Original Research ajog.

org

GYNECOLOGY
Medical management of ectopic pregnancy with
single-dose and 2-dose methotrexate protocols: human
chorionic gonadotropin trends and patient outcomes
Michelle C. Mergenthal, MD; Suneeta Senapati, MD; Jarcy Zee, PhD; Lynne Allen-Taylor, PhD;
Paul G. Whittaker, DPhil; Peter Takacs, MD; Mary D. Sammel, ScD; Kurt T. Barnhart, MD

BACKGROUND: Ectopic pregnancy, although rare, is an important
cause of female morbidity and mortality and early, effective treatment is
critical. Systemic methotrexate has become widely accepted as a safe and
effective alternative to surgery in the stable patient. As the number and
timing of methotrexate doses differ in the 3 main medical treatment
regimens, one might expect trends in serum human chorionic gonado-
tropin and time to resolution to vary depending on protocol. Furthermore,
human chorionic gonadotropin trends and time to resolution may predict
ultimate treatment success.
OBJECTIVE: This study hypothesized that the 2-dose methotrexate
protocol would be associated with a faster initial decline in serum human
chorionic gonadotropin levels and a shorter time to resolution compared to
the single-dose protocol.
STUDY DESIGN: A prospective multicenter cohort study included
clinical data from women who received medical management for ectopic
pregnancy. Rates of human chorionic gonadotropin change and successful
pregnancy resolution were assessed. Propensity score modeling addressed
confounding by indication, the potential for differential assignment of
patients with better prognosis to the single-dose methotrexate protocol.
RESULTS: In all, 162 ectopic pregnancies were in the final analysis;
114 (70%) were treated with the single-dose methotrexate and 48
(30%) with the 2-dose protocol. Site, race, ethnicity, and reported pain
level were associated with differential protocol allocation (P < .001,
P ¼ .011, P < .001, and P ¼ .035, respectively). Women had similar
initial human chorionic gonadotropin levels in either protocol but the
mean rate of decline of human chorionic gonadotropin from day 0 (day
of administration of first dose of methotrexate) to day 7 was significantly
more rapid in women who received the single-dose protocol compared
to those treated with the 2-dose protocol (mean change e31.3% vs
e10.4%, P ¼ .037, adjusted for propensity score and site). The 2
protocols had no significant differences in success rate or time to
resolution.
CONCLUSION: In a racially and geographically diverse group of
women, the single- and double-dose methotrexate protocols had com-
parable outcomes. The more rapid human chorionic gonadotropin initial
decline in the single-dose group suggested these patients were probably
at lower risk for ectopic rupture than those getting the 2-dose protocol. A
prospective randomized controlled design is needed to remove con-
founding by indication.
Key words: ectopic pregnancy, human chorionic gonadotropin,
methotrexate, protocol comparisons

