- Email: [email protected]
Serum activin B concentration as predictive biomarker for ectopic pregnancy
Serum activin B concentration as predictive biomarker for ectopic pregnancy Pooja Dhiman, G.P. Senthilkumar, Soundravally Rajendiran, K. Sivaraman, S. Soundararaghavan, Maheshwari Kulandhasamy PII: DOI: Reference:
S0009-9120(16)00090-4 doi: 10.1016/j.clinbiochem.2015.11.024 CLB 9253
To appear in:
Clinical Biochemistry
Received date: Revised date: Accepted date:
3 September 2015 16 November 2015 18 November 2015
Please cite this article as: Dhiman Pooja, Senthilkumar GP, Rajendiran Soundravally, Sivaraman K, Soundararaghavan S, Kulandhasamy Maheshwari, Serum activin B concentration as predictive biomarker for ectopic pregnancy, Clinical Biochemistry (2016), doi: 10.1016/j.clinbiochem.2015.11.024
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Title Page Full title: Serum activin B concentration as predictive biomarker for ectopic
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pregnancy
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Short title: Activin B in ectopic pregnancy
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Authors
1. Dr. Pooja Dhiman*, MD, Department of Biochemistry, 2nd floor, New Academic block, Jawaharlal Institute of Postgraduate Medical Education & Research, Puducherry-
NU
605006, India.
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2. GP Senthilkumar*, Phd, Department of Biochemistry, 2 nd floor, New Academic block, Jawaharlal Institute of Postgraduate Medical Education & Research, Puducherry-
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605006, India.
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3. Dr. Soundravally Rajendiran(corresponding author), MD, Associate Professor, Department of Biochemistry, 2nd floor, New Academic block, Jawaharlal Institute of
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Postgraduate Medical Education & Research, Puducherry-605006, India. [email protected]
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4. Sivaraman K, MSc, Department of Biochemistry, 2nd floor, New Academic block, Jawaharlal Institute of Postgraduate Medical Education & Research, Puducherry605006, India.
5. Dr. Soundararaghavan S, MS, Department of Obstetrics & Gynaecology, Jawaharlal Institute of Postgraduate Medical Education & Research, Puducherry-06, India. 6. Dr. Maheshwari Kulandhasamy, MD, Department of Biochemistry, 2nd floor, New Academic block, Jawaharlal Institute of Postgraduate Medical Education & Research, Puducherry-605006, India. *Share equal authorship
ACCEPTED MANUSCRIPT Abstract We evaluated the diagnostic accuracy of activin B in discriminating tubal ectopic pregnancy
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(tEP) from intrauterine miscarriages (IUM), and normal viable intrauterine pregnancy (IUP).
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We included 28 women with tEP, 31 women with IUM, and 29 normal IUP, confirmed both
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by clinical examination and ultrasonography. Serum activin B concentration was measured at the time of admission using ELISA kit. The median serum activin B concentration were
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found to be significantly decreased in both tEP (p =0.004) and IUM (p =0.022) compared to normal IUP. When compared between tEP and IUM, activin B concentrations did not differ
MA
significantly. ROC analysis of activin B and free β-hCG demonstrated AUC of 0.722 and 0.805, respectively to discriminate tEP from viable IUP. The model including both activin B
D
and free β-hCG improved the discriminating potential with greater AUC (0.824), and
TE
specificity (93%) than individual one. To discriminate tEP from IUM, activin B, free β-hCG and combination of both performed poorly. We conclude that serum activin B concentration
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is lower in tubal ectopic pregnancy, and can discriminate it from normal pregnancy with moderate accuracy. It also shows improved diagnostic potential along with free β-hCG, but
AC
cannot distinguish tEP from IUM reliably.
Keywords- ectopic pregnancy; intrauterine miscarriage; activin B; diagnostic accuracy Corresponding author: Dr. Soundravally Rajendiran, Associate Professor, Department of Biochemistry, JIPMER, Puducherry-06, India;Email [email protected];Phone no 919442214256
ACCEPTED MANUSCRIPT Introduction Ectopic pregnancy, a disorder of early pregnancy, is associated with high morbidity and
T
mortality, predominantly due to delay in diagnosis. This delay stems from the fact that the
IP
current modality of diagnosis, that is transvaginal ultrasonography and serial free beta human
SC R
gonadotropin (free β-hCG) estimations are not entirely reliable (1). These fallacies of the available diagnostic tests have led to the search of reliable biomarkers for diagnosing
NU
ectopic pregnancy, to prevent life threatening complications from abdominal bleeding, and to minimize the need of surgical interventions. Various biomarkers have been investigated in
MA
the diagnosis of ectopic pregnancy as single and multiple markers, which include markers of normal implantation, abnormal corpus luteum and inflammatory markers (2).
Activins,
D
dimeric proteins of transforming growth factor-beta (TGF-b) family, first isolated from
TE
follicular fluid, have demostrated growth factor like actions (3). They are produced by many
CE P
organs including pituitary gland, gonads, placenta, adrenal,spleen etc. These molecules bind to their transmembrane receptors and bring about downstream phosphorylation of
AC
different proteins to alter gene expression, leading to increased decidualization of the endometrium during pregnancy (4). Consequently low serum activin concentration have been associated with ectopic pregnancy (4-8). Recent studies have mainly focussed on the role of serum activin A as a potential marker of ectopic pregnancy(3), and limited literature is available regarding the role of activin B, inspite of the strong experimental evidence of the differential effect of activin B on the decidua (5). We hypothesize that activin B concentration is low in tubal ectopic pregnancy (tEP) than normal intrauterine pregnancy(IUP) and intrauterine miscarriage (IUM),
and therefore can be useful in its
diagnosis. Hence, we evaluated the role of serum activin B in discriminating and predicting tEP from normal IUP and IUM.
