Keywords
ectopic pregnancy - frozen section - pregnancy of unknown location - β-hCG - endometrial
thickness
Introduction
Ectopic pregnancy can be identified early by the widespread use of transvaginal ultrasonography
with high frequency probes, together with the use of sensitive quantitative measurements
of serum human chorionic gonadotropin (hCG).[1]
[2]
[3]
[4]
[5]
[6]
[7] Confirmation of the presence of intrauterine pregnancy by transvaginal ultrasonography
is a highly accurate method of exclusion of an ectopic pregnancy, and in most cases
has replaced diagnostic curettage as a cornerstone for ruling out or ruling in an
ectopic pregnancy.[8] However, in certain situations ultrasonography might result in diagnostic uncertainty,
in which case an endometrial biopsy may still be necessary to distinguish between
abnormal intrauterine and ectopic pregnancies.
Frozen section evaluation of uterine curetting is performed in certain instances of
pregnancy of unknown location (PUL), to rule out ectopic pregnancy. If chorionic villi
are identified in utero, an ectopic gestation is essentially ruled out (except for rare cases of heterotopic
pregnancies) and no further treatment is needed. If villi are not detected, this may
reflect an ectopic pregnancy, although the patient may also have expelled all intrauterine
villi. The absence of chorionic villi on endometrial frozen section may lead to laparoscopy,
laparotomy, salpingectomy or methotrexate therapy, despite the fact that an ectopic
pregnancy is not always present.[8] Therefore, diagnosis using the frozen section technique performed on endometrial
material shortly after curettage and after conception products have been identified
can dramatically decrease the time needed to rule out the presence of an ectopic pregnancy.
Such diagnosis also avoids the undesired administration of methotrexate to women with
intrauterine pregnancy.
The accuracy of endometrial biopsies using the frozen section technique has not been
thoroughly evaluated. No systematic reviews have been conducted on this topic.
We found three relevant articles in PubMed and MedLine databases. Two of them highly
recommended the use of frozen section examination as an accurate method for identifying
products of conception in endometrial curetting. In their analysis of 87 cases in
which frozen section assessment of an endometrial curettage specimen was performed,
Spandorfer et al[9] found that 81 (93.1%) were accurate. Only one woman was misdiagnosed and did not
have an ectopic pregnancy. In a study conducted at our medical center, frozen section
analysis had a sensitivity of 78%, specificity of 98%, positive predictive value of
95% and a negative predictive value of 93%.[10] In that study of 70 frozen sections, 63 (90%) were accurate. Of 50 specimens interpreted
as negative on frozen sections, 6 (12%) contained conception products on final pathologic
review. One of the 20 (5%) specimens interpreted as positive by a frozen section failed
to demonstrate products of conception on a final pathologic section. The sensitivity
of frozen sections in the diagnosis of ectopic pregnancy was 76%; specificity, 98%;
positive predictive value, 95%; negative predictive value, 88%, and accuracy, 90%.
The accuracy of frozen section diagnoses was analyzed and stratified by preoperative
serum hCG concentration. The cut-off point used for serum hCG was 1,000 IU/l.[10] The percentage of inaccurate frozen section diagnoses was greater in the subgroup
with higher serum hCG values (12.5%) than in the subgroup with lower values (9.3%).
However, the difference between the two groups was not statistically significant.
In contrast to these two studies, Heller et al[11] concluded that frozen section evaluation of uterine curetting can produce false
negative diagnosis, and that this should be considered in the operative planning of
women with suspected ectopic pregnancy. They reviewed 36 cases, of which 13 showed
evidence of intrauterine pregnancy on final permanent sections. Five false negatives
were identified in which no villi had been identified on a frozen section, but villi
or evidence of an implantation site had been noted on final pathologic sections. The
sensitivity of frozen sections in the diagnosis of ectopic pregnancy was 62%.
The purpose of the current study was to determine the accuracy of frozen section analysis
in cases of pregnancy of unknown location, and the relation of this assessment of
accuracy to β-hCG level and endometrial thickness.
