The impact of the yttrium oxide nano particles Y₂O₃ on the in vitro fertilization and in vitro culture media in a mouse model

2.1 Preparation and Characterization of Yttrium Oxide Nanoparticles (Y₂O₃ NPs)

The Y₂O₃ NPs purchased from Sigma Aldrich as nano powder material < 50 nm particle size, Selvaraj et al.2014 Siddhardha et al., 2020 Transmission Electronic Microscopy (TEM) completed the nanoparticle characterization for the size, shape, and phase (TEM) and X-Ray Diffraction (XRD) in King Abdullah Institute for Nanotechnology (KAIN) (Mourdikoudis et al. 2018). The nano powder was dissolved in Milli Q water (1 mg/1ml) inside a sonicator's glass tube and used for all experiments as a stock solution (Grana Dávila et al. 2018). Before the nanoparticles were added to the IVF & IVC dishes media, the ultrasonication method sonicator (Ultasonic,FS20H, Fisher Scientific, Park Lane Pittsburg, PA, USA) was followed (40 Waves × for 10 min) to avoid nanoparticle agglomeration before exposure to the cells (Alarifi et al., 2017; Takeo, and Nakagata, 2018). The nanoparticles were immediately added to IVF dishes: 2.5 μl in the drop of the first treatment group and 5 μl in the second treatment group drop. In the IVC, dishes were supplemented with 4.5 μl in the drops of the first treatment group and 9 μl in the second treatment group's drops.

2.2 Animal treatment

Research Ethical Community at the King Saud University approved No.SE-19-136 was obtained after submission of the experimental protocol and animal handling procedure. The laboratory Mus musculus mice strain SWR/J, male between the ages of 3 and 6 months, and females between the ages of 6 and 8 weeks.

2.3 Spermatozoa Collection

The mice males (15 SWR/J, 30–35 gm weight) was euthanasia sacrificed method of exposure to 80 % CO₂ / 20 % O₂ for 120 s in a sealed chamber then dissected and the two cauda epididymides removed. Then two cauda epididymides transferred to paraffin oil in the sperm collection dish, which contains two drops of about 400 μl of human tubular fluid (HTF) medium (Irvine Scientific -Corporate 2511 Daimler Street Santa Ana, CA 92705, USA. Catalog #90125) covered with mineral paraffin oil Sigma M5310. Prior to beginning sperm collection, the dish was set up in an incubator with 5 % CO2 and 37℃ temperature control. This process should be completed at least 30 min before the collection begins.(CO2 Lab Incubator Heraeus Instruments, Function line, Thermo Scientific). An incision was made in each cauda epididymis under the oil, the suspension of sperm was allowed to capacitate by placing it in an incubator (37℃, 5 % CO2 in air) for 60 min before fertilization (Takeo, and Nakagata, 2018).

2.4 Oocytes Collection: Hormonal treatment for superovulation

The Female mice (45 SWR/J, 25–30 gm. weight) were injected intraperitoneally (IP) with 7.5 IU of equine chorionic gonadotropin (eCG) (Serigan 6000 IU, Ovejero, Ctran. Leon-Vilecha, Spain) between 4:00 to 5:00 pm of Day 1, after 48 h, the same mice receive 7.5 IU (IP) injection of human chorionic gonadotropin (hCG) (Choriomon 5000 IU, Instiut Biochimique SA 6903 Lugano, Switzerland). Ovulation occurred approximately 15–17 h after the hCG injection (Takeo, and Nakagata, 2018; Alhimaidi et al.2022). The cumulus - oocytes complexes (COCs) were collected from the oviducal ampullae at 15 h after hCG injection. The female mice were sacrificed as the same way male were treated, then dissected, and the two oviductal ampullae were removed, transferred to the oocyte collection petri dish (BD Falcon 353001), containing a drop (200 μl.) of (HTF) medium with five more small drops. The fertilization dishes and oocyte collection dishes were set up at least 30 min before the oocyte collection. They were then put in an incubator that was set to 37℃ and had 5 % CO2 in the air. Directly after ampullae collection, an incision was made in each ampulla by using the needle of insulin syringe to open the ampulla and drag (COCs) into the HTF medium drops.(Takeo, and Nakagata, 2018; Alhimaidi et al.2022).

