Pre‐implantation genetic testing: Past, present, future

Abstract Background Pre‐implantation genetic testing (PGT) has been performed worldwide since it was first used by Handyside et al in the United Kingdom to sex embryos in 1990. Until about 2010, cleavage stage embryo biopsy and fluorescent in situ hybridization (FISH) were mainstream; however, in 2012, blastocyst biopsy (trophectoderm; TE biopsy) became mainstream. In addition, array comparative genomic hybridization (aCGH) was used for analysis and further evolved to next‐generation sequencing (NGS), which is used worldwide. Methods PGT for reciprocal balanced translocation and Robertsonian translocation (PGT‐SR) was approved in Japan for habitual abortion to reduce pregnancy loss, and since 2008, we have been performing PGT‐SR using cleavage stage embryos and FISH. In 2014, we performed TE biopsy and NGS analysis. Main findings In this paper, I separately described the details of our methods and clinical results of FISH and NGS. NGS is superior to FISH because it can detect all chromosomes. Conclusion TE biopsy and NGS, which have recently become mainstream, have stable outcomes, because TE biopsy yields more cells and fewer mosaics than the cleavage stage. As a result, diagnoses are more reliable, resulting in higher pregnancy rates and lower abortion rates.

The first successful PGT for Tay-Sachs disease (intron 12) was performed in the United States in 1995. 2 A single blastomere was removed from a cleavage stage embryo, and its DNA was amplified by polymerase chain reaction (PCR). A restriction enzyme was then used to distinguish between affected and unaffected embryos, after which embryo transfer resulted in a healthy child. 1 3. Outcomes at our clinic

| Cleavage stage embryo biopsy
Embryo biopsy poses two questions: at what stage to biopsy cells and the number of biopsies. For diagnosis, harvesting multiple cells is advantageous. In a mouse experiment, we examined which stage cells could be harvested from, and how many cells could be harvested. 4 In both the four-cell and eight-cell stages, harvesting a single blastomere did not affect subsequent development into blastocysts, post-embryo transfer implantation rates in recipients, or live birth rates (Table 1). In addition, biopsy did not affect abnormalities or deformities. The study was continued to the next generation, which also did not demonstrate any adverse effects associated with biopsy. 4 Although blastomere removal was demonstrated not to affect subsequent development in animal experiments, does the same hold true for human embryos? With human embryos, a biopsy provides fewer cells than an animal embryo, and performing a biopsy prior to compaction is considered to result in less damage to the embryo. Therefore, the 8-cell stage is considered suitable for embryo biopsy. 5 We have long performed embryo biopsy primarily by extrusion, as we believe that not directly aspirating the sample blastomere results in less damage to the DNA. Our embryo biopsy procedure is described in detail below: Extrusion 4 : This method involves using a pipette to inject culture medium into the embryo to increase internal pressure and, thus, extrude a single blastomere from the opening in the zona pellucida. First, the zona pellucida is punctured with a special needle, and the tip of the needle is hit with a holding pipette to create an opening in the zona pellucida. Next, after the culture medium is suctioned, the zona pellucida is penetrated again from the 3 o' clock position, and the culture medium is injected into the embryo. Finally, a single blastomere is extruded from the cleavage in the zona pellucida at the 12 o' clock position (arrow) ( Figure 1).

| P G T FOR BAL AN CED RECIPRO C AL TR AN S LO C ATI ON S US ING FIS H (OUTCOME S AT OUR CLINI C)
In embryo biopsies, we have long conducted PGT primarily using Example of diagnosis in interphase nuclei: The fixation of blastomeres and the actual conditions of the FISH method will be described.