Methods
We performed a retrospective analysis of the Department of Obstetrics and Gynecology
database in Galilee Medical Center from January 2009 to December 2014. In women with
PUL, a diagnostic curettage was performed and the material from the curetting was
sent for frozen section examination. PUL was defined as the situation of a positive
pregnancy test with no signs of intra- or extrauterine pregnancy on transvaginal sonography.[12] Curettage was performed only in cases with a plateau of β-hCG levels in at least
two consecutive examinations; or β-hCG of more than 2,000 mIU/ml in 2 sequential measurements,
48 hours apart, a rise of less than 1.66 in β levels (48 h/0 < 1.66) and no evidence
of intra or extrauterine pregnancy.
In the department of pathology, frozen sections were prepared from endometrial curetting,
and blood clots were separated from the specimen macroscopically before freezing.
The tissue was embedded in optimal cutting temperature aqueous medium and then it
was frozen in liquid isopentane at -25°C. The frozen tissue was cut on a cryostat,
and 5 to 9 μm sections of tissue were transferred to a glass slide at room temperature,
stained with hematoxylin and eosin, and a cover slip was applied. The remaining frozen
material was thawed and processed for routine paraffin fixation. Cases with negative
result for chorionic villi have the samples supplied with β hCG, Vimentin and Pan
Keratin A1/A3 immunohistochemical staining (Zymed#, Zymed Laboratories Inc., San Francisco,
CA, USA). The entire frozen section process took no longer than 20 minutes, including
microscopic inspection of the section.
The main variables investigated were frozen section diagnosis and final pathologic
diagnosis, both variables according to the presence or absence of products of conception.
Frozen section diagnosis was considered accurate if it concurred with the final pathologic
diagnosis. Clinical and epidemiological data were also investigated, including age,
week of gestation, β-hCG level, and endometrial thickness before the curettage took
place.
Quantitative data, such as age, week of gestation, β-hCG level, and endometrial thickness,
are described by mean and standard deviation, and by median and range. Qualitative
data are presented as frequencies and percentages. This includes calculations of accuracy,
sensitivity, specificity, and positive and negative predictive values. Accuracy, which
was assessed as the concurrence or discrepancy between frozen section diagnosis and
final pathologic diagnosis, was calculated for subgroups according to-β hCG level
determined by receiver operator characteristic (ROC) curves. The cut-off point used
was a serum hCG level of 1,000 IU/l. This level was chosen based on the ‘discriminatory
zone’ values reported in the literature. This concept was developed by Kadar et al[13] to determine the serum hCG level at which a sac should be seen on ultrasound examination.
According to this concept, if the serum hCG level is above the ‘discriminatory zone’
and an intrauterine gestational sac is not seen, the pregnancy is abnormal, has aborted,
or is in an ectopic location. Using vaginal sonography to detect the sac, the range
of ‘discriminatory zones’ reported extends from 600 to 1,025 mIU/ml.[14]
[15]
[16]
[17]
The t-test was used to examine the relation between the accuracy of frozen section
and both β hCG level and endometrial thickness. A statistically significant result
was defined if p < 0.05. The study was approved by the Institutional Review Board (Helsinki Committee)
of Galilee Medical Center.
Results
Data of 106 women were analyzed. [Table 1] presents demographic and clinical data. Frozen sections were positive for products
of conception in 27 (26%), whereas final pathological examination was positive in
33 (31%) ([Fig. 1]). The accuracy of frozen section analysis in detecting products of conception, when
compared with the final pathological diagnosis, was 88.7%. The correlation between
frozen section and final pathological diagnosis was statistically significant (p < 0.001), with phi = 0.729 (high strength). Of the 106 frozen section studies the
diagnosis was accurate in 94 (88.7%) and inaccurate in 12 (11.3%). Of the 79 specimens
interpreted as negative on frozen sections (no products of conception noted), 9 (11.4%)
were found to contain conception products on final pathologic review. Three of the
27 (11.1%) specimens interpreted as positive by a frozen section failed to demonstrate
products of conception on a final pathologic section. All three women were not discharged
and received followed-up because of the low percentage, but yet very dangerous misdiagnosis
of a normal intrauterine pregnancy in cases of ectopic pregnancy.
