**2.2. Markers of abnormal corpus luteal function**

These are the markers for the evaluation of luteal function by measuring secreted products from the ovary. As the continuous function of the corpus luteum is essential for the mainte‐ nance of early pregnancy, these functions may be suboptimal in ectopic pregnancy, and can be measured for diagnostic usage.

## **1.** Progesterone

**8.** A disintegrin and metalloprotease-12 (ADAM-12)

marker still requires large prospective cohorts for validation.

biomarker discovery with further studies being conducted.

**9.** Placental micro-RNA

56 Contemporary Gynecologic Practice

**10.** Placental mRNA

detecting early pregnancy failures.

between IUP and ectopic [Unpublished data].

**11.** Alpha feto protein (AFP)

**12.** Cell free fetal DNA

has been limited.

ADAM-12 is a glycoprotein primarily produced by syncytiotrophoblast, with a role in syncytial fusion [47]. ADAM-12 was found to be significantly decreased in patients with EP when compared to viable IUP in a cohort of 199 patients [48]. Horne et al observed that when measured in isolation, ADAM-12 levels had limited value as a diagnostic biomarker for EP [49], whereas Yang et al observed low levels of ADAM12 in complete spontaneous abortion and ectopic pregnancy compared to normal pregnancies [50]. Overall, this promising new

At least 31micro-RNA expressed from placenta have been isolated with various functions in gene regulation[51,52]. Zhao et al found that microRNA miR-323-3p was found to be lower in patients with EP as compared to those with normal IUP, yielding a sensitivity of 30% and a specificity of 90% in the diagnosis of ectopic pregnancy [53]. This is another promising area of

Placental mRNAs secreted by trophoblasts can be altered in early pregnancy failure, and therefore have been studied recently for their diagnostic potential in EP. A recent case-control study conducted by Takacs et al demonstrated that patients with EP have significantly lower copy numbers of hCG and hPL mRNA in plasma compared to viable IUP [54]. Placental mRNA have also been studied for viability and chromosomal aneuploidy [55,56]. Evaluation of changes in placental mRNA expression may serve as a potential biomarker in future for

AFP is a produced from both yolk sac and fetal liver with a role analogous to adult albumin [57]. Grosskinsky et al found AFP to be elevated in EP, whereas Kuscu et al study contradicted these findings [58, 59]. In our experience, we observed AFP concentration to be elevated in women with miscarriages when compared to normal IUP, but found it to be decreased in women with tubal ectopic compared to both IUP and miscarriages. ROC analysis in our study revealed that AFP was able to discriminate between miscarriage and ectopic, as well as

Cell free fetal DNA escaping into the maternal circulation has also been evaluated as a marker of early pregnancy failure. Lazar et al estimated the cell-free foetal DNA of the Sry gene, and observed its concentration to be significantly higher in those with a tubal ectopic pregnancy. At a cut-off of more than 80 GE/ml, cell free fetal DNA was able to differentiate a tubal ectopic from an intrauterine pregnancy with sensitivity of 84%, a specificity of 76% [60]. As the technology at present is cumbersome, its utility as a useful serum biomarker in a clinical setup

Maternal progesterone is initially produced by corpus luteum followed by placenta, and ensures appropriate development of the endometrium, uterine growth, adequate uterine blood supply, and preparation of the uterus for labor [60]. Progesterone levels are found to be low in both ectopic pregnancy and other abnormal IUP, when compared to viable IUP [62]. Extensive studies have been performed for the use of progesterone both as a single marker and in multiple marker settings to diagnose EP. A recent meta-analysis of 26 cohort studies, including 9436 pregnant women observed that at a cut-off values from 3.2 to 6 ng/mL, progesterone predicted a non-viable pregnancy with sensitivity of 74.6% and a specificity of 98.4%, although it was not able to distinguish EP from other abnormal IUP [63]. Similar results were observed by the meta analysis performed by Mol et al with a progesterone cut-off of <5ng/ ml [64]. In our experience of the cohort of women, progesterone concentration was significantly lower in woman with ectopic pregnancy and women with miscarriages compared to patients with normal IUP. We observed a cut-off of <4.6ng/ml, to have a sensitivity of 93.3% and a specificity of 96.7% in differentiating EP from viable IUP, and a a cut-off of <12ng/ml, to have a sensitivity of 92.8% and a specificity of 90% in differentiating miscarriages from viable IUP, but found progesterone to be unable to discriminate between EP and other abnormal IUP [Unpublished data]. Also, high progesterone does not rule out EP in a patient with inconclusive ultrasound findings. Therefore, further large scale studies are required to study the utility of progesterone as a single marker or as a part of multiple markers to diagnose EP with acceptable sensitivity and specificity.