| Select probes
The probe is divided into CEP that stains centromere, a subtelomerespecific probe that stains telomeres at the ends, and a Locus-specific probe that stains a specific site, depending on the portion to be stained.
The probe used, a balanced or unbalanced type, is a combination of at least three probes according to the karyotype of the balanced reciprocal translocation carrier. The balanced type is alternate segregation and becomes normal or carrier. indicates alternate segregation and that the embryo will be normal or a carrier. All other ratios of colors indicate an unbalanced translocation. Figure 3 shows images in three color FISH of the balanced type. Table 2 shows the clinical outcomes for FISH at our clinic.

| CURRENT TROPHEC TODERM B I OPSY ( TE B I OPSY ) IN THE B L A S TO C YS T S TAG E
For chromosomal analysis, 6 Kokkali et al recommended performing embryo biopsy in the blastocyst stage rather than the cleavage stage, in which mosaicism is common and few cells can be obtained. 7 Therefore, the most common method of embryo biopsy is TE biopsy.
Our TE biopsy procedure and sampling method are described below.
After preparing in advance, a dish (Falcon 351007) for the number of embryos to biopsied, a holding pipette (medion international CC), and a biopsy pipette (sunlight medical) with an inner diameter of 25 μm were installed, and then, the blastocyst was transferred to the dish.
The biopsy pipette was previously washed with polyvinyl pyrrolidone (PVP) (Orgio) to prevent cell attachment, and the inside was coated.
Embryos that grew into hatched blastocysts were fixed with a holding pipette, TE cells were aspirated into the biopsy pipette while avoiding inner cell mass (ICM), and laser irradiation (pulse 300 μs) (laser perforator LYKOS (Brown Technology)) was performed. The cells were collected, the obtained cell sample was discharged from a biopsy pipette, the number of cells was confirmed, and the cells were collected.
Next, the sampling will be described. As a preliminary preparation, we entered the sample number in the dish (Falcon 351007) for washing the sample cells and 0.2-mL tube (Eppendorf PCR tubes 0.2 mL) for storing the sample.
In order to prevent cell stickiness, Pasteur, which aspirates the sample, has its tip lightly washed with PVP beforehand.
After washing the sample three times with PBS, it was moved to the bottom of the 0.2 mL tube, and the tube lid was closed.
The actual NGS procedures are as follows: First, the whole sample subjected to biopsy was subjected to whole-genome amplification (WGA) using a Veriseq PGS kit (Illumina) and a thermal cycler (Mastercycler Nexus, Eppendorf). The obtained sample was quantified and diluted to 0.2 ng/μL. The diluted sample was amplified with DHA tag and polymerase chain reaction (PCR).
After amplification, cleanup and normalization were performed, as well as load library, pooling, and loading. The next day, the obtained data were subjected to chart analysis using BlueFuse Multi Software (Illumina).
Thawed blastocysts were recovered using a time-lapse incubator. For embryos in which blastocoel expansion was confirmed, TE biopsy was performed via either (a) aspiration or (b) a novel extrusion method.

| Aspiration
The LYKOS laser perforation system (Hamilton Thorne, Inc) was used at a pulse of 120 μs to create an opening in the zona pellu-

| Novel extrusion method
After recovery, laser irradiation at a pulse of 120 µs was used to create a small opening in the zona pellucida of embryos that developed into expanded blastocysts ( Figure 5). The culture medium was injected through the opening, and cells were extruded from the opening. These extruded cells were subjected to TE biopsy, as in our aspiration method.
After TE biopsy, embryos were transferred to culture medium.
After preservation of the biopsy cells was complete, they were frozen with Cryotip Vitrification (Kitazato).
A basic study at our clinic revealed no differences in post-refreezing/rethawing recovery rates between aspiration and our novel extrusion method (Table 3).
In habitual abortion (balanced reciprocal translocation carriers, Robertsonian translocation), chromosomes are now analyzed using NGS rather than FISH. With FISH, only specific areas of chromosomes could be detected. However, NGS enables the detection of not only the translocation area but also of all chromosomes. Figure 6 shows examples of the actual charts of NGS. Tables 4 and 5 show clinical outcomes using NGS for PGT-SR using NGS and for PGT-M at our clinic.

