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CsA promotes trophoblast invasion accompanied by changes in leukaemic inhibitory factor and fibroblast growth factor in peri-implantation blastocysts

Published online by Cambridge University Press:  21 December 2023

Dan Li Open the ORCID record for Dan Li [Opens in a new window] ,
Qiuling Jie ,
Qi Li ,
Ping Long ,
Zhen Wang ,
Jiaxing Wang ,
Shengnan Tian ,
Menglan Wu ,
Yanlin Ma  and
Yuanhua Huang
Dan Li
Affiliation:
Hainan Provincial Key Laboratory for Human Reproductive Medicine and Genetic Research, The First Affiliated Hospital of Hainan Medical University, Hainan Medical University, China Hainan Medical University, China Department of Reproductive Medicine, Haikou Women & Children Hospital, China
Qiuling Jie
Affiliation:
Hainan Provincial Key Laboratory for Human Reproductive Medicine and Genetic Research, The First Affiliated Hospital of Hainan Medical University, Hainan Medical University, China Hainan Medical University, China Hainan Provincial Clinical Research Center for Thalassemia, The First Affiliated Hospital of Hainan Medical University, Hainan Medical University, China Department of Reproductive Medicine, The First Affiliated Hospital of Hainan Medical University, Hainan Medical University, China Haikou Key Laboratory for Preservation of Human Genetic Resource, The First Affiliated Hospital of Hainan Medical University, Hainan Medical University, China
Qi Li
Affiliation:
Hainan Provincial Key Laboratory for Human Reproductive Medicine and Genetic Research, The First Affiliated Hospital of Hainan Medical University, Hainan Medical University, China Hainan Provincial Clinical Research Center for Thalassemia, The First Affiliated Hospital of Hainan Medical University, Hainan Medical University, China Department of Reproductive Medicine, The First Affiliated Hospital of Hainan Medical University, Hainan Medical University, China Haikou Key Laboratory for Preservation of Human Genetic Resource, The First Affiliated Hospital of Hainan Medical University, Hainan Medical University, China
Ping Long
Affiliation:
Guizhou Qiannan People’s Hospital, China
Zhen Wang
Affiliation:
Hainan Provincial Key Laboratory for Human Reproductive Medicine and Genetic Research, The First Affiliated Hospital of Hainan Medical University, Hainan Medical University, China Department of Reproductive Medicine, The First Affiliated Hospital of Hainan Medical University, Hainan Medical University, China Haikou Key Laboratory for Preservation of Human Genetic Resource, The First Affiliated Hospital of Hainan Medical University, Hainan Medical University, China
Jiaxing Wang
Affiliation:
Hainan Medical University, China
Shengnan Tian
Affiliation:
Hainan Medical University, China
Menglan Wu
Affiliation:
Department of Reproductive Medicine, The First Affiliated Hospital of Zhengzhou University, China
Yanlin Ma
Affiliation:
Hainan Provincial Key Laboratory for Human Reproductive Medicine and Genetic Research, The First Affiliated Hospital of Hainan Medical University, Hainan Medical University, China Hainan Medical University, China Hainan Provincial Clinical Research Center for Thalassemia, The First Affiliated Hospital of Hainan Medical University, Hainan Medical University, China Department of Reproductive Medicine, The First Affiliated Hospital of Hainan Medical University, Hainan Medical University, China Haikou Key Laboratory for Preservation of Human Genetic Resource, The First Affiliated Hospital of Hainan Medical University, Hainan Medical University, China
Yuanhua Huang*
Affiliation:
Hainan Provincial Key Laboratory for Human Reproductive Medicine and Genetic Research, The First Affiliated Hospital of Hainan Medical University, Hainan Medical University, China Hainan Medical University, China Hainan Provincial Clinical Research Center for Thalassemia, The First Affiliated Hospital of Hainan Medical University, Hainan Medical University, China Department of Reproductive Medicine, The First Affiliated Hospital of Hainan Medical University, Hainan Medical University, China Haikou Key Laboratory for Preservation of Human Genetic Resource, The First Affiliated Hospital of Hainan Medical University, Hainan Medical University, China
*
Corresponding author: Yuanhua Huang; Email: [email protected]
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Summary

During the early stages of human pregnancy, successful implantation of embryonic trophoblast cells into the endometrium depends on good communication between trophoblast cells and the endometrium. Abnormal trophoblast cell function can cause embryo implantation failure. In this study, we added cyclosporine A (CsA) to the culture medium to observe the effect of CsA on embryonic trophoblast cells and the related mechanism. We observed that CsA promoted the migration and invasion of embryonic trophoblast cells. CsA promoted the expression of leukaemic inhibitory factor (LIF) and fibroblast growth factor (FGF). In addition, CsA promoted the secretion and volume increase in vesicles in the CsA-treated group compared with the control group. Therefore, CsA may promote the adhesion and invasion of trophoblast cells through LIF and FGF and promote the vesicle dynamic process, which is conducive to embryo implantation.