ACCEPTED MANUSCRIPT
Material and Methods
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a. Study design: This is a study of diagnostic accuracy. This prospective case-control
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study was carried out in JIPMER hospital, Puducherry in the Department of Biochemistry in collaboration with the Department of Obstetrics and Gynaecology between January 2011 to December 2012.
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b. Subjects: After seeking approval of Institute’s ethics committee and written informed consent from the subjects, patients who presented to the hospital with bleeding per
MA
vaginum, abdominal pain and positive free β-hCG, were recruited as subjects. To further confirm the diagnosis of the patients for subsequent management, transvaginal
D
ultrasonography scan was done by a group of obstetricians trained in the management
TE
of early pregnancy failure. Women who demostrated the evidence of embryo in the
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fallopian tube and absence of intrauterine gestational sac on ultrasound, were included in the case group called tEP (n=28). Diagnosis of tEP was further confirmed in all cases by diagnostic laproscopy. All women in this group presented with impending
AC
rupture. Women who had ultrasonography evidence of intrauterine pregnancy and absence of vital signs of embryo at admission were included in the control group called IUM (n=31). We also included another control group consists of women with normal intrauterine pregnancy (IUP) (n=29). Clinical conditions which can affect the serum concentrations of activin B such as hydatiforme mole, history of missed abortion, subjects on medications based on progesterone, non tubal EP were excluded from this study. c. Blood sampling: Five millilitre of their venous blood samples was collected at the time of admission. The freshly seperated serum was stored at -70 o C until use.
ACCEPTED MANUSCRIPT d. Methods: Serum activin B concentrations were analysed using commercially available ELISA kit (Uscn Life Science Inc., USA). The assay was specific for human serum
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activin B using activin beta homodimer antibody, with the lower limit of detection
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being 6.4 pg/ml. Capture antibody was monoclonal antibody and detection antibody
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used was polyclonal antibody derived from rabbit.The inter-assay and intra-assay CV was <12% and <10% respectively. Total duration of assay was five hours. Briefly, all reagents, samples and standards were prepared and 100µL was added to each well,
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which was precoated with antibody specific to activin B. After an incubation of two
Following an
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hours at 37 o C, 100µL of biotin-conjugated antibody specific to activin B was added. incubation of 1 hour at 37
o
C, 100µL of avidin conjugated to
Horseradish Peroxidase (HRP), and subsequently TMB substrate was added . This
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was followed by an incubation of 15-25 minutes (37 o C). Lastly, 50µL stop solution was added and the plate was read at 450nm. Serum free β-hCG was measured by the
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principle of chemiluminescence in Siemen’s Advia Centaur (Siemens diagnostics, Germany).
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e. Statistical analysis: Statistical analysis was performed using the SPSS version 16 (SPSS, Chicago, IL, USA).
P< 0.05 was considered as statistically significant.
Results were compared using Non-parametric Kruskal–Wallis tests to identify differences in the concentrations of activin B among the three groups. The performance of serum activin B as a biomarker for ectopic pregnancy was evaluated by Receiver operator curve analysis (ROC) using Medcalc®, Belgium.
Results The study groups were matched for age and weeks of pregnancy. The median values with inter quartile range of serum activin B and free β-hCG are depicted in table 1. Activin B
ACCEPTED MANUSCRIPT concentration was significantly lower in woman with tEP compared to patients with normal IUP (P=0.004), and in women with intrauterine miscarriages compared to IUP (P =0.022). No
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statistically significant difference was observed between intrauterine miscarriage and tEP.
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ROC analysis of activin B and free β-hCG demonstrated AUC of 0.722 and 0.805
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respectively to discriminate tEP from viable IUP demonstrating moderate sensitivity and specificity (Table 2). The model including both the activin B and free β-hCG improved the discriminating potential with greater AUC (0.824), and specificity (93%) than individual one.
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(Fig 1 and Table 2). To discriminate tEP from intra uterine miscarriage, activin B, free β-
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hCG, and combination of both performed poor with p values of 0.65, 0.16 and 0.39 respectively. Correlation analysis between free β-hCG and activin B in different study groups
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was r= 0.081(p=0.675) in IUP, r=0.192 (p=0.333)in tEP and r= 0.146(p=0.441)in IUM,
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Discussion
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indicating weak correlation, which was statistically insignificant.
Reliable serum biomarkers for tubal ectopic pregnancy are in immense need for this potentially life threatening condition. This study addressed the above issue using single point measurement of serum activin B. Activins, a dimer of TGF-β family of proteins, functions to promote normal decidualization of the endometrium during pregnancy (4). Since tEP is associated with poor decidualization, serum activin concentrations are expected to be low in this condition. Though the functional aspect of activin A on endometrium have been described in detail, molecular action of activin B is yet to be explored. It has also been established in the previous studies that activin A and activin B function separately in the
ACCEPTED MANUSCRIPT tissues (9). Therefore, it would be prudent to study the diagnostic accuracy of activin B as a biomarker in discriminating tEP from normal IUP and IUM .
