Three-Dimensional Live Imaging of Bovine Preimplantation Embryos: A New Method for IVF Embryo Evaluation

Conception rates for transferred bovine embryos are lower than those for artificial insemination. Embryo transfer (ET) is widely used in cattle but many of the transferred embryos fail to develop, thus, a more effective method for selecting bovine embryos suitable for ET is required. To evaluate the developmental potential of bovine preimplantation embryos (2-cell stage embryos and blastocysts), we have used the non-invasive method of optical coherence tomography (OCT) to obtain live images. The images were used to evaluate 22 parameters of blastocysts, such as the volume of the inner cell mass and the thicknesses of the trophectoderm (TE). Bovine embryos were obtained by in vitro fertilization (IVF) of the cumulus-oocyte complexes aspirated by ovum pick-up from Japanese Black cattle. The quality of the blastocysts was examined under an inverted microscope and all were confirmed to be Code1 according to the International Embryo Transfer Society standards for embryo evaluation. The OCT images of embryos were taken at the 2-cell and blastocyst stages prior to the transfer. In OCT, the embryos were irradiated with near-infrared light for a few minutes to capture three-dimensional images. Nuclei of the 2-cell stage embryos were clearly observed by OCT, and polynuclear cells at the 2-cell stage were also clearly found. With OCT, we were able to observe embryos at the blastocyst stage and evaluate their parameters. The conception rate following OCT (15/30; 50%) is typical for ETs and no newborn calves showed neonatal overgrowth or died, indicating that the OCT did not adversely affect the ET. A principal components analysis was unable to identify the parameters associated with successful pregnancy, while by using hierarchical clustering analysis, TE volume has been suggested to be one of the parameters for the evaluation of bovine embryo. The present results show that OCT imaging can be used to investigate time-dependent changes of IVF embryos. With further improvements, it should be useful for selecting high-quality embryos for transfer.


INTRODUCTION
Bovine embryo transfer (ET) has been widely used to produce calf in combination with other reproductive technologies, such as in vitro fertilization (IVF). However, the conception rate of ET using IVF embryos (30-40%) is lower than that of using embryos produced in vivo (around 50%) (1)(2)(3)(4). Embryos for transfer are usually selected by observation under a conventional optical microscope at the time of transfer, and embryo quality is subjectively assigned as one of the codes according to the International Embryo Technology Society (IETS) standards for embryo evaluation (5,6).
In human artificial reproductive technology (ART), embryos are evaluated based on the Veeck and Gardner classification (7,8) and time-lapse cinematography (TLC) with a visible light microscope, which has recently become a popular technology. Morphokinetic parameters, such as the number of pronuclei or nuclei, timing of cleavage, and the number of blastomeres, are used as potential indicators that may improve the success of ART (9). Furthermore, ART success rate has been improved by comprehensive chromosomal screening using techniques such as array comparative genomic hybridization, quantitative single nucleotide polymorphism arrays, and next-generation sequencing (10,11). To evaluate in vitro developed bovine embryos, TLC has been used to determine the time of the first cleavage and the subsequent number of blastomeres, and the number of blastomeres at the onset of the lag-phase (4,(12)(13)(14). However, so far, live bovine embryos have not been evaluated based on their three-dimensional (3D) structure. A morphological grading system in human ART was first described by Gardner and Schoolcraft (15). According to this system, three parameters (degree of blastocoel expansion, size and compactness of ICM, and the cohesiveness and number of TE) are graded. Based on these criteria, an additional consensus on embryo assessment was agreed including new references for each parameter (16,17). In this consensus, ICM grade is suggested to be more important for determining the implantation potential of a blastocyst. To select the best blastocyst when performing ET on Day 5, several parameters have been suggested to contribute to the implantation potential of blastocyst. Some investigators have shown that the timing of blastocoel development and the grade of expansion are important parameters for implantation (18)(19)(20). Other investigators have suggested that the size and shape of ICM are related to implantation (21)(22)(23). Either a positive association or no association of TE cells with implantation has been reported (22,(24)(25)(26). Morphological grading, while common for human blastocysts, is difficult for bovine blastocyst because of their dark cytoplasm (27,28).
Optical coherence tomography (OCT) has been developed for non-invasive, cross-sectional imaging in biological systems (29)(30)(31), and is presently used in ophthalmology, especially for funduscopic examination of the retina. OCT can be used to measure 3D images with high spatial resolution, because it can scan small biological structures, such as micro vessel structures during in vitro angiogenesis (31). Recently, OCT imaging of mouse (32)(33)(34) and porcine (34) early-stage embryo has been reported. In the mouse blastocysts, their nucleoli were also clearly visualized by OCT (34). In cattle, blastocysts have been imaged by OCT, and their cytoplasm movements that are potentially associated with viability were monitored, suggesting that OCT can be used for the measurement of the damage after cryopreservation (35). However, the quantification of the structures of bovine blastocysts for evaluating embryo quality has not been reported. We have recently developed a technique for the 3D imaging of bovine blastocysts and used it to evaluate 22 parameters including the volumes of the ICM, TE, zona pellucida (ZP) and blastocoel of an embryo (36). Here, we used this technique to compare the characteristics of embryos that did or did not develop to term in order to identify the parameters associated with successful ET. Furthermore, in the blastomere observation, the shape, size, cytoplasm color, even distribution of cytoplasm, and number of nucleus have been suggested to be related to the developmental potential of bovine embryos (4,(12)(13)(14). Because bovine and porcine early embryos contain much more lipid than human or mouse embryos, pronucleus formation in early embryos cannot be confirmed under a microscope, which made it difficult to evaluate their quality (27,28,37,38). Thus, we have also tried to obtain 3D images of early-stage bovine embryos.