Keywords

Cyclosporine AFibroblast growth factorInvasionLeukaemic inhibitory factorTrophoblast
Type
Research Article
Information
Zygote , Volume 32 , Issue 1 , February 2024 , pp. 71 - 76
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Copyright
© The Author(s), 2023. Published by Cambridge University Press

Introduction

Infertility is a global reproductive health problem (Rooney and Domar, Reference Rooney and Domar2018). In vitro fertilization–embryo transfer (IVF–ET) has been accepted by the majority of couples with infertility since the first ‘test tube baby’ was born (Johnson, Reference Johnson2019). Embryo implantation is a complex and important process after ET. Adhesion and invasion of trophoblast cells are critical to this process, and it is a complex but well balanced control system. The trophoblast cells in contact with the mother are mainly extravillous trophoblast cells, which can be divided into proliferative and invasive phenotypes. The proliferative phenotype has proliferation and division activity. Trophoblast cells with the invasive phenotype stop proliferating, start to differentiate and have the ability to invade, but this is inconsistent with the continuous aggressiveness of tumour cells (Carvajal et al., Reference Carvajal, Gutiérrez, Morselli and Leiva2021). Invasive extravillous trophoblast cells further differentiate into vascular endothelial and stromal-phenotype trophoblast cells. Vascular endothelial-phenotype extravillous trophoblast cells invade and destroy the vascular wall and enter the vascular cavity, gradually replacing vascular endothelial cells, and finally completing the process of embryo implantation into the decidua (Tani et al., Reference Tani, Mitsui, Mishima, Ohira, Maki, Eto, Hayata, Nakamura and Masuyama2021). Stromal-phenotype extravillous trophoblast cells secrete proteolytic enzymes to invade the decidua and the decidual–muscular interface, therefore enabling preimplantation embryos to acquire invasive ability (Huang et al., Reference Huang, Ma, Ma, Mao, Zhang, Du and Li2014). Enhancing the adhesion and invasion of trophoblast cells is an important strategy to improve the success rate of embryo preimplantation (Park et al., Reference Park, Mani, Clair, Olson, Paurus, Ansong, Blundell, Young, Kanter, Gordon, Yi, Mainigi and Huh2022). However, the success rate of IVF is not high as a result of embryo implantation disorder (Menkhorst et al., Reference Menkhorst, Winship, Van Sinderen and Dimitriadis2016; Pantos et al., Reference Pantos, Grigoriadis, Maziotis, Pistola, Xystra, Pantou, Kokkali, Pappas, Lambropoulou, Sfakianoudis and Simopoulou2022). Multiple factors can affect the adhesion and invasion of preimplantation trophoblasts. So far, there have been many studies in this area, but they have not been comprehensive, such as studies observing the molecular morphology from the ultrastructure and the roles of related factors.

In 2002, Junwang found that the intracellular transport function of vesicles could upregulate integrin binding activity and enhance trophoblast cell migration and invasion (Wang et al., Reference Wang, Mayernik and Armant2002). Vesicles are commonly used as indicators of trophoblast invasiveness. LIF mainly regulates cell proliferation and differentiation and significantly promotes trophoblast invasion in early pregnancy (Hamelin-Morrissette et al., Reference Hamelin-Morrissette, Dallagi, Girouard, Ravelojaona, Oufqir, Vaillancourt, Van Themsche, Carrier and Reyes-Moreno2020). FGF is a key factor in embryo implantation (Isaac and Pfeffer, Reference Isaac and Pfeffer2021; Molè et al., Reference Molè, Coorens, Shahbazi, Weberling, Weatherbee, Gantner, Sancho-Serra, Richardson, Drinkwater, Syed, Engley, Snell, Christie, Elder, Campbell, Fishel, Behjati, Vento-Tormo and Zernicka-Goetz2021). FGF can promote angiogenesis, regulate the local action of trophoblast cells, and play an important role in the regulation of placenta formation and function in buffalo (Devi et al., Reference Devi, Kumar, Konyak, Bharati, Bhimte, Pandey, Kumar, Paul, Kala, Samad, Verma, Singh, Bag, Sarkar and Chouhan2020). It can also promote the proliferation of trophoblast development in early equine embryos, which has also been demonstrated by Bonometti et al. (Reference Bonometti, Menarim, Reinholt, Ealy and Johnson2019). It also can improve embryo quality (Serrano Albal et al., Reference Serrano Albal, Silvestri, Kiazim, Vining, Zak, Walling, Haigh, Harvey, Harvey and Griffin2022). Therefore, FGF plays a major role in early embryo development and successful implantation (Mor et al., Reference Mor, Mondal, Reddy, Nandi and Gupta2018). Yamada et al. (Reference Yamada, Ohtsuki, Shiga, Green, Matsuno and Imakawa2022) found that mesenchymal markers such as vimentin (VIM) are highly expressed during the differentiation of trophoblast cells into invasive extracellular villus trophoblast cells in early pregnancy. Acetylation of VIM promotes trophoblastic epithelial-mesenchymal transition and enhances invasiveness, which suggests that VIM plays an important role in the processes of trophoblast differentiation and migration (Xiong et al., Reference Xiong, Ye, Chen, Fu, Li, Xu, Yu, Wen, Gao, Fu, Qi, Kilby, Saffery, Baker and Tong2021).