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Our study hypothesizes that activin B concentration is lower in tEP than normal pregnancy
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and IUM, and hence can be used as a diagnostic tool. We observed a lower concentration of
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activin B in women with tEP compared to those with normal IUP supporting our hypothesis. This result may be explained by the fact that activin B is involved in decidualization of endometrium in pregnancy, and in ectopic pregnancy this process is hindered causing
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decreased local and systemic concentration of activin B. Our results are in concordance with
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Horne et al, who found decreased expression of activin B in endometrium along with decreased serum concentration of activin B with normal concentration of progesterone in 11
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women with tEP(5). The same study also demonstrated a positive association of activin B
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with decidualisation of endometrium, which in turn reinforce its functional significance in the endometrial development. We observed the mean value of activin B in tEP to be 4 pg/ml,
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which is lower than the concentration reported in Horne et al study. This difference in results can be attributed either due to difference in gestational age of women in two studies(between
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6-8 weeks in the Horne et al study & 4-6 weeks in our study),or the use of different ELISA kit for the estimation of serum activin B(activin B ELISA that incorporates the use of monoclonal antibody 46A/F ,as both capture and detection antibody with a sodium dodecyl sulfate and heat pretreatment of samples in the study by Horne et al, whereas commercial ELISA kit(Uscn Life Science Inc., USA) using monoclonal capture antibody and polyclonal detection antibody, with no sample pretreatment in our study). It has been reported that the expression of activin B increases with increase in decidualisation of endometrium by an invitro experiment (5). We observed lower serum concentration of activin B in intrauterine miscarriages when compared to IUP, which is in line with results reported by Horne et al. Although decreased
ACCEPTED MANUSCRIPT expression of activin A in endometrium of women with recurrent miscarriages have been observed (10), there is paucity of reports on the status of activin B expression in miscarriages
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except for the indirect evidence from Horne et al report which demonstrated a decreased
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decidualisation status in miscarriage, which in turn correlates with the expression of activin
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B(5). We did not find any statistically significant difference in the concentration of activin B between tEP and IUM, thereby limiting the use of activin B in the diagnosis of tEP. Serum free β-hCG concentration was found to be significantly lower in both tEP and IUM
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than normal pregnancy . This result is expected in tEP as there is restricted growth of
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placenta due to limited space availability in the tubal musculature. In IUM, loss of placental tissue is the major etiological factor for decreased concentration of this hormone in the
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circulation. This study did not find any significant difference in serum free β-hCG
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concentration between tEP and IUM and similar findings has been reported previously (12). A significant positive correlation was observed between free β-hCG and activin B in all
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groups, but individual groups failed to establish any signifiact correlation between the two biomarkers. The probable reason for this result may be the small sample size in individual
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gropus. The model constructed using both free β-hCG and activin B measurment demostrated higher specificity than a single parameter in differentiating tEP from IUP, but was not significant in differentiating IUM from tEP. Further diagnostic potential of this model should be explored in larger cohort. Recently, the role of serum activin A as a biomarker for ectopic pregnancy have been explored with some varied results (4-8,11). The study conducted by Elito et al did not demonstrate any significant differences in activin A concentration between tEP and normal pregnancy. The reason could be the smaller sample size as it included only five cases of ectopic pregnancy. In our study, ROC analysis of serum activin B as a biomarker for ectopic pregnancy shows that at a value of less than 23.3 pg/ml , activin B had a sensitivity of 82%,
ACCEPTED MANUSCRIPT and specificity of 62% in discriminating tEP from normal IUP. ROC analysis did not show the utility of activin B in differentiating IUM from tEP. This is a first study of the kind to
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define a discriminative zone of activin B for tubal ectopic pregnancy, though fair number of
tEP from normal pregnancy with
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around 50 ng/ml has been reported to discriminate
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studies have described the diagnostic accuracy of activin A. The activin A concentration of
moderate accuracy (4,6) . This value is higher than the cut off of activin B defined in the current study. The difference could be attributed to the variations in the gestational age of the
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groups studied.
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Although preliminary, the findings demonstrate serum activin B to be a promising biomarker for tubal ectopic pregnancy. The limitations of the present study are the inability to measure
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serial changes in serum activin B concentration. Whether doubling time of activin B can be
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used in the diagnosis of tEP and IUM, similar to free β-hCG in the diagnosis of IUP needs to be evaluated. Also, the study could not include any cases of unruptured tEP since the
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institute is tertiary hospital, cases are referred once complications set in. When compared to free β-hCG, activin B has multiple source of origin(placenta, ovary, spleen, adrenals), and its
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assay is time consuming as compared to free β-hCG,which limits application in emergency. Our results indicate that serum activin B may have a promising role in the differentiation of ectopic pregnancy from normal gestation, but can not distinguish miscarriages from ectopic. These results further warrent larger prospective studies in various populations. Conclusion: Serum activin B concentrations are decreased in tubal ectopic pregnancy and discriminate tEP from normal pregnancy with moderate accuracy and also shows improved diagnostic potential along with free β-hCG, but cannot distinguish tEP from IUM reliably.
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ACCEPTED MANUSCRIPT
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References
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1. Jurkovic D, Wilkinson H. Diagnosis and management of ectopic pregnancy. British Medical Journal.2011;342:d3397.
