Four categories were established: euploidy (<30% aneuploid cells), low degree mosaicism (30–50%), high degree mosaicism (>50–70%), and aneuploidy (>70%). To classify samples in these categories, we defined the thresholds of different degrees of mosaicism. These thresholds were calculated using our cell line models to mimic different levels of mosaicism. The mean difference values for each category were 0.05 (SD = 0.04), 0.33 (SD = 0.08), 0.52 (SD = 0.09), 0.72 (SD = 0.12), 0.94 (SD = 0.08) for the 0%, 30%, 50%, 70%, and 100% categories, respectively. Figure 2A displays the distributions and confidence intervals of the different CNV thresholds when considering all chromosomes at different percentages of mosaicism (0%, 30%, 50%, 70% and 100%). The distribution obtained using all chromosomes is very similar to the distribution obtained when each chromosome was analyzed separately using the same method (Figure 2B).

**Figure 2.** (**A**) Difference values for all chromosomes by known percentage of mosaicism. (**B**) All samples by selected chromosomes and proportion.

To check if our algorithm was correctly diagnosing percent mosaicism, 18 samples with different percentages of mosaicism (40%, 60% and 100%) generated using two cell lines with trisomy in chromosome 8 and 9 (GM00425 and NA09287, respectively) were analyzed. 100% of the samples amplified correctly (18/18), and 94.4% (17/18) were correctly categorized; only one 40% mosaic sample was mis-categorized as being high mosaic instead of low. All QC criteria were met (Table 1).

Finally, to estimate the percentage of mosaicism in TE biopsies, we applied the algorithm retrospectively to 14, 108 TE biopsies analyzed in 10 different diagnostic laboratories from our group. The overall percentage of mosaicism was 5%, with 3.66% (SD = 0.86) of samples classified as low-degree mosaicism and 1.34% (SD = 0.36) samples as high-degree mosaicism. The differences among laboratories were not significant (*p* < 0.05). These data are in concordance with previously reported percentages [21].

#### *3.2. Phase II: PGT-SR*

For these samples, the amplification rate was 100% (48/48). Samples were sequenced in four 520 chips, with only 12 samples per run. The average number of reads per sample was 305,287 (156,970–604,356; SD = 120,052) and the MAPD mean value was 0.145 (0.106–0.3; SD = 0.037).

All deletions were detected (48/48), including the smallest, setting the detection limit to 5.6 Mb and making the concordance rate and sensitivity/specificity 100% (48/48) (Table 1).

#### **4. Discussion**

In recent years, several sequencing platforms have been applied to PGT-A, and initial publications [10–15] have highlighted the need for a proper validation of each platform, mostly for mosaicism levels and resolution for de novo and inherited Del/Dup. Here, we describe an improved, mostly automatized, fast, and accurate protocol for detecting whole uniform aneuploidies, de novo Del/Dup (≥10 Mb), unbalanced Del/Dup up to 6 Mb in carriers of structural rearrangements, as well as mosaic aneuploidies. A key advantage of NGS is that portions of the protocols can be automated minimizing the contamination risk, mismatch of samples, time, and cost. In this study, introducing automated library preparation with the Ion Chef™ increased the robustness and reproducibility of the NGS protocol. To our knowledge, this is the first study to extensively validate a semiautomated NGS protocol with the Ion Chef + S5 sequencer for PGT-A, PGT-SR (≥ 6 Mb), and mosaicism detection using a proprietary algorithm.

To validate our PGT-A strategy for detecting whole uniform chromosome aneuploidies and large Del/Dup, we used cell lines of known karyotypes. These cell lines have been utilized previously by other groups to validate PGT-A with both aCGH and NGS techniques [1], but, to our knowledge, our study was the most comprehensive regarding the number of different cell lines and different chromosomes affected (14 chromosomes divided between nine cell lines for whole uniform chromosome aneuploidy and six cell lines for large segmentals ≥ 10 Mb). Other authors, e.g., Kung et al. (2015) [1] and Goodrich et al. (2017) [28], used six and four cell lines, respectively, and only for whole-chromosome aneuploidies and without the benefit of the automation steps used in this study.

Our PGT-A protocol was highly effective at detecting segmentals (≥ 10 Mb) in pools of 5-6 cells, mimicking TE biopsy. In 100% of samples, we not only correctly determined whole uniform chromosome aneuploidies, but also detected Del/Dup. These results are consistent with or improve upon those obtained in previous works, e.g., the Fiorentino et al. (2014) study [29], where authors set the size of de novo detectable segmentals to 14 Mb using the Illumina NGS platform.

