The therapeutic effects and optimal timing of granulocyte colony stimulating factor intrauterine administration during IVF-ET.

Most of embryos fail to produce live offspring during In Vitro Fertilization-Embryo Transfer (IVF-ET) procedure. There is a dearth of research activity addressing this problem despite the significant population of women suffering from repeated implantation failure after transfer of high-quality of embryos. As a clinically accessible option, granulocyte colony stimulating factor (G-CSF) is often used for the treatment to improve the rates of embryo implantation. However, there are currently no evidence-based standardized protocol for the clinical use of G-CSF. G-CSF was administered into one side of mouse uterine horns and saline was infused into the other side of horns as a control. Intrauterine G-CSF administration showed maximal effects 24 h after administration in enhancing endometrial receptivity and subsequent increase of angiogenesis by demonstrating elevated integrin ß3 and OPN and reduced cytotoxicity of NK cells. Furthermore, G-CSF administration 24 h prior to embryo transfer promoted the stability of attached embryos at the early stage of implantation in vitro. Our findings suggest as new consensus criteria providing a potential therapeutic strategy of the clinical use of G-CSF to achieve maximal effects of IVF-ET for patients who are suffering from repeated implantation failure with the problems with endometrial receptivity.

Three-dimensional microengineered vascularised endometrium-on-a-chip.

STUDY QUESTION: Can we reconstitute physiologically relevant 3-dimensional (3D) microengineered endometrium in-vitro model? SUMMARY ANSWER: Our representative microengineered vascularised endometrium on-a-chip closely recapitulates the endometrial microenvironment that consists of three distinct layers including epithelial cells, stromal fibroblasts and endothelial cells in a 3D extracellular matrix in a spatiotemporal manner. WHAT IS KNOWN ALREADY: Organ-on-a-chip, a multi-channel 3D microfluidic cell culture system, is widely used to investigate physiologically relevant responses of organ systems. STUDY DESIGN, SIZE, DURATION: The device consists of five microchannels that are arrayed in parallel and partitioned by array of micropost. Two central channels are for 3D culture and morphogenesis of stromal fibroblast and endothelial cells. In addition, the outermost channel is for the culture of additional endometrial stromal fibroblasts that secrete biochemical cues to induce directional pro-angiogenic responses of endothelial cells. To seed endometrial epithelial cells, on Day 8, Ishikawa cells were introduced to one of the two medium channels to adhere on the gel surface. After that, the microengineered endometrium was cultured for an additional 5-6 days (total ∼ 14 days) for the purpose of each experiment. PARTICIPANTS/MATERIALS, SETTING, METHODS: Microfluidic 3D cultures were maintained in endothelial growth Medium 2 with or without oestradiol and progesterone. Some cultures additionally received exogenous pro-angiogenic factors. For the three distinct layers of microengineered endometrium-on-a-chip, the epithelium, stroma and blood vessel characteristics and drug response of each distinct layer in the microfluidic model were assessed morphologically and biochemically. The quantitative measurement of endometrial drug delivery was evaluated by the permeability coefficients. MAIN RESULTS AND THE ROLE OF CHANCE: We established microengineered vascularised endometrium-on-chip, which consists of three distinct layers: epithelium, stroma and blood vessels. Our endometrium model faithfully recapitulates in-vivo endometrial vasculo-angiogenesis and hormonal responses displaying key features of the proliferative and secretory phases of the menstrual cycle. Furthermore, the effect of the emergency contraception drug levonorgestrel was evaluated in our model demonstrating increased endometrial permeability and blood vessel regression in a dose-dependent manner. We finally provided a proof of concept of the multi-layered endometrium model for embryo implantation, which aids a better understanding of the molecular and cellular mechanisms underlying this process. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This report is largely an in-vitro study and it would be beneficial to validate our findings using human primary endometrial cells. WIDER IMPLICATIONS OF THE FINDINGS: Our 3D microengineered vascularised endometrium-on-a-chip provides a new in-vitro approach to drug screening and drug discovery by mimicking the complicated behaviours of human endometrium. Thus, we suggest our model as a tool for addressing critical challenges and unsolved problems in female diseases, such as endometriosis, uterine cancer and female infertility, in a personalised manner. STUDY FUNDING/COMPETING INTEREST(S): This work is supported by funding from the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) to Y.J.K. (No. 2018R1C1B6003), to J.A. (No. 2020R1I1A1A01074136) and to H.S.K. (No. 2020R1C1C100787212). The authors report no conflicts of interest.

CXCL12 enhances pregnancy outcome via improvement of endometrial receptivity in mice.

Successful pregnancy inevitably depends on the implantation of a competent embryo into a receptive endometrium. Although many substances have been suggested to improve the rate of embryo implantation targeting enhancement of endometrial receptivity, currently there rarely are effective evidence-based treatments to prevent or cure this condition. Here we strongly suggest minimally-invasive intra-uterine administration of embryo-secreted chemokine CXCL12 as an effective therapeutic intervention. Chemokine CXCL12 derived from pre- and peri-implanting embryos significantly enhances the rates of embryo attachment and promoted endothelial vessel formation and sprouting in vitro. Consistently, intra-uterine CXCL12 administration in C57BL/6 mice improved endometrial receptivity showing increased integrin ß3 and its ligand osteopontin, and induced endometrial angiogenesis displaying increased numbers of vessel formation near the lining of endometrial epithelial layer with higher CD31 and CD34 expression. Furthermore, intra-uterine CXCL12 application dramatically promoted the rates of embryo implantation with no morphologically retarded embryos. Thus, our present study provides a novel evidence that improved uterine endometrial receptivity and enhanced angiogenesis induced by embryo-derived chemokine CXCL12 may aid to develop a minimally-invasive therapeutic strategy for clinical treatment or supplement for the patients with repeated implantation failure with less risk.

