**2. Markers related to embryo**

**1.1. Problem statement**

52 Contemporary Gynecologic Practice

**1.2. Application area**

**1.3. Research course**

**1.4. Method used**

**1.5. Status**

intrauterine pregnancy (IUP).

at the first visit of the patient, is the need of the hour.

mortality associated with early pregnancy failure.

that assessed diagnostic accuracy were included.

both these categories are described

Tubal ectopic pregnancy is an important cause of early pregnancy failure attributing signifi‐ cantly to morbidity and mortality, in absence of expeditious diagnosis. Currently, the diagnosis of ectopic pregnancy is based on combined ultrasonograhic findings and serial β-hCG levels. However, low positivity rates in early gestational age and a need for repeated testing are major limitations of these diagnostic procedures, leading to added risk of tubal rupture and intraabdominal bleed. Therefore, a circulating serum based biomarker, which could differentiate tubal ectopic from viable intrauterine pregnancy and other abnormal gestation (miscarriages)

A circulating serum based biomarker which could accurately predict the outcome of preg‐ nancy such as ectopic pregnancy, miscarriage and viable intrauterine pregnancy. This may be used to diagnose tubal ectopic, decide the course of management (medical or surgical), and may aid in monitoring the prognosis and response to treatment in patients. This advancement in clinical diagnostic may help in significantly bringing down the maternal morbidity and

Due to paucity of suitable animal models of tubal ectopic pregnancy, biomarker researches have mainly been carried out in cohorts of women with tubal ectopic, miscarriages and viable

Comprehensive literature search was carried out for identifying studies on women with gestational age of less than 12 weeks in which serum markers were used to predict the outcome of pregnancy. The databases used were Medline, Embase, CINAHIL and Cochrane library, using key work search ectopic pregnancy", "tubal pregnancy" alone and in combination with "diagnosis", "screening" and "biomarkers". Only the articles published in English were included and references of all selected articles were also thoroughly searched. All the studies

The factors deciding the fate of first trimester pregnancy, and hence the potential biomarkers for tubal ectopic pregnancy can be systematically categorized into two groups-Markers related to implantation site & milieu and Markers related to embryo (Fig 1). Important biomarkers in

#### **2.1. Markers of abnormal trophoblast function**

Theses are the molecules secreted from the conceptus. It would be probable that the pregnancy implanted in the ectopic environment will have abnormal growth kinetics that can be reflected in disarrayed trophoblast function. The following markers have been described in this section:

**1.** Beta-human chorionic gonadotropin (β-hCG)

3 It is a glycoprotein hormone produced by trophoblast which maintains corpus luteum. Currently, β-hCG is the only biomarker used clinically in the diagnosis of ectopic pregnan‐ cy[4]. A doubling of serum β-hCG over 48 hours is suggestive of viable intrauterine pregnancy [5]. If the levels remain static or fall, it is suggestive of early pregnancy failure, and the diagnosis need to be confirmed by more invasive procedures like endometrial curettage or laproscopy. To prevent unnecessary invasive procedures, studies suggested that the minimum rise of βhCG over 48 hours to predict normal viable intrauterine pregnancy can be reduced to 53% [6] or 35% [7,8]. Another important concept in serum β-hCG estimation is the discriminatory zone, that is the minimum β-hCG concentration at which an intrauterine gestational sac could be reliably identified on ultrasound. The current discriminatory zone lies between 1500-3000 mIU/L [9]. Recently, a series of mathematical models incorporating β-hCG ratio (serum β-hCG at 48 hours/ serum β-hCG at 0 hours) have been developed to predict early pregnancy outcome, although it has not been sufficiently validated [10-13]. Another retrospective cohort study observed that in addition to the two values of serum β-hCG at 0 and 48 hours, third β-hCG evaluation on day 4 improved the accuracy of ectopic pregnancy prediction by 9.3% [14]. The role of β-hCG has also been studied to predict response to therapy. A cut-off value of 2121 mIU/L of the initial serum β-hCG, has shown a specificity of 76.4% and a sensitivity of 80.56% in predicting response to methotrexate therapy, with poor response below the cut-off [15]. Similarly, a cut-off of more than 1790mIU/ml was found to predict methotrexate treatment failure by another group [16]. Presently, serial monitoring of β-hCG is in use for the diagnostic triage of first trimester bleeding, but further studies are being conducted to use it in the prognostic protocol of EP.