2. Rausch ME, Barnhart KT. Serum biomarkers for detecting ectopic pregnancy.
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Clinical Obstetrics and Gynecology 2012;55:418-23.
Possible
physiological
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3. Luisi S, Florio P, Reis FM, Petraglia F. Expression and secretion of activin A: and
clinical
implications. European
Journal
of
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Endocrinology. 2001;145:225–36.
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4. Jones RL, Salamonsen LA, Zhao YC, Ethier JF, Drummond AE, Findlay JK. Expression of activin receptors, follistatin and betaglycan by human endometrial
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stromal cells; consistent with a role for activins during decidualization. Molecular Human Reproduction 2002; 8:363–374.
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5. Horne AW, van den Driesche S, King AE, Burgess S, Myers M, Ludlow H, Lourenco P, Ghazal P, Williams AR, Critchley HO, Duncan WC. Endometrial inhibin/activin beta-B subunit expression is related to decidualization and is reduced in tubal ectopic pregnancy. Journal of Clinical Endocrinology and Metabolism. 2008;93(6):2375-82. 6. Prakash A, Li TC, Tuckerman E, Laird S, Wells M, Ledger WL. A study of luteal phase expression of inhibin, activin, and follistatin subunits in the endometrium of women with recurrent miscarriage. Fertility and Sterility. 2006;86(6):1723-30. 7. Florio
P, Severi
FM, Bocchi
C, Luisi
S, Mazzini
M, Dannerò
S, Torricelli
M, Petraglia F. Single serum activin a testing to predict ectopic pregnancy. Journal of Clinical Endocrinology and Metabolism. 2007;92(5):1748-53.
ACCEPTED MANUSCRIPT 8. Warrick J, Gronowski A, Moffett C, Zhao Q, Bishop E, Woodworth A. Serum activin A does not predict ectopic pregnancy as a single measurement test, alone or as
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part of a multi-marker panel including progesterone and hCG. Clinica Chimica
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Acta. 2012;413 (7-8):707-11.
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9. Thompson TB, Cook RW, Chapman SC, Jardetzky TS, Woodruff TK. Beta A versus beta B: is it merely a matter of expression? Molecular Cell Endocrinology. 2004 ;225(1-2):9-17.
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10. Daponte A, Deligeoroglou E, Garas A, Pournaras S, Hadjichristodoulou C, Messinis
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IE. Activin A and follistatin as biomarkers for ectopic pregnancy and missed abortion. Disease Markers (United States), 2013;35 (5):497-50.
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11. Elito Junior J, Gustavo Oliveira L, Octavio Fernandes Silva M, Araujo Junior E,
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Camano L. Serum activin A concentrations and tubal ectopic pregnancy. Iran Journal of Reproductive Medicine 2014;12:227-28.
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12. Letterie GS, Hibbert M. Serial serum human chorionic gonadotropin (hCG) levels in ectopic pregnancy and first trimester miscarriage. Arch Gynecol Obstet.
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2000;263(4):168-9.
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Table 1: General characteristics, serum concentration of free β-hCG and activin B in tubal
Intrauterine
Normal
pregnancy(n=28)
miscarriages(n=31)
pregnancy(n=29)
Maternal Age (yrs)
24.3±3.2
24.2±4
25±5
GestationalAge (wks)
4.6±1.5
5.2±1.2
5.4±2.1
20115±28424*
56213±52530
free β-hCG (IU/L)
11415±19724* (20-33520)
(0.02-130875)
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(Min-Max)
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Tubal ectopic
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Parameters
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ectopic pregnancy, Intrauterine miscarriages and normal viable intrauterine pregnancy.
4.0(0.0-17.6)*
(Min-Max)
(6.4-104.5)
5.9(0.0-21.0)* (6.4-1816.7)
37.5 (6.4-55.6) (6.4-364.1)
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Activin B (pg/mL)
(200-200000)
Variables are expressed in mean ± standard deviation except for Activin B which is expressed
*
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in Median with interquartile ranges. indicates p value <0.05 in comparison to Normal Pregnancy. Normally distributed data was
compared by one way ANOVA with post hoc tukey test and non normal data with KruskalWallis test followed by bonferrini correction.
ACCEPTED MANUSCRIPT
Table 2: ROC analysis of serum concentration of free β-hCG,activin B,
and a model
Parameters
Area
Under Cutoff
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including both activin B & free β-hCG in tubal ectopic pregnancy.
Sensitivity
Activin B
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the Curve 0.722
<23.3
82
0.805
<5100
free β-hCG
(Activin B
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hCG)
0.001
79
79
0.679-0.898
<0.0001
>0.7045
68
93
0.700-0.912
0.0298
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0.824
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Model
& free β-
0.588-0.833
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(IU/L)
interval
62
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(pg/mL)
Specificity 95% Confidence P value
prediction equation for the model = 1 /1+ e-L Where L = - 0.016249 x Activin B – 0.000031144 x free β-hCG
ACCEPTED MANUSCRIPT Figure 1: Receiver operating curve (ROC) of serum Activin B, free β-hCG and Model including both in discriminating ectopic pregnancy from normal viable intrauterine
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pregnancy.
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ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Highlights Serum activin B concentration is low tEP and IUM compared to normal IUP.
When compared between tEP and IUM, activin B concentrations do not differ
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Serum activin B concentration can discriminate tEP from normal pregnancy with
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significantly.
moderate accuracy.
