Elsevier

Life Sciences

Volume 260, 1 November 2020, 118439
Life Sciences

The therapeutic effects and underlying mechanisms of the intrauterine perfusion of granulocyte colony-stimulating factor on a thin-endometrium rat model

https://doi.org/10.1016/j.lfs.2020.118439Get rights and content

Highlights

  • Intrauterine perfusion of G-CSF could significantly increase the thickness of endometrium in thin-endometrium rat model.

  • Cytokeratin 19 and Vimentin were augmented in G-CSF treatment group when compared to control group.

  • G-CSF treatment could stimulate the expression of VEGF and LIF in thin-endometrium rat model.

  • G-CSF might improve endometrial receptivity through stimulating endometrial proliferation and angiogenesis.

Abstract

Aims

This study aims to investigate the effects of intrauterine perfusion of granulocyte colony-stimulating factor (G-CSF) on a thin-endometrium rat model.

Main methods

Twenty rats in two groups of 10 were used. Group I was perfused with normal saline (NS) in the right uterine horn and 95% ethanol in the left one. Group II was bilaterally perfused with 95% ethanol into the uterine horns. After three estrous cycles, Group II was perfused with NS in the right uterine horn and G-CSF (30 μg/kg) in the left one. Hematoxylin–eosin (HE) and immunohistochemistry (IHC) staining were used to detect changes in endometrial thickness and expression of cytokeratin 19 (CK19) and vimentin (Vim). The relative expression levels of vascular endothelial growth factor (Vegf) and leukemia inhibitory factor (Lif) were also tested via reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) and Western-blot analyses.

Key findings

G-CSF treatment significantly increased the thickness of the endometrium in the 95% ethanol-induced thin-endometrium rat model. The expression levels of endometrial glandular epithelial cell marker for CK19 and stromal cell marker Vim were augmented in the G-CSF-treated group compared with the control group. Moreover, G-CSF treatment stimulated the expression of VEGF and LIF in the 95% ethanol-induced thin-endometrium rat model.

Significance

G-CSF intrauterine perfusion improved endometrial receptivity in the thin-endometrium rat model by stimulating endometrial proliferation and angiogenesis.

Introduction

The ultimate goal of assisted reproductive technology is to achieve successful clinical pregnancies and healthy live births after embryo implantation [1]. A thin endometrium is generally defined as the cut-off value of 7 mm in the luteal middle phase, which is critical for embryo implantation and pregnancy maintenance [2,3]. The pathogenesis of a thin endometrium is very complicated. The suppression of vascular endothelial growth factor (VEGF) decreases the endometrial blood supply and leads to a thin endometrium [4]. In addition, decreased estrogen receptor expression can jeopardize the stimulating effect of estrogen on endometrial proliferation [5]. Endometrial thickness can be used as an endometrial receptivity marker that will predict the outcome of in vitro fertilization and embryo transfer (IVF-ET) [6]. Thus, the management of a thin endometrium is one of the most difficult and critical issues in clinical work.

Given that a thin endometrium usually leads to repeated implantation failure and cycle cancellation, many traditional methods, such as extended estrogen administration, vasoactive medicines, and endometrial scraping, have been used to increase the endometrial thickness. However, none of these strategies have shown consistent effects on patients; thus, new therapeutic approaches to improve the endometrial thickness are very desirable. In recent years, several clinical studies have shown that the intrauterine perfusion of granulocyte colony-stimulating factor (G-CSF) can improve the endometrial thickness, clinical pregnancy rate, and embryo implantation rate [[7], [8], [9]]. However, the efficacy of G-CSF administration to infertile women with thin endometria has been contradictory in some studies [[10], [11], [12]]. To prevent clinicians from becoming confused by these inconsistent conclusions and provide evidence for clinical decision support, we published the first meta-analysis and systematic review on this topic in 2017 [13]. Although the encouraging results from our meta-analysis suggest that the intrauterine perfusion of G-CSF can achieve good clinical prognosis in women with thin endometria, the underlying mechanisms remain unclear.