#### **2.** Hyperglycosylated hCG

Hyperglycosylated hCG (hCG-H) is a variant of hCG with four instead of two oligosaccharides. The site of production is first trimester invading extravillous cytotrophoblast, where it prevents trophoblast cell apoptosis [17,18]. Low proportions of hCG-H (< 50% of total hCG) was shown to predict failed pregnancy [19,20]. Another study identified a cut-off of 13µg/L for identifying failed pregnancy with 73% sensitivity and 98% specificity [21]. On the other hand, Butler et al did not find a role of hCG-H in the diagnostic algorithm for EP [22]. Recent study explored the role of hCG-H in predicting ongoing pregnancies after in vitro fertilization and found that day 9 level of hCG-H > 110pg/ml was 96% specific for ongoing pregnancy [23]. Conflicting results regarding the use of hCG-H as a marker of preeclampsia have also been reported [24,25]. Since, there is paucity of data on use of either concentration or proportion in distinguishing ectopic pregnancy from miscarriage, further studies are required to validate its usefulness as a biomarker for EP.

#### **3.** Activin A

Activin A is a dimeric glycoprotein of TGF-β superfamily, with a role in cytotrophoblast invasion [26]. A study by Florio et al found Activin A level at a cut-off of ≤0.37ng/mL to have 100% sensitivity and 99.6% specificity for the diagnosis of EP [27]. Rausch et al found Activin A to have 80% sensitivity and 72% specificity as a single marker in a cohort of patients with EP [28]. Daponte et al also observed activin A concentrations to be significantly lower in women with EP and women with missed abortion, compared to IUP, and reported that at a threshold value of 505 pg/mL, activin A had 87.9% sensitivity and 100% specificity for discriminating an ectopic pregnancy from viable pregnancies [29]. On the contrary, Activin-A was not found useful in diagnosing EP [30, 31]. Similarly, Elito et al demonstrated that serum activin A levels could not discriminate between an EP from a normal intrauterine pregnancy when an adnexal mass was found by transvaginal scan [32]. Therefore, A validation of this biomarker in a larger cohort would be encouraged before makes it a tool for diagnostics.

**4.** Follistatin (FS)

cy[4]. A doubling of serum β-hCG over 48 hours is suggestive of viable intrauterine pregnancy [5]. If the levels remain static or fall, it is suggestive of early pregnancy failure, and the diagnosis need to be confirmed by more invasive procedures like endometrial curettage or laproscopy. To prevent unnecessary invasive procedures, studies suggested that the minimum rise of βhCG over 48 hours to predict normal viable intrauterine pregnancy can be reduced to 53% [6] or 35% [7,8]. Another important concept in serum β-hCG estimation is the discriminatory zone, that is the minimum β-hCG concentration at which an intrauterine gestational sac could be reliably identified on ultrasound. The current discriminatory zone lies between 1500-3000 mIU/L [9]. Recently, a series of mathematical models incorporating β-hCG ratio (serum β-hCG at 48 hours/ serum β-hCG at 0 hours) have been developed to predict early pregnancy outcome, although it has not been sufficiently validated [10-13]. Another retrospective cohort study observed that in addition to the two values of serum β-hCG at 0 and 48 hours, third β-hCG evaluation on day 4 improved the accuracy of ectopic pregnancy prediction by 9.3% [14]. The role of β-hCG has also been studied to predict response to therapy. A cut-off value of 2121 mIU/L of the initial serum β-hCG, has shown a specificity of 76.4% and a sensitivity of 80.56% in predicting response to methotrexate therapy, with poor response below the cut-off [15]. Similarly, a cut-off of more than 1790mIU/ml was found to predict methotrexate treatment failure by another group [16]. Presently, serial monitoring of β-hCG is in use for the diagnostic triage of first trimester bleeding, but further studies are being conducted to use it in the