Our protocol was also highly effective at predicting mosaicism. We established thresholds for this using multiple mixtures of gDNA from cell lines with known karyotype, mimicking different mosaic percentages. Other groups have used distinct strategies combining different numbers of euploid and aneuploid individual cells [28,30]. Our gDNA approach allowed us to test mosaicism for more chromosomes than commercial cell lines (eight in total) can test, covering potential variability among chromosomes, since it is known that amplification can be biased by GC content [31]. Using the sequencing information from our samples, we developed a proprietary algorithm allowing the automated assignation of mosaic embryos to different categories, avoiding the subjectivity of both the scientist performing the diagnosis and the laboratory where the analysis has taken place. For this, we divided mosaic embryos into two categories: low and high. Such categorization is clinically relevant since implantation and ongoing pregnancy rates after the transfer of mosaic embryos relates to the degree of mosaicism, with low mosaic embryos exhibiting better implantation rates [17,20,24,32,33]. Nevertheless, euploid embryos should be chosen first for transfer, and the transfer of mosaic embryos should be coupled with pre- and post-genetic counselling, including the option of a new IVF cycle if there are no euploid embryos in the current cycle, to yield a better prognosis [34]. Segmental aneuploidies which are not uniformly present in blastocysts and have low predictive value in IVF/PGT-A [35,36] were not considered when determining mosaicism in embryos.

Our algorithm to detect mosaicism is robust even when considering samples from different laboratories. We retrospectively analyzed the incidence of mosaicism in TE biopsies from 10 diagnostic Igenomix laboratories, finding an average incidence of 5% (3.6% low and 1.4% high), with no significant differences among laboratories, demonstrating the consistency of the algorithm. A wide range of mosaicism has been reported by different authors, suggesting that mosaicism may be over-diagnosed and highlighting the need to set thresholds for the degree of mosaicism that can be detected in a TE biopsy based upon the background signal that can interfere with the interpretation of results [37]. Interestingly, the different percentages of mosaic embryos reported in the bibliography are linked with

the cutoffs used [38]. Some laboratories broadly defined mosaicism as being between 20% and 80% admixed aneuploid and euploid DNA and others, including us, used thresholds of 30% and 70%. Using the 20–80% range detected mosaicism in up to 17% of embryos, whereas the 30–70% threshold range decreased the mosaicism rate to 5%. Miscarriage rates are similar in both scenarios, indicating the 20–80% range may overdiagnoses mosaicism [38].

Finally, our protocol was successfully modified to create the PGT-SR protocol for detecting smaller Del/Dup (≥6 Mb). For this, we used four different cells lines with deletions in different chromosomes and with different breakpoints to assay the robustness of the technique in dealing with different chromosomal conditions. Other groups have reported the detection of deletions as small as 5 Mb in embryos using a similar platform [39]. However, these samples were amplified twice, first with Sureplex (Illumina, San Diego, CA, USA) and then during WGA, and therefore more DNA (100 g) than seen in regular protocols was used to prepare the libraries [39]. In an additional study, the authors reported automatic calling of deletions as small as 10 Mb but detected fragments around 5 Mb when data were examined manually, during which subjectivity could alter the diagnosis [40]. Additionally, the abovementioned studies all used TE biopsies not cell lines, likely contributing to the variation in the fragment sizes detected.

#### **5. Conclusions**

Our study indicates that the NGS platform Ion Chef plus S5 sequencer from ThermoFisher is a reliable tool for testing the chromosomal complement of preimplantation embryos, detecting whole uniform aneuploidies, segmentals (≥10 Mb), small rearrangements (Del/Dup ≥ 6 Mb), and degree of mosaicism. Part of the protocol is automated, remarkably reducing user error and the subjectivity often seen in manual PGT-A evaluation. Our automated algorithm allows for accurate, unbiased, and reproducible diagnoses for PGT-A and PGT-SR application. The next steps would be trying to enhance the detection of small rearrangements by improving the resolution to 6 Mb and moreover, to improve the accuracy of the diagnostic algorithm of mosaicism including data from the chromosomal analysis of the products of conception and livebirths.

**Author Contributions:** C.R., C.S., and C.M.G.-P. participated in conceptualizing the study; Methodology was developed by L.R., L.N.-S., and C.M.G.-P.; Most experiments were performed by L.M. and L.N.-S.; R.N., L.N.-S., and C.M.G.-P. curated and analyzed the data; Software application and visualization were carried out by R.N. and J.J., C.R. was involved in supervising and administering the project. The manuscript was written by C.M.G.-P. and L.N.-S. and reviewed by L.R., C.S. and C.R. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study has been funded by Igenomix.

