Retrieval of trophoblast cells from the cervical canal for prediction of abnormal pregnancy: a pilot study

Abstract

Background: Fetal cells are shed from the regressing chorionic villi and it is possible to retrieve extravillous cytotrophoblast cells by transcervical sampling. The abundance of trophoblast cells in transcervical samples suggests that this non-invasive approach could distinguish between normal and abnormal pregnancies, such as an ectopic pregnancy (EP) and blighted ovum (BO). We aim to identify and quantify fetal trophoblast cells in the cervical canal during the first trimester to assess their usefulness to predict an abnormal pregnancy.

Methods: Patients, age 18-45, presenting with a normal intrauterine pregnancy (IUP; n = 37), diagnosis of EP (n = 10) or BO (n = 5) were enrolled for collection of transcervical specimens using a cytobrush and fixative rinse. Non-pregnant, nulliparous women (n = 7) were included as negative controls. Cells were cleared of mucus by acidification, prepared on microscope slides and labeled with a monoclonal antibody recognizing the trophoblast marker, human leukocyte antigen (HLA)-G. HLA-G positive and negative cells were counted to calculate the ratio of trophoblast cells to total cervical cells.

Results: Trophoblast cells were observed in 35/37 normal IUP, 6/10 EP and 4/5 BO specimens. The average frequency of HLA-G positive cells in the normal IUP cervical samples was approximately 1 in 2000, which was 4-fold higher than samples from patients with EP or BO (P < 0.001). Receiver operating characteristic analysis showed that EP and BO pregnancies were distinguishable from normal pregnancies with 93% sensitivity, 95% specificity, 97% positive predictive value and 87% negative predictive value.

Conclusions: This pilot study presents evidence that trophoblast cells can be reliably obtained and identified among cervical cells in the first trimester by immunohistochemical staining for HLA-G, and suggests for the first time that abnormal pregnancies may be predictable based on the abundance of trophoblast cells in the cervical canal.

Figure 1

Identification of cytotrophoblast cells in cervical samples by immunofluorescence microscopy. Cervical cells were labeled with antibodies against HLA-G (A, C; red) and cytokeratin 7 (B, D; green), as well as with DAPI to stain nuclei (shown in all panels; blue). Cells were identified as cytotrophoblasts by their labeling for both HLA-G and cytokeratin (indicated with arrows). Resident cervical cells appear as DAPI stained cells that were not labeled by either antibody (indicated by asterisks). Bar, 50 µm.

Figure 2

Immunohistochemical detection of cytotrophoblast cells in cervical specimens labeled with anti-HLA-G. Slide preparations were made from cervical samples obtained from nulliparous women who were not pregnant (row A), as well as from women with normal intrauterine pregnancy (row B), tubal ectopic pregnancy (row C) or blighted ovum (row D). Each panel displays cells from a different patient. Antibody binding appears as a dark brown precipitate around HLA-G positive cells (indicated with arrows). Cells were counterstained with hematoxylin. Bar, 100 µm.

Figure 3

Frequency of trophoblast cells in cervical samples. Cervical specimens collected from non-pregnant women (n = 7) or pregnant women with a normal intrauterine pregnancy (IUP; n = 37), ectopic pregnancy (EP; n = 10) or blighted ovum (BO; n = 5) were labeled with antibody to HLA-G and counterstained with hematoxylin. The number of trophoblast cells per total cervical cells (trophoblast frequency) was determined and the means with SE (error bars) are shown for each group. Bars labeled with different letters are significantly different (P < 0.05).

Figure 4

Receiver operating characteristic curve for the predictive value of endocervical trophoblast cell frequency. The ability of trophoblast cell frequency to predict a normal intrauterine pregnancy was tested against abnormal pregnancies (ectopic pregnancy or blighted ovum).

Figure 5

Influence of gestational age on the endocervical trophoblast cell frequency. Trophoblast frequencies for individual women with normal intrauterine pregnancy (open circles), ectopic pregnancy (closed diamonds) or blighted ovum (closed triangles) are shown with respect to the gestational age at the time of collection. A linear regression line drawn through the normal intrauterine pregnancy values is shown and had an R² value of 0.005.