Cyclosporin A (CsA) is in a class of immunosuppressive agents that induces the immune tolerance of the maternal immune system to the embryo (Ling et al., Reference Ling, Huang, Chen, Mao and Zhang2017). Many studies have shown that CsA can enhance the adhesion and invasion of trophoblast cells (Wang et al., Reference Wang, Long, Tian, Zu, Liu, Wu, Mao, Li, Ma and Huang2023). However, the mechanism by which CsA improves the adhesion and invasion of trophoblast cells is unclear. In the present study, we aimed to elucidate the potential role of CsA in the pathogenesis of embryo implantation and its underlying molecular mechanisms. We examined the effect of CsA on trophoblast cell function and the underlying mechanism. We also used electron microscopy to observe trophoblast vesicles at different stages to determine whether CsA influences the ultrastructure of vesicles. We found that CsA regulated trophoblast cell migration and invasion by regulating the expression of LIF and FGF. Furthermore, CsA enhanced the dynamics of trophoblast cell vesicles during peri-implantation. Therefore, CsA may play a potential pathogenic role in embryo implantation in IVF–ET.

Materials and methods

Animals

Four-week-old ICR female mice and 8-week-old ICR male mice were purchased from Changsha Tianqin Biotechnology Co., Ltd. The study protocol was approved by the Ethics Committee of the First Affiliated Hospital of Hainan Medical University. Superovulation was performed on each female mouse by intraperitoneal injection of 5–8 IU of pregnant mare serum gonadotropin (Animal Drug Factory, Hangzhou, China) from 02:00 to 03:00 on the first day. Then, 5–8 IU of human chorionic gonadotrophin (Livzon Pharmaceutical Group Inc., Guang Dong, China) were injected from 13:00 to 14:00 on the third day, and the mating rate of females and males was 2:1. The vaginal plug was examined before 09:00 on the fourth day. The day when vaginal plugs were found was marked as Day 0.5 post mating (0.5 dpc). The use of animals was approved by the Ethics Committee of Hainan Medical College.

Preimplantation embryo culture

The mouse embryos were removed in the morning at 1.5 dpc (Huang et al., Reference Huang, Ma, Ma, Mao, Zhang, Du and Li2014). The embryos were cultured in an incubator (COOK, Indiana, America) containing 5% CO2, 5% O2 and 90% N2 at 37°C with cleavage-phase medium G1 (Vitrolife, Stockholm, Sweden) at 3 dpc and then transferred to blastocyst medium G2 (Vitrolife, Stockholm, Sweden) at different CsA (Solarbio, Beijing, China) concentrations. The concentration of CsA in the control group was 0 μM. The concentration of CsA in the experimental group was 1 µM, which has been confirmed to promote embryonic adhesion and invasion, which is beneficial to embryo implantation (Huang et al., Reference Huang, Ma, Ma, Mao, Zhang, Du and Li2014).

Migration area assay of preimplantation embryos

The laminin stock (Sigma, St. Louis, MO, USA) solution was diluted to 40 µg/ml with ddH2O, divided into sterile Eppendorf (EP) tubes and stored in a −20°C freezer. Then, 1.5 ml of the diluted liquid was placed into a culture dish. The Petri dish was placed in an incubator and preheated for 30 min, and then the excess liquid was removed. Then, 1.5 ml of blastocyst medium with or without CsA was added before the laminin had completely dried. Finally, the samples were covered with 2 ml of mineral oil. Embryos at 3 dpc were transferred to a trophoblast culture medium and placed onto Petri dishes precoated with laminin. The spreading of the preimplantation embryo at the bottom of the dish was observed under an inverted microscope (Nikon, Tokyo, Japan) and photographed at 7.5 dpc. ImageJ software was used to analyze the spreading area.

Transmission electron microscopy (TEM) of preimplantation embryos

Mouse embryos were also analyzed by TEM to investigate the number and area of vesicles of trophoblast cells. First, embryos at 5 dpc and 6 dpc were treated with or without CsA and fixed for 2 h in a fixative solution (2% glutaraldehyde, 2% paraformaldehyde; Solarbio, Beijing, China) prepared in 0.1 M phosphate-buffered saline at room temperature. Subsequently, samples were postfixed in 1% osmium tetroxide solution prepared in the same buffer and stained overnight in 1% uranyl acetate aqueous solution at 4°C. After dehydration in a series of alcohol concentrations, the samples were infiltrated with graded propylene oxide/epoxy resin solution for 1 day and then embedded in epoxy resin. After hardening in the oven at 65°C for 2 days, the samples were cut using a Richert–Jung UltracutE ultramicrotome into 70-nm slices. TEM samples were prepared by drying a 5-µl drop of nanoparticle suspension on a carbon film-coated copper grid. TEM micrographs were collected with an electron microscope (Hitachi Limited, Tokyo, Japan).