2.5 In vitro Fertilization IVF

The fertilization dishes were prepared and placed in an incubator (37℃, 5 % CO2 in air) for at least 30 h to allow the medium equilibration. For the insemination one million sperm/ml from sperm suspension was added to COCs drop. After insemination, the fertilization dishes placed in an incubator (37℃, 5 % CO₂ in air) for 6 h Takeo, and Nakagata, 2018; Alhimaidi et al.2022).

2.6 Embryo Culture

The potassium simple optimization medium (KSOM)(TBS-8071 Lot no 200110, Tri-bioscience, Sunnyvale CA, USA). a culture dishes were pre-prepared for a minimum of 30 min prior to use, and that has been acclimated in a CO₂ incubator. After 6 h of fertilization, the oocytes were transferred into a 60 mm petri dishes containing hyaluronidase enzyme covered with mineral paraffin oil to remove cumulus cells mechanically using a glass pasture pipette, and the denuded oocytes transferred to the culture medium, then the fertilized oocytes cultured for 4–5 days until the blastocyst stage reached in a 5 % CO2 incubator at 37 °C (Takeo, and Nakagata, 2018; Alhimaidi et al.2022).

2.7 Experimental Design Y₂O₃ Nano particles treatments

After oocytes collection, oocytes were divided into three major groups, the control group and 2 treatment groups, depending on the Y₂O₃ nanoparticles concentration, 1st group (25 μg/ml), and 2nd group (50 μg/ml). The treatment starts with IVF and during the embryo culture medium as stated before. Then the fertilized oocytes cultured until the blastocyst stage which approximately reached at 4th-5th day after fertilization in a 5 % CO2 incubator at 37 °C (Takeo, and Nakagata, 2018; Bakhtari et al., 2020).

2.8 Statistical analysis

The IVF, cleaved embryos rates data, and blastocyst rate was tested by the Package for social sciences (SPSS) Software version 20. Before analysis, the normality and equal variance were tested using Histogram, Box Plot, and then compared by one way-ANOVA, followed by multiple pairwise comparisons using the Tukey’s test. Data presented as mean ± SEM. Differences considered significant if the P-value was less than (0.05). All the experiments were replicated at least three times.

3.1 Characterization of Yttrium Oxide Nanoparticles (Y₂O₃ NPs)

3.1.1

3.1.1 By the transmission electronic microscope (TEM) for (Y₂O₃ NPs)

The shapes of yttrium oxide Y2O3 mostly cuboidal, and the size of crystallite is ∼ 18 ± 5 nm (∼500 nm with nano-agglomeration) (Fig. 1 (a) and (b)) showing a crystallinity surface (Fig. 1 a) at low resolution and (Fig. 1 b) at higher resolution.

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Fig. 1

(a) and (b). Transmission electron micrograph (TEM) micrographs of nano-yttrium oxide Y₂O₃ NPs: (a) low- and (b) high-resolution image.

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3.1.2

3.1.2 By the X rays diffractions (XRD) analysis results

The X rays diffractions XRD patterns of the yttrium oxide Y2O3 nano powders (Fig. 2) obtained by using the database of International Centre for Diffraction Data (ICDD) powder diffraction file database card file No. 89-5591. The yttrium oxide Y2O3was crystalline and had no second-phase or impurity peaks. Scherrer's equation used to calculate the average yttrium -oxide Y2O3 nano powders crystallites size (L) from the peak at 2θ = 29.08° (Balzar, and Popa, 2002):

(1)

$$L=\frac{k\lambda }{\beta \cos \theta }$$ where k, λ, β, θ are the shape factor (0.94), X-ray wavelength (0.15419 nm), line broadening at half the maximum intensity [full width at half maximum (FWHM)] in radians, and the Bragg angle, respectively.