| WHAT IS THE RI G HT CELL S TAG E FOR THE EMB RYO B I OPSY ?
Polar bodies have long been used for analysis. Although they offer the advantages of minimal invasiveness and lack of mosaics in chromosomal analysis, they only yield maternal information and are therefore not frequently used today. Next, cleavage stage embryos, which have been used in analysis longer than embryos of any other stage.
As described earlier, we have long used embryos at the 8-cell stage in the diagnosis of balanced reciprocal translocation with 3 biopsy requires greater time and effort. In addition, in a blastocyst biopsy, analysis takes more than 24 hours, thus requiring the embryo to be frozen. However, in our experiment, post-biopsy embryo survival rates were similar to those of normal embryos, the recovery rate was high, and freezing did not pose any problems.
One problem that has been raised in PGT is the potential for misdiagnosis due to mosaic embryos. 8  as mosaics. Table 6 shows the advantages and disadvantages of biopsy in each embryonic stage.

| Blastocoelic fluid aspiration 11
Blastocoelic fluid aspiration was first reported by Magli et al, 11    a high discordance of 52% between blastocoelic fluid and inner cell mass-TE, leading them to conclude that blastocoelic fluid is not well suited for clinical application. The above findings suggest that further study is required before blastocoelic fluid can be clinically applied to PGT.

| Spent media
Several studies have recently attempted to conduct PGT using spent embryo culture media. [15][16][17][18][19] Figure 7 shows the general procedure for collecting the spent embryo culture media. While blastocoelic fluid collection is also minimally invasive, spent media analysis does not require biopsy of the embryo itself, and anyone can collect spent media. Thus, spent media analysis is likely to be studied further in the future. In spent media analysis, the greatest concern is contamination of maternal cells.
In the analysis of medium used to culture embryos from day 3 to 5, Lane et al 17 observed a low concordance with TE biopsy, which they considered the result of contamination of maternal cells (granulosa cells).
In a recent study, Rubio et al 18 reported that embryo free DNA analysis was 78.2% concordant with the corresponding TE biopsy, and no significant differences were detected among multiple centers ranging from 72.5% to 86.3%.
Moreover, they reported that concordance rates exceeded 86% when all defined steps in the culture laboratory were controlled to minimize the impact of maternal and operator contamination.
Ho et al 19 compared concordance with TE biopsy for ploidy and sex in two groups of embryos: one that underwent assisted hatching (AH) on days 3 and 5, and one that did not undergo AH (Table 7).
Concordance was found to be higher on day 5 than on day 3, while embryos that did not undergo AH were surprisingly found to exhibit a higher concordance. Although AH on day 3 was generally considered necessary in spent medium analysis, Ho et al 19 reported that not only is AH not necessary, not performing AH, in fact, yields results that are more favorable. At any rate, blastocyst biopsy appears to be necessary to obtain large numbers of cells for PGT-A. Some aspects of minimally invasive embryo biopsy may still need to be examined before it can be completely applied in clinical practice. In the field of assisted reproductive technology, further study may be necessary to determine whether PGT-A is truly effective for obtaining live births.

ACK N OWLED G M ENTS
I would like to express my profound gratitude to the embryology staff of Takeuchi Ladies Clinic Center for Reproductive Medicine for their technical expertise and tremendous assistance in the drafting of the present review manuscript.

D I SCLOS U R E S
I, Kazuhiro Takeuchi, declare that I have no conflicts of interest.
Human rights statement and informed consent: All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national), and with the Helsinki Declaration of 1964 and its later amendments.
Informed consent was obtained from all patients in this study.
Approval by the ethics committee: All procedures that involved human participants were carried out in accordance with the ethical standards of the institutional review board of the Takeuchi ladies clinic. Total includes PGD and PGS for data I-XIII, as well as social sexing cycles for data I-XII (705 cycles). From data XIII onwards, details of social sexing cycles were no longer reported.