Subject selection

We conducted a case-control study examining archived placental tissue and patient records from pregnancies with and without Trisomy 13 for the past 10 years. Cases (Trisomy 13 placentas) and control (Trisomy 21 and euploid) placentas of patients undergoing chorionic villous sampling at Beth Israel Deaconess Medical Center (BIDMC, Boston, MA) were included. The Institutional Review Board at BIDMC approved this study (2009-P-000413; BIDMC).

The following clinical data were obtained from each patient’s medical record: the subject’s age, estimated gestational age, medical history, body mass index, karyotype, and blood pressure. All pregnancies included in this study resulted in elective termination or spontaneous miscarriage. In the euploid group, 3 patients had elective terminations and 3 had spontaneous abortions (2 patients at 13 weeks 4 days and 1 patient at 13 weeks 3 days). The elective terminations were for the following reasons: 1 after rupture of membranes after chorionic villous sampling, 1 for omphalocele, and 1 for cystic fibrosis. In the Trisomy 21 group, 9 patients had elective terminations (for Trisomy 21), and 2 patients had spontaneous abortions at 18 weeks 0 days and 13 weeks 1 day. All of the Trisomy 13 patients were elective terminations for Trisomy 13. All cases and controls were confirmed by karyotype from chorionic villous sampling performed early in the pregnancy.

The placental tissue was collected at the time of pregnancy termination and processed in the standard fashion through the Pathology Department. The average storage time for placental tissue at our institution is 10 years. The pathologic diagnosis was available for all samples. Control subjects who smoke, had multiple gestations, and those with medical comorbidities known as risk factors for preeclampsia such as diabetes mellitus, systemic lupus erythematosis, chronic hypertension, and chronic renal disease were excluded. Placental weight was unavailable for both cases and controls.

Morphologic evaluation

Formalin-fixed, paraffin-embedded tissues were processed and stained with hematoxylin and eosin (H&E) using routine techniques. Immunoperoxidase studies to identify sFlt-1 expression were performed using a goat antihuman vascular endothelial growth factor receptor-1/Flt-1 antibody that identifies the N-terminal region of both sFlt-1 and membrane-bound Flt-1 (catalog number AF321; R&D Systems, Minneapolis, MN). A single preparation was made using a primary antibody dilution of 1:200 as described in a previous study. Binding of the antibody to its antigenic sites in sections was detected using ImmPRESS anti-goat staining kit (catalog number MP-7405; Vector Laboratories, Burlingame, CA). Antigen retrieval was performed using citrate buffer pH 6.0 and pressure heating. The antigen-antibody reaction sites were visualized using 3,3-diamonobenzidine for 5 minutes, and subsequently, sections were counterstained with Harris hematoxylin.

Evaluation of the H&E and immunoperoxidase sections was performed by a single pathologist (I.E.S.) in a blinded fashion as previously reported. Grading of villous trophoblast sFlt-1 staining was done using the semiquantitative ordinal scale as follows: 1+ (strong focal less than trophoblast staining), 2+ (<50% of the villous trophoblast showing staining), 3+ (51-90% staining), and 4+ (91-100% staining). Weak staining was considered negative. The median staining intensity grade was compared among the groups using the Kruskal-Wallis test. Individual groups were compared with each other using the Wilcoxon rank sum test. A P < .05 was considered significant. Statistical analysis was performed with the use of software (Stata 11 SE; StataCorp LP, College Station, TX).