Non-invasive Intrauterine Administration of Botulinum Toxin A Enhances Endometrial Angiogenesis and Improves the Rates of Embryo Implantation.

Endometrial angiogenesis plays crucial roles in determining the endometrial receptivity. Defects in endometrial receptivity often cause repeated implantation failure, which is one of the major unmet needs for infertility and contributes a major barrier to the assisted reproductive technology. Despite the numerous extensive research work, there are currently no effective evidence-based treatments to prevent or cure this condition. As a non-invasive treatment strategy, botulinum toxin A (BoTA) was administered into one side of mouse uterine horns, and saline was infused into the other side of horns for the control. Impact of BoTA was assessed in the endometrium at 3 or 8 days after infusion. We demonstrated that BoTA administration enhances the capacity of endothelial cell tube formation and sprouting. The intrauterine BoTA administration significantly induced endometrial angiogenesis displaying increased numbers of vessel formation and expression levels of related marker genes. Moreover, BoTA intrauterine application promoted the endometrial receptivity, and the rates of embryo implantation were improved with BoTA treatment with no morphologically retarded embryos. Intrauterine BoTA treatment has a beneficial effect on vascular reconstruction of functional endometrium prior to embryo implantation by increasing endometrial blood flow near the uterine cavity suggesting BoTA treatment as a potential therapeutic strategy for patients who are suffering from repeated implantation failure with the problems with endometrial receptivity.

Eupatilin treatment inhibits transforming growth factor beta-induced endometrial fibrosis in vitro.

OBJECTIVE: Endometrial fibrosis, the primary pathological feature of intrauterine adhesion, may lead to disruption of endometrial tissue structure, menstrual abnormalities, infertility, and recurrent pregnancy loss. At present, no ideal therapeutic strategy exists for this fibrotic disease. Eupatilin, a major pharmacologically active flavone from Artemisia, has been previously reported to act as a potent inducer of dedifferentiation of fibrotic tissue in the liver and lung. However, the effects of eupatilin on endometrial fibrosis have not yet been investigated. In this study, we present the first report on the impact of eupatilin treatment on transforming growth factor beta (TGF-ß)-induced endometrial fibrosis. METHODS: The efficacy of eupatilin on TGF-ß-induced endometrial fibrosis was assessed by examining changes in morphology and the expression levels of fibrosis markers using immunofluorescence staining and quantitative real-time reverse-transcription polymerase chain reaction. RESULTS: Eupatilin treatment significantly reduced the fibrotic activity of TGF-ß-induced endometrial fibrosis in Ishikawa cells, which displayed more circular shapes and formed more colonies. Additionally, the effects of eupatilin on fibrotic markers including alpha-smooth muscle actin, hypoxia-inducible factor 1 alpha, collagen type I alpha 1 chain, and matrix metalloproteinase-2, were evaluated in TGF-ß-induced endometrial fibrosis. The expression of these markers was highly upregulated by TGF-ß pretreatment and recovered to the levels of control cells in response to eupatilin treatment. CONCLUSION: Our findings suggest that suppression of TGF-ß-induced signaling by eupatilin might be an effective therapeutic strategy for the treatment of endometrial fibrosis.

Endometrial profilin 1: a key player in embryo-endometrial crosstalk.

OBJECTIVE: Despite extensive research on implantation failure, little is known about the molecular mechanisms underlying the crosstalk between the embryo and the maternal endometrium, which is critical for successful pregnancy. Profilin 1 (PFN1), which is expressed both in the embryo and in the endometrial epithelium, acts as a potent regulator of actin polymerization and the cytoskeletal network. In this study, we identified the specific role of endometrial PFN1 during embryo implantation. METHODS: Morphological alterations depending on the status of PFN1 expression were assessed in PFN1-depleted or control cells grown on Matrigel-coated cover glass. Day-5 mouse embryos were cocultured with Ishikawa cells. Comparisons of the rates of F-actin formation and embryo attachment were performed by measuring the stability of the attached embryo onto PFN1-depleted or control cells. RESULTS: Depletion of PFN1 in endometrial epithelial cells induced a significant reduction in cell-cell adhesion displaying less formation of colonies and a more circular cell shape. Mouse embryos co-cultured with PFN1-depleted cells failed to form actin cytoskeletal networks, whereas more F-actin formation in the direction of surrounding PFN1-intact endometrial epithelial cells was detected. Furthermore, significantly lower embryo attachment stability was observed in PFN1-depleted cells than in control cells. This may have been due to reduced endometrial receptivity caused by impaired actin cytoskeletal networks associated with PFN1 deficiency. CONCLUSION: These observations definitively demonstrate an important role of PFN1 in mediating cell-cell adhesion during the initial stage of embryo implantation and suggest a potential therapeutic target or novel biomarker for patients suffering from implantation failure.

Neogenin regulates mitochondrial activity in pre-implantation mouse embryos.

• Mitochondrial activation signaling pathways are not clearly identified. • Embryonic mitochondria activity is important for a successful pregnancy and live birth. • Neogenin is a multi-functional receptor that contributes to embryo development. • Neogenin as a receptor is related to mitochondrial activation and replication. • Neogenin could activate mitochondria in pre-implantation embryo development.