Introduction
Ectopic pregnancy accounts for 1.5-2%1
of all pregnancies and is an important
cause of morbidity and mortality in
women of reproductive age. Early and
effective treatment either with surgical
or medical management is critical. Sys-
temic methotrexate was first recognized
as a medical treatment for unruptured
ectopic pregnancy in 1982 by Tanaka
et al,2 and it has since become widely
accepted as a safe and effective alterna-
tive to surgery in the stable patient.3,4
Currently, there are 3 main treatment
Cite this article as: Mergenthal MC, Senapati S, Zee J,
et al. Medical management of ectopic pregnancy with
single-dose and 2-dose methotrexate protocols: human
chorionic gonadotropin trends and patient outcomes. Am
J Obstet Gynecol 2016;215:590.e1-5.
0002-9378/$36.00
ª 2016 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.ajog.2016.06.040
regimens for management of ectopic
pregnancy with methotrexate: the mul-
tidose protocol, the single-dose proto-
col,5 and the 2-dose protocol.6 As the
number and timing of methotrexate
doses differ in these protocols, one may
expect trends in serum human chorionic
gonadotropin (hCG) and time to reso-
lution to vary depending on protocol. By
extension, hCG trends and time to res-
olution may predict ultimate treatment
success. As such, this study aimed to
evaluate the association between meth-
otrexate protocol (single dose vs 2
doses), hCG trends, and time to resolu-
tion of ectopic pregnancy with the
hypothesis that the 2-dose protocol
would be associated with faster initial
decline in serum hCG levels and a
shorter time to resolution.
Materials and Methods
This prospective cohort study included
clinical data collected at 3 academic
medical centers from Aug. 1, 2007,
through June 30, 2009: the University of
Pennsylvania, the University of Miami,
and the University of Southern Califor-
nia. The study was approved by the
institutional review board at each of
these institutions. Informed consent
was obtained from all individual partic-
ipants included in the study. Subjects
were initially encountered both as
emergency room and as emergency
walk-in consultations, but all metho-
trexate was given on an obstetric outpa-
tient basis. None of the subjects
conceived using assisted reproductive
technologies. Women who: (1) presented
with first-trimester vaginal bleeding,
pelvic pain, or both; (2) were diagnosed
with ectopic pregnancy; and (3) under-
went medical management with either
the single-dose or 2-dose methotrexate
protocols were included and followed up
to assess treatment outcome. Diagnosis
of ectopic pregnancy was made by