**Acknowledgments:** Authors would like to thank all Igenomix laboratory staff around the world for sharing valuable data.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Review* **Preimplantation Genetic Testing for Monogenic Disorders**

#### **Martine De Rycke \* and Veerle Berckmoes**

Center for Medical Genetics, Universitair Ziekenhuis Brussel, Laarbeeklaan 101, 1090 Brussels, Belgium<EMAIL_ADDRESS>
**\*** Correspondence<EMAIL_ADDRESS>
Received: 4 July 2020; Accepted: 29 July 2020; Published: 31 July 2020

**Abstract:** Preimplantation genetic testing (PGT) has evolved into a well-established alternative to invasive prenatal diagnosis, even though genetic testing of single or few cells is quite challenging. PGT-M is in theory available for any monogenic disorder for which the disease-causing locus has been unequivocally identified. In practice, the list of indications for which PGT is allowed may vary substantially from country to country, depending on PGT regulation. Technically, the switch from multiplex PCR to robust generic workflows with whole genome amplification followed by SNP array or NGS represents a major improvement of the last decade: the waiting time for the couples has been substantially reduced since the customized preclinical workup can be omitted and the workload for the laboratories has decreased. Another evolution is that the generic methods now allow for concurrent analysis of PGT-M and PGT-A. As innovative algorithms are being developed and the cost of sequencing continues to decline, the field of PGT moves forward to a sequencing-based, all-in-one solution for PGT-M, PGT-SR, and PGT-A. This will generate a vast amount of complex genetic data entailing new challenges for genetic counseling. In this review, we summarize the state-of-the-art for PGT-M and reflect on its future.

**Keywords:** preimplantation genetic testing; monogenic disease; multiplex PCR; SNP array; NGS

#### **1. Introduction**

Preimplantation genetic testing (PGT) can be performed for monogenic disorders or single gene defects (PGT-M), for chromosomal structural rearrangements (PGT-SR), and for aneuploidy detection (PGT-A) [1]. PGT involves the biopsy of a single or few cells from in vitro fertilized embryos and testing of the biopsied samples for genetic aberrations followed by the selective transfer of embryos unaffected for the condition under study. Although genetic testing of single or few cells is challenging and the overall procedure is quite complex, PGT has evolved from an experimental procedure in the early 1990s to a well-established alternative to invasive prenatal diagnosis and possible therapeutic termination of pregnancy. The first report on children born after PGT was published by Handyside and colleagues in 1990, describing the use of PCR amplification for the detection of repetitive Y-sequences for gender determination in families with X-linked diseases [2]. The single cell simplex PCR applied in this earliest PGT-M approach was soon replaced by multiplex PCR testing, in which closely linked informative short tandem repeat (STR) markers are co-amplified, with or without the pathogenic variant amplicon. Single cell biopsy at day 3 followed by multiplex PCR became the method of choice for the detection of monogenic disorders [3]. Biopsy at the blastocyst stage followed by genome-wide technologies began to replace this gold-standard method over the last decade. The genome-wide methods yield data on genotyping as well as on chromosome copy number, allowing for concurrent analysis of PGT-M and PGT-A [4–8].

As PGT and especially PGT-M is technically complex, transport PGT was implemented, a procedure in which embryo samples biopsied in a satellite IVF laboratory are transported to a genetics unit for testing. Transport PGT has the advantage that testing can be performed by experienced teams in genetic laboratories specialized in single cell molecular diagnostics. Transport PGT is a service that has expanded substantially, despite the challenges related to transport and collaborations over (long) distances [9].

PGT guidelines and recommendations for good practice have been designed by several international societies such as the PGD International Society (PGDIS), the American Society for Reproductive Medicine (ASRM), and the European Society for Human Reproduction and Embryology (ESHRE) PGT Consortium. The latter society has recently updated and extended four sets of recommendations, covering guidance on the organization of PGT service as well as technical guidance on embryo biopsy and genetic testing [9–12].

In this review, we present an overview of current PGT-M practice from patient inclusion to baby follow-up and reflect on future developments.

#### **2. Indications for PGT-M**

The provision of PGT is legally restricted in many countries, yet, policies and regulations differ [13]. Some countries have quite restrictive laws with a clear line between acceptable and unacceptable indications with specific mechanisms in place for delineating which indications are eligible for PGT. For instance, the Human Fertilization and Embryology Authority oversees the acceptable use of PGT in the UK. In France, l'Agence de la Biomédecine is charged with the regulation of PGT. In other countries, the law is more liberal. A minority of countries have no government regulation. The USA, for example, has no established restrictions on PGT practice and as such, PGT is also used for nonmedical reasons such as social sexing [14]. A recent overview of regulatory frameworks in 43 European countries shows only two countries where PGT is not allowed (Malta and Bosnia & Herzegovina) [15]. The main concern raised from the dawn of PGT has been the fear for eugenics. Many countries have a legislation banning any form of eugenic selection, allowing to select against high risk and serious disorders in PGT-M and PGT-SR but excluding the selection or enhancement of non-pathological characteristics in humans. PGT-A fails to meet the standard of a 'high risk and serious disorder' and is therefore not permitted in 11 out of 43 European countries [15]. As the field is rapidly progressing, it is essential to continue the scientific, ethical, and legal debate about embryo selection and to make amendments when necessary. For instance, the implementation of preconception carrier screening is likely to bring more requests for double or triple conditions, which may not all meet the 'high risk and serious disorder' standard. It is clear that this should be balanced with thorough discussions and ethical reflections [16]. PGT-M can in theory be offered for all (combinations of) monogenic disorders for which the disease-causing loci have been unequivocally identified. These loci are nuclear (X-linked, autosomal, dominantly or recessively inherited) or mitochondrial (maternally inherited) and involve (likely) pathogenic genetic variant(s) (class 4–5) [17].

