Ectopic pregnancy (EP) is a form of pregnancy in which the zygote is implanted outside of the normal uterine cavity and accounts for approximately 1.5 to 2% of all pregnancies; over 95% of EPs are located in the fallopian tubes (FTs) [1]. The maternal morbidity and mortality arising from cases of EP has created a significant health and economic burden worldwide. Our knowledge of the underlying causes of this condition has increased over recent years and the most accepted hypothesis is retention of the fertilized egg within the FTs due to alterations in the tubal condition and impaired embryo-tubal transport, thus resulting in early embryo implantation. Over recent years, the rates of medical abortion, artificial abortion and previous chronic pelvic inflammatory disease (PID) as increased; this is associated with a mild increase in the incidence of EP [2]. The exact mechanism underlying EP induced by artificial abortion remains unclear. If the cervix is pulled excessively during artificial abortion, it can easily damage the uterosacral ligament and may damage nerves on the ligament surface. Excessive suction or curettage can also readily cause damage to the nerves between the endometrium and the myometrium. In a previous study, Neamtu et al. [3] suggested that the motility of the FTs was influenced by both nerves and hormones. Several neuropeptides, such as neuropeptide Y-immunoreactivity (NPY), have been detected in nerve fibers in the FTs [4]. For example, Jankovic et al. [5] detected some nerves fibers and neuropeptides in the FTs which were considered to regulate the surface epithelium, vasculature and smooth muscle, thus controlling the beat activity of cilia and the contractility of the smooth muscle in the FTs [6]. Moreover, the abnormal distribution of nerve fibers in the FTs may result in dysfunctional motion in the tube; this condition is closely related to EP.

Based on studies published by the pioneers mentioned above, we hypothesized that EP was also associated with nerve fiber damage. In our study, we detected damaged nerve fibers in the FTs of patients by immunohistochemical staining methods using highly specific biomarkers of nerve fibers, a polyclonal rabbit protein gene product 9.5 (PGP9.5) antibody and an S100 antibody.

The purpose of our study was to compare the distribution and density of nerve fibers in FTs between patients with and without EP. Our aim was to acquire a better knowledge of tubal pathologies such that we can improve the reproductive and sexual health of women.

No statistical differences were detected between the two groups in terms of body mass index (BMI), age, or the number of previous abortions (p $\mathrm{>}$ 0.05, Table 1). PGP9.5-immunoreactive nerve fibers were detected in all 25 patients of group B in the mucosal, muscular and serosal layers of the oviduct ampulla (100%, 25/25). PGP9.5-immunoreactive nerve fibers were also detected in women with EP, accounting for 84%, 88% and 84% in the mucosal, muscular and serosal layers of the ampulla tissue, respectively (Figs. 1,2). However, we found no statistical difference between the two groups in terms of the proportion of PGP9.5-immunoreactive nerve fibers in the mucosal, muscular and serosal layers of the oviduct ampulla (p $>$ 0.05, Table 1). There were significant differences in the density of PGP9.5-immunoreactive nerve fibers in women with EP when compared to women without FP in the serosal (6.22 $\pm$ 2.95/mm${}^2$ versus 11.42 $\pm$ 3.24/mm${}^2$, p = 0.006; Table 1), muscular (10.58 $\pm$ 3.19/mm${}^2$ versus 18.49 $\pm$ 3.82/mm${}^2$, p = 0.008; Table 1), and mucosal layer (5.98 $\pm$ 2.32/mm${}^2$ versus 10.21 $\pm$ 3.02/mm${}^2$, p = 0.002; Table 1).

Fig. 1.

PGP9.5 (a 40$\mathrm{\times }$, b 200$\mathrm{\times }$) and S100 (c 40$\mathrm{\times }$, d 200$\mathrm{\times }$) nerve fibers in the oviduct ampulla of women without tubal ectopic.

Fig. 2.

PGP9.5 (a 100$\mathrm{\times }$, b 200$\mathrm{\times }$) and S100 (c 100$\mathrm{\times }$, d 200$\mathrm{\times }$) nerve fibers in the oviduct ampulla of women with tubal ectopic.

Similar to PGP9.5, there were no significant differences between the two groups in the serosal, muscular and mucosal layers in the density of S100-immunoreactive fibers in the oviduct ampulla (p $>$ 0.05; Figs. 1,2). However, there was a significant reduction in the density of S100-immunoreactive nerve fibers in the mucosal, muscular and serosal layers of the ampulla in group B (the EP group; p 0.05, Table 1).

