated with a cotton roll, the supragingival plaque was removed with curettes but without contacting the gingival margin. The gingival sulcus was then gently dried with an air syringe. GCF was collected using paper strips. The strips were placed into the sulci/pocket until mild resistance was sensed and left in place for 30 seconds. Strips contaminated by saliva or blood were excluded from the study. After GCF collection, the strips were placed into an Eppendorf tube containing 100 μl of phosphate-buffered saline with 0.05% (v/v) Tween-20. GCF was eluted from the strips by centrifugation (10,000 g for 5 min). This procedure was repeated twice. GCF samples were obtained from four periodontal sulcus/pockets (1 x quadrant) at the most affected periodontal site, representative of the periodontal diagnosis of the patient, and then the strips were pooled to make one sample119.