#### **2.** Oestradiol

Oestradiol (E2) is another important steroid secreted from the corpus luteum of pregnancy in response to hCG and could serve as a marker of pregnancy dynamics. Guillaume at al observed in his preliminary study that all of the EP patients had an E2 level of less than 650 pg/ml giving a sensitivity of 100% and specificity of 99% [65]. Witt et al however, did not find any difference in serum E2 when women with EP were compared with those with nonviable intrauterine pregnancies [46]. Mantzavinos et al on the other hand reported that E2 concentrations rose continuously in viable pregnancies, whereas in EP the values plateaued after the sixth week and declined after the eighth week of gestation [66]. However, as the concentrations of serum oestradiol have a considerable overlap among various clinical studies, this marker has not been put into clinical use.

#### **3.** Inhibin A

Inhibin A is a dimeric protein produced from corpus luteum [67]. Seifer et al observed lower levels of inhibin-A in EP compared to normal viable IUP [68]. Further, Segel et reported that at a cut-off of 50 pg/mL, Inhibin-A had 100% sensitivity and specificity for diagnosing EP compared to viable IUPs, but found decrease in both sensitivity and specificity when patients of EP were compared with those of other abnormal IUP [69]. Similarly, Rausch et al found Inhibin A to have a sensitivity of 83% and a sensitivity of 79% at a lower cut-off of 23.67pg/ml in discriminating EP from viable IUP [28]. In contrast to the above findings, Chetty et al found Inhibin-A to be unable to discriminate EP from viable IUP in their cohort of 109 patients [70]. It can be concluded that although Inhibin-A is a promising marker for the early pregnancy viability, further studies need to be carried out to use it as a biomarker in the detection of EP.

#### **4.** Relaxin and renin

Relaxin is a peptide hormone produced by the corpus luteum of pregnancy, which is elevated shortly after conception and remain steady until the 15th week of gestation [71]. Garcia et al observed a lower levels of serum relaxin concentrations in patients with EP than that in those with a viable IUP [72]. On the other hand, Witt et al found relaxin to be poorly discriminatory as a biomarker of ectopic pregnancy [46].

Renin is another peptide produced by ovaries and the production rises after pregnancy [73]. Meunier et al reported that active renin was significantly decreased in women with EP when compared with those with an ongoing IUP or spontaneous miscarriage, however because of its low specificity and sensitivity, the use of renin in the clinical setting has been limited [74].

#### **2.3. Markers of angiogenesis**

**1.** Vascular Endothelial Growth Factor (VEGF)

VEGF plays an important role in the angiogenesis of the fetomaternal unit [75]. It has been reported that maternal serum VEGF concentrations are associated with depth of trophoblastic penetration into the tubal wall [76]. A case control study observed a cut-off of 200pg/ml had a sensitivity of 60% and a specificity of 90 % in predicting EP [77]. Similar findings were observed by Felemban et al [78]. Daponte et al observed a sensitivity of 78% and a specificity of 100 % in predicting EP at a cut-off of 174.5pg/ml. [41]. Recently Fernandes et al reported that serum level of VEGF was significantly higher in women with EP, and when threshold concentrations of serum VEGF level > 200 pg/ml were used, an EP could be distinguished from a normal pregnancy with a sensitivity of 51.4%, a specificity of 90.9% [79]. On the contrary, Rausch et al found no difference in the levels of VEGF between EP, abnormal IUP and viable IUP [28]. The variations in the study results of VEGF shows that further investigations are required to ascertain its suitability as a biomarker for EP.

**2.** Placental like growth factor (PlGF)

PIGF is a pro-angiogenic growth factor produced by the trophoblast at the site of implantation [80]. Horne et al reported the expression of PIGF m-RNA to be lower in trophoblastic cells in patients with EP as compared to those with viable IUP [81]. Daponte et al later observed that at a PIGF level of more than 15.73pg/ml could differentiate viable pregnancy from early pregnancy failures including EP and abnormal IUP with reasonable sensitivity and specificity, but could not differentiate among them [82]. Recent report by Martínez-Ruiz et al also didn't find significant differences for PlGF between EP and viable IUP [83]. Therefore, there is a need of validation of this marker in a larger cohort.