590.e1 American Journal of Obstetrics & Gynecology NOVEMBER 2016

ajog.org
ultrasound, abnormal serum hCG trend,
and/or by the absence of products of
conception after uterine evacuation
according to American Congress of
Obstetricians and Gynecologists guide-
lines.4 Women with nontubal ectopic
pregnancies (ie, interstitial/cornual,
cesarean delivery scar, cervical, intra-
abdominal, or ovarian) and heterotopic
pregnancies were excluded from the
analysis. Women initially treated via sal-
pingostomy or who had initial serum
hCG levels >10,000 mIU/mL were also
excluded. Single-dose methotrexate was
administered in accordance with the
protocol originally described by Stovall
et al5 in 1991. In brief, methotrexate is
administered intramuscularly (IM) at
a dose based on body surface area
(50 mg/m2) on day 0. Serum hCG is then
measured on posttreatment days 4 and 7.
If at least a 15% decrease in hCG is
observed between days 4-7, these women
are then followed up with weekly hCG
measurements until the result is negative.
If the decline between days 4-7 is <15%,
a second IM dose of methotrexate
(50 mg/m2) is administered on day 7.
Repeat hCG measurements are then
obtained and if, during follow-up, hCG
levels plateau or increase, methotrexate
may be repeated.5
The 2-dose methotrexate protocol was
administered as described by Barnhart
et al6 in 2007. According to this protocol,
IM methotrexate of 50 mg/m2 is
administered on days 0 and 4. As in the
single-dose protocol, hCG is measured
on days 4 and 7; if hCG does not decline
at least 15% between days 4-7, a third
dose of methotrexate is administered.
Patients who receive a third dose of
methotrexate return on day 11 for
another hCG measurement. If the hCG
level decreases by at least 15% between
days 7-11, weekly hCG measurements
are performed until a negative result is
obtained. Otherwise, a fourth dose
of methotrexate is administered, and
another hCG level is obtained on day 14.
If there is at least a 15% decrease between
days 11-14, weekly hCG measurements
are performed until a negative result is
obtained. If at least a 15% decrease does
not occur, the patient is referred for
surgical management.6
GYNECOLOGY
Baseline characteristics including
clinical site, age, race, ethnicity,
gravidity, parity, weight, body mass in-
dex, history of ectopic pregnancy, and
history of spontaneous abortion were
collected at initial presentation together
with gestational age, initial hCG value,
presence of pain and bleeding, ultra-
sound characteristics, method of diag-
nosis, and treatment outcome. Serum
hCG values were collected from the day
of the initial methotrexate dose (day 0, or
T1), the first assessment after the initial
dose (day 4 or T2), and the second
assessment after the initial dose (day 7 or
T3). The changes in hCG from day 0-4,
day 4-7, and day 0-7 were obtained by
calculating percent change between
each of the 2 time points.
The primary outcome of interest was
percent change in hCG from day 0-4,
day 4-7, and day 0-7. Secondary
outcomes included treatment success
rates and time to successful resolution.
Successful resolution was defined as
achieving an hCG level of <5 mIU/mL,
and lack of resolution was defined as
needing definitive surgical management
after treatment with methotrexate.
Those who had lack of resolution or
were lost to follow-up were considered
censored for the event outcome of time
to successful resolution. A sensitivity
analysis was performed to assess the
impact of loss to follow-up by defining
successful resolution as a final hCG
level of <100 mIU/mL, and lack of
resolution as either needing definitive
surgical management after treatment
with methotrexate or having a final
hCG level of >100 mIU/mL.
Propensity score modeling was uti-
lized to address possible confounding by
indication, ie, differential assignment of
patients with better prognosis to the
single-dose methotrexate protocol. A
logistic model was developed to predict
whether a patient was more likely to
receive one intervention (ie, the 2-dose
protocol) over the other (ie, the single-
dose protocol). Forward stepwise vari-
able selection was used to evaluate all
available covariates (eg, clinical site, pa-
tient demographics, medical history) to
develop the final propensity score model,
which included initial hCG value, site,
NOVEMBER 2016 American Journal of Obstetrics & Gynecology
Original Research
parity, method of ectopic pregnancy
diagnosis, and ultrasound impression.
Based on the model’s predicted proba-
bilities of receiving the 2-dose protocol,
each subject was assigned a propensity
score, which was used in subsequent
regression modeling.6,7
Subjects were analyzed on the basis of
treatment received, since intent to treat
was not recorded. Differences in baseline
characteristics between the 2 protocols
were assessed by t tests (all continuous
variables) and c2 and proportion tests
(all categorical variables). Associations
between percent change in hCG out-
comes, treatment protocol, and cate-
gorical covariates of interest were
evaluated using linear regression models
after adjustment for the propensity
score. Similarly, Cox proportional haz-
ards models were used to assess protocol
and covariate differences in time to
successful resolution. Models were also
adjusted for any covariates that were still
unbalanced across the protocols after
propensity score adjustment, the only
one of which was site of treatment. Sta-
tistical analyses were performed with
SAS 9.2 (SAS Institute, Cary, NC) and
STATA 12 (StataCorp LP, College
Station, TX).
Results
In all, 162 ectopic pregnancies were
included in the final analysis; 114
(70.4%) were treated with the single-
dose methotrexate protocol and 48
(29.6%) with the 2-dose protocol. At day
7, data were available on 106 patients
treated with the single-dose metho-
trexate protocol and 42 with the 2-dose
protocol (retention 93% and 88%,
respectively). Baseline characteristics of
the 2 groups are described in Table 1.
The use of single-dose vs 2-dose protocol
was significantly associated with site,
race, ethnicity, and reported pain level (P
< .001, P ¼ .011, P < .001, and P ¼ .035,
respectively). Patients treated at the
University of Pennsylvania were signifi-
cantly more likely to receive the 2-dose
vs single-dose protocol (79.6% vs
20.4%, P < .001), whereas patients
treated at the University of Miami and
the University of Southern California
were more likely to receive the
590.e2
Original Research GYNECOLOGY ajog.org