It also shows improved diagnostic potential along with β-hCG, but cannot distinguish tEP
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from IUM reliably.
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S0009-9120(16)00090-4 doi: 10.1016/j.clinbiochem.2015.11.024 CLB 9253
To appear in:
Clinical Biochemistry
Received date: Revised date: Accepted date:
3 September 2015 16 November 2015 18 November 2015
Please cite this article as: Dhiman Pooja, Senthilkumar GP, Rajendiran Soundravally, Sivaraman K, Soundararaghavan S, Kulandhasamy Maheshwari, Serum activin B concentration as predictive biomarker for ectopic pregnancy, Clinical Biochemistry (2016), doi: 10.1016/j.clinbiochem.2015.11.024
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Title Page Full title: Serum activin B concentration as predictive biomarker for ectopic
T
pregnancy
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Short title: Activin B in ectopic pregnancy
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Authors
1. Dr. Pooja Dhiman*, MD, Department of Biochemistry, 2nd floor, New Academic block, Jawaharlal Institute of Postgraduate Medical Education & Research, Puducherry-
NU
605006, India.
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2. GP Senthilkumar*, Phd, Department of Biochemistry, 2 nd floor, New Academic block, Jawaharlal Institute of Postgraduate Medical Education & Research, Puducherry-
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605006, India.
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3. Dr. Soundravally Rajendiran(corresponding author), MD, Associate Professor, Department of Biochemistry, 2nd floor, New Academic block, Jawaharlal Institute of
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Postgraduate Medical Education & Research, Puducherry-605006, India. [email protected]
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4. Sivaraman K, MSc, Department of Biochemistry, 2nd floor, New Academic block, Jawaharlal Institute of Postgraduate Medical Education & Research, Puducherry605006, India.
5. Dr. Soundararaghavan S, MS, Department of Obstetrics & Gynaecology, Jawaharlal Institute of Postgraduate Medical Education & Research, Puducherry-06, India. 6. Dr. Maheshwari Kulandhasamy, MD, Department of Biochemistry, 2nd floor, New Academic block, Jawaharlal Institute of Postgraduate Medical Education & Research, Puducherry-605006, India. *Share equal authorship
ACCEPTED MANUSCRIPT Abstract We evaluated the diagnostic accuracy of activin B in discriminating tubal ectopic pregnancy
T
(tEP) from intrauterine miscarriages (IUM), and normal viable intrauterine pregnancy (IUP).
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We included 28 women with tEP, 31 women with IUM, and 29 normal IUP, confirmed both
SC R
by clinical examination and ultrasonography. Serum activin B concentration was measured at the time of admission using ELISA kit. The median serum activin B concentration were
NU
found to be significantly decreased in both tEP (p =0.004) and IUM (p =0.022) compared to normal IUP. When compared between tEP and IUM, activin B concentrations did not differ
MA
significantly. ROC analysis of activin B and free β-hCG demonstrated AUC of 0.722 and 0.805, respectively to discriminate tEP from viable IUP. The model including both activin B
D
and free β-hCG improved the discriminating potential with greater AUC (0.824), and
TE
specificity (93%) than individual one. To discriminate tEP from IUM, activin B, free β-hCG and combination of both performed poorly. We conclude that serum activin B concentration
CE P
is lower in tubal ectopic pregnancy, and can discriminate it from normal pregnancy with moderate accuracy. It also shows improved diagnostic potential along with free β-hCG, but
AC
cannot distinguish tEP from IUM reliably.
Keywords- ectopic pregnancy; intrauterine miscarriage; activin B; diagnostic accuracy Corresponding author: Dr. Soundravally Rajendiran, Associate Professor, Department of Biochemistry, JIPMER, Puducherry-06, India;Email [email protected];Phone no 919442214256
ACCEPTED MANUSCRIPT Introduction Ectopic pregnancy, a disorder of early pregnancy, is associated with high morbidity and
T
mortality, predominantly due to delay in diagnosis. This delay stems from the fact that the
IP
current modality of diagnosis, that is transvaginal ultrasonography and serial free beta human
SC R
gonadotropin (free β-hCG) estimations are not entirely reliable (1). These fallacies of the available diagnostic tests have led to the search of reliable biomarkers for diagnosing
NU
ectopic pregnancy, to prevent life threatening complications from abdominal bleeding, and to minimize the need of surgical interventions. Various biomarkers have been investigated in
MA
the diagnosis of ectopic pregnancy as single and multiple markers, which include markers of normal implantation, abnormal corpus luteum and inflammatory markers (2).
Activins,
D
dimeric proteins of transforming growth factor-beta (TGF-b) family, first isolated from
TE
follicular fluid, have demostrated growth factor like actions (3). They are produced by many
CE P
organs including pituitary gland, gonads, placenta, adrenal,spleen etc. These molecules bind to their transmembrane receptors and bring about downstream phosphorylation of
AC
different proteins to alter gene expression, leading to increased decidualization of the endometrium during pregnancy (4). Consequently low serum activin concentration have been associated with ectopic pregnancy (4-8). Recent studies have mainly focussed on the role of serum activin A as a potential marker of ectopic pregnancy(3), and limited literature is available regarding the role of activin B, inspite of the strong experimental evidence of the differential effect of activin B on the decidua (5). We hypothesize that activin B concentration is low in tubal ectopic pregnancy (tEP) than normal intrauterine pregnancy(IUP) and intrauterine miscarriage (IUM),
and therefore can be useful in its
diagnosis. Hence, we evaluated the role of serum activin B in discriminating and predicting tEP from normal IUP and IUM.