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Fig. 2

X- Ray Diffraction (XRD) patterns of Yttrium-oxide Y₂O₃ NPs nano powders.

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The crystallite size was 11.14 nm, which is slightly lower than the TEM imaging value.

3.2 The In vitro Fertilization (IVF) and in vitro Culture of mouse embryos

The total number of ova collected from mice control group female were 443 ova, the 1st treated group Y₂O₃ (25ug/ml) 472 ova and the 2nd treated group Y₂O₃ (50ug/ml) 237 ova. The results of different concentrations (25 µg/ml and 50 µg/ml) of Y₂O₃ NPs assessed on early embryonic development of mice oocytes are presented in Table 1. There were no significant differences within and between the treated and the control groups in comparing each cell stage of early embryo development from 1 cell till morula stage (Fig. 3-a, b,and c). While the mean of the blastocyst stage showed significant differences (P < 0.05) between the control and the 1st treated group compared to 2nd treated group (mean ± SE = 56.50 ± 5.67 and 73.75 ± 10.30 compared to 20.50 ± 2.72), respectively Table 1 and (Fig. 3.d). In addition, the result of the different development stages which showed some variations between the three groups, such as in the 2nd treated group most of the ova remained in 1 cell stage and at the degenerated ova compared to the 1st treated and control groups (Fig. 3 a, b, and c). In addition, (Fig. 3.d) compared among the 3 groups in the totally different early embryo development stages, the 1st treated group embryo cleavage rate 363/443, 76.9 % = 80.0 + SE 7.34), the 2nd treated group (shoed the lowest rate 150/237, 63.3 % = 72.92 + SE 8.42), compared to the control group (354/443, 79.9 % =81.24 + SE 5.06). While the blastula stages, which showed some significant variations in blastula stage especially in the 1st treated group showed the highest rate (296/363. 81.5 % = 89.90 + SE 11.29) compared to the 2nd treated group (82/150,63 % = 66.47 + SE 16.51) and the control group 226/354 63.8 % = 65.01 + SE 9.33) at P (<0.05). Fig. 4 illustrates the picture of some different developmental embryo’s stages.

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Fig. 3

a, b, c and d.) The effect of Y ₂O ₃ NPs (a) treated with 25ug/ml, or (b) 50 ug/ml, compared to (c) control on early mice embryonic devolvement and (d) blastula rate. * Significant at (P < 0.05).

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Fig. 4

The in vitro fertilization (IVF) of mice early embryonic cleavage stages and Blastula. Tow cell stage b) 4 cell stage c) blastula stage.

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4.1 Characterization of Yttrium Oxide Nanoparticles (Y₂O₃ NPs)

In the current study, which may be considered as the first time studied the potential role of Y2O3 NPs in vitro fertilization and early stage of development in vitro on mice embryo. Nanoparticles often show different properties from the original material it synthesizes by (Mourdikoudis et al. 2018). The nanoparticle's characterization is necessary to know the probabilities and the application of the new material. The characterization parameters that excessively studied were the size and the shape of nanoparticles (Mourdikoudis et al. 2018). Our nanoparticle characterization result showed a cubic shape and a size 18 ± 5 nm by TEM. While x-ray diffraction showed 11.14 nm size of the nanoparticle, which is smaller than the size of the TEM. The difference in nanoparticles' size due to the calculation method between TEM and XRD techniques (Andelman et al., 2010). In a previous study, the nanoparticle's shape was cubic, and the size was 35 ± 10 nm (Akhter et al. 2020). In the present study, the x-ray diffraction result showed impurity peaks. In a previous study, the breakdown of peaks occurs when the particle size is small, so the average size of yttrium oxide nanoparticles by the XRD technology was between 3 nm and 4 nm (Andelman et al., 2010). The Y2O3 NPs reported in previous studies as antioxidative and protective against oxidative stress damage in differential cells type and retinal origin (Hossain et al.2015; Khaksar et al., 2017; Grana et al. 2018) which agreed with our study. Additionally, Y2O3 NPs therapeutic effect was reported in oxidative stress-related disease (Song,et al., 2019).