single-dose vs 2-dose protocol (97.5% vs

TABLE 1
2.5%, P <.001 and 78.1% vs 21.9%, P ¼
Baseline characteristics of study population across 3 sites
.002, respectively). Caucasian patients
(83.6% vs 16.4%, P < .001), those who Single dose Two-dose
reported race as “other” (69.2% vs MTX, n ¼ 114 MTX, n ¼ 48
30.8%, P ¼ .050), and Hispanic patients Age, y, mean ( SD) 30.6 (6.3) 30.1 (6.9)
(87% vs 13%, P < .001) were signifi- a
Site (%)
cantly more likely to receive the single-
dose vs 2-dose protocol. Patients who University of Miamia 79 (97.5) 2 (2.5)
a
reported no pain, mild pain, or moder- University of Pennsylvania 10 (20.4) 39 (79.6)
ate pain at presentation were more likely University of Southern California b
25 (78.1) 7 (21.9)
to receive the single-dose protocol over c
Race (%)
the 2-dose protocol (P ¼ .035, P < .001,
and P ¼ .001, respectively). There were African American 45 (60.0) 30 (40.0)
c
no significant differences with respect Other 18 (69.2) 8 (30.8)
to age, gravidity, parity, body mass Caucasian a
51 (83.6) 10 (16.4)
index, history of ectopic pregnancy, a
Ethnicity (%)
history of spontaneous abortion, gesta-
tional age, or severity of vaginal bleeding Hispanica 60 (87.0) 9 (13.0)
at presentation between the 2 protocol Non-Hispanic 54 (58.1) 39 (41.9)
groups. Gravidity, mean ( SD) 2.9 (1.9) 2.9 (1.6)
Women had similar initial hCG
Parity, mean ( SD) 0.9 (1.0) 1.0 (1.1)
levels in either protocol (Table 1). When
we examined hCG trends by treat- BMI, mean ( SD) 30.1 (6.4) 29.1 (5.5)
ment protocol using linear regression Prior ectopic (%) 22 (78.6) 6 (21.4)
modeling and adjusting for propensity Prior SAB (%) 38 (71.7) 15 (28.3)
score and site (Table 2), we found that
the mean decline of hCG from day Gestational age, wk, mean ( SD) 6.0 (2.8) 6.3 (2.1)
0 (day of administration of first dose of Initial hCG, mIU/mL, mean ( SD) 1684 (1922) 1839 (2112)
methotrexate) to day 4 was significantly Bleeding (%)
greater in women who were treated with
None 25 (21.9) 14 (29.2)
the single-dose protocol compared to
those treated with the 2-dose protocol Mild 73 (64.0) 30 (62.5)
(mean change e10.8% vs þ5.14%, P ¼ Moderate/severe 16 (14.0) 4 (8.3)
.031). Of note, the average serum hCG c
Pain (%)
level decreased from day 0-4 in the group
Nonec 29 (65.9) 15 (34.1)
that received the single-dose protocol, a
while it increased in the group that Mild 43 (82.7) 9 (17.3)
b
received the 2-dose protocol during Moderate 36 (73.5) 13 (26.5)
the same time period. The hCG levels Severe 3 (37.5) 5 (62.5)
also declined faster in the single-dose Data are mean (SD) or n (%).
compared to the 2-dose group from BMI, body mass index; hCG, human chorionic gonadotropin; MTX, methotrexate; SAB, spontaneous abortion.
day 0-7 (mean change e31.3% vs a
P <.001; b P <.01; c P <.05 significant statistical difference between protocols (Student t test or c2 tests).
e10.4%, P ¼ .037). There was no sig- Mergenthal et al. One- and 2-dose methotrexate protocols for ectopic pregnancy. Am J Obstet Gynecol 2016.
nificant difference in percent change
from day 4-7 between single-dose and
2-dose protocols after adjusting for
propensity score and site (mean change (Table 3). Time to resolution of hCG A sensitivity analysis was performed
e31.8% vs e10.6%, P ¼ .110). from the serum for women in each to assess the impact of loss to follow-up.
Rates of treatment success were com- protocol is presented in the Figure. In this analysis, resolution was defined as
parable between the 2 groups (83% for There were also no distinguishing factors a final hCG level of <100 mIU/mL, and
single-dose vs 79% for 2-dose, P ¼ .53). between successful and failed therapy lack of resolution as either needing
There was no significant difference among the women of each treatment definitive surgical management after
between the 2 groups in time to resolu- group. The number of women needing treatment with methotrexate or having
tion (hazard ratio, 0.441; 95% confi- urgent intervention was not different a final hCG level of >100 mIU/mL. As
dence interval, 0.139e1.401; P ¼ .165) between the 2 groups. in the original analysis, there was no