The requests can be for rare or for more common diseases. The more frequent indications for which PGT-M are currently applied are cystic fibrosis and hereditary hemoglobinopathies for the autosomal recessive disorders, and myotonic dystrophy type 1, neurofibromatosis, Huntington's disease, and hereditary cancer syndromes for the autosomal dominant disorders. For the X-linked disorders, PGT is mainly carried out for Duchenne's muscular dystrophy, hemophilia, and fragile X syndrome (unpublished data from the ESHRE PGT consortium). The advantages of specific DNA diagnosis over sexing for recessive X-linked disorders are twofold: healthy male embryos are not discarded and female carrier embryos can be identified and possibly used for transfer, according to the patient's wishes and the center's policy. PGT with sex selection for non-medical reasons such as family balancing is prohibited in most countries.

Some special indications have raised further ethical concerns. For instance, Human Leucocyte Antigen (HLA) matching of preimplantation embryos is an exceptional indication as it is not a pathological condition. PGT is applied to select an embryo that is HLA compatible with an affected sibling who will need a bone marrow transplantation in the future. Hematopoietic stem cells from the cord blood at birth or later from bone marrow of the PGT baby are used to transplant and cure the affected sibling. HLA typing alone is carried out for couples having a child with an acquired hematological malignancy, but mostly, HLA typing is combined with PGT for a monogenic disorder, commonly immunodeficiencies and hemoglobinopathies [18]. The selection of HLA-matched embryos has evoked many ethical debates. The possible instrumentalization of the child to be born is the main issue raised in these discussions. As a result, the regulation of PGT and HLA matching varies in different countries around the world. Local and national legislation usually allow the use of PGT to avoid the transmission of diseases for which no treatment exists, but only a subset of frameworks is permissive for PGT and HLA typing.

In families with a history of late-onset neurodegenerative disorders such as Huntington's disease, individuals at risk who want to avoid pre-symptomatic testing but wish for their own biological unaffected children may opt for PGT with exclusion testing [19]. Exclusion testing recognizes the right of the parent not to know whether they are themselves affected while enabling them to have children not affected by the disease. Genetic implication counseling is a necessary part of the procedure [11]. Exclusion testing is indirect, based only on genetic markers, and involves the transfer of embryos carrying the haplotype derived from the non-affected grandparent. Embryos which have inherited the haplotype of the affected grandparent will be discarded as they have a 50% chance of being affected. These embryos have as well a 50% chance of being healthy for the disease under study. This fact together with the fact that about half of the couples will have an unnecessary IVF/PGT treatment with exposure to side effects and risks for the female and embryo, may be considered unethical. PGT with exclusion testing is therefore prohibited in some countries. The alternative with direct testing and non-disclosure of the results is not recommended as it obligates extreme confidentiality and may impose unethical behavior on the practitioners (for instance, fake embryo transfers) [20].

PGT for mitochondrial (mt) DNA pathogenic variants is offered in only few centers worldwide. The majority of mitochondrial DNA (mtDNA) pathogenic variants implicated in diseases show heteroplasmy, which is the co-existence of wildtype and pathogenic variant mtDNA in a single cell. The mtDNA pathogenic variant load (proportion of pathogenic variant mtDNA) may vary over time and differ from one cell type to another. Clinical symptoms manifest once a particular pathogenic variant load threshold has been exceeded. Because of a genetic bottleneck during oogenesis, the proportion of pathogenic variant mtDNA inherited from one generation to the next varies widely. PGT can be applied to select for embryos with a mtDNA pathogenic variant load below the threshold of clinical expression. It is an ethically difficult indication group as the approach reduces the risk for an affected child rather than eliminating it and this requires case-by-case counseling [21].

#### **3. Genetic and Reproductive Counseling—Preclinical Workup**

Before starting a clinical cycle, extensive genetic and reproductive counseling is provided to the prospective parent(s). Psychological support may be offered as well. Parents are asked to sign informed consent and blood samples are collected for preclinical workup. For PGT-M this usually includes blood samples and genetic reports from relevant first-degree family members. The preclinical reproductive workup and ovarian stimulation is similar as for patients undergoing conventional IVF. The preclinical genetic workup requires a conventional karyotype of both partners. This can be complemented by screening tests for carriership of common genetic variants for cystic fibrosis, spinomuscular atrophy, or hemoglobinopathies. In the near future, these individual screening tests will be most likely replaced by extended carrier screening. Genetic reports should be available at the intake of a PGT request. The preclinical genetic workup for the monogenic disorder depends on the test methodology (targeted versus genome-wide testing) and the strategy (an indirect test based on genetic markers versus a direct test including the detection of the pathogenic variant).