Hyperglycosylated hCG (hCG-H) is a variant of hCG with four instead of two oligosaccharides. The site of production is first trimester invading extravillous cytotrophoblast, where it prevents trophoblast cell apoptosis [17,18]. Low proportions of hCG-H (< 50% of total hCG) was shown to predict failed pregnancy [19,20]. Another study identified a cut-off of 13µg/L for identifying failed pregnancy with 73% sensitivity and 98% specificity [21]. On the other hand, Butler et al did not find a role of hCG-H in the diagnostic algorithm for EP [22]. Recent study explored the role of hCG-H in predicting ongoing pregnancies after in vitro fertilization and found that day 9 level of hCG-H > 110pg/ml was 96% specific for ongoing pregnancy [23]. Conflicting results regarding the use of hCG-H as a marker of preeclampsia have also been reported [24,25]. Since, there is paucity of data on use of either concentration or proportion in distinguishing ectopic pregnancy from miscarriage, further studies are required to validate its

Activin A is a dimeric glycoprotein of TGF-β superfamily, with a role in cytotrophoblast invasion [26]. A study by Florio et al found Activin A level at a cut-off of ≤0.37ng/mL to have 100% sensitivity and 99.6% specificity for the diagnosis of EP [27]. Rausch et al found Activin A to have 80% sensitivity and 72% specificity as a single marker in a cohort of patients with EP [28]. Daponte et al also observed activin A concentrations to be significantly lower in women with EP and women with missed abortion, compared to IUP, and reported that at a threshold value of 505 pg/mL, activin A had 87.9% sensitivity and 100% specificity for

prognostic protocol of EP.

54 Contemporary Gynecologic Practice

**2.** Hyperglycosylated hCG

usefulness as a biomarker for EP.

**3.** Activin A

Follistatin is a circulating protein produced from human placenta, with rising serum concen‐ tration throughout pregnancy [33, 34]. Follistatin is primarily involved in modulating the biological activity of activin by binding to activin by high-affinity, which can neutralize the majority of the actions of activin [35]. Daponte et al reported that the concentration of FS was significantly lower in EP and MA compared to women with a viable IUP, and found that FS was able to discriminate IUP from EP, But not miscarriage from EP [29].

**5.** Pregnancy associated plasma protein-A (PAPPA)

PAPPA is a glycoprotein produced by trophoblast. Normally, PAPPA is up-regulated by progesterone, which promotes the adhesion and proliferation potential of trophoblastic cells [36]. It has been extensively studied and used as a marker of screening for first trimester aneuploidy [37]. Mueller et al found decreased levels of PAPPA in patients with ectopic pregnancy when compared to normal viable intrauterine pregnancy [38]. Dumps et al found a cut-off of <14.3 ng/ml to have a sensitivity of 64.5% with a 99% specificity for pregnancy failure [39] But the levels were found to be lower in miscarriages making the discrimination between EP and other abnormal intrauterine pregnancy dissatisfactory. On the other hand Ugurlu et al and Daponte et al observed that PAPPA can differentiate abnormal pregnancy from the viable normal IUP [40, 41]. Inconsistency in these results prompts us to study this marker in greater details.

**6.** Pregnancy specific beta glycoprotein-1 (SP-1)

Pregnancy specific beta glycoprotein-1 (SP-1) is a secreted protein produced from syncytio‐ trophoblast, thought to be involved in immunomodulation [42]. In addition, recently it has been observed that SP-1 induce transforming growth factor beta 1 (TGFB1), which among its other diverse functions inhibits T-cell function and has proangiogenic properties [43]. SP1 has also been reported as a first trimester maternal serum marker of small for gestational age (SGA) and preterm delivery [44]. For early pregnancy failure, Tornehave et al found lower levels of SP-1 to be suggestive of ectopic pregnancy [45]. Witt et al found a sensitivity of 65 % and a specificity of 74 % by taking a cut-off of 103.3µg/ml [46]. Further studies are needed to validate the use of SP-1 in EP.

**7.** Human placental lactogen (hPL)

Human placental lactogen is a circulating protein produced by the trophoblasts. Mueller et al found hPL levels to be decreased in ectopic pregnancy [38], but Rausch et al and Daponte et al found no difference in hPL levels between Ectopic pregnancy, abnormal IUP and normal viable IUP [28,41]. hPL as a biomarker of EP has to be evaluated in larger cohort of patients.