Immunofluorescence assay

LIF, FGF and VIM were detected with mouse anti-LIF, FGF and VIM antibodies, respectively (Abcam, Cambridge, MA, USA). Embryos at 5.5 dpc were fixed with 4% formaldehyde for 10 min, permeabilized with 1% Triton-X100 (Ameresco, Solon, OH, USA) for 30 min, and then blocked in 5% fetal bovine serum for 1 h at room temperature. The embryos were then incubated in the primary antibody at a dilution of 1:200 in a blocking solution at 4°C overnight. The next day, the embryos were stained with an anti-mouse Alexa 488 or anti-rabbit Alexa 568 secondary antibody (Abcam, Cambridge, MA, USA) according to the primary antibody at a 1:500 dilution in PBST for 1 h at room temperature. The embryos were then stained for 10 min at room temperature with DAPI. Imaging was performed using laser confocal microscopy (Olympus, Tokyo, Japan), and the relative expression levels were measured by software.

Statistical analysis

Statistical analysis was performed using SPSS 23.0 software. Each experiment was repeated at least three times. Measurement data are expressed as the mean ± standard deviation (x ± s). A P-value < 0.05 was considered to indicate a significant difference.

Results

CsA promotes the adhesion ability of trophoblast cells

To test the effect of CsA on the stretching growth of preimplantation embryos, mouse embryos were cultured with CsA at a concentration of 1 μM (CsA group; Fig. 1C, D) or without CsA (control group; Fig. 1A, B) at 7.5 dpc. The results demonstrated that the adhesion rate and the stretching area of hatched embryos were significantly increased with CsA treatment (P < 0.05; Fig. 1E).

Figure 1. CsA promotes the adhesion ability of trophoblast cells. The migration area of embryonic trophoblast cells. Blastocysts were cultured in medium without CsA on 7.5 dpc (A, ×20. B, ×60). Blastocysts were cultured in medium containing CsA at concentration of 1 μM on 7.5 dpc (C, ×20. D, ×60). The distribution of embryonic trophoblast cells at the bottom of laminin-coated dish was observed under an inverted microscope. Spreading area was analyzed using ImageJ software (E), the area of the CsA group was 1.75 times of the control group, and there was a statistical difference. *P < 0.05.

CsA regulates the expression of LIF and FGF in embryos

Our previous study showed that CsA enhances the invasion of trophoblast cells in vitro (Wang et al., Reference Wang, Long, Tian, Zu, Liu, Wu, Mao, Li, Ma and Huang2023). To confirm the mechanism in vivo, the expression of LIF, FGF and VIM was analyzed in mouse embryos by immunofluorescence. The results showed that LIF, FGF and VIM were expressed in the cytoplasm. The fluorescence intensity of LIF, FGF, and VIM was greater in the CsA group compared with the control group (Fig. 2A). The fluorescence intensity was further measured with software, and the results showed that the protein expression of LIF and FGF was significantly upregulated in embryonic tissues in the experimental group compared with the control group, similar to the above results (Fig. 2B). However, there was no significant difference in the expression of VIM (Fig. 2B). These results indicated that CsA may regulate LIF and FGF in the process of early embryo invasion.

Figure 2. CsA regulates the expression of LIF and FGF in embryos. Effect of CsA on the expression of FGF, LIF and Vimentin. The 5.5 dpc blastocysts were cultured in medium containing CsA (A, ×40). The protein level of FGF, LIF and Vimentin were detected using fluorescence microscopy (A, ×40). Embryos were observed using confocal laser scanning microscope and their relative quantification was measured using software (B). The relative quantification of LIF in the CsA group was 4.7 times of the control group (B). The relative quantification of FGF in the CsA group was 3.8 times of the control group (B). *P < 0.05.

CsA changes the dynamics of the vesicles of trophoblast cells in embryos

Based on the apparent role of vesicles in regulating trophoblast adhesion during embryo implantation, TEM was used to observe the ultrastructure of vesicles in trophoblast cells from 5 dpc embryos in the control group (Fig. 3A) and experimental group (Fig. 3C) and 6 dpc embryos in the control group (Fig. 3B) and experimental group (Fig. 3D). The results showed that the number of intracellular vesicles was obviously increased in 5 dpc embryos in the experimental group (Fig. 3E). However, the area of intracellular vesicles showed no significant difference from that in the control group (Fig. 3F). Next, the change in vesicles was further observed in 6 dpc embryos. The results showed that the number of vesicles was not different between the two groups (Fig. 3E). However, the area of vesicles was significantly increased in the experimental group (Fig. 3F). Furthermore, a morphometric evaluation of vesicles within the cytoplasm of trophoblast cells confirmed a significant increase between 5 dpc and 6 dpc (Fig. 3F).

Figure 3. CsA changes the dynamic of vesicle of trophoblast cells in embryo. The regulation of CsA on vesicles in blastocysts. The ultrastructure of vesicles was observed using electron microscopy on 5 dpc embryo in control group (A) and CsA group (C). Embryo in control group (B) and CsA group (D) on 6 dpc. (Magnification ×10,000). The red arrows refer to vesicles. The number and size of the vesicles were measured in the control group and the experimental group on 5 dpc and 6 dpc (E, F). The number of vesicles in the CsA group is 3.93 times that of the control group on 5 dpc (E). The average area of vesicles in the CsA group is 3.43 times that of the control group on 6 dpc (F). The average area of vesicles at the top of 6 dpc embryonic trophoblasts in the control group was 3.6 times that of 5 dpc. The average area of 6 dpc vesicles in the test group was 8.93 times that of 5 dpc (F). * P < 0.05.

