There is a need for a sensitive and specific diagnostic biomarker that can facilitate early diagnosis and permit timely treatment of tubal EP
[14, 15]. Currently, the limited specificity and sensitivity of circulating biomarkers for the early diagnosis of tubal EP has greatly restricted their reliability.
The present study has significant strengths. First, this study shows in an independent and well-defined sample set that individual measurements of serum P4 levels were lower in women with tubal EP than gestational age-matched women with IUP, but this difference was not statistically significant. Sub-group analysis, however, provided a much more striking result. When samples were compared at a serum β-hCG level of less than 1000 mIU/mL, serum P4 levels differed significantly between the tubal EP and gestational age-matched IUP groups. These results suggest that using a combination of serum P4 and β-hCG levels can possibly differentiate a certain group of women with tubal EP from those with normal IUP. More importantly, we show that individual measurements of serum E2 or VEGF-A levels have significant advantages over serum P4 levels for distinguishing tubal EP from gestational age-matched IUP. Our data, together with that provided by others
[15, 33], show that circulating E2 levels are higher in women with tubal EP than non-pregnant women in the mid-secretive phase of the menstrual cycle, but are lower than in gestational age-matched women with IUP. These findings indicate that abnormal E2 levels may interrupt the tubal microenvironment and ultimately lead to improper embryo implantation in the Fallopian tube. In line with previous studies
[12, 18–23]despite differences in the study populations, we have found a significant induction in VEGF-A levels in women with EP relative to gestational age-matched women with IUP. Using an increased cut-off of 200 pg/mL, we have shown that all women with tubal EP (n = 33) had a serum VEGF-A level more than 200 pg/mL, which is consistent with previous studies
[18, 21]. The data indicate that a serum VEGF-A level of greater than 200 pg/mL can be used to discriminate a tubal EP from a normal IUP in women. Additionally, we found that serum E2 and VEGF-A concentration measurements generated superior diagnostic accuracy. Taken together, these data suggest that E2 and VEGF-A can be useful biomarkers for the early diagnosis of tubal EP.
The second important finding of the present study is that there are different correlations between the levels of steroid hormones and VEGF-A, PIGF, and ADAM12 in both the early stage of IUP and in tubal EP. This suggests that their in vivo biological functions should not be considered independently of each other. We show that, in contrast to the early stage of IUP, serum E2 levels do not correlate with serum VEGF-A levels but do correlate with serum PIGF levels in tubal EP. We also show that serum T levels correlate with serum VEGF-A levels and that serum P4 levels correlate with serum ADAM12 levels in tubal EP. Although highly suggestive, these significantly correlated data cannot prove that steroid hormones are responsible for the changes in the levels of VEGF-A, PIGF, and ADAM12 in the Fallopian tube. Embryonic implantation in the uterus is a complex developmental process that is likely mediated by a variety of molecules such as steroid hormones, growth factors, and cytokines
. However, little is known about the interactions of these molecules, especially in terms of tubal implantation
[5, 9]. Previous studies have demonstrated the use of combinations of circulating proteins for the diagnosis of EP
[12, 22], and we show here that the E2: VEGF-A and E2: PIGF ratios can aid in distinguishing tubal EP from normal IUP. It will be of interest in future studies to validate the results from our study with additional cohorts and to determine whether alternative combinations of these markers may demonstrate improved performance.
Possible mechanisms responsible for the regulation of VEGF/PIGF expression, secretion, and signaling pathways during the development of tubal EP
To grow to the point of causing harm, a tubal EP requires the development of a local blood supply and angiogenesis at the tubal implantation site. It is thus reasonable to expand upon clinical research on circulating VEGF isoforms to understand the mechanisms by which their synthesis is regulated and to understand their biological functions during Fallopian tubal implantation. Such research would allow for a better understanding of the actual diagnostic values of VEGF-A and PIGF. In the human Fallopian tube, VEGF is localized in the epithelial cells, smooth muscle cells, and blood vessel cells in a region-specific manner
[35, 36], and significantly higher levels of tubal VEGF and VEGF receptor mRNAs are detected in women with a hydrosalpinx
, which is defined as tubal dilation and abnormal fluid accumulation
. Moreover, in vitro studies have shown an increase in VEGF and soluble VEGF receptor secretion in human tubal epithelial cells and stromal fibroblasts in response to hypoxic stimulation
. It has been shown that VEGF-A and VEGF receptor mRNAs are significantly increased in the implantation site compared to non-implantation sites of human Fallopian tubes
, and these are associated with trophoblastic invasion into the tubal wall in vivo
 suggesting that the development of tubal EP could contribute to the elevation of circulating VEGF-A levels. Moreover, ligation of the Fallopian tube, which mimics the tubal occlusion that likely induces an EP in women, increases VEGF protein expression in rat Fallopian tubes
