Exploring the presence of markers of decidualization in the fallopian tubes: a systematic review

Abstract The fallopian tubes (FTs) are part of the female upper genital tract. The healthy FT provides the biological environment for successful fertilization and facilitates the subsequent movement of the conceptus to the endometrial cavity. However, when the FT is damaged, as with salpingitis, pyosalpinx, and hydrosalpinx, it may increase the risk of an ectopic pregnancy, a life-threatening condition. Decidualization refers to a multifactorial process by which the endometrium changes to permit blastocyst implantation. The decidualization reaction is vital for endometrial receptivity during the window of implantation. To date, no comprehensive review that collates evidence on decidualization in the human FT has been conducted. Therefore, the aim of this review is to compile the current evidence on cellular decidualization occurring in the healthy and pathological FT in women of reproductive age. A literature search was conducted using five databases and identified 746 articles, 24 of which were analyzed based on inclusion and exclusion criteria. The available evidence indicates that the FT are able to undergo decidual changes under specific circumstances; however, the exact mechanism by which this occurs is poorly understood. Further research is needed to elucidate the mechanism by which decidualization can occur in the FT.


Introduction
The fallopian tubes (FTs) are part of the female upper genital tract. In health, the FTs provide the biological environment for successful fertilization and facilitate the transport of the conceptus from the distal part of the FT to the endometrium [1]. However, pathological processes that cause tubal damage increase the chance of ectopic pregnancy (EP), a lifethreatening condition [2]. EP is defined as the implantation of a blastocyst outside the endometrial lining of the uterus [3]. While EPs only occur in 2% of all pregnancies, they account for 8-9% of maternal mortality; over 95% of EPs are located in the FT, with the majority implanting in the ampulla [3,4]. Intriguingly, tubal EP (tEP) appears to be restricted to primates and does not occur in other mammals. This distinction may be due to differences in uterine and tubal anatomy in primates, which allow for mixing of luminal fluids and thus potentially promote a more permissive environment for implantation in the FT [5]. Risk factors for tEP include, but are not limited to, previous tubal surgery, existing tubal pathology, and infection of the genital tract [6]. However, many EPs occur in women without any known risk factors [3]. In non-idiopathic tEP cases, the conventional postulation that ectopic implantation is a direct consequence of tubal damage has not been fully confirmed by the available evidence [3].
Pelvic inflammatory disease (PID) is the infection of the upper genital tract, which may manifest as pathologies of the FT [7]. These include salpingitis, pyosalpinx, and hydrosalpinx [7,8]. Salpingitis refers to inflammatory and edematous FTs after ascending infection [7]. Pyosalpinx refers to a FT that is distended with pus due to obstruction following infection, inflammation, and subsequent formation of adhesions around the FT [7]. Hydrosalpinx describes a distended, fluid-filled FT that occurs as a result of tubal obstruction [7].
The tubal mucosa, termed the endosalpinx, possesses a distinct profile of hormone receptor expression across the menstrual cycle, yet does not demonstrate the same dynamic changes in proliferative activity in response to hormones as the eutopic endometrium ( Figure 1A) [9]. Normal embryo implantation occurs in the endometrium, and decidualization is considered a prerequisite for establishing a pregnancy [10]. A 2010 review exploring possible functional mechanisms by which risk factors predispose a tEP concluded that such molecular pathways have yet to be fully elucidated [10].
Decidualization, otherwise known as the decidual reaction, refers to a multifactorial process through which the endometrial stratum functionalis changes to allow a blastocyst to interact with the endometrium and implant. Decidualization includes both morphological and functional changes, of which the two most important are the differentiation of endometrial stromal cells to decidual cells, and leukocyte recruitment [11,12]. Decidual transformation of stromal cells is primarily mediated by progesterone, which promotes intracellular accumulation of cyclic adenosine monophosphate (cAMP) [12]. Progesterone and cAMP regulate a network of signaling pathways; cAMP-mediated protein kinase A (PKA) is critical for decidualization, and exchange protein directly activated by cAMP (EPAC) can potentiate this process [13]. Downstream regulators of progesterone/cAMP signaling include forkhead box O1 (FOXO1), signal transducers and activators of transcription (STAT5), and CCAAT/enhancerbinding protein β (C/EBPβ). Decidual cells secrete factors that regulate embryo implantation and placentation, including insulin-like growth factor binding protein-1 (IGFBP-1) and prolactin (PRL) [11,12]. Leukocytes play vital roles in decidual remodeling and immune tolerance of the endometrium during pregnancy establishment ( Figure 1B) [14]. Humans are among the few viviparous species in which the endometrium will begin the process of decidualization during the postovulatory secretory phase of the menstrual cycle, independent of the presence of a conceptus [11,12]. The decidual reaction is key to the endometrium being receptive, thus sanctioning the window of implantation, the timeframe within which the blastocyst can attach to and invade the superficial uterine wall [15]. An abnormal decidual response can lead to aberrations in placentation and, thus, both early and late gestational problems, such as recurrent implantation failure and preeclampsia [16].
To date, no comprehensive review has explored the available evidence on decidualization in the FT. Here, we conduct a systematic review to explore the potential for a decidual response in healthy and diseased FTs.

