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. 2023 Oct 19:11:1260886.
doi: 10.3389/fbioe.2023.1260886. eCollection 2023.

Advancing bovine in vitro fertilization through 3D printing: the effect of the 3D printed materials

Affiliations

Advancing bovine in vitro fertilization through 3D printing: the effect of the 3D printed materials

Ramses Belda-Perez et al. Front Bioeng Biotechnol. .

Abstract

Nowadays there is an increasing demand for assisted reproductive technologies due to the growth of infertility problems. Naturally, fertilization occurs in the oviduct, where the oviductal epithelial cells (OECs) secrete many molecules that affect the embryo's metabolism and protect it from oxidative stress. When the OECs are grown in 3D culture systems, they maintain a great part of their functional characteristics, making them an excellent model for in vitro fertilization (IVF) studies. In this work, we aimed to evaluate the suitability of different 3D-printing processes in conjunction with the corresponding set of commercially available biomaterials: extrusion-based processing using polylactic acid (PLA) and polycaprolactone (PCL) and stereolithography or digital-light processing using polyethylene-glycol-diacrylate (PEGDA) with different stiffness (PEGDA500, PEGDA200, PEGDA PhotoInk). All the 3D-printed scaffolds were used to support IVF process in a bovine embryo assay. Following fertilization, embryo development and quality were assessed in terms of cleavage, blastocyst rate at days 7 and 8, total cell number (TCN), inner cell mass/trophectoderm ratio (ICN/TE), and apoptotic cell ratio (ACR). We found a detrimental effect on cleavage and blastocyst rates when the IVF was performed on any medium conditioned by most of the materials available for digital-light processing (PEGDA200, PEGDA500). The observed negative effect could be possibly due to some leaked compound used to print and stabilize the scaffolds, which was not so evident however with PEGDA PhotoInk. On the other hand, all the extrusion-based processable materials did not cause any detrimental effect on cleavage or blastocyst rates. The principal component analysis reveals that embryos produced in presence of 3D-printed scaffolds produced via extrusion exhibit the highest similarity with the control embryos considering cleavage, blastocyst rates, TCN, ICN/TE and ACR per embryo. Conversely, all the photo-cross linkable materials or medium conditioned by PLA, lead to the highest dissimilarities. Since the use of PCL scaffolds, as well as its conditioned medium, bring to embryos that are more similar to the control group. Our results suggest that extrusion-based 3D printing of PCL could be the best option to be used for new IVF devices, possibly including the support of OECs, to enhance bovine embryo development.

Keywords: IVF; PCL; PEGDA; PLA; biomaterials; bovine; embryo culture.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Experimental design. (A) IVF in normal Fert-TALP medium, without conditioning or scaffold, (B) IVF in Fert-TALP medium conditioned by scaffold for 24 h, and (C) IVF performed in presence of the rinsed scaffold.
FIGURE 2
FIGURE 2
Effect of different materials on the percentage of cleaved embryos. The histograms show the cleavage rate when the IVF was performed with an unconditioned medium (CTRL), with medium conditioned by each material (rinse groups) or when different scaffolds were present (scaffold groups). The data show significant differences (p < 0.05) in CTRL vs. Scaffold PEGDA500, CTRL vs. Rinse PEGDA500 and CTRL vs. Rinse PEGDA200. The data are presented as the mean ± SD of 11 independent experiments. Data were analyzed using Dunnett’s test. **p < 0.01, ***p < 0.005, ****p < 0.0001 versus control.
FIGURE 3
FIGURE 3
Effect of different materials on blastocyst rate at day 7. The histograms show the blastocyst rate at day 7 when the IVF was performed with unconditioned medium (CTRL), with medium conditioned by each material (rinse groups) or when different scaffolds were present (scaffold groups). The data show significant differences (p < 0.05) in CTRL vs. Scaffold PEGDA500, CTRL vs. Rinse PEGDA500 and CTRL vs. Rinse PEGDA200. The data are presented as the mean of 11 independent experiments. Data were analyzed using the Dunnett’s test. **p < 0.01, ***p < 0.005 versus control.
FIGURE 4
FIGURE 4
Effect of different materials scaffolds on blastocyst rate at day 8. The histograms show the blastocyst rate at day 8 when the IVF was performed with unconditioned medium (CTRL), with medium conditioned by each material (rinse groups) or when different scaffolds were present (scaffold groups). The data shows significant differences (p < 0.05) in CTRL vs. Scaffold PEGDA500, CTRL vs. Rinse PEGDA500 and CTRL vs. Rinse PEGDA200. The data are presented as mean of 11 independent experiments. Data were analyzed using the Dunnett’s test. *p < 0.05, **p < 0.01 versus control.
FIGURE 5
FIGURE 5
Representative confocal image of blastocyst at day 8. Fluorescent image of differential apoptotic staining (A–D). At day 8, bovine blastocysts were fixed, dyed with Hoechst 33342 for nuclei (B), immune-stained for CDX2 for the trophectoderm (C), and for active caspase-3 for the apoptosis (D). In (A) an overlay (B–D) is provided.
FIGURE 6
FIGURE 6
Principal component analysis performed by assessing the different parameters studied (Cleavage, blastocyst rate at days 7 and 8, TCN, ICM/TE and ACR). Principal component analysis shows no separation among groups. However, we observed that the closest groups to control are scaffold PLA, scaffold PCL and rinse PCL.

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