ACCEPTED MANUSCRIPT
Material and Methods
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a. Study design: This is a study of diagnostic accuracy. This prospective case-control
SC R
study was carried out in JIPMER hospital, Puducherry in the Department of Biochemistry in collaboration with the Department of Obstetrics and Gynaecology between January 2011 to December 2012.
NU
b. Subjects: After seeking approval of Institute’s ethics committee and written informed consent from the subjects, patients who presented to the hospital with bleeding per
MA
vaginum, abdominal pain and positive free β-hCG, were recruited as subjects. To further confirm the diagnosis of the patients for subsequent management, transvaginal
D
ultrasonography scan was done by a group of obstetricians trained in the management
TE
of early pregnancy failure. Women who demostrated the evidence of embryo in the
CE P
fallopian tube and absence of intrauterine gestational sac on ultrasound, were included in the case group called tEP (n=28). Diagnosis of tEP was further confirmed in all cases by diagnostic laproscopy. All women in this group presented with impending
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rupture. Women who had ultrasonography evidence of intrauterine pregnancy and absence of vital signs of embryo at admission were included in the control group called IUM (n=31). We also included another control group consists of women with normal intrauterine pregnancy (IUP) (n=29). Clinical conditions which can affect the serum concentrations of activin B such as hydatiforme mole, history of missed abortion, subjects on medications based on progesterone, non tubal EP were excluded from this study. c. Blood sampling: Five millilitre of their venous blood samples was collected at the time of admission. The freshly seperated serum was stored at -70 o C until use.
ACCEPTED MANUSCRIPT d. Methods: Serum activin B concentrations were analysed using commercially available ELISA kit (Uscn Life Science Inc., USA). The assay was specific for human serum
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activin B using activin beta homodimer antibody, with the lower limit of detection
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being 6.4 pg/ml. Capture antibody was monoclonal antibody and detection antibody
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used was polyclonal antibody derived from rabbit.The inter-assay and intra-assay CV was <12% and <10% respectively. Total duration of assay was five hours. Briefly, all reagents, samples and standards were prepared and 100µL was added to each well,
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which was precoated with antibody specific to activin B. After an incubation of two
Following an
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hours at 37 o C, 100µL of biotin-conjugated antibody specific to activin B was added. incubation of 1 hour at 37
o
C, 100µL of avidin conjugated to
Horseradish Peroxidase (HRP), and subsequently TMB substrate was added . This
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was followed by an incubation of 15-25 minutes (37 o C). Lastly, 50µL stop solution was added and the plate was read at 450nm. Serum free β-hCG was measured by the
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principle of chemiluminescence in Siemen’s Advia Centaur (Siemens diagnostics, Germany).
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e. Statistical analysis: Statistical analysis was performed using the SPSS version 16 (SPSS, Chicago, IL, USA).
P< 0.05 was considered as statistically significant.
Results were compared using Non-parametric Kruskal–Wallis tests to identify differences in the concentrations of activin B among the three groups. The performance of serum activin B as a biomarker for ectopic pregnancy was evaluated by Receiver operator curve analysis (ROC) using Medcalc®, Belgium.
Results The study groups were matched for age and weeks of pregnancy. The median values with inter quartile range of serum activin B and free β-hCG are depicted in table 1. Activin B
ACCEPTED MANUSCRIPT concentration was significantly lower in woman with tEP compared to patients with normal IUP (P=0.004), and in women with intrauterine miscarriages compared to IUP (P =0.022). No
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statistically significant difference was observed between intrauterine miscarriage and tEP.
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ROC analysis of activin B and free β-hCG demonstrated AUC of 0.722 and 0.805
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respectively to discriminate tEP from viable IUP demonstrating moderate sensitivity and specificity (Table 2). The model including both the activin B and free β-hCG improved the discriminating potential with greater AUC (0.824), and specificity (93%) than individual one.
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(Fig 1 and Table 2). To discriminate tEP from intra uterine miscarriage, activin B, free β-
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hCG, and combination of both performed poor with p values of 0.65, 0.16 and 0.39 respectively. Correlation analysis between free β-hCG and activin B in different study groups
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was r= 0.081(p=0.675) in IUP, r=0.192 (p=0.333)in tEP and r= 0.146(p=0.441)in IUM,
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Discussion
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indicating weak correlation, which was statistically insignificant.
Reliable serum biomarkers for tubal ectopic pregnancy are in immense need for this potentially life threatening condition. This study addressed the above issue using single point measurement of serum activin B. Activins, a dimer of TGF-β family of proteins, functions to promote normal decidualization of the endometrium during pregnancy (4). Since tEP is associated with poor decidualization, serum activin concentrations are expected to be low in this condition. Though the functional aspect of activin A on endometrium have been described in detail, molecular action of activin B is yet to be explored. It has also been established in the previous studies that activin A and activin B function separately in the
ACCEPTED MANUSCRIPT tissues (9). Therefore, it would be prudent to study the diagnostic accuracy of activin B as a biomarker in discriminating tEP from normal IUP and IUM .