#### **4. IVF, Embryo Biopsy, Transfer, and Cryopreservation**

#### *4.1. IVF and Current Embryo Biopsy Methods*

Fertilization by ICSI rather than regular IVF is recommended for PGT treatment, in order to avoid contamination from remaining cumulus cells or residual sperm cells attached to the zona pellucida. Certain PGT indications may be associated with reduced spermatogenesis, and present with lowered fertilization rates. For instance, it has been known that the *PKD1* and *PKD2* genes underlying autosomal dominant polycystic kidney disease (ADPKD) play a role in the male reproductive system. Males affected with ADPKD may present with lower sperm motility and lower sperm concentration. A recent study showed that fertilization rates and live birth delivery rates tended to be lower for couples with the male partner affected with ADPKD, compared to couples with the female partner affected with ADPKD, although the higher female age in the former group was a confounding factor [22]. In some centers, oocyte or embryo vitrification before biopsy is applied as a systematic approach to accumulate a larger number of embryos for testing while in other cases it is applied as a rescue strategy in a minority of cycles, when in need for rapid interventions to preserve fertility such as in cases of cancer treatment [23–25].

Biopsy can be performed at different developmental stages. All present methods are invasive. Biopsy of the first and second polar body (both are required for an accurate diagnosis) is currently applied in only a minority of centers (unpublished data from the ESHRE PGT consortium of 2016 and 2017). An advantage is that the removal of polar bodies has no detrimental effect on embryonic development, however the most important limitation is that only the maternal genetic contribution can be evaluated.

Cleavage-stage embryo biopsy has been the gold-standard for many years. It implies zona opening (mechanically, chemically, or by using laser energy) and blastomere removal, mainly by aspiration, on day 3 of preimplantation development. Major disadvantages of day 3 biopsy are the limited amount of DNA available for testing and the negative impact of the removal of embryonic cells. It was shown that a two-cell removal at the cleavage stage harms embryonic development and implantation potential more than the removal of one cell [26]. Therefore, the removal of a single cell at the cleavage stage has been recommended. Cleavage-stage biopsy leaves sufficient time for genetic analysis before fresh embryo transfer on day 5. If available, supernumerary genetically transferable embryos can be cryopreserved for later use.

Blastocyst or trophectoderm (TE) biopsy is at present the most widely used technique [27]. Laser energy is used to open the zona pellucida, either on day 3/4 or on day 5. TE cells are aspirated and excised with a laser from herniating blastocysts, or aspirated in combination with mechanical dissection from blastocysts, usually on day 5/6. TE biopsy provides more cells (ideally five to eight cells) for genetic analysis providing a better accuracy [28]. This embryonic stage is also considered less sensitive to possible embryo damage as the inner cell mass from which the fetus originates is left intact. A paired clinical trial showed that implantation rates of 50% in the nonbiopsied group were diminished to 30% in the cleavage-stage biopsy group, while similar implantation rates were obtained for the blastocyst-biopsy group versus the nonbiopsied group [29]. Another benefit of TE biopsy is the lower level of chromosomal mosaicism at this stage as compared to the cleavage stage. The problem of limited time for analysis in the case of a fresh embryo transfer at day 5/6 is overcome by vitrification and embryo transfer in a deferred cycle.

#### *4.2. Current Developments and Future Sampling Methods*

An alternative method is morula-stage biopsy, performed on day 4, after artificial decompaction using Ca/Mg-free medium [30]. This option is attractive as more cells are obtained compared with cleavage-stage biopsy, cells are intact in contrast to TE biopsy where cells may be damaged and fresh embryo transfer is still possible. A drawback is the inability to distinguish between inner cell mass and TE cells. Whether or not the removal of several cells at day 4 has a negative impact on embryo development and implantation remains largely unknown. Irani and colleagues relied on morula biopsy at day 6 for slowly developing embryos in order to enlarge the cohort of available embryos for testing. Of note were findings of lower implantation and live birth rates as well as higher rates of complex aneuploidy [31].

Another option is blastocentesis, which is the aspiration of blastocoel fluid (BF) containing cell-free DNA (cfDNA) from the blastocoel cavity with a fine needle [32]. This procedure is considered less invasive than TE biopsy and results in embryo collapse which is a manipulation applied in routine vitrification practice to maximize embryo survival post-vitrification [33]. A true non-invasive alternative is to rely on cfDNA present in the spent blastocyst medium (SBM) [34]. Several studies showed that the karyotype concordance between blastocoel fluid samples and inner cell mass and/or TE cells varied widely, indicating that genetic analysis following blastocentesis is insufficiently accurate for clinical PGT-A/PGT-SR [35]. Moreover, the diagnostic efficiency was low with high amplification failure rates, making blastocentesis also unsuitable for PGT-M.

