Immunohistochemical Localization of Placental Hormones as Markers for ... and human placental lactogen (hPL) in tissue obtained by endometrial curetting to.
International Journal of Surgical Pathology http://ijs.sagepub.com/
Immunohistochemical Localization of Placental Hormones as Markers for Differentiating Uterine Abortion vs Ectopic Pregnancy Janelle K. Strom, Chhanda Bewtra and Gilles R. G. Monif INT J SURG PATHOL 1993 1: 51 DOI: 10.1177/106689699300100107 The online version of this article can be found at: http://ijs.sagepub.com/content/1/1/51
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Immunohistochemical Localization of Placental Hormones as Markers for Differentiating Uterine Abortion vs Ectopic Pregnancy Janelle K. Strom, M.D., Chhanda Bewtra, M.D.,* and Gilles R. G. Monif, M.D.
The ability of immunohistochemical staining for human chorionic gonadotropin (hCG) and human placental lactogen (hPL) in tissue obtained by endometrial curetting to distinguish first trimester spontaneous abortions from ectopic pregnancy (EP) was evaluated. Material from 25 patients with uterine bleeding during the first trimester and whose specimens did not contain chorionic villi (CV) on routine histology was stained, and positive results for one or both placental hormones were correlated with subsequent clinical confirmation. Results showed immunohistochemical presence of trophoblasts containing hCG and/or hPL in eight (75%) of the intrauterine pregnancy cases (IUP) with no CV identifiable in routine histological analysis. Human chorionic gonadotropin and hPL were positive in all (100%) cases of IUP with CV (positive control) and negative (0%) in all EP (negative control) cases (P 0.044). Increased acute inflammation (66% vs 30%, P .163), vascular wall thickening and occlusion vs P and decreased Arias-Stella cell changes (50% vs 61%, P 53.8%, (83% .25), .859) in IUP cases were noted, although none were significant. While the specificities of hCG/hPL positivity and CV are 100% in diagnosing IUP, the sensitivity of CV alone was 45.4% and hCG/hPL positivity was 81.7%. Therefore, in suspected IUP cases, where no CV are seen, hCG and hPL immunostaining may aid in differentiation from EP. Int J Surg Pathol 1(1): 51-56, 1993 Key words: human chorionic gonadotropin, human placental lactogen, ectopic pregnancy, abortion. =
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The differentiation of abnormal uterine bleeding due to spontaneous intrauterine abortion from that due to ectopic pregnancy (EP) is contingent on the demonstration of fetal or placental tissue (chorionic villi [CV] and/or trophoblasts) within passed or curetted endometrial specimens. While the demonstration of CV is diagnostic, its absence cannot be used to definitely exclude intrauterine gestation.22 The presence of trophoblasts (cytotrophoblasts, syncytiotrophoblasts, or intermediate trophoblasts) in From the
the curettings also indicates intrauterine gestation. Kurman et al. have described the intermediate trophoblast as a mononucleate trophoblastic cell that infiltrates the endomyometrium at the placental site and represents a transitional stage between cytotrophoblast and syncytiotrophoblast. 3.4 However, the presence of the intermediate trophoblast may not always be obvious using routine hematoxylin and eosin (H&E) stain because its appearance may resemble that of native stromal cells showing decidual change or smooth muscle cells.’-’ Most other histological criteria associated with intrauterine pregnancies (IUP) have been of limited value in differentiating these two conditions. Variable amounts of hemorrhage, inflammation, and de-
Departments of Obstetrics and Gynecology and * PaSchool of Medicine, Omaha, Ne-
thology, Creighton University braska.
Reprint requests: Janelle K. Strom, M.D., 1520 Northway Drive, St. Cloud, MN 56303.
51
52 cidual and Arias-Stella cell changes can be seen in the endometrial curettings of both EP and IUP. Blood vessels in the endometrium in patients with known IUP have notably thickened hyalinized walls at the implantation site. Intrauterine gestation has also been correlated with the presence of vessel luminal occlusion and intimal cell proliferation. 6,7 The development and utilization of immunohistological techniques have enhanced the ability to differentiate between these EP and IUP.8 Angel et al. localized human placental lactogen (hPL) in intermediate trophoblasts of endometrial specimens lacking demonstrable CV that were collected from women with JUp.2 In none of their immunopositive hPL cases was ectopic pregnancy the outcome. Other markers of trophoblasts, including keratin, epithelial membrane antigen, and placental alkaline phosphatase, have also been used. The most promising of these markers is keratin, which appears to be more sensitive but not as specific as hPL.9 Endometrial glandular cells may also stain keratin-positive. This report analyzes the ability of these specific immunohistochemical (human chorionic gonadotropin [hCG] and hPL) and histological features to differentiate between intrauterine and extrauterine gestations.