590.e3 American Journal of Obstetrics & Gynecology NOVEMBER 2016

ajog.org GYNECOLOGY Original Research

minimize differences in outcome be-

TABLE 2
tween groups due to confounding.
Human chorionic gonadotropin trends by methotrexate protocol
Adjustment with the propensity score
Single-dose Two-dose demonstrated that the magnitude of
methotrexate methotrexate differences in initial hCG trends, before
Unadjusted Adjusted
Geometric mean (95% confidence interval) P valuea P valueb the second dose was given, was even
greater than without adjustment
% D T1eT2 e10.8 (e20.2 to e1.3) 5.1 (e15.4 to 25.7) .109 .031
(Table 2). This suggested that differences
% D T2eT3 e31.8 (e37.4 to e26.1) e10.6 (e50.7 to 29.5) .123 .110 in hCG trends prior to treatment was
% D T1eT3 e31.3 (e41.8 to e20.7) e10.4 (e32.0 to 11.2) .055 .037 not due to differences in population
% D, percent change between time points; T1, day 0; T2, day 4; T3, day 7. characteristics (which are included in the
a
Comparison value from linear regression modeling; b Adjusted for propensity score and site. propensity score) but rather confound-
Mergenthal et al. One- and 2-dose methotrexate protocols for ectopic pregnancy. Am J Obstet Gynecol 2016. ing by indication (choice or protocol
by the clinician). A propensity score
adjustment cannot control for the like-
significant difference between the 2 prognosis. Evidence for this is the dra- lihood that clinicians who are aware of
groups in time to resolution (hazard matic difference (between groups) in the entire clinical picture choose a
ratio, 0.531; 95% confidence interval, hCG values between day 0 (day of 2-dose protocol for women at higher risk
0.232e1.215; P ¼ .134) (Table 3). methotrexate administration) and day 4. based on clinical signs and symptoms,
In women who received the 2-dose despite a similar baseline hCG level. For
Comment protocol, the day-4 hCG value (ob- example, the trend in hCG levels prior
We had hypothesized that women tained prior to administration of the to treatment of methotrexate was not
who received the 2-dose protocol second dose of methotrexate) was actu- available in our analysis, but could affect
would experience a greater hCG ally increased, which suggested that these both the choice of single- vs 2-dose
decline and a faster time to ultimate women had a more aggressive ectopic methotrexate and the trajectory of
successful resolution when compared pregnancy. Ultimately, there was no hCG after treatment. Alternatively it is
to those receiving the single-dose pro- difference in success rates or time to conceivable that clinicians noted the
tocol (who receive only 1 dose of resolution between the 2 groups, sug- dramatic decline in hCG between days
methotrexate in the first 7 days), gesting that the 2-dose protocol may be 0-4 and thus withheld the second dose
because patients in the 2-dose protocol attenuating the differences in prognosis of methotrexate (even though they
receive more methotrexate, sooner. and subsequent risk of failure. may have intended to use the 2-dose
However, we did not observe this effect. To address the issue of confounding protocol).
Instead, counterintuitively, we found by indication, we developed a propensity One of the major strengths of our
that hCG levels declined more rapidly score model to predict whether a patient study was that data were collected at 3
from day 0-4 and from day 0-7 in those was more likely to receive one metho- large institutions located in different
receiving the single-dose protocol. This trexate protocol over the other, and then regions of the country, and our subjects
unexpected finding was noted despite applied that model to subsequent ana- represented a racially and ethnically
similar initial hCG levels, and no lyses. The incorporation of propensity diverse group of women. We would
difference in success rate or time to score modeling to study the association expect this to increase the generaliz-
successful resolution. between methotrexate protocol and ability of our results. Our study also
These findings suggest the occurrence various outcomes is unique in the could have been impacted by informa-
of confounding by indication in that ectopic pregnancy literature. It would be tion bias; however, the risk of this was
the single-dose protocol may have anticipated that controlling for pro- minimized through use of a clinical
been reserved for patients with better pensity in a multivariable analysis would database in which data were prospec-
tively collected. Another potential limi-
tation is that not all women were
TABLE 3 followed up until hCG was completely
Time to resolution, adjusted for propensity score cleared from the serum, although there
were no reports of failed treatment with
N N (eventsa) HR 95% CI P very low hCG values. When assessing
Original analysis 162 68 0.441 0.139e1.401 .165 time to resolution, this study treated
Sensitivity analysis 162 133 0.531 0.232e1.215 .134 those who were lost to follow-up as
CI, confidence interval; HR, hazard ratio.
censored. However, the sensitivity anal-
a
Successes.
ysis suggests that these findings are
Mergenthal et al. One- and 2-dose methotrexate protocols for ectopic pregnancy. Am J Obstet Gynecol 2016. robust to variation in final hCG level
up to 100 mIU/mL.