Analysis of cfDNA of spent embryo culture medium collected at the blastocyst stage seems a more promising sampling method. A recent study of Capalbo and colleagues compared blastocoel fluid and spent culture medium samples with TE cells as template for PGT-M [35]. TE samples showed 100% amplification and a high genotype concordance rate (99.8%). Blastocoel fluid samples gave high amplification failure (72.6%) with low genotype concordance (13.3%). SBM samples performed better with low amplification failure (10.3%) and an intermediate accordance rate of 59.5%. The lower diagnostic accuracy rate was due to contamination derived from maternal DNA due to incomplete oocyte denudation and from exogenous DNA present in supplements of culture media. As contamination is a major risk factor for genetic misdiagnosis, it is necessary to further optimize current protocols to favor embryo-specific analysis and allow discrimination between embryonic and non-embryonic DNA. This will provide more reliable and accurate testing for PGT-M in the short-term. Future research should also focus on the origin of the cell-free DNA to determine whether collected samples reflect the real genetic status of the embryo [36]. Substituting the laser-based biopsy methods by non-invasive sampling would definitely transform the field of PGT as it represents a safer option for the embryo and it would make the overall treatment less expensive.

#### *4.3. Embryo Transfer and Cryopreservation*

More and more PGT centers carry out a single embryo transfer (SET), a policy that can be linked with mechanisms of reimbursement and legislation, but also with the acknowledgement that SET is associated with a safer clinical outcome for any ensuing pregnancy. The substitution of slow-freezing by vitrification greatly contributed to the widespread use of SET. Vitrification has been shown to be superior to slow-freezing in terms of survival rate for both cleavage-stage embryos and blastocysts [37]. The evidence overall indicates that clinical outcomes after elective frozen embryo transfer are equivalent with the outcome after fresh embryo transfer for the general population (normo-ovulatory patients) and prove even better for specific subgroups such as patients at high risk of ovarian hyperstimulation syndrome (OHSS) [38].

The implementation of vitrification also changed the overall timeline of a PGT cycle. For many years, embryo biopsy on day 3, and testing and embryo transfer on day 5/6 were carried out within the timeframe of one cycle; the transfer was a 'fresh embryo transfer' and any surplus embryo from that cycle was cryopreserved. An alternative timeline emerged for cases for which no fresh embryo transfer was possible (high risk of OHSS or endometrium problems). During this freeze-all strategy, all genetically suitable embryos are vitrified and transfer was scheduled in a later cycle. Current comprehensive genetic testing is mostly linked with TE biopsy and a freeze-all strategy. This enables a more efficient and cost-effective laboratory organization as the larger time windows make it possible to pool and co-process samples of multiple patients. Once the genetic testing results have been disclosed, only genetically suitable embryos remain cryopreserved.

#### **5. Diagnostic Methods**

#### *5.1. Early Methods of PGT-M*

An overview of past, present, and future methods for PGT-M is summarized in Figure 1. At the start of PGT-M in the early 1990s, single cell simplex PCR amplification was applied. From these earliest approaches, contamination and allele drop out (ADO) surfaced as important issues that could lead to misdiagnosis [39]. Contamination with extraneous DNA or carry-over from previous amplification reactions was and still is a major problem, given the high number of amplification cycles that is required to increase the minute amount of DNA. It can be minimized by taking rigorous prevention measures. ADO originates from the unequal amplification of alleles present in a heterozygous sample (called preferential amplification) to the point where an allele remains undetected. Control for ADO relies on optimized methods for cell lysis and amplification and on sensitive methods for allele detection. By far, the most important measure to control and detect ADO as well as contamination was the inclusion of closely linked informative short tandem repeat (STR) markers in the PCR reaction. The co-amplification of STR markers with or without the pathogenic variant amplicon(s) at the level of a single or few cells yields a more accurate test and this so-called haplotyping approach has been the gold-standard for over two decades [27,40]. A major limitation of this approach is that the development and validation of a single cell multiplex PCR is labor intensive for the laboratory and couples face a long waiting time.