Methods
This systematic review was reported in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) statement [17] and was preceded by a prospectively written protocol registered with PROSPERO (registration number: CRD42022333468) [18].

Search strategy and selection criteria
A comprehensive literature search was conducted on 29 September 2022. Scopus, PubMed, CINAHL, EMBASE and EMCARE were searched for relevant published material. The search strategy included the following Medical Subject Heading (MeSH) terms, keywords, and their combinations: ("Fallopian tube" OR "Oviduct" OR "Uterine tube") AND ("Decidua"). No filters were applied to the search, and wildcards were incorporated to encompass various word endings where appropriate. All search results were uploaded into Rayyan [18], an electronic systematic review software enabling enhanced title and abstract screening. Duplicated literature was removed, and two independent reviewers performed a title and abstract screen according to the inclusion and exclusion criteria. Studies that met the following criteria were included: (1) concerning the decidualization of the human FT in health or benign pathology, (2) population of pre-menopausal or pregnant women, (3) publications in the English language. The exclusion criteria included (1) exclusive focus on malignant pathology; (2) animal studies; and (3) secondary, non-electronic, and gray literature. Following screening, full-text reviews were conducted by two independent reviewers, and a third reviewer was recruited for the resolution of any disagreements.

Data extraction and analysis
Data from all eligible studies were extracted and recorded into an Excel spreadsheet recording the following: author, year of publication, study aim, sample size, comparator groups, experimental technique, relevant results, and author conclusions. Given the heterogeneity of both the methods and results of included studies, statistical meta-analysis was not feasible. Therefore, data have been presented thematically.

Quality assessment
Risk-of-bias assessment was conducted by two independent reviewers (FA and CHR) using two well-established scoring tools. The Newcastle-Ottawa Scale (NOS) [19] was used for case-control and cohort studies and evaluated each study based on three domains: selection, comparability, and outcome. Each study receives a score between 0 and 9, which categorizes as either good, fair, or poor. In addition, a modified version of the NOS proposed by Murad et al. [20] was used for case series, which consists of eight questions across four domains: selection, ascertainment, causality, and reporting. Although a score between 0 and 8 can be attributed to each study based on binary responses to each question, Murad et al. suggest that numerical representation of methodological quality is not always recommended when certain questions are deemed more essential than others. Therefore, in this study, a judgment of methodological quality for each paper was made based on questions 1, 2, 3, 4, 6, and 8. The risk-of-bias assessment is detailed in Table 1 and Table 2.

Results
The literature search identified 746 unique articles; 354 remained after removing duplicate studies. Eligibility screening of these publications based on the assessment of their title and abstract, following the predetermined inclusion and exclusion criteria, led to the exclusion of a further 169 publications. The remaining 185 full-text articles were sought for retrieval, where, following evaluation, an additional 161 articles were excluded. Subsequently, 24 studies are included in the present review. This selection process is illustrated by a PRISMA flow diagram in Figure 2. Table 3 provides a summary of all studies included in this systematic review.