Discussion

Normal migration and invasion of trophoblast cells are the key processes of embryo implantation (Mishra et al., Reference Mishra, Ashary, Sharma and Modi2021). During the embryo preimplantation period, trophoblastic cells grow rapidly, forming hairy projections called villi on the surface of the embryo. This layer of cells then gradually differentiates into trophoblast columns. Trophoblastic layers in the cell column proliferate through syncytial trophoblastic layers, and the adjacent trophoblastic layers fuse with each other to form trophoblastic shells. In these cell columns, proliferating but noninvasive cytotrophoblast cells become invasive and begin to enter the parent tissue, eventually forming EVTs (Moser and Huppertz, Reference Moser and Huppertz2017), which invade the vascular walls and lumens of the uterine placental bed spiral artery. These functions of trophoblast cells are similar to those of cancer cells (Kshitiz et al., Reference Kshitiz, Afzal, Maziarz, Hamidzadeh, Liang, Erkenbrack, Kim, Haeger, Pfarrer, Hoang, Ott, Spencer, Pavličev, Antczak, Levchenko and Wagner2019). The process of human trophoblast invasion is complex and susceptible to various factors.

CsA can improve the invasion and adhesion of trophoblasts in early pregnancy and improve pregnancy outcomes (He et al., Reference He, Li, Wu, Ruan, Teng, Li and Tang2020; Huang et al., Reference Huang, Lu, Li, Zhang, Xie, Luo, Wei, Ma and Huang2020). It can also inhibit the apoptosis of trophoblasts and increase the capacities of trophoblast proliferation and invasion (Wang et al., Reference Wang, Ge and Zhou2021). Our previous studies have found that CsA at a low dose promotes the adhesion and invasion ability of embryonic trophoblast cells, ultimately improving the invasiveness of early embryos and promoting embryo implantation at a concentration of 1 µM (Huang et al., Reference Huang, Ma, Ma, Mao, Zhang, Du and Li2014). The clinical efficacy of CsA has also been verified (Ling et al., Reference Ling, Huang, Chen, Mao and Zhang2017). It has been reported that the expansion area reflects the invasiveness of an embryo (Yu et al., Reference Yu, Wang, Liu, Wang and Yan2017). In this study, we observed that the migration of the embryo and the invasion of trophoblast cells were significantly improved in the CsA group compared with the control group.

LIF is a haematopoietic differentiation factor that acts through autocrine or paracrine signalling. LIF is the first factor used in the development and differentiation of mouse embryos (Smith et al., Reference Smith, Heath, Donaldson, Wong, Moreau, Stahl and Rogers1988). LIF can induce strong attachment and adhesion of the activated blastocyst trophectoderm to the uterine wall (Fukui et al., Reference Fukui, Hirota, Matsuo, Gebril, Akaeda, Hiraoka and Osuga2019). It has also been reported that LIF may be related to embryo survival and nutrition of implanted embryos (Massimiani et al., Reference Massimiani, Lacconi, La Civita, Ticconi, Rago and Campagnolo2019). FGF can improve the attachment and outgrowth of trophoblast cells by increasing the expression of Oct4, Sox2, c-Myc and Bcl2 (Alzahrani, Reference Alzahrani2019). In this study, we found that CsA upregulated the protein levels of LIF and FGF, which are closely related to trophoblast invasion function. This suggests that CsA affects villi and that the effect of pretrophoblast invasiveness may be closely related to LIF and FGF.

In addition, there is another important mechanism by which embryo adhesion is induced. Vesicles play a major role in this process (Wang et al., Reference Wang, Mayernik and Armant2002). Vesicles are directly produced and shed by the plasma membrane, which contains large amounts of protein, nucleic acids, RNA transcripts and other substances. There are surface receptors on the membrane that can be degraded by lysosomes that fuse with the plasma membrane and release their contents to other cells, thereby stimulating signalling activity and resulting in phenotypic and functional changes in the recipient cells. Vesicles can activate trophoblast signalling pathways through interaction with fibronectin and strengthen trophoblast migration function (Desrochers et al., Reference Desrochers, Bordeleau, Reinhart-King, Cerione and Antonyak2016). In this experiment, we found that the secretion of vesicles significantly increased after CsA treatment and, as the embryo developed, the vesicles gradually fused into larger vesicles. At 6 dpc compared with 5 dpc, the average area of vesicles increased significantly, which was presumed to be related to the larger area of small vesicles after fusion. This finding indicates that, with the development of embryos, the growth of vesicles is a dynamic process, and the gradual growth of vesicles is not only related to the growth and development of each individual vesicle but also possibly to fusion between vesicles. We believe that CsA can promote vesicle secretion and volume increases. Upon growth to a certain stage, the vesicle membrane ruptures and releases its contents to play its biological role, therefore affecting the function of trophoblasts around the preimplantation embryo.