presence or absence of hCG and/or hPL positive cells, (2) presence of decidua, (3) the presence or absence
of endometrial vascular changes, (4) the Arias-Stella phenomenon, and (5) inflammation. The last three features were graded semiquantitatively. Normal arterioles were graded as 0. Arterioles with slight thickening of their walls or minimal intimal proliferation were grade 1. Those showing pronounced wall thickening with a lumen to wall ratio of 2 :1 1 or eccentric intimal cell proliferation were grade 2. Severe luminal occlusion, with a lumen to wall ratio of 1:1, and a moderate degree of intimal cell proliferation with foam cells present in the intimal were grade 3 (Fig. 1). Inflammation was assessed as acute (presence of polymorphonuclear leukocytes) or chronic (presence of plasma cells) and graded as mild (less than 25% of curetted tissue containing inflammatory cells) or marked (more than 75% of curetted tissue containing inflammatory cells). Arias-Stella cell changes were graded 0-3. No Arias-Stella cell phenomenon was graded 0. Grade 1 showed moderate
Materials and Methods
Specimen
Selection
Twenty-five endometrial specimens were retrospectively identified during the period between 1984 and 1990. The specimens were obtained from women who had uterine bleeding during the first trimester and whose specimens did not contain CV when analyzed by routine H&E stains. Thirteen of these 25 cases had documented ectopic (tubal) pregnancies. In each case, the entire specimen had been submitted for analysis in multiple containers and stained initially with H&E. This group of specimens
compared with endometrial samples obtained from 10 patients with IUP undergoing spontaneous first trimester abortion with demonstrated CV on H&E stains. All samples were randomized and analyzed by two independent observers (C. B. and J. S.). was
Study
Parameters
The clinicopathologic analysis focused on those in which curettings from a documented intrauterine gestation without CV were compared with curettings from patients with known EP. All specimens were examined for the following features: (1) cases
1. Grade 3 intermediate changes demonstrating severe luminal occlusion due to intimal cell hyperplasia. (H&E)
Fig.
53 increase of mucus secretion in surface or glandular epithelial cells with minimal nuclear hyperplasia. Grade 2 showed more pronounced and more frequent nuclear hyperchromasia and mucus secretion in cytoplasm of endometrium glandular cells. Grade 3 showed very pronounced nuclear hyperchromasia with &dquo;dysplastic&dquo; characteristics and mucus hypersecretion by epithelial cells.
Immunochemical
Staining
All slides were reviewed and the block that showed the maximum amount of viable tissue and the minimal amount of blood, inflammation, and necrotic debris was chosen for immunohistochemical staining. One block per case was chosen for immunohistochemical staining of hCG and hPL. Four-millimeterthick sections were cut and mounted on slides coated with poly-L-lysine, dewaxed in xylene, and rehydrated in a series of graded alcoholic baths. Endogenous peroxidase was blocked by preincubation in a 1:67 dilution of goat serum for hCG and rabbit serum for hPL (Vector Lab). The primary antibodies (polyclonal rabbit antihuman hCG and goat antihuman hPL, Dako Corp.) were applied for 40 minutes followed by biotinylated antibodies ( 1:10 goat antirabbit hCG and rabbit antigoat hPL) for 20 minutes. Quenching of endogenous peroxidase was achieved by adding freshly prepared 3% H2 02 for 10 minutes. The avidin-biotin complex was developed by adding 1:135 diluted avidin complexed with biotinylated peroxidase for 20 minutes followed by 1: 35 dilution of 3-amino-9-ethyl carbazol (Vector Lab) in 2%
H202 for 15 minutes. All incubations were performed in a humidified chamber at room temperature and were followed by two 2-minute rinses with phosphate-buffered saline (0.01 M, pH 7.2 ) .1 ° Finally, the sections were counterstained with hematoxylin, cleared with xylene, and mounted in glycerol gelatin (Sigma Co.) A reddish brown intracytoplasmic staining was considered positive (Figs. 2, 3). Normal term placenta was used as positive control, and omission of primary antibodies was used as negative control. All samples were reviewed in a blinded fashion by J. S. and then confirmed by J. S. and C. B. As the criteria were agreed upon in the beginning, there were few significant disagreements. Minor subjective disagreements were resolved by detailed reexamination. After specimen analysis, clinical data for each case were retrospectively reviewed by clinic chart review or telephone contact of the patient’s
physician. Statistical
Analysis
Data were analyzed by Fisher’s exact test or, when applicable, chi-square test with Yates’ correction.
Results
Twenty-five endometrial samples without histological evidence of CV were analyzed. Thirteen of the 25 cases were proven EP. The remaining 12 specimens were from women who clinically had resolved ...&dquo;.
Fig. 2. Human chorionic gonadotropin stain. Darkly stained
areas
ity. Positive trophoblastic
represent positivareas represent tissue.
&dquo;’..
54
Fig.
3. Human
placental lactogen
stain.