Decidualization associated with tubal ectopic pregnancy
Decidualization associated with tEP has been described in several studies. Nine included papers observed a decidual reaction in the FT containing the tEP at the site of implantation, away from the site of implantation within the same tube, or at both sites [25,[27][28][29][30][31][32][33][34][35]. One study has also demonstrated decidualization in the contralateral FT in women with tEP [25]. However, Floridon et al. (1999) detected tubal decidualization only in two cases of tEP with localized endometriosis from a total of 50 tEP specimens. None of the above studies described the decidualization to be as extensive as would be expected at the implantation site in a normal IUP. Ordi et al. (2006), Goffin et al. (2006), and Vassiliadou et al. (1998) analyzed a total of 41 tEP specimens and concluded an absence of a decidual reaction at the site of implantation [21,36,37]. Interestingly, Randall et al. (1987) demonstrated that cells, which initially resembled decidual cells at the site of implantation, were in fact of cytotrophoblastic origin [32].

Leukocyte infiltration in the fallopian tube
A study by Von Rango et al. (2001) indicated that the number of CD45 + leukocytes increased in the tubal mucosa from non-pregnant to tEP and suggest it to be a consequence of increased numbers of CD68 + macrophages [38]. In tEP, there is a marked lack of CD56 + uterine natural killer (uNK) cells, which are thought to limit trophoblast invasion in normal IUPs [21,[37][38][39]. Ordi et al. (2006) found that increased recruitment of uNK cells in decidual tissue is a common phenomenon regardless of location and that this process is mediated by hormones rather than the presence of an implanting blastocyst [21]. In addition, Von Rango et al. (2001) stated that while uNK cells are not necessary for successful implantation, they may limit trophoblast invasion; thus, the absence of uNK cells in the FT is proposed to allow for the increased trophoblastic invasion seen in tEP [38].
The most abundant leukocytes identified in tEP were macrophages and T cells [37][38][39]. When comparing the leukocyte populations at the tEP implantation site with the matched intrauterine decidua, the numbers of T cells and macrophages were similar [39]. Basta et al. (2010) reported a significantly lower percentage of T regulatory cells in the subpopulation of CD4 + T lymphocytes in the decidual of tEP compared to the secretory phase eutopic endometrium [30].