Ethics Statement
Animal handling and experimental procedures were carried out following the Guidelines for Proper Conduct of Animal Experiments by the Science Council of Japan (http://www.scj.go. jp/ja/info/kohyo/pdf/kohyo-20-k16-2e.pdf).

IVF
Frozen semen of Japanese Black bulls stored in straws was thawed in water (37 • C, 40 sec), and then centrifuged twice in IVF100 (Research Institute for the Functional Peptides, Yamagata, Japan; 600 × g, 5 min). After centrifugation, spermatozoa were removed from the pellet, and added to IVF100 to obtain a suspension with

In vitro Culture (IVC)
After insemination, oocytes were completely denuded from the cumulus cells and spermatozoa by pipetting with a glass pipette in IVC medium: potassium simplex optimized medium with amino acid (KSOMaa Evolve Bovine; Zenith Biotech, Bangkok, Thailand) supplemented with 5% NCS and 0.6 mg/mL of L-carnitine (C0158, Sigma-Aldrich). Subsequently, presumptive zygotes were washed three times with IVC medium and cultured in 100 µL droplets of IVC medium for 48 h. Each droplet contained approximately 20 presumptive zygotes. Average value of cleavage rate was 74.0% (179/242). At 48 h post-insemination (hpi), embryos with more than four cells were transferred from the 35 mm dishes to well-of-the-well (WOW) dishes (LinKID micro25, Dai Nippon Printing Co., Ltd., Tokyo, Japan) as described (12). WOW dishes, which have 25 microwells (5 columns × 5 rows) in a circular wall in the center of a 35 mm dish, can culture up to 25 embryos each with a single drop of medium and track individual embryos throughout the culture. IVC medium and mineral oil were pre-cultured for at least 12 h in glass bottles separately at 38.5 • C in 5% CO 2 , 5% O 2 , and 90% N 2 in humidified air, and the pre-cultured IVC medium (100 µL) was placed within the circular wall and covered with the pre-cultured mineral oil. At 168 to 180 hpi, embryos that had developed to or beyond the blastocyst stage were observed under an inverted microscope. Finally, 123 embryos had developed to the blastocyst stage (50.8%), and 80 blastocysts had been cryopreserved.