It is reported that over 98% of EPs occur in the FTs and that a proportion of these patients are difficult to diagnose until symptoms occur, mostly because of intra-abdominal bleeding [6]. The FTs is the most common site for EP, although other reported sites include the cornua uteri, cervix, ovary, broad ligament, and the interstitial portion of the uterus [1]. The current therapies for EP are drug therapy (mifepristone or methotrexate) and surgical interventions; however, acquiring a more enhanced knowledge of the pathogenesis of EP could avoid these situations by providing better prediction and prevention for at-risk patients [11]. In a previous study, Shao et al. [12] performed animal experiments related to FT biology and provided an enhanced knowledge of the mechanisms underlying EP; however, such studies cannot be performed in humans. Thus, the most significant area of confusion relates to why some women develop EP while others do not. Understanding the molecular and cellular mechanisms underlying the implantation of embryos in the FTs will lead to the development of better diagnostics and therapies for EPs [13].

The uterine nerves pass through the pelvic cavity and then through the uterosacral-cardinal ligament complex and uterine vessel before entering the uterus. The myometrium receives sparse innervation from endometrial-myometrial and sub-serosal nerve fibers, which also supply plexus to the muscle layers of the FTs. Furthermore, the distal FTs receive additional innervation from the ovarian nerves through the mesosalpinx. Therefore, injuries to the endometrial-myometrial nerves would have significant impact on the nervous supply to the FTs [14, 15]. A reduction of nerve fibers in the FTs may affect the secretory function of epithelial cells and the activity of ciliary cells; these events may play a significant role in the pathogenesis of EP [6].

S100 is an immunohistochemical marker of Schwann cells and represents a reliable and sensitive marker of nerve damage [16]. In a previous study, Bacchi and Fleury [17] visualized peripheral nerves in tuberculoid granulomas by staining for S100. In another study, Zhang et al. [10] reported that PGP9.5 was a highly specific pan-neuronal marker that stained both sensory nerve fibers and autonomic nerve fibers. S100 and PGP9.5 may, therefore, be considered as reliable diagnostic markers for nerve damage in the FTs. However, only a few of the existing have attempted to determine the role of S100 in the diagnosis of nerve damage in EP. Our present study showed that nerve fibers that were positive for S100 and PGP9.5 staining were present in the serosal, muscular and mucosal layers of the oviduct ampulla. Furthermore, when compared with women without EP, S100- and PGP9.5-immunoreactivity were both significantly reduced in each layer of the ampulla tissue in cases with EP. Moreover, Zhu et al. [18] reported the presence of PGP9.5-positive nerve fibers in smooth muscle strands, beneath the surface epithelium of the ampulla tissue, and in the vascular smooth muscle, thus suggesting that these may supply the surface epithelium, the smooth muscle, and the vasculature of the FTs. Abnormalities of the nerve plexus may be involved in the mechanisms that facilitate movement of the gametes; this could result in the pathogenesis of EP. On the other hand, the reduction of PGP9.5 and S100 in the mucosal and muscular layers of the FTs suggests that injury to the nerve fibers in the ampulla tissue may negatively impact on the release of transmitters including acetylcholine (ACh), adrenaline, noradrenaline, and the modulation of the nerve reflex. These events may reduce oviduct motility by influencing the vascular muscle contractility, the tubal smooth muscle, ciliary beat activity and the secretory processes of the mucosal epithelial cells [3], thus resulting in the pathogenesis of FP.

However, our study was retrospective, thus creating a notable limitation. Furthermore, most tissues were fixed and stored for approximately 1–2 years prior to analysis. This may be the reason why some tissues were negative for PGP9.5 and S100 staining. Further prospective studies need to be performed to overcome these limitations.

Our analysis showed that the density of nerve fibers stained with S100 and PGP9.5 in the oviduct ampulla tissue were significantly reduced in patients with EP. Our results suggested that nerve injury in the walls of the FTs may represent one of the causes of EP although further prospective studies are needed to confirm these findings.

The datasets generated and/or analyzed during the current study are not publicly available, because we still have some research on ectopic pregnancy in the future, but are available from the corresponding author on reasonable request.

FT: substantial contributions to the conception; FT, LJi: design of the work; LJin, MH: the acquisition, analysis, and interpretation of data for the work; LJi, LJin: drafting the manuscript; FT, MH: revising it critically for important intellectual content. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

The study was approved by the Ethics Committee of Affiliated Jinhua Hospital, Zhejiang University School of Medicine (ethics approval number: 2022-16). The Statements of Informed Consent was provided and written by the patients before surgery.

Thanks are due to Mingjun Shao for valuable discussion.

This research received no external funding.

The authors declare no conflict of interest.