In summary, our study extends previous observations and reveals new functions and mechanisms of CsA. CsA can increase the invasion and migration capacity of trophoblasts before the villi appear in the implantation stage. Mechanistically, CsA upregulates LIF and FGF expression to improve the invasion of trophoblasts and increase the secretion and development of vesicles. These findings provide novel insights into the mechanisms underlying the occurrence of embryo implantation and are of great significance for the prevention and treatment of infertility and for assisted reproduction.

Financial support

This study was supported by the Major Science and Technology Programme of Hainan Province (No. ZDKJ2017007), the Hainan Provincial Natural Science Foundation of China (No. 2019CXTD408) and the National Natural Science Foundation of China (No. 81460236).

Competing interests

The authors declare no conflicts of interest.

Ethical standards

The animal experiments were approved by the Ethics Committee for Reproductive Medicine.

Footnotes

*

These authors contributed equally.

References

Alzahrani, F. A. (2019). Synergistic effect of basic fibroblast growth factor (bFGF) and epidermal growth factor on derivation of camel (Camelus dromedarius) trophoblast stem cells. Zygote, 27(4), 255–258. doi: 10.1017/S0967199419000169 CrossRefGoogle ScholarPubMed
Bonometti, S., Menarim, B. C., Reinholt, B. M., Ealy, A. D. and Johnson, S. E. (2019). Growth factor modulation of equine trophoblast mitosis and prostaglandin gene expression. Journal of Animal Science, 97(2), 865–873. doi: 10.1093/jas/sky473 CrossRefGoogle ScholarPubMed
Carvajal, L., Gutiérrez, J., Morselli, E. and Leiva, A. (2021). Autophagy process in trophoblast cells invasion and differentiation: Similitude and differences with cancer cells. Frontiers in Oncology, 11, 637594. doi: 10.3389/fonc.2021.637594 CrossRefGoogle ScholarPubMed
Desrochers, L. M., Bordeleau, F., Reinhart-King, C. A., Cerione, R. A. and Antonyak, M. A. (2016). Microvesicles provide a mechanism for intercellular communication by embryonic stem cells during embryo implantation. Nature Communications, 7, 11958. doi: 10.1038/ncomms11958 CrossRefGoogle ScholarPubMed
Devi, H. L., Kumar, S., Konyak, Y. Y., Bharati, J., Bhimte, A., Pandey, Y., Kumar, K., Paul, A., Kala, A., Samad, H. A., Verma, M. R., Singh, G., Bag, S., Sarkar, M. and Chouhan, V. S. (2020). Expression and functional role of fibroblast growth factors (FGF) in placenta during different stages of pregnancy in water buffalo (Bubalus bubalis). Theriogenology, 143, 98–112. doi: 10.1016/j.theriogenology.2019.11.034 CrossRefGoogle ScholarPubMed
Fukui, Y., Hirota, Y., Matsuo, M., Gebril, M., Akaeda, S., Hiraoka, T. and Osuga, Y. (2019). Uterine receptivity, embryo attachment, and embryo invasion: Multistep processes in embryo implantation. Reproductive Medicine and Biology, 18(3), 234–240. doi: 10.1002/rmb2.12280 CrossRefGoogle ScholarPubMed
Hamelin-Morrissette, J., Dallagi, A., Girouard, J., Ravelojaona, M., Oufqir, Y., Vaillancourt, C., Van Themsche, C., Carrier, C. and Reyes-Moreno, C. (2020). Leukemia inhibitory factor regulates the activation of inflammatory signals in macrophages and trophoblast cells. Molecular Immunology, 120, 32–42. doi: 10.1016/j.molimm.2020.01.021 CrossRefGoogle ScholarPubMed
He, B., Li, Q. Y., Wu, Y. Y., Ruan, J. L., Teng, X. M., Li, D. J. and Tang, C. L. (2020). Cyclosporin A protects JEG-3 cells against oxidative stress-induced apoptosis by inhibiting the p53 and JNK/p38 signaling pathways. Reproductive Biology and Endocrinology: RB&E, 18(1), 100. doi: 10.1186/s12958-020-00658-0 CrossRefGoogle ScholarPubMed
Huang, Y. H., Ma, Y. L., Ma, L., Mao, J. L., Zhang, Y., Du, M. R. and Li, D. J. (2014). Cyclosporine A improves adhesion and invasion of mouse preimplantation embryos via upregulating integrin β3 and matrix metalloproteinase-9. International Journal of Clinical and Experimental Pathology, 7(4), 1379–1388.Google ScholarPubMed
Huang, W., Lu, W., Li, Q., Zhang, Y., Xie, B., Luo, S., Wei, Y., Ma, Y. and Huang, Y. (2020). Effects of cyclosporine A on proliferation, invasion and migration of HTR-8/SVneo human extravillous trophoblasts. Biochemical and Biophysical Research Communications, 533(4), 645–650. doi: 10.1016/j.bbrc.2020.09.072 CrossRefGoogle ScholarPubMed
Isaac, E. and Pfeffer, P. L. (2021). Growing cattle embryos beyond Day 8 – An investigation of media components. Theriogenology, 161, 273–284. doi: 10.1016/j.theriogenology.2020.12.010 CrossRefGoogle ScholarPubMed
Johnson, M. H. (2019). A short history of in vitro fertilization (IVF). International Journal of Developmental Biology, 63(3–4), 83–92. doi: 10.1387/ijdb.180364mj CrossRefGoogle Scholar
Kshitiz, Afzal, J., Maziarz, J. D., Hamidzadeh, A., Liang, C., Erkenbrack, E. M., Kim, H. N., Haeger, J. D., Pfarrer, C., Hoang, T., Ott, T., Spencer, T., Pavličev, M., Antczak, D. F., Levchenko, A. and Wagner, G. P. (2019). Evolution of placental invasion and cancer metastasis are causally linked. Nature Ecology & Evolution, 3, 1743–1753. doi: 10.1038/s41559-019-1046-4 CrossRefGoogle Scholar
Ling, Y., Huang, Y., Chen, C., Mao, J. and Zhang, H. (2017). Low dose cyclosporin A treatment increases live birth rate of unexplained recurrent abortion – Initial cohort study. Clinical and Experimental Obstetrics and Gynecology, 44(2), 230–235. doi: 10.12891/ceog3375.2017 CrossRefGoogle ScholarPubMed