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Our study hypothesizes that activin B concentration is lower in tEP than normal pregnancy
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and IUM, and hence can be used as a diagnostic tool. We observed a lower concentration of
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activin B in women with tEP compared to those with normal IUP supporting our hypothesis. This result may be explained by the fact that activin B is involved in decidualization of endometrium in pregnancy, and in ectopic pregnancy this process is hindered causing
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decreased local and systemic concentration of activin B. Our results are in concordance with
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Horne et al, who found decreased expression of activin B in endometrium along with decreased serum concentration of activin B with normal concentration of progesterone in 11
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women with tEP(5). The same study also demonstrated a positive association of activin B
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with decidualisation of endometrium, which in turn reinforce its functional significance in the endometrial development. We observed the mean value of activin B in tEP to be 4 pg/ml,
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which is lower than the concentration reported in Horne et al study. This difference in results can be attributed either due to difference in gestational age of women in two studies(between
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6-8 weeks in the Horne et al study & 4-6 weeks in our study),or the use of different ELISA kit for the estimation of serum activin B(activin B ELISA that incorporates the use of monoclonal antibody 46A/F ,as both capture and detection antibody with a sodium dodecyl sulfate and heat pretreatment of samples in the study by Horne et al, whereas commercial ELISA kit(Uscn Life Science Inc., USA) using monoclonal capture antibody and polyclonal detection antibody, with no sample pretreatment in our study). It has been reported that the expression of activin B increases with increase in decidualisation of endometrium by an invitro experiment (5). We observed lower serum concentration of activin B in intrauterine miscarriages when compared to IUP, which is in line with results reported by Horne et al. Although decreased
ACCEPTED MANUSCRIPT expression of activin A in endometrium of women with recurrent miscarriages have been observed (10), there is paucity of reports on the status of activin B expression in miscarriages
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except for the indirect evidence from Horne et al report which demonstrated a decreased
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decidualisation status in miscarriage, which in turn correlates with the expression of activin
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B(5). We did not find any statistically significant difference in the concentration of activin B between tEP and IUM, thereby limiting the use of activin B in the diagnosis of tEP. Serum free β-hCG concentration was found to be significantly lower in both tEP and IUM
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than normal pregnancy . This result is expected in tEP as there is restricted growth of
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placenta due to limited space availability in the tubal musculature. In IUM, loss of placental tissue is the major etiological factor for decreased concentration of this hormone in the
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circulation. This study did not find any significant difference in serum free β-hCG
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concentration between tEP and IUM and similar findings has been reported previously (12). A significant positive correlation was observed between free β-hCG and activin B in all
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groups, but individual groups failed to establish any signifiact correlation between the two biomarkers. The probable reason for this result may be the small sample size in individual
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gropus. The model constructed using both free β-hCG and activin B measurment demostrated higher specificity than a single parameter in differentiating tEP from IUP, but was not significant in differentiating IUM from tEP. Further diagnostic potential of this model should be explored in larger cohort. Recently, the role of serum activin A as a biomarker for ectopic pregnancy have been explored with some varied results (4-8,11). The study conducted by Elito et al did not demonstrate any significant differences in activin A concentration between tEP and normal pregnancy. The reason could be the smaller sample size as it included only five cases of ectopic pregnancy. In our study, ROC analysis of serum activin B as a biomarker for ectopic pregnancy shows that at a value of less than 23.3 pg/ml , activin B had a sensitivity of 82%,
ACCEPTED MANUSCRIPT and specificity of 62% in discriminating tEP from normal IUP. ROC analysis did not show the utility of activin B in differentiating IUM from tEP. This is a first study of the kind to
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define a discriminative zone of activin B for tubal ectopic pregnancy, though fair number of
tEP from normal pregnancy with
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around 50 ng/ml has been reported to discriminate
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studies have described the diagnostic accuracy of activin A. The activin A concentration of
moderate accuracy (4,6) . This value is higher than the cut off of activin B defined in the current study. The difference could be attributed to the variations in the gestational age of the
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groups studied.
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Although preliminary, the findings demonstrate serum activin B to be a promising biomarker for tubal ectopic pregnancy. The limitations of the present study are the inability to measure
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serial changes in serum activin B concentration. Whether doubling time of activin B can be
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used in the diagnosis of tEP and IUM, similar to free β-hCG in the diagnosis of IUP needs to be evaluated. Also, the study could not include any cases of unruptured tEP since the
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institute is tertiary hospital, cases are referred once complications set in. When compared to free β-hCG, activin B has multiple source of origin(placenta, ovary, spleen, adrenals), and its
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assay is time consuming as compared to free β-hCG,which limits application in emergency. Our results indicate that serum activin B may have a promising role in the differentiation of ectopic pregnancy from normal gestation, but can not distinguish miscarriages from ectopic. These results further warrent larger prospective studies in various populations. Conclusion: Serum activin B concentrations are decreased in tubal ectopic pregnancy and discriminate tEP from normal pregnancy with moderate accuracy and also shows improved diagnostic potential along with free β-hCG, but cannot distinguish tEP from IUM reliably.
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References
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1. Jurkovic D, Wilkinson H. Diagnosis and management of ectopic pregnancy. British Medical Journal.2011;342:d3397.
2. Rausch ME, Barnhart KT. Serum biomarkers for detecting ectopic pregnancy.
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Clinical Obstetrics and Gynecology 2012;55:418-23.