## **3.** Angiopoietins

at a cut-off of 50 pg/mL, Inhibin-A had 100% sensitivity and specificity for diagnosing EP compared to viable IUPs, but found decrease in both sensitivity and specificity when patients of EP were compared with those of other abnormal IUP [69]. Similarly, Rausch et al found Inhibin A to have a sensitivity of 83% and a sensitivity of 79% at a lower cut-off of 23.67pg/ml in discriminating EP from viable IUP [28]. In contrast to the above findings, Chetty et al found Inhibin-A to be unable to discriminate EP from viable IUP in their cohort of 109 patients [70]. It can be concluded that although Inhibin-A is a promising marker for the early pregnancy viability, further studies need to be carried out to use it as a biomarker in the detection of EP.

Relaxin is a peptide hormone produced by the corpus luteum of pregnancy, which is elevated shortly after conception and remain steady until the 15th week of gestation [71]. Garcia et al observed a lower levels of serum relaxin concentrations in patients with EP than that in those with a viable IUP [72]. On the other hand, Witt et al found relaxin to be poorly discriminatory

Renin is another peptide produced by ovaries and the production rises after pregnancy [73]. Meunier et al reported that active renin was significantly decreased in women with EP when compared with those with an ongoing IUP or spontaneous miscarriage, however because of its low specificity and sensitivity, the use of renin in the clinical setting has been limited [74].

VEGF plays an important role in the angiogenesis of the fetomaternal unit [75]. It has been reported that maternal serum VEGF concentrations are associated with depth of trophoblastic penetration into the tubal wall [76]. A case control study observed a cut-off of 200pg/ml had a sensitivity of 60% and a specificity of 90 % in predicting EP [77]. Similar findings were observed by Felemban et al [78]. Daponte et al observed a sensitivity of 78% and a specificity of 100 % in predicting EP at a cut-off of 174.5pg/ml. [41]. Recently Fernandes et al reported that serum level of VEGF was significantly higher in women with EP, and when threshold concentrations of serum VEGF level > 200 pg/ml were used, an EP could be distinguished from a normal pregnancy with a sensitivity of 51.4%, a specificity of 90.9% [79]. On the contrary, Rausch et al found no difference in the levels of VEGF between EP, abnormal IUP and viable IUP [28]. The variations in the study results of VEGF shows that further investigations are required to

PIGF is a pro-angiogenic growth factor produced by the trophoblast at the site of implantation [80]. Horne et al reported the expression of PIGF m-RNA to be lower in trophoblastic cells in patients with EP as compared to those with viable IUP [81]. Daponte et al later observed that at a PIGF level of more than 15.73pg/ml could differentiate viable pregnancy from early pregnancy failures including EP and abnormal IUP with reasonable sensitivity and specificity, but could not differentiate among them [82]. Recent report by Martínez-Ruiz et al also didn't

**4.** Relaxin and renin

58 Contemporary Gynecologic Practice

as a biomarker of ectopic pregnancy [46].

**1.** Vascular Endothelial Growth Factor (VEGF)

ascertain its suitability as a biomarker for EP.

**2.** Placental like growth factor (PlGF)

**2.3. Markers of angiogenesis**

Angiopoietins are proteins which belong to the family of angiogenic proteins, which has been shown to be critically involved in the process of placental maturation and growth from early pregnancy. Angiopoietin-1 (Ang-1) and Ang-2 are two critical regulators with different functions of vascular development and angiogenesis [84, 85]. Daponte et al reported Ang-1 and Ang-2 concentrations and their ratio to be lower in EP compared to IUP. They also found the trophoblastic Ang-1 mRNA expression levels to be lower in EP compared to IUP, while Ang-2 mRNA was found to be higher in EP than in IUP [86]. Schneuer et al in their recent study suggested that the lower Ang-1/Ang-2 ratio in first trimester is associated with most adverse pregnancy outcomes, but do not predict outcomes any better than clinical and maternal risk factor information [87].
