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

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 marker still requires large prospective cohorts for validation.

#### **9.** Placental micro-RNA

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 biomarker discovery with further studies being conducted.

## **10.** Placental mRNA

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 detecting early pregnancy failures.

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

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study revealed that AFP was able to discriminate between miscarriage and ectopic, as well as between IUP and ectopic [Unpublished data].

#### **12.** Cell free fetal DNA

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 has been limited.