4.2 In-vitro Fertilization

May be for the first time, the current study evaluated the role of adding the Y₂O₃ NPs in the culture media of in vitro mouse embryos development. The present study results showed that exposure of sperm and oocyte to Y₂O₃ NPs for 5 h in fertilization medium and culture medium for 5th days, indicate some significant changes in cleavage and blastocyst rates. The result of this study showed some variations compared with the previous study, that showed the (Y₂O₃ NPs) (35 ± 10 nm) did not work as an antioxidant enzyme-like activity, non-oxidative, and biocompatible in the human breast carcinoma MCF-7 and fibroblast HT-1080 cells (Akhter et al. 2020). The biocompatible behavior was due to the high surface reactivity of (Y₂O₃ NPs) that interact with the culture media component and formation NP-protein corona, which is an aid in biocompatibility (Akhter et al. 2020). While the other studies showed the antioxidative properties of Y₂O₃ NPs, in mice macrophage cell line and hepatic failure of murine model Y₂O₃ NPs (40––100 nm in size) which showed antioxidant and anti-inflammatory in vitro studied, and oxidative inhibition stress, inflammatory response, and cell apoptosis, liver injury (Song,et al., 2019). Owing to the high free energy of Y₂O₃ NPs and normalize inflammatory mediator expression by impeding of ROS/NF-κB pathway (Song,et al., 2019). As stated by previous study reported that NPs of Y₂O₃ (zeta sizer;175.34 ± 15.5 nm) work as an antioxidative, anti-inflammatory, and antifibrotic effects on macrophages cells and isoproterenol-induced cardiotoxicity mice remodel. Due to the free radical scavenging activity of nano-yttria and the inhibition of cardiotoxicity, pro-inflammatory cytokines, and protective effect in histo-architecture and rhythm of the heart (Godugu,et al., 2018). The anti-pancreatitis activity of Y₂O NPs at a sizer; 159.2 ± 7.5 nm) via inhibited mitochondrial and endoplasmic reticulum stress by the decrease of the Nrf2/NFκB pathway (Khurana et al., 2019). The protective effect observed in photoreceptor cells in the murine model by pro-treatment with Y₂O₃ NPs (∼10–14 nm in diameter) (Mitra, 2014). The neuroprotective and antioxidative effects of nano-yttria (5.61–18.2 nm) showed in PC12 cells only and in combination with CeO₂ nanoparticles (Ghaznavi et al., 2015). In rat hippocampal, the nanoyttria showed protective effect only and combined with CeO₂ nanoparticles (Schubert et al., 2006). The antiapoptotic effect of the combination of Y₂O₃ and CeO₂ nanoparticles was reported in β-cells of isolated rat pancreatic islets (Hosseini et al., 2013). Another previous study showed recovery in the rat pancreas model after treatment of Y₂O₃NPs only and combined with CeO₂ NPs by inhibition oxidative stress and apoptosis (Khaksar et al., 2017). The synergetic effect of Y₂O₃ NPs (30–50 nm in size) was observed in human cells line combined with nano-zinc oxide (Grana 2018). On the other hand, Y₂O₃ NPs (41 ± 5 nm in size) have been reported to be cytotoxic in the HEK293 cells and inhibit cell proliferation 50 % at concentration 108 μg/ml The result of the current study showed that Y2O3 NPs (18 ± 5 nm) have a non-antioxidative effect and non-cytotoxic effect in the culture media of early embryonic development in a mouse model, which agree with Rajakumar, et al study about the safety using Y2O3 NPs Biomedical Applications.