Fig. 1 Results of frozen section and final pathological examination.
Table 1
Demographic and clinical data of 106 women with suspected ectopic pregnancies
|
Variables
|
Number of records found
N (%)
|
Mean ± SD
|
Range
|
|
Minimum
|
Maximum
|
|
Age (years)
|
(99%) 105
|
31.4 ± 5.6
|
20
|
44
|
|
Week of gestation
|
(93%) 99
|
6.2 ± 1.6
|
3
|
12
|
|
Beta-hCG level (mIU/ml)
|
(100%) 106
|
6,394 ± 22,988.4
|
5
|
159,000
|
|
Endometrial thickness (mm)
|
(81%) 86
|
11.75 ± 6
|
1
|
26
|
Abbreviation: SD, standard deviation.
As presented in [Table 2], the sensitivity of frozen section analysis in the diagnosis of ectopic pregnancy
was 72.7%, specificity 95.9%, positive predictive value 88.9% (24/27) and negative
predictive value 88.6% (70/79) ([Table 2]). The median β-hCG level was lower for cases in which the frozen section diagnosis
was accurate than for cases of inaccuracy ([Table 3]). This correlation was statically significant (p = 0.01). Endometrial thickness was greater for cases that showed discrepancy between
frozen section analysis and pathological diagnosis; however, the difference was not
statically significant ([Table 4]). More frozen section diagnoses were accurate in cases with preoperative serum hCG
concentration < 1,000 mIU/ml than in cases with hCG concentration > 1,000 mIU/ml,
p < 0.001 ([Table 5]). According to ROC: area under curve = 0.696, p = 0.001, confidence interval (CI) was 95% (0.583, 0.809). For β-hCG level = 1,000
mIU/ml, the sensitivity of accurate diagnosis was 61%, and the specificity was 70%.
Table 2
Results of frozen section and pathological examination
|
Pathological diagnosis
|
|
Negative
|
Positive
|
|
Frozen section diagnosis
|
Negative
|
Number of cases
|
70
|
9
|
|
%
|
95.9%
|
27.3%
|
|
Positive
|
Number of cases
|
3
|
24
|
|
%
|
4.1%
|
72.7%
|
|
Total
|
Number of cases
|
73
|
33
|
|
%
|
100%
|
100%
|
Table 3
Beta-hCG level according to the accuracy of frozen section and pathological results
|
Accuracy of FS and pathological diagnosis
|
Number of cases
|
Median
|
Minimum
|
Maximum
|
P¤
|
|
Valid
|
Missing
|
|
Beta-hCG level
|
Inaccurate
|
12
|
0
|
1,551.5
|
422
|
10,115
|
0.01
|
|
Accurate
|
94
|
0
|
493.52
|
5
|
159,000
|
1-sided
|
Abbreviation: FS, Frozen section.
¤ P-value according to Wilcoxon rank sum test.
Table 4
Endometrial thickness according to the accuracy of frozen section and pathological
results
|
Accuracy of FS and pathological diagnosis
|
Number of cases
|
Minimum
|
Maximum
|
P¤
|
|
Valid
|
Missing
|
Mean ± SD
|
|
Endometrial thickness
|
Inaccurate
|
11
|
1
|
14.5 ± 5.4
|
8
|
25
|
0.101
|
|
Accurate
|
75
|
19
|
11.3 ± 6
|
1
|
26
|
2-sided
|
Abbreviation: FS, Frozen section; SD, standard deviation.
¤ P-value according to Wilcoxon rank sum test.