OCT Observations
IVF embryos were cultured for seven days (by this time, they reached the expanded blastocyst stage) and examined under an inverted microscope at 27-31 hpi (at the 2-cell stage; n = 15) and at 168 to 180 hpi (at the blastocyst stage; n = 30). In blastocysts, only the embryos that were independently classified as Code1 according to the IETS standards by three skilled observers were used. OCT imaging was done as described previously (31,36). Unstained live embryos were imaged by OCT using the Cell3iMager Estier (SCREEN Holdings Co., Ltd, Kyoto, Japan). The imaging system of Cell3iMager Estier is outlined in Figure 1. The system is equipped with a super luminescent diode (SLD; center wavelength: 890 nm, N.A. = 0.3). The SLD output is coupled to a single-mode optical fiber and split at an optical fiber coupler into the sample and reference arms. The reflections from the two arms are combined at the coupler and detected by the spectrometer. The 3D image data of the blastocysts were constructed from individual 2D ×-z crosssectional images, which were obtained by a series of longitudinal scans obtained by laterally translating the optical beam position. The data acquisition window was 200-300 × 200-300 × 200-300 µm, and the voxel size was 1 × 1 × 1 µm. The OCT system scans light source positions in the x-axis direction while the shifting scanning line positions on the y-axis to obtain a signal on the x-y plane at a focus position on the z-axis. By repeating this scanning while shifting the z-axis focus positions, 3D images of the embryos were acquired. The lengths of the imaging range on the x-, y-, and z-axis were 300, 300, and 200 µm, respectively. The exposure time was 150 µs, and scanning an entire embryo was completed in a few minutes. OCT provided cross-sectional images with a slice thickness of 1 µm.

Image Analysis
We recently reported using OCT to image bovine embryos (36). After the 3D images were captured by OCT (Figure 1), the 3D images were analyzed in an automized way (Figures 2, 3). Process (i) The 3D images of bovine embryos were binarized (Figures 2,  3). For each image, vectors were drawn at equal angles in the elevation direction (−90-90 • ) and the azimuth direction (−180-180 • ) from the center of the embryo to the outer surface and the inner surface (the outermost of blastocoel). The thicknesses of the embryo along each vector (T All ) were then measured (Figure 3). Process (ii) To distinguish the ICM from T All , an appropriate threshold (TH icm ; the cut-off value for distinguishing ICM from T All ) was set by Otsu's method (41, 42) (Figure 3). The parts where the thickness from the outer edge of the embryo region was lower than TH icm were excluded from the binarized images. The largest object among the remaining objects after the exclusion was defined as ICM. T ALL was separated into the thickness information of the area corresponding to ICM (T ICM ) and the thickness information areas other than ICM (T other ). Process (iii) Based on the T other value, the average thickness (TH m ) was evaluated, and the threshold (TH t ) was set by Otsu's method (41,42) (Figure 3). The threshold (TH zp ) that separates TE and ZP was set by TH m -(TH t -TH m ) (in case of TH t > TH m ) or by TH m + (TH m -TH t ) (in case of TH m ≧ TH t ). The region where the thickness of T other was lower than TH zp was defined as ZP, and the remaining region after removing TH zp from T other was defined as TE. The unfulfilled region, surrounded by the embryo parts, such as ICM, ZP, and TE, in the binarized image was defined as the blastocoel. The volumes of the defined ICM, TE, ZP, and blastocoel were evaluated. The means, medians, standard deviations, minimum, maximum, and range of the thickness of ICM were evaluated from T ICM . The thickness of TE was evaluated from the thickness information, which was obtained by subtracting TH zp from T other . Summary statistics related to the thickness of ZP was set by TH zp .