Our results, together with those from other laboratories, provide a basis for proposing a working model for regulation and activation of VEGF isoforms and their receptor signaling pathways during tubal implantation. Disruption of the local environment such as a low oxygen level, possibly due to embryo implantation in the Fallopian tube, induces elevated levels of hypoxia-inducible factors (HIFs)
. This leads to tubal VEGF-A synthesis and secretion resulting in the abnormal levels of circulating VEGF-A seen in women with EP. HIF, a critical hypoxia sensor, is a heterodimeric complex composed of three alpha subunits (HIF-1α, HIF-2α, and HIF3α) and a stable beta subunit (HIF-1β, also known as aryl hydrocarbon receptor nuclear translocator (ARNT))
. Hypoxic HIF activity is controlled primarily through post-translational modification and stabilization of the HIF-1α and HIF-2α subunits, and HIF-1β expression levels constitute important determinants of hypoxia responsiveness
. It is well known that heterodimeric complexes of HIF-1α/β translocate to the nucleus and activate several hypoxia-associated genes, including VEGF
. VEGF-A can form homodimers with itself and/or heterodimers with PIGF, and acts by binding to its two cell surface tyrosine kinase receptors, FLT-1 (VEGFR1) and KDR/FLK-1 (VEGFR2). PIGF binds only to FLT-1 (VEGFR1)
. Thus, implantation in the Fallopian tube leads to the coordinated activation of a transcriptional cascade in response to the presence of excessive hypoxia. As a result, tubal VEGF-A levels are increased and this activates the VEGF signaling cascades in an autocrine manner. On the other hand, increased levels of tubal VEGF causes them to be released into the circulation, and this leads to activation of the VEGF signaling cascades in a paracrine manner. Both autocrine and paracrine regulation may result in tubal fluid secretion, vascular defects, angiogenic dysfunction, and tubal wall damage. During normal pregnancy, VEGF isoforms are involved in building the placenta
. VEGF-A is expressed in the human placenta throughout gestation
, and placenta-specific PIGF has a similar role as VEGF during normal IUP
. We show that serum PIGF levels are significantly increased in the middle and late stages of IUP compared to the early stage of IUP. In contrast to VEGF-A, however, we were unable to find a significant difference in serum PIGF levels between women with tubal EP and gestational age-matched women with IUP. Some previous studies have indicated that serum PIFG levels are elevated in tubal EP compared to IUP
[23, 24]. This discrepancy may be due to the different ethnic backgrounds of the patients analyzed, the pooling of blood samples from different gestational-aged women with acute and chronic clinical presentation of EP, and the use of a non-gestational age-matched IUP as a comparison control.
E2 has traditionally been considered the major hormonal regulator of the Fallopian tube
. E2 exerts its biological effects by binding to the estrogen receptor (ER), which exists as two different subtypes, ERα and ERβ. Both ER subtypes are expressed in normal human Fallopian tubes
[5, 44–46]. Moreover, ERα is frequently lost in the implantation and non-implantation site (our unpublished data) of the Fallopian tube in women who have suffered from EP
[44, 47]. Although the VEGF gene promoter harbors the estrogen response element
, whether E2 is able to directly regulate VEGF-A expression via the activation of ER signaling in human Fallopian tubes is not fully understood. On the other hand, animal studies suggest that E2 and hypoxia can cooperate to regulate the same target in the Fallopian tube. For example, the expression of erythropoietin, a potent anti-inflammatory cytokine, is increased by both E2 and hypoxia in mouse Fallopian tubes both in vivo and in vitro
. Treatment with E2 followed by hypoxic stimulation significantly reduces VEGF-A protein synthesis and release in human endometrial tissues in vitro
. Because the C-terminal domain of HIF-1β, a potent coactivator of ER-dependent transcription, is essential for the enhancement of ER transcription
, E2-dependent regulation of VEGF expression and secretion may be indirect through HIF isoforms during the Fallopian tubal implantation. Furthermore, in vitro experiments have shown that insulin-like growth factor-1 and interleukin-1β directly regulate VEGF and VEGFR expression in human tubal epithelial cells and stromal fibroblasts
[51, 52]. Collectively, these observations show that different molecules contribute to the regulation of VEGF synthesis and secretion, but their precise roles in tubal EP are not clearly understood. No existing mouse models can establish causative roles for factors implicated in the pathogenesis of EP
[1, 9], thus new animal models will need to be developed to specifically address these issues.
Although placenta-specific ADAM12 has been previously reported to distinguish EP from IUP
[12, 26], our findings are in agreement with a recent finding by Horne et al.
 that demonstrated the limited utility of single serum ADAM12 measurements as an early diagnostic biomarker for tubal EP.
Limitations to the study
A limitation of the study is the relatively small sizes of the patient groups, and these may not accurately represent the actual biomarker levels during the development and onset of tubal EP. Thus additional large-scale studies are needed to determine the changes in selected biomarker levels before onset of tubal EP to validate their utility in diagnosing tubal EP. Because it is ethically impossible to obtain tissues from early IUP and post-surgery EP patients, another limitation is that our study lacks data using Western blotting analysis and immunohistochemistry for determining VEGF-A, PIGF, and ADAM12 expression levels in these patients.