**Figure 1.** Overview of past, present, and future methods for PGT-M. The timeline is divided over past, present, and future sections without precise timepoints and starts in the early 1990s with blastomere biopsy and simplex PCR amplification for detection of the genetic variant in PGT-M. If the amplicon did not generate a difference in fragment length, post-PCR reactions were applied to allow low- and high-risk allele discrimination. These methods are currently still employed, for instance for detection of (de novo) genetic variants following WGA and SNP array. The addition of STR markers flanking the region of interest increased the accuracy of the PCR test: with every informative marker added, the diagnosis is confirmed and contamination and ADO pitfalls can be detected. The use of commercial PCR multiplex kits facilitated the development of single cell PCR reactions and duplex or triplex PCRs became multiplexes of 10 or more amplicons. Single cell multiplex PCR has been the gold standard for over two decades, alongside blastomere biopsy at day 3 and fresh embryo transfer on day 5/6. TE biopsy is currently the norm for embryo biopsy and is linked with the freeze-all strategy. The substitution of slow-freezing by vitrification greatly contributed to the widespread use of SET. WGA represents a technical milestone. The method of single or few cell WGA followed by regular PCR of multiple STRs with or without the genetic variant amplicon is a more universal method with a reduced

workup as the adaptation/validation of PCR reactions to the level of single or few cells can be omitted. The implementation of WGA followed by genome-wide SNP array or NGS represents a truly generic method, making it possible to combine haplotyping results for PGT-M with genome-wide chromosome copy number PGT-A data. Both platforms require a sample of a valid reference family member for haplotyping. Genotype-based NGS is regarded as the most powerful platform for future PGT as it will allow an all-in-one solution for direct genotyping and chromosome aberration assessment. Whether TE biopsy will be replaced by non-invasive sampling methods for PGT in the future requires further investigations. TA: targeted amplification: i.e., PCR-based amplification of the region of interest, either the genetic variant and/or a genetic marker(s); WGA: whole genome amplification: WGA products are used in downstream amplification reactions, either TA (regular PCR) or via genome-wide platforms (SNP array or NGS); STR: short tandem repeat marker; SNP: single nucleotide polymorphism marker; TE: trophectoderm; cfDNA: cell-free DNA; SET: single embryo transfer.

#### *5.2. Whole Genome Amplification Approaches*

The implementation of single or few cell whole genome amplification (WGA) was a technical improvement which stimulated the development of more generic approaches. The first WGA methods were PCR-based and suffered from incomplete genome coverage and amplification bias. The use of *Taq* DNA polymerase yielded an average fragment length of 400–500 bp (with a maximum size of 3 kb) and introduced many DNA sequence errors [41]. A multiple displacement amplification (MDA) method relying on isothermal strand displacement amplification was developed at the single cell level more than a decade ago [41]. In a MDA reaction, random exonuclease-resistant primers anneal to the denatured target DNA and a DNA polymerase with strand-displacement activity such as *Phi29* elongating the primers in an isothermal reaction at 30 ◦C. Additional priming events can occur on each displaced strand leading to a network of branched DNA strands over 10 kb. Because of the proofreading activity of the *Phi29* polymerase, the error rate of MDA-based WGA is much lower compared with *Taq* DNA polymerase-based methods, but the non-linear amplification yields over- or under-representation of genomic regions [42]. Afterwards, WGA methods combining MDA and PCR amplification were introduced. Both the Rubicon PicoPLEX and the MALBAC (Multiple Annealing and Looping Based Amplification Cycles) protocol initiate with DNA fragmentation and a pre-amplification MDA reaction using hybrid primers, followed by PCR [43,44].

None of the WGA methods are producing a true linear representation of the single or few cell genome and the results vary in ADO, preferential amplification rate, coverage, and nucleotide copy errors. As a consequence, a specific WGA method is chosen in function of the downstream application: the Rubicon PicoPLEX protocol is currently the method of choice for the detection of chromosomal copy number because of the reduced amplification bias while MDA is preferred for haplotyping applications in case of monogenic disorders because of the better genome coverage and low error rates [45–47]. Final data interpretation has to take into account bias and artefacts introduced from WGA. Also artefacts from the cell cycle phase should be considered. Analysis of TE samples containing cells in different cell cycle stages may overcome this problem while the WGA representation bias may be partially filtered out by computational algorithms but can not be completely eliminated.

WGA followed by standard PCR reactions of a multitude of STR markers flanking the region(s) of interest is simpler and requires a shorter validation, compared to single or few cell multiplex PCR [48]. WGA followed by genome-wide methods based on single nucleotide polymorphism (SNP) array or next-generation sequencing (NGS), represents a truly comprehensive approach.