ET and Pregnancy Diagnosis
The OCT-imaged embryos were transferred to 30 recipient cows [47.5 ± 25.5-month-old Holstein (18 lactating cows) and Japanese Black cows (10 cows; two cows received ET twice)]

Data Analysis
Hierarchical clustering analysis was performed using 13 blastocoel-related and ZP-related parameters of bovine blastocysts. Metrics and linkage criteria for hierarchical clustering were Pearson's correlation and unweighted average linkage. The data was normalized so that the average = 0 and

3D Imaging of Bovine IVF Embryos at the 2-Cell Stage
With OCT, it was possible to non-invasively obtain live 3D images of a 2-cell embryo. OCT images also showed the nuclei (Figures 4B,C), which made it easy to find binuclear blastomeres (blue and green in Figures 5B,C). Of the 15 embryos examined by OCT, two were binuclear and were not used for transfer.

PCA of 22 Morphological Parameters of Bovine Blastocysts
A PCA identified three principal components, PC1, PC2, and PC3. PC1 was related to the thickness of ZP and TE, which accounted for 41.00% of the variance; PC2 was related to the volumes of parts, which accounted for 29.81% of the variance; and PC3 was related to the thickness of ICM and TE, which accounted for 13.52% of the variance (Figure 9B). Figure 9A shows plots of PC1 vs. PC2 (a), PC1 vs. PC3 (b), and PC3 vs. PC2 (c). None of the plots clearly separated the P and NP embryos.

Hierarchical Clustering Analysis of the Morphological Parameters of Bovine Blastocysts
The hierarchical clustering analysis based on the blastocoelrelated and ZP-related parameters (Figure 10) showed two clusters with a threshold of Dissimilarity = 2.0, and these clusters were also found separated into low (Cluster 1) and high (Cluster 2) blastocoel volume clusters on blastocoel volume-TE + ZP thickness plane (Figure 11). In Cluster 1, no significant difference was found in any of the 22 parameters between the P and NP embryos (Figure 12), while in Cluster 2, TE volume was significantly lower in the P embryos (p < 0.05) (Figure 12, 13).
No difference between the P and NP embryos in any of the 22 parameters was common to both clusters (Figures 12, 13).