Cellular markers of decidualization
The studies included in this review investigated various cellular markers of decidualization. In particular, two studies by Refaat et al. (2008Refaat et al. ( , 2011) employed immunohistochemistry and quantitative reverse transcription polymerase chain reaction to quantify the expression of activins in tEP. These studies suggest that activins have a paracrine and autocrine action in the FT; in the endometrium, decidualization is facilitated by activins increasing the expression of matrix metalloproteinases [40,41]. The increased expression of activins in tEP, when compared to secretory phase tubes, is considered pathological. However, it is also suggestive of tubal decidualization because activins are raised in the cycling endometrium during the luteal phase [40,41]. Refaat et al. (2008) also investigated the action of follistatin in tEP. Their findings indicated that the expression of activins and follistatin might play an important role in the pathogenesis of ectopic implantation but not necessarily in determining the site of implantation. The authors propose that increased expression of activin-A in the FT could increase nitric oxide production, which may induce a pathological relaxation in the smooth muscle of the FT. This muscular relaxation would prevent adequate movement of an embryo, which in turn could increase the chance of a tEP [40,41]. This theory is supported by a similar finding, whereby the embryo was located in the same place as a decidual polyp in the tube; Wist et al. (1954) suggest that the tubal obstruction prevented the embryo from moving through the tube and therefore caused a tEP [33]. Wist et al. (1954) proposed that the decidual reaction should be considered a result of pregnancy but not the cause of the tEP [33].
The expression of mucin-1 (MUC1) in tubal epithelial cells fluctuates throughout the menstrual cycle [42]. In the luteal phase, increased MUC1 expression in tubal epithelial cells may act as a protective mechanism against ectopic implantation, which might include an anti-adhesive effect and/or facilitate transport [42]. In tEP, decreased MUC1 expression indicates feature changes in the tubal epithelium [42].
Fibronectin is a ligand for integrins that is present at the implantation site in the endometrium and has a key role in embryo implantation following its adhesion to the maternal tissue. Integrins and fibronectin, which are considered necessary for uterine implantation, have also been shown to be present in tEP, indicating that they may have a role in tubal implantation [27]. Kuroda et al. (2004) observed the total loss of alpha-smooth muscle actin (αSMA) and CD34 + stromal cells in both IUP and tEP compared to non-pregnant endometrial and tubal tissues. Loss of αSMA + and CD34 + stromal cells may therefore indicate decidualization-specific changes in tEP [31].
A study by Ji et al. (2013) compared estrogen receptor (ER) and progesterone receptor (PR) expression between normal mid-secretory non-pregnant tubes with tEP, both at the site of implantation and at distant regions of the same tube. They reported a decrease in the expression of ER and PR at the site of implantation compared to other tubal regions of the pregnant FT and secretory phase non-pregnant tubes; the expression of ER and PR in the latter two groups was similar. Expression of ER and PR was mainly confined to the epithelial nuclei and sparsely in the tubal stroma [43]. Land and Arends (1992) suggest that the absence of sufficient decidualization in tubal pregnancies may be explained by the lack of PR in the FT [28], yet the action of progesterone via PR is known to reduce the expression level of its own receptor and ER [44].