G-CSF was first cloned and purified from mice in 1983 and then outside the human body three years later [14]. G-CSF plays a significant immunological role by normally inducing neutrophilic granulocyte to proliferate and differentiate [15]. G-CSF promotes the number of regulatory T cells, activates dendritic cells, and suppresses the cytotoxic effects of natural killer cells [[16], [17], [18]]. In the reproductive system, G-CSF contributes to follicular development, ovulation, and embryo implantation [[19], [20], [21]]. G-CSF can also promote endometrial vascular remodeling, local immunoregulation, and cell adhesion to treat recurrent implantation failure [22]. Although the direct effect of G-CSF on thin endometria is still unproven, the potential mechanism could be related to some of the downstream immunological factors for endometrial rehabilitation and regeneration that help in repairing the damaged glands and stroma of thin endometria. Some studies have suggested that G-CSF also plays an important role in improving endometrial receptivity by affecting the invasion of trophoblasts cells through the upregulation of metalloproteinase-2 (MMP-2) and VEGF [23], promoting the implantation process by decreasing CD16 and CD56, and increasing the expression of leukemia inhibitory factor (LIF) [12]. Meanwhile, the results of previous studies suggested that the possible mechanism of endometrial regeneration promoted by G-CSF is mobilizing bone marrow mesenchymal stem cells to the peripheral blood supply and then to differentiate them into epithelial cells [24,25]. In clinical practice, G-CSF is usually administered to patients with thin endometria by intrauterine perfusion rather than intravenous injection. Therefore, the exploration of the specific molecular biological mechanisms of G-CSF on the local endometrium is particularly meaningful.

In this study, a thin-endometrium rat model was established by 95% ethanol bathing followed by treatment with or without G-CSF intrauterine perfusion. Finally, the endometria of the rats were collected for morphological and molecular examinations. The aim of the present study is to explore the potential mechanisms of the therapeutic effects of G-CSF on rats with thin endometria.

Section snippets

Animal model

Twenty female Sprague–Dawley (SD) rats weighing 180–220 g at 6–8 weeks old were purchased from the Laboratory Animal Center of Guangxi Medical University. The estrous stage of the rats was defined by vaginal smear cytology. All of the procedures in the animal experiments were reviewed and approved by the Animal Care and Welfare Committee of Guangxi Medical University (No. 201712011) and were in accordance with the UK Animals (Scientific Procedures) Act, 1986.

The 95% ethanol-induced

Results

Among the 20 rats included in the experiments, 18 successfully recovered from the procedure (the number of uterine horns in each group was 9) and two died from an overdose of anesthesia (Fig. 1).

Discussion

Given that optimal endometrium thickness is critical for pregnancy, the preparation of endometrium has been considered crucial for embryo implantation. G-CSF, as a new approach of enhancing blood supply and improving the receptivity of endometrium, has been widely used in clinical practice. However, mechanism research about G-CSF based on a reliable and reproducible animal model of thin endometrium is urgently required due to difficulties in obtaining human thin-endometrium specimens.

In this

Conclusions

The intrauterine perfusion of G-CSF can improve the endometrial thickness in 95% ethanol-induced thin endometrium rat model through the promotion of angiogenesis by VEGF augmentation and the improvement of endometrial receptivity by LIF augmentation. However, the detailed mechanisms and biological pathways remain unclear. We first administrated G-CSF by intrauterine perfusion, which is close to the clinical practice of G-CSF administration. Further studies should focus on comparing the

Acknowledgment

This study was supported by grants from the National Natural Science Foundation of China (NO. 81871172) and the Natural Science Foundation of Guangxi Zhuang Autonomous Region (NO. 2018GXNSFDA050017 and NO. 2019GXNSFFA245013) and the Guangxi Medical University Training Program for Distinguished Young Scholars to Yihua Yang. The authors also thank Derong Li for nice help on animal modeling and John M. for English revision.

Declaration of competing interest

The authors declare that there are no conflicts of interest.

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