Objective
Increased levels of soluble fms-like tyrosine kinase (sFlt-1) in Trisomy 13 pregnancies are thought to be mediated by the placenta. This study aimed to compare sFlt-1 expression in Trisomy 13 (n = 7) placentas with that in control placentas (Trisomy 21, n = 11, and euploid, n = 6).
Study Design
This was a retrospective case-control study analyzing paraffin-embedded placental blocks that were stained with hematoxylin and eosin and antibodies to sFlt-1. Their staining intensity was compared using a semiquantitative technique. The Kruskal-Wallis test and Wilcox rank sum test were used for statistical analysis.
Results
The median staining was significantly higher in Trisomy 13 compared with control specimens ( P = .008) (for Trisomy 13 vs Trisomy 21, P = .003, and Trisomy 13 vs euploid, P = .004).
Conclusion
Our study demonstrates that Trisomy 13 placentas express more sFlt-1 than control placentas. These results strengthen the hypothesis that the increased incidence of preeclampsia in Trisomy 13 pregnancies is secondary to placental up-regulation of sFlt-1.
Trisomy 13, also known as Patau syndrome, occurs in about 1 in every 5000 live births. It is a devastating clinical entity characterized by an extra copy of chromosome 13 and is defined by a wide array of congenital malformations with very high rates of fetal deaths and less than 10% survival ex utero. The different types of associated congenital malformations vary widely, but the most recognizable ones are holoprosencephaly, cleft lip and palate, polydactyly, and rocker bottom feet. Trisomy 13 is largely caused by nondisjunction of chromosome 13 but can also be the result of a balanced translocation.
Studies have shown that pregnancies complicated by Trisomy 13 have an increased incidence of preeclampsia (PE), with an estimated increased risk ranging from 2- to 20-fold with 25-40% of Trisomy 13 pregnancies being afflicted by the disease.
PE is a pregnancy-specific disorder characterized by hypertension and proteinuria. The placenta has long been implicated in its pathogenesis because PE resolves with the delivery of the placenta and can occur in women with molar pregnancies. Patients with PE are thought to have abnormal placental implantation and subsequent release of circulating factors, which cause systemic vascular dysfunction and are responsible for the maternal syndrome. There is growing evidence that the vascular injury is the result of an imbalance of angiogenic proteins released by the placenta. Specifically, increased levels of circulating antiangiogenic proteins such as soluble fms-like tyrosine kinase (sFlt-1) are seen in women with PE at the time of clinical disease.
The association of pregnancies with Trisomy 13 and PE has not been clearly understood; however, a recent study showed that women with Trisomy 13 have similar angiogenic abnormalities as women with PE. Furthermore, this study found that in early pregnancy, women with Trisomy 13 gestations had higher median circulating sFlt-1/placental growth factor (PlGF) ratios as compared with normal controls. In contrast, the median sFlt-1/PlGF ratio in Trisomy 18 and 21 were not significantly different from control samples. Interestingly, the locus for the sFlt-1 gene lies on chromosome 13 (13q12). This suggests the possibility that the extra copy of chromosome 13 may lead to increased production of sFlt-1 and therefore increased release into the circulation of women carrying Trisomy 13 pregnancies, thereby accounting for their increased risk of PE. Other chromosomal abnormalities, such as Trisomy 18 or Trisomy 21, have PE rates that are similar to the general population.
We hypothesized that the increased levels of circulating sFlt-1/PlGF seen in women with Trisomy 13 pregnancies originates from the placenta, and we aimed to test whether these placentas have increased staining for sFlt-1 as compared with Trisomy 21 and euploid placentas.
Materials and Methods
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).
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