Table 5
The accuracy of frozen section diagnosis according to preoperative serum hCG concentration:
higher hCG concentration was associated with higher accurate diagnosis of the frozen
section (p < 0.001)
|
hCG level (mIU/ml)
|
Number of cases
|
Diagnoses concur
%
|
Diagnoses differ
%
|
|
< 1000
|
64
|
60
93.7%
|
4
6.3%
|
|
> 1000
|
42
|
34
81%
|
8
19%
|
Discussion
The management of PUL was thoroughly revised by Barnhart et al.[18] In their review, several management protocols were suggested, including histologic
confirmation of the diagnosis in some cases.[18] Our study supports the use of frozen section technique as a rapid and accurate method
of identifying products of conception on endometrial curettage. The 88.7% accuracy
of frozen section analysis we reported is similar to that demonstrated by Barak et
al.[10] The positive and negative predictive values (88.9% and 88.6%, respectively), though
high, are of limited strength due to the retrospective design of the analysis.
Of the incorrect diagnosis in our study, most were false negatives (9 of 106 cases),
in which women were erroneously diagnosed by the frozen section technique with the
absence of intrauterine pregnancy. In all nine cases, women started methotrexate therapy.
Although the single dose methotrexate protocol has fewer side-effects than the earlier
multiple dose therapy, this antimetabolite treatment is not completely safe and has
its drawbacks. Such drawbacks are bone marrow suppression and delaying the next pregnancy
to ensure that the medication has been cleared from the organism, since methotrexate
is associated with birth defects. Thus, methotrexate should be initiated only after
the confirmation of a pathological diagnosis of ectopic pregnancy or after measuring
β-hCG level at least once more after the procedure.
There were three false-positive diagnoses among our cohort, in which women were erroneously
thought to have villi in the frozen section specimen and were thus misdiagnosed as
not having an ectopic pregnancy. A possible explanation for this error is that part
of a trophoblastic villus from the tubal pregnancy was shed into the uterus. As the
amount of tissue may have been minimal, it could have been included in the frozen
section specimen only, and not in the final pathology material. A false positive result
poses the highest risk since a woman could be discharged from the hospital with an
ectopic pregnancy that could rupture and be fatal. Therefore, even if the frozen section
detects products of conception, and the woman is hemodynamically stable, a thorough
monitoring of β-hCG should be done to ensure a downward trend. The occurrence of false
positive and false negative results emphasizes the need to confirm the frozen section
diagnosis with a definitive pathologic examination.
Beta-hCG was lower among those subjects with accurate frozen section diagnosis; this
included both true positive (24 of 106) and true negative (70 of 106) cases. A possible
explanation is that true negative cases comprise 75% of the accurate cases (70 of
94 cases); since this represents the absence of intrauterine pregnancy, β-hCG level
is expected to be low. To improve the accuracy of the pregnancy location Seeber et
al.[19] suggested the use of a redefined hCG curves, and yet 12% of the patients with ectopic
pregnancy were not diagnosed. The results of this study[19] also emphasize the need to rely on more than one criterion to diagnose ectopic pregnancy,
such as progesterone plasma concentrations.[20]
We initially hypothesized that greater endometrial thickness will have a significant
correlation with frozen section diagnosis, because thickened endometrium will provide
more material for frozen section evaluation. Our results did not show such relationship.
We suggest, as an explanation for this result, that endometrial thickness, as measured
by ultrasound, includes in most cases blood clots and not only products of conception.
The frozen section method provides a rapid means of diagnosing ectopic pregnancy.
Advantages of this technique include early institution of therapy and reduction in
the hospitalization period, with its inherent costs in cases of suspected abnormal
intrauterine pregnancy. The patient can be discharged once the diagnosis of conception
products is obtained by frozen section, and the uterus is evacuated. However, the
adherence to strict criteria of diagnosing an abnormal intrauterine pregnancy or ectopic
pregnancy is important to avoid the unintended termination of a wanted pregnancy.[21]
Conclusion
In conclusion, frozen section examination is a rapid and mostly accurate method of
diagnosing products of conception in endometrial curetting. We report a statically
significant correlation between lower β-hCG levels and high accuracy of the frozen
section technique. Such a correlation was not displayed between endometrial thickness
and the accuracy of the frozen section technique. The results of our study suggest
that implementation of this technique in the clinical management of an ectopic pregnancy
enables more rapid diagnosis, facilitates earlier institution of treatment and shortens
the period of hospitalization.