Larger, dark-staining cells represent intermediate trophoblasts.
IUP. Ten endometrial samples of early abortions exhibiting CV were included as positive controls. Histological comparison of IUP and EP showed no differences in the presence or absence of suspected intermediate trophoblast-like cells on H&E or Arias-
Stella cell
changes. Histologic comparisons of CV-negative IUP and EP cases showed increased decidualization (91.6% vs 46%, P .0046), acute inflammation (66% vs 30%, P = .1632), vascular wall thickening and occlusion (83% vs 53.8%, P = .25), and decreased Arias-Stella cell changes (50% vs 61 %, P = .859) in IUP cases (Table 1). Increased decidualization in IUP specimens versus EP specimens was significant. All other histological parameters were not significant. The presence of severe (grade 3) vascular changes in 25% (3/12) of the specimens appeared to differentiate IUP from EP. However, the sample size precluded valid statistical analysis. Immunohistochemical staining for hPL and hCG was positive in 100% (10/10) of the endometrial samples with CV and negative in 100% ( 13/13) of the
100% specificity for EP. In the 12 samples from patients who had no CV present, 7 showed presence of both hPL- and hCG-positive cells (Table 2). One case showed only hPL-positive cells. Overall, the sensitivity for documenting IUP in specimens without CV was 67% when hCG and/or hPL were used.
=
proven EP group
(P = .00168). This demonstrated
_
Discussion
When IUP cannot be differentiated from EP on clinical grounds (ultrasonagram, physical examination), or by the presence of CV, the identification of a single intermediate trophoblastic cell can be helpful. Because of the difficulty in differentiating intermediate trophoblasts from degenerative decidual and smooth muscle cells using conventional H&E stains, the ability of these cells to stain positively for hCG and hPL appears to be of value in identifying first trimester abortion in patients in whom spontaneous abortion cannot be distinguished from EP on the basis of physical examination, ultrasonography, or
histological findings.
55 Table 1.
Comparison of Immunohistochemical
to
Criteria Used to Differentiate Intrauterine Abortion From Ectopic Pregnancy in Specimens Without Chorionic Villi
Histological
hPL, human placental lactogen; hCG, human chorionic gonad-
otropin.
Intermediate trophoblasts are large mononucleate multinucleate cells with abundant amphophilic cytoplasm. The intermediate trophoblast appears to be the predominant form of trophoblast at the placental implantation site. It is also the only type of trophoblastic cell that has been shown to invade the walls of spiral arterioles and thus is thought to play a major role in the development of uteroplacental circulation.’-’ During normal pregnancy, hCG is prominent in first trimester villous syncytiotrophoblast but thereafter declines steadily, so that by term, little staining is seen. In contrast, hPL within villous syncytiotrophoblast increases throughout the pregnancy.3,4 In our study, the intermediate trophoblasts or
Table 2. Immunohistochemical Comparison of Intrauterine Contents
(extra villous) stained positive for both hCG and hPL in the majority of cases. These immunohistochemical stains had a specificity of 100% and sensitivity of 67%. The lack of positive stains in four of the intrauterine gestations was probably due to previously passed tissue or inadequate sampling.’ The presence or absence of Arias- Stella cells and inflammation was not helpful in differentiating between EP and IUP. Well-formed decidua was present more often in IUP than EP. Most of the minor vascular changes seen in gestational endometrium were not shown to be significant between IUP and EP; however, severe degree of vascular changes may be correlated with IUP. This observation is also consistent with previous studies by Lichtig et al.~ Besides hCG and hPL, other immunostains may also be employed. Keratin’ is an extremely sensitive stain for trophoblasts, although it is not as specific. The trophoblasts and glandular cells stain strongly for keratin, whereas decidual and smooth muscle cells do not. Immunohistochemical stains can help distinguish endometrial tissue from EP versus those from jUp.2 Immunohistochemistry is expensive and takes approximately 1-2 days. The technique of &dquo;deeper sectioning&dquo; the blocks does sometimes reveal diagnostic CV. Many of our cases had multiple deeper sections cut from selected blocks. This method is more cost effective and should be used before resorting to immunohistochemistry. In practical situations, the majority of cases can be diagnosed correctly by sampling the entire tissue and using deeper sections in selected or representative blocks. Immunohistochemistry should be reserved for those cases where the above methods fail to reveal CV. These stains may also be used to confirm the intermediate trophoblast-like cells in implantation sites. Fragments of definite implantation site plaques or invading trophoblasts are also helpful, but not often present. None of our cases showed such definitive foci. In conclusion, our data show that the positivity of immunohistochemical stains for hPL and hCG on endometrial tissue is highly specific for IUP and can be used to rule out EP when CV are absent.
.
hPL, human placental lactogen; hCG, human chorionic gonad-
otropin.
,
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References
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..
-..-
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56 DW, Scully RE.
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