Massimiani, M., Lacconi, V., La Civita, F., Ticconi, C., Rago, R. and Campagnolo, L. (2019). Molecular signaling regulating endometrium-blastocyst crosstalk. International Journal of Molecular Sciences, 21(1). doi: 10.3390/ijms21010023 CrossRefGoogle ScholarPubMed
Menkhorst, E., Winship, A., Van Sinderen, M. and Dimitriadis, E. (2016). Human extravillous trophoblast invasion: Intrinsic and extrinsic regulation. Reproduction, Fertility, and Development, 28(4), 406–415. doi: 10.1071/RD14208 CrossRefGoogle ScholarPubMed
Mishra, A., Ashary, N., Sharma, R. and Modi, D. (2021). Extracellular vesicles in embryo implantation and disorders of the endometrium. American Journal of Reproductive Immunology, 85(2), e13360. doi: 10.1111/aji.13360 CrossRefGoogle ScholarPubMed
Molè, M. A., Coorens, T. H. H., Shahbazi, M. N., Weberling, A., Weatherbee, B. A. T., Gantner, C. W., Sancho-Serra, C., Richardson, L., Drinkwater, A., Syed, N., Engley, S., Snell, P., Christie, L., Elder, K., Campbell, A., Fishel, S., Behjati, S., Vento-Tormo, R. and Zernicka-Goetz, M. (2021). A single cell characterisation of human embryogenesis identifies pluripotency transitions and putative anterior hypoblast centre. Nature Communications, 12(1), 3679. doi: 10.1038/s41467-021-23758-w CrossRefGoogle ScholarPubMed
Mor, A., Mondal, S., Reddy, I. J., Nandi, S. and Gupta, P. (2018). Molecular cloning and expression of FGF2 gene in pre-implantation developmental stages of in vitro-produced sheep embryos. Reproduction in Domestic Animals = Zuchthygiene, 53(4), 895–903. doi: 10.1111/rda.13182 Google ScholarPubMed
Moser, G. and Huppertz, B. (2017). Implantation and extravillous trophoblast invasion: From rare archival specimens to modern biobanking. Placenta, 56, 19–26. doi: 10.1016/j.placenta.2017.02.007 CrossRefGoogle ScholarPubMed
Pantos, K., Grigoriadis, S., Maziotis, E., Pistola, K., Xystra, P., Pantou, A., Kokkali, G., Pappas, A., Lambropoulou, M., Sfakianoudis, K. and Simopoulou, M. (2022). The role of interleukins in recurrent implantation failure: A comprehensive review of the literature. International Journal of Molecular Sciences, 23(4). doi: 10.3390/ijms23042198 CrossRefGoogle ScholarPubMed
Park, J. Y., Mani, S., Clair, G., Olson, H. M., Paurus, V. L., Ansong, C. K., Blundell, C., Young, R., Kanter, J., Gordon, S., Yi, A. Y., Mainigi, M. and Huh, D. D. (2022). A microphysiological model of human trophoblast invasion during implantation. Nature Communications, 13(1), 1252. doi: 10.1038/s41467-022-28663-4 CrossRefGoogle ScholarPubMed
Rooney, K. L. and Domar, A. D. (2018). The relationship between stress and infertility. Dialogues in Clinical Neuroscience, 20(1), 41–47. doi: 10.31887/DCNS.2018.20.1/klrooney CrossRefGoogle ScholarPubMed
Serrano Albal, M., Silvestri, G., Kiazim, L. G., Vining, L. M., Zak, L. J., Walling, G. A., Haigh, A. M., Harvey, S. C., Harvey, K. E. and Griffin, D. K. (2022). Supplementation of porcine in vitro maturation medium with FGF2, LIF, and IGF1 enhances cytoplasmic maturation in prepubertal gilts oocytes and improves embryo quality. Zygote, 30(6), 801–808. doi: 10.1017/S0967199422000284 CrossRefGoogle ScholarPubMed
Smith, A. G., Heath, J. K., Donaldson, D. D., Wong, G. G., Moreau, J., Stahl, M. and Rogers, D. (1988). Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature, 336(6200), 688–690. doi: 10.1038/336688a0 CrossRefGoogle ScholarPubMed
Tani, K., Mitsui, T., Mishima, S., Ohira, A., Maki, J., Eto, E., Hayata, K., Nakamura, K. and Masuyama, H. (2021). EG-VEGF induces invasion of a human trophoblast cell line via PROKR2. Acta Medica Okayama, 75(6), 677–684. doi: 10.18926/AMO/62806 Google ScholarPubMed
Wang, J., Mayernik, L. and Armant, D. R. (2002). Integrin signaling regulates blastocyst adhesion to fibronectin at implantation: Intracellular calcium transients and vesicle trafficking in primary trophoblast cells. Developmental Biology, 245(2), 270–279. doi: 10.1006/dbio.2002.0644 CrossRefGoogle ScholarPubMed
Wang, N., Ge, H. and Zhou, S. (2021). Cyclosporine A to treat unexplained recurrent spontaneous abortions: A prospective, randomized, double-blind, placebo-controlled, single-center trial. International Journal of Women’s Health, 13, 1243–1250. doi: 10.2147/IJWH.S330921 CrossRefGoogle ScholarPubMed
Wang, J., Long, P., Tian, S., Zu, W., Liu, J., Wu, B., Mao, J., Li, D., Ma, Y. and Huang, Y. (2023). Cyclosporin A promotes invasion and migration of extravillous trophoblast cells derived from human-induced pluripotent stem cells and human embryonic stem cells. Stem Cells and Development, 32(3–4), 60–74. doi: 10.1089/scd.2022.0144 CrossRefGoogle ScholarPubMed
Xiong, L., Ye, X., Chen, Z., Fu, H., Li, S., Xu, P., Yu, J., Wen, L., Gao, R., Fu, Y., Qi, H., Kilby, M. D., Saffery, R., Baker, P. N. and Tong, C. (2021). Advanced maternal age-associated SIRT1 deficiency compromises trophoblast epithelial-mesenchymal transition through an increase in vimentin acetylation. Aging Cell, 20(10), e13491. doi: 10.1111/acel.13491 CrossRefGoogle ScholarPubMed
Yamada, A., Ohtsuki, K., Shiga, N., Green, J. A., Matsuno, Y. and Imakawa, K. (2022). Epithelial-mesenchymal transition and bi- and multi-nucleated trophoblast cell formation in ovine conceptuses during the peri-implantation period. Journal of Reproduction and Development, 68(2), 110–117. doi: 10.1262/jrd.2021-088 CrossRefGoogle ScholarPubMed
Yu, M., Wang, J., Liu, S., Wang, X. and Yan, Q. (2017). Novel function of pregnancy-associated plasma protein A: Promotes endometrium receptivity by up-regulating N-fucosylation. Scientific Reports, 7(1), 5315. doi: 10.1038/s41598-017-04735-0 CrossRefGoogle ScholarPubMed
Figure 0