Possible
physiological
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3. Luisi S, Florio P, Reis FM, Petraglia F. Expression and secretion of activin A: and
clinical
implications. European
Journal
of
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Endocrinology. 2001;145:225–36.
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4. Jones RL, Salamonsen LA, Zhao YC, Ethier JF, Drummond AE, Findlay JK. Expression of activin receptors, follistatin and betaglycan by human endometrial
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stromal cells; consistent with a role for activins during decidualization. Molecular Human Reproduction 2002; 8:363–374.
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5. Horne AW, van den Driesche S, King AE, Burgess S, Myers M, Ludlow H, Lourenco P, Ghazal P, Williams AR, Critchley HO, Duncan WC. Endometrial inhibin/activin beta-B subunit expression is related to decidualization and is reduced in tubal ectopic pregnancy. Journal of Clinical Endocrinology and Metabolism. 2008;93(6):2375-82. 6. Prakash A, Li TC, Tuckerman E, Laird S, Wells M, Ledger WL. A study of luteal phase expression of inhibin, activin, and follistatin subunits in the endometrium of women with recurrent miscarriage. Fertility and Sterility. 2006;86(6):1723-30. 7. Florio
P, Severi
FM, Bocchi
C, Luisi
S, Mazzini
M, Dannerò
S, Torricelli
M, Petraglia F. Single serum activin a testing to predict ectopic pregnancy. Journal of Clinical Endocrinology and Metabolism. 2007;92(5):1748-53.
ACCEPTED MANUSCRIPT 8. Warrick J, Gronowski A, Moffett C, Zhao Q, Bishop E, Woodworth A. Serum activin A does not predict ectopic pregnancy as a single measurement test, alone or as
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part of a multi-marker panel including progesterone and hCG. Clinica Chimica
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Acta. 2012;413 (7-8):707-11.
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9. Thompson TB, Cook RW, Chapman SC, Jardetzky TS, Woodruff TK. Beta A versus beta B: is it merely a matter of expression? Molecular Cell Endocrinology. 2004 ;225(1-2):9-17.
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10. Daponte A, Deligeoroglou E, Garas A, Pournaras S, Hadjichristodoulou C, Messinis
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IE. Activin A and follistatin as biomarkers for ectopic pregnancy and missed abortion. Disease Markers (United States), 2013;35 (5):497-50.
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11. Elito Junior J, Gustavo Oliveira L, Octavio Fernandes Silva M, Araujo Junior E,
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Camano L. Serum activin A concentrations and tubal ectopic pregnancy. Iran Journal of Reproductive Medicine 2014;12:227-28.
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12. Letterie GS, Hibbert M. Serial serum human chorionic gonadotropin (hCG) levels in ectopic pregnancy and first trimester miscarriage. Arch Gynecol Obstet.
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2000;263(4):168-9.
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Table 1: General characteristics, serum concentration of free β-hCG and activin B in tubal
Intrauterine
Normal
pregnancy(n=28)
miscarriages(n=31)
pregnancy(n=29)
Maternal Age (yrs)
24.3±3.2
24.2±4
25±5
GestationalAge (wks)
4.6±1.5
5.2±1.2
5.4±2.1
20115±28424*
56213±52530
free β-hCG (IU/L)
11415±19724* (20-33520)
(0.02-130875)
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(Min-Max)
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Tubal ectopic
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Parameters
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ectopic pregnancy, Intrauterine miscarriages and normal viable intrauterine pregnancy.
4.0(0.0-17.6)*
(Min-Max)
(6.4-104.5)
5.9(0.0-21.0)* (6.4-1816.7)
37.5 (6.4-55.6) (6.4-364.1)
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Activin B (pg/mL)
(200-200000)
Variables are expressed in mean ± standard deviation except for Activin B which is expressed
*
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in Median with interquartile ranges. indicates p value <0.05 in comparison to Normal Pregnancy. Normally distributed data was
compared by one way ANOVA with post hoc tukey test and non normal data with KruskalWallis test followed by bonferrini correction.
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Table 2: ROC analysis of serum concentration of free β-hCG,activin B,
and a model
Parameters
Area
Under Cutoff
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including both activin B & free β-hCG in tubal ectopic pregnancy.
Sensitivity
Activin B
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the Curve 0.722
<23.3
82
0.805
<5100
free β-hCG
(Activin B
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hCG)
0.001
79
79
0.679-0.898
<0.0001
>0.7045
68
93
0.700-0.912
0.0298
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0.824
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Model
& free β-
0.588-0.833
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(IU/L)
interval
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(pg/mL)
Specificity 95% Confidence P value
prediction equation for the model = 1 /1+ e-L Where L = - 0.016249 x Activin B – 0.000031144 x free β-hCG
ACCEPTED MANUSCRIPT Figure 1: Receiver operating curve (ROC) of serum Activin B, free β-hCG and Model including both in discriminating ectopic pregnancy from normal viable intrauterine
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pregnancy.
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ACCEPTED MANUSCRIPT Highlights Serum activin B concentration is low tEP and IUM compared to normal IUP.
When compared between tEP and IUM, activin B concentrations do not differ
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Serum activin B concentration can discriminate tEP from normal pregnancy with
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significantly.
moderate accuracy.
It also shows improved diagnostic potential along with β-hCG, but cannot distinguish tEP
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from IUM reliably.
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