The therapeutic effects and underlying mechanisms of the intrauterine perfusion of granulocyte colony-stimulating factor on a thin-endometrium rat model
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.
References (36)
- et al.
Outcome of assisted reproduction treatment in patients with endometrial thickness less than 7 mm
Reprod. BioMed. Online
(2009) - et al.
Pathophysiological features of thin endometrium
Fertil. Steril.
(2006) - et al.
Two protocols to treat thin endometrium with granulocyte colony-stimulating factor during frozen embryo transfer cycles
Reprod. BioMed. Online
(2015) - et al.
The granulocyte colony-stimulating factor (G-CSF) upregulates metalloproteinase-2 and VEGF through PI3K/Akt and Erk1/2 activation in human trophoblast Swan 71 cells
Placenta.
(2014) - et al.
Efficacy of ethanol sclerotherapy for ovarian endometriomas
Int. J. Gynecol. Obstet.
(2001) - et al.
A case of keratin-positive acute myeloid leukemia: a possible role for cytokeratin 19 as a specific epithelial marker
Pathol
(2000) - et al.
Pathophysiologic features of “thin” endometrium
Fertil. Steril.
(2009) - et al.
Decidua in human implantation
Hum. Reprod.
(1995) - et al.
Endometrial thickness and pregnancy rates after IVF: a systematic review and meta-analysis
Hum. Reprod. Update
(2014) - et al.
Steroid receptors ERalpha, ERbeta, PR-A and PR-B are differentially expressed in normal and atrophic human endometrium
Histol. Histopathol.
(2007)
A retrospective cross-sectional study: fresh cycle endometrial thickness is a sensitive predictor of inadequate endometrial thickness in frozen embryo transfer cycles
Reprod. Biol. Endocrinol.
A pilot cohort study of granulocyte colony-stimulating factor in the treatment of unresponsive thin endometrium resistant to standard therapies
Hum. Reprod.
A prospective randomized controlled study (RCT) of intra-uterine administration of granulocyte colony-stimulating factor (G-CSF) before embryo-transfer on resistant thin endometrium in IVF cycles
Hum. Reprod.
Granulocyte colony-stimulating factor administration for infertile women with thin endometrium in frozen embryo transfer program
Reprod. Sci.
Granulocyte colony stimulating factor (G-CSF): does it really improve the endometrial thickness in women with persistent thin endometrial thickness undergoing frozen endometrial transfer?
Hum.Reprod.
Transvaginal perfusion of G-CSF for infertile women with thin endometrium in frozen ET program: a non-randomized clinical trial
Iran. J. Reprod.
Efficacy of intrauterine perfusion of granulocyte colony-stimulating factor (G-CSF) for infertile women with thin endometrium: a systematic review and meta-analysis
Am. J. Reprod. Immunol.
Molecular cloning and expression of cDNA for human granulocyte colony-stimulating factor
Nature
Cited by (14)
Green tea catechin EGCG could prevent obesity-related precocious puberty through NKB/NK3R signaling pathway
2022, Journal of Nutritional BiochemistryCitation Excerpt :The stained sections were scanned with the Pannoramic Digital Slide scanner (Ningbo Jiangfeng Bioinformatics Technology Co., Ltd, China) and photographed using Pannoramic Viewer software (K-Viewer, version 1.5.5.6). Endometrial thickness was measured by the K-Viewer software, as described previously [24]. Primary antibodies against NKB (1:200, Abcam) were used to immunostain ovarian tissues, according to the manufacturer's recommendations.
Granulocyte colony-stimulating factor in reproductive-related disease: Function, regulation and therapeutic effect
2022, Biomedicine and PharmacotherapyCitation Excerpt :The possible roles of G-CSF in reproduction have been supported by previous studies, demonstrating that systematic or local G-CSF treatment could improve reproductive outcome, but the specific mechanism is unclear. Results from experimental rats showed that G-CSF administration could induce a thicker endometrium and higher expression levels of vimentin, cytokeratin 19, vascular endothelial growth factor (VEGF), leukemia inhibitory factor (LIF), proliferative cell nuclear antigen (PCNA) and VEGF receptor-2, which promoted endometrial proliferation, angiogenesis and DNA fragmentation [98–100]. Results from experimental porcine showed that G-CSF supplementation upregulated the expression of genes related to anti-apoptosis (BCL2), proliferation (PCNA) and pluripotency (POU5F1) in the blastocyst samples [101].
The Effect of Granulocyte Colony Stimulating Factor (G-CSF) on the Outcome of Assisted Reproduction Technology in Thin Endometrium Women: A Meta-Analysis
2023, Clinical and Experimental Obstetrics and GynecologyNetwork Pharmacology Analysis of the Mechanisms Underlying the Therapeutic Effects of Yangjing Zhongyu Tang on Thin Endometrium
2023, Drug Design, Development and TherapyFibroblast growth factor 1 ameliorates thin endometrium in rats through activation of the autophagic pathway
2023, Frontiers in Pharmacology