DISCUSSION
The present study describes the 3D images of bovine embryos at the 2-cell and blastocyst stages obtained by OCT. Blastomere nuclei at the 2-cell stage were also clearly visualized by the same system. At the blastocyst stage, 22 morphological parameters were evaluated based on the 3D OCT images. The transfer of 30 bovine embryos after being imaged by OCT resulted in 15 pregnancies (pregnancy rate: 50%) and 12 births (birth rate: 40%), which was typical for the ET attempts (1)(2)(3)(4). Bovine blastocysts appeared healthy after a long-term (over 18 h) capture by OCT for monitoring their micro-scale movements (35). These results indicate that OCT can be used to evaluate embryos before ET. As described previously, OCT can capture the inside structure of mammalian embryos (32)(33)(34)(35)(36). In addition, the present study has made it possible to quantify several parts of bovine blastocysts including its inside structure, such as the blastocoel, which could not be visualized by conventional microscopy. A PCA of the measured parameters was unable to find the critical parameters associated with pregnancy. To find the critical parameters for pregnancy, a greater number of transfers of OCTimaged embryos is needed. As bovine embryos expand into blastocysts, the thickness of the ZP decreases (5). In addition, we detected the blastocoel-related parameters (volume and diameter), which were originally quantified in our recent study by OCT (36). Thus, we conducted a hierarchical clustering analysis based on the blastocoel-related and ZP-related parameters. The hierarchical clustering analysis (Figure 10) shows two clusters with a threshold of Dissimilarity = 2.0, and these clusters were also found separated into two clusters with low (Cluster 1) or high (Cluster 2) blastocoel volumes on a blastocoel volume-TE + ZP thickness plane. While no difference common to both clusters were found in any of the 22 parameters between the P and NP embryos, TE volume in Cluster 2 was significantly lower FIGURE 12 | P-values in the comparison of 22 parameters evaluated from the optical coherence tomography (OCT)-scanned 3D images of bovine blastocysts between pregnancy and non-pregnancy in each cluster (pregnancy: n = 15, non-pregnancy: n = 15). Statistical significance was analyzed using the Mann-Whitney U test. A value of p < 0.05 was considered statistically significant.
in the P embryos than in the NP embryos. In Cluster 2, blastocoel volumes were relatively high and the TE + ZP thicknesses were relatively low (Figure 11), suggesting that embryos in Cluster 2 were well-expanded. These results imply that a low TE volume could be one of the parameters for selecting embryo for ET especially in well-expanded blastocysts. However, since in Cluster 1, there was no significant difference in TE volume between the P and NP embryos, more precise methods for the quantification of TE-related parameters and/or a combination of parameters based on the increased number of OCT images are needed to find the critical parameters for the evaluation of bovine embryos for ET. Furthermore, embryo evaluation is more effective if the OCT measurements are done in parallel with time lapse imaging to evaluate other developmental landmarks, such as the timing of embryo cleavage, timing of each developmental stage, and evaluating cell number.
The present study imaged blastomere nuclei at the 2-cell stage. Karnowski et al. (34) reported that OCT could visualize not only nuclei but also pronuclei and nucleoli in mouse early embryos and blastocysts. They also used OCT to show time dependent changes in these nuclear architectures (34). In cattle, TLC analysis has revealed that the time-dependent changes, such as the time of the first cleavage and the subsequent number of blastomeres, and the number of blastomeres at the onset of the lag-phase are useful for selecting embryos with a development potential (4,(12)(13)(14). Since we also visualized binuclear cells in a blastomere at the 2-cell stage, which is known as a negative indicator for development (43), OCT should also be useful for weeding out poor quality embryos. Previous reports also indicate that morphological indices also help to select high quality bovine embryos (4,13,43). Furthermore, the structure of bovine blastocyst has been precisely quantified in the present study. Together, the above findings suggest that detecting time-dependent structural changes of early-stage bovine embryo by OCT could improve evaluations at the blastocyst stage.
The present study reports the first normal deliveries of calves following the transfer of OCT-analyzed bovine embryos. FIGURE 13 | Comparison of TE volume evaluated from the optical coherence tomography (OCT)-scanned 3D images of bovine blastocysts between pregnancy (P) and non-pregnancy (NP) in each cluster (pregnancy: n = 15, non-pregnancy: n = 15). Statistical significance was analyzed using the Mann-Whitney U test. A value of p < 0.05 was considered statistically significant.
The present conception rate (50%) and the birth rate (40%) following OCT are typical for ETs, indicating that OCT did not adversely affect ET. Although a PCA was unable to identify the parameters associated with pregnancy, TE-related parameters may be useful for evaluating bovine embryos. At present, OCT imaging should be useful for investigating the time-dependent changes of IVF embryos, and with further improvements, be useful for the selection of high-quality embryos for transfer.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

ETHICS STATEMENT
Ethical review and approval was not required for the animal study because the presented results were based on the bovine blastocyst images captured in culture conditions, so that the present experiments did not have any stress to animals. Animal handling and experimental procedures in farms were carried out following the Guidelines for Proper Conduct of Animal Experiments by Science Council of Japan (http://www.scj.go. jp/ja/info/kohyo/pdf/kohyo-20-k16-2e.pdf). Written informed consent was obtained from the owners for the participation of their animals in this study.

AUTHOR CONTRIBUTIONS
RN: conceptualization, resources, data curation, supervision, project administration, and writing-review and editing. YM: methodology, investigation, and writing-original draft preparation. RH, YK, MK, and KU: methodology, investigation, and writing-review and editing. MH and TO: writingreview and editing. All authors have read and agreed with the manuscript for publication.