Discussion
The objective of this review was to compile the available evidence regarding the potential of the FT to undergo decidualization. We found that the FT has the ability to undergo stromal decidualization under specific circumstances [31,45]. However, unlike the endometrium, where decidualization is a hallmark of the secretory phase, decidualization in the FT appears to be a relatively rare occurrence, and it is unclear how or why it transpires.
In the endometrium, decidualization is modulated by cyclic fluctuations in the ovarian steroid hormones, estrogen and progesterone [46]. In the absence of an embryo, the superficial endometrial layer is shed during menses. Endometrial decidua can be characterized by morphological changes, phenotypic markers, and a unique immune cell profile.
In the luteal phase, the rise in progesterone levels stimulates a chain of reactions in the endometrial stromal cells, causing an upregulation of multiple genes, including the classical markers of decidual cells, PRL and IGFBP-1 [11]. Endometrial receptivity also involves the presentation of adhesion molecules and simultaneous loss of inhibitory factors that prevent embryo attachment [47]. The phenotypical changes of the endometrium include vascular remodeling, an influx of uNK cells, and the differentiation of stromal cells to a hypertrophic, secretory phenotype [12,48]. There is a fivefold increase in leukocytes during the secretory phase, of which the most notable change is the significant increase in uNK cells; uNK cells account for approximately 70% of the total leukocyte population [21,47,49].
As identified in this review, the FT have the ability to decidualize under specific circumstances. One explanation is that the FT is more sensitive to the higher concentrations of progesterone produced by the placenta compared to the relatively moderate levels produced by the corpus luteum, which is why decidual changes in the FT associated with IUP are present post-partum [25].
It is important to note that the hormone responsiveness of the tubal mucosa is proposed to be different to that of endometrial cells. The dynamic changes in the expression of steroid hormone receptors are not observed in healthy pre-menopausal tubes when compared with the eutopic endometrium [9]. The relative hormone resistance of the tubal mucosal would prevent initiation of decidualization, but in the event that the cells become sensitized, possibly via prolonged and sustained exposure to high progesterone levels, decidualization may occur and promote tEP.
Furthermore, there is a paucity of studies investigating the FT in early IUP to confirm that this is only a late pregnancy event. For obvious reasons, access to such material is limited. The developing fetus and placenta of ongoing IUPs produce many endocrine agents that may influence the tubal mucosa, potentially inducing decidual changes [11,12,14]. Intriguingly, no studies have investigated tubal changes following exogenous progestogen administration, which typically induces a decidualization response in the endometrium. Therefore, further studies are needed to conclude on the decidualization potential of healthy FT.
The receptive endometrium describes the stage at which an embryo can implant, and it can have degrees and types of abnormality [50]. In parallel, extravillous trophoblasts are thought to switch from a differentiating phenotype to an invasive phenotype, which is believed to occur independently of the maternal environment [36], meaning that the embryo could begin to invade any tissue that it is in contact with when this change occurs. This postulation is acceptable, considering the observation of rare ectopic pregnancies in the abdomen. Evidence suggests that tubal implantation may occur due to stagnation of the embryo in the FT; such immobility will allow prolonged exposure of the FT to the secretory products of an embryo, which may induce a local decidual reaction in the FT, encouraging tubal implantation [33]. However, stagnation of the embryo in the FT in the study by Wist et al. (1954) occurred due to the presence of a decidual polyp [33]. Interestingly, pseudoxanthomatous salpingitis manifests histological similarities to decidualization [51]. As the study by Wist et al. was published in 1954, it can be speculated that the multiple decidual polyps were, in fact, expanded plicae due to the presence of numerous histiocytes [33,51].
Activins are important autocrine/paracrine regulators that stimulate and facilitate endometrial decidualization, which is crucial for successful implantation [52]. They are secreted by newly decidualized cells, promoting the spread of decidualization throughout the endometrium [40,[53][54][55]. The presence of specific molecular markers, such as activins in the FT, affects tubal mobility via altering smooth muscle contractility and/or ciliary beat activity, leading to tubal transport failure and, consequently, blastocyst retaining within the tube, which overexposed the tubal epithelium to the embryonic chorionic gonadotrophin, and ultimately induces tubal epithelial receptivity [9,[38][39][40].
Although the immune cell profile of the FT may have similarities to that of the endometrium, there are stark differences, such as the increased number of T cells and the lack of uNK cells in the FT. The absence of uNK cells in the FT may allow over-invasion of the extravillous trophoblasts [36], which could be a reason for frequent rupture of the FT observed in tEP. Unlike the endometrium, the immune cell profile of the FT does not appear to change in response to an embryo implanting. This is again likely to reflect the relative resistance of the tubal mucosa to steroid hormones [9]. Von Rango et al. (2001) identified T cells, followed by macrophages, as the most abundant leukocytes in the healthy FT [38]. This immune profile is similar to that of the proliferative phase endometrium described by Vallvé-Juanico et al. (2019), whereby the most abundant leukocytes are T cells, followed by macrophages and uNK cells [56], although uNK cells are virtually absent from tubal mucosa [38,57].
In summary, there is insufficient evidence to define the decidualization potential of healthy and pathological FT in full. Furthermore, no studies have explored decidualization reactions in damaged FT, such as hydrosalpinx after infection. Therefore, it remains challenging to find a causal relationship between factors influencing tEP. This uncertainty creates a causality dilemma, in which it is difficult to confirm what came first: the embryo expressing the invasive phenotype or a pre-existing receptive FT. In addition, the association of ectopic pregnancy with many risk factors, such as previous FT surgery and PID, is well established; however, this review did not identify any literature exploring decidualization in such cohorts. Furthermore, there is a lack of studies regarding decidualization in the FT during specific stages of the menstrual cycle.

Conclusions
The FT can undergo decidual changes under specific circumstances. These may include prolonged exposure to high levels of progesterone, placental products, and prolonged exposure to a conceptus. The presence of decidual cells in tEP is poorly understood, and many questions are left unanswered. Further research surrounding the decidualization of the FT at different stages of the menstrual cycle and following damage would help to bridge the gap of knowledge in understanding the pathophysiology of the FT. In addition, receptivity markers, the proliferation of the tubal mucosa, and the immune profile of normal and damaged tubes could be explored in FT across the menstrual cycle. Such studies could provide greater insight into the mechanisms of aberrant embryo implantation at ectopic sites.