Figure 1. CsA promotes the adhesion ability of trophoblast cells. The migration area of embryonic trophoblast cells. Blastocysts were cultured in medium without CsA on 7.5 dpc (A, ×20. B, ×60). Blastocysts were cultured in medium containing CsA at concentration of 1 μM on 7.5 dpc (C, ×20. D, ×60). The distribution of embryonic trophoblast cells at the bottom of laminin-coated dish was observed under an inverted microscope. Spreading area was analyzed using ImageJ software (E), the area of the CsA group was 1.75 times of the control group, and there was a statistical difference. *P < 0.05.

Figure 1

Figure 2. CsA regulates the expression of LIF and FGF in embryos. Effect of CsA on the expression of FGF, LIF and Vimentin. The 5.5 dpc blastocysts were cultured in medium containing CsA (A, ×40). The protein level of FGF, LIF and Vimentin were detected using fluorescence microscopy (A, ×40). Embryos were observed using confocal laser scanning microscope and their relative quantification was measured using software (B). The relative quantification of LIF in the CsA group was 4.7 times of the control group (B). The relative quantification of FGF in the CsA group was 3.8 times of the control group (B). *P < 0.05.

Figure 2

Figure 3. CsA changes the dynamic of vesicle of trophoblast cells in embryo. The regulation of CsA on vesicles in blastocysts. The ultrastructure of vesicles was observed using electron microscopy on 5 dpc embryo in control group (A) and CsA group (C). Embryo in control group (B) and CsA group (D) on 6 dpc. (Magnification ×10,000). The red arrows refer to vesicles. The number and size of the vesicles were measured in the control group and the experimental group on 5 dpc and 6 dpc (E, F). The number of vesicles in the CsA group is 3.93 times that of the control group on 5 dpc (E). The average area of vesicles in the CsA group is 3.43 times that of the control group on 6 dpc (F). The average area of vesicles at the top of 6 dpc embryonic trophoblasts in the control group was 3.6 times that of 5 dpc. The average area of 6 dpc vesicles in the test group was 8.93 times that of 5 dpc (F). * P < 0.05.