NOVEMBER 2016 American Journal of Obstetrics & Gynecology 590.e4

Original Research GYNECOLOGY
FIGURE
Cox model survival estimates of time to resolution
Broken line indicates single-dose methotrexate protocol, solid line indicates 2-dose protocol.
Mergenthal et al. One- and 2-dose methotrexate protocols for ectopic pregnancy. Am J Obstet Gynecol 2016.
Many of the limitations of the current
study could be successfully addressed
with a randomized controlled trial
comparing hCG trends in single-dose
and 2-dose methotrexate protocols as
a possible mechanism for improved
success. Small randomized trials exist
that looked at the efficacy between
single-dose and 2-dose protocols.7-9
Individually all report no differences in
treatment success based on protocol, but
all report a trend toward better outcome
with the 2-dose protocol. When com-
bined, the superiority of the 2-dose
protocol approaches statistical signifi-
cance (83.5% vs 88.5% success: relative
risk, 0.94 confidence interval;
0.86e1.03).7 Two of these studies also
find a reduced time to resolution with
590.e5 American Journal of Obstetrics & Gynecology NOVEMBER 2016
the 2-dose protocol.7,8 Overall our
finding of similar success rates and
time to resolution for the 2 protocols,
despite confounding by indication,
is consistent with the results of these
randomized trials7-9 and supports
the use of the 2-dose protocol as
primary medical management of ectopic
pregnancy. n Medicine, University of Pennsylvania, Philadelphia, PA;
Acknowledgment
The authors acknowledge contributions by
Karine Chung, MD, MSCE (Department of Ob-
stetrics and Gynecology, University of Southern
California), to data collection and interpretation.
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Author and article information
From the Departments of Obstetrics and Gynecology (Drs
Mergenthal, Senapati, Whittaker, Sammel, and Barnhart)
and Biostatistics and Epidemiology (Dr Zee, Taylor,
Whittaker, Sammel, and Barnhart), Perelman School of
and Department of Obstetrics and Gynecology, Eastern
Virginia Medical School, Norfolk, VA (Dr Takacs).
Received May 16, 2016; revised June 18, 2016;
accepted June 20, 2016.
This study was funded by National Institutes of
Health grants K24HD060687 (K.T.B., M.D.S.) and
R01HD076279 (K.T.B., M.D.S.). The sponsor had no role
in data collection, analysis, interpretation, report writing,
or decision to publish.
The authors report no conflict of interest.
Corresponding author: Kurt T. Barnhart, MD.
kbarnhart@obgyn.upenn.edu