#### *5.3. SNP Array for PGT-M*

SNP arrays are high density oligo arrays containing up to several million probes, which allow genotyping of hundreds of thousands of selected SNPs across all chromosomes in a single reaction. SNPs are mostly biallelic, alleles are indicated as A and B and genotypes are homozygous AA or

BB, or heterozygous AB. The commercially available SNP arrays use different methods for SNP genotyping: hybridization to SNP allele-specific probes or single base extension reactions are often applied [49]. The arrays are scanned and SNP genotypes are called based on the total fluorescence and the ratio of hybridization intensities for A and B (allele frequencies) (for example AB is called in case of similar intensities of an intermediate level). Targeted multiplex PCR and SNP array share the same principle of linkage-based testing for PGT-M, but the SNP array workflow is much more standardized and uniform, without the need for a locus-specific preclinical workup. This reduces the laboratory workload and the waiting time for the couples substantially. A drawback of SNP arrays is the high cost of equipment and consumables which seems to hamper their widespread clinical use. The SNP array platform is especially powerful for double indications (for instance two monogenic disorders or a monogenic disorder plus HLA matching) as whole genome haplotyping is accomplished from a single data set. Haplotyping via SNP array can also be applied for balanced translocations or inversions. Analysis reaches a high resolution and can distinguish normal from balanced translocation carriers. The major requirements for SNP array application are that the chromosomal or monogenic aberration(s) is/are inherited and relevant family samples are available for haplotyping.

Different SNP genotyping algorithms are available. Handyside and coworkers developed karyomapping, a family-based computational phasing approach for reconstruction of SNP haplotypes which flank the pathogenic variant(s) [5,50]. It is applicable for both SNP arrays and NGS. The commercially available karyomapping algorithm uses discrete diploid SNP calls (assuming AA, BB, AB, or No call as possible states for each SNP) together with basic Mendelian laws and requires a close relative for phasing.

#### *5.4. PGT-M for De Novo Pathogenic Variants*

In case of a de novo pathogenic variant(s) or when relevant DNA samples of family members cannot be obtained, it is necessary to include the genetic variant detection in the test strategy. This is feasible in targeted—as well as in genome-wide—methods, but the validation step of the latter methods is much easier. Many types of genetic variants can be detected by PCR supplemented with a post-PCR reaction if necessary. Deletion or duplication variants of a few nucleotides can be detected directly via fragment length difference. For single nucleotide substitutions, different strategies of allele discrimination have been developed, e.g., minisequencing [51]. The direct detection of complex and/or larger gene rearrangements has so far been difficult since the exact breakpoints of the rearrangement are frequently unknown. High-resolution characterization of breakpoints located outside highly repetitive regions is now achievable with long-read nanopore sequencing [52].

Since high-risk and low-risk haplotypes cannot be established during workup, segregation must be determined and diagnosis can be obtained during the first PGT cycle(s), provided that sufficient embryos are available with at least one embryo diagnosed as affected and with the pathogenic variant consistently detected in the presence of the same parental haplotype.

#### *5.5. SNP Array for Concurrent PGT-M and PGT-A*

SNP array can allow for simultaneous analysis of PGT-M and PGT-A as both SNP genotype and chromosome copy number info are obtained from the raw data set. As such, SNP arrays can reveal the presence of aneuploidies, polyploidies, and uniparental disomy [53]. Two measures provide evidence about the copy number state: the log R ratio (the log2 transformed value of the normalized intensity of the SNP) and the B allele frequency (BAF, which is the signal intensity of the B allele over the total signal intensity for a SNP). BAF values of 0, 0.5, and 1 represent a normal copy number (n = 2) but aberrations will cause a decrease or increase of the total intensity and allele frequencies. SNP arrays at the level of a single or few cells yield a lot of noise because of WGA pitfalls and therefore demand particularly well-developed algorithms for data interpretation. Genotyping algorithms using discrete diploid SNP calls such as karyomapping will yield errors across regions with copy number variations (true or WGA-induced) and therefore have restrictions in the detection of copy number aberrations. So far, few well-developed algorithms providing an all-in-one solution for data interpretation have been published. Some PGT centers rely on in-house developed extended or novel algorithms, to overcome this limitation. For instance, haplarithmisis is a computational pipeline that primarily relies on continuous BAFs and allows haplotype and copy number detection as well as determination of the parental origin of the chromosomal anomaly [6].

The true clinical benefit of PGT-A remains a topic of discussion, neither is there a consensus about adding PGT-A routinely to PGT-M cases, yet, concurrent PGT-A and PGT-M is increasingly regarded as an acceptable option, since it does not entail additional procedures or higher risks for the couple or future child.

#### *5.6. NGS for Concurrent PGT-M and PGT-A*

NGS involves DNA fragmentation and preparation of a library of templates using adapters containing barcodes for a more affordable analysis with multiple samples in a single run. The single molecule templates are then sequenced in parallel from one end or from both ends, either directly (third-generation) or after prior clonal amplification (second-generation) and the sequence reads are mapped to a reference genome. A crucial parameter is the genome coverage or read depth referring to the number of reads that is found at a given genomic position. A relatively low average coverage has been demonstrated as sufficient for accurate numerical chromosome analysis [54]. For monogenic disorders, sequencing at high coverage is required, which at this time is still too expensive for routine clinical applications at a whole-genome scale, and therefore, various strategies, aiming at affordable and rapid protocols are being developed. These protocols often provide a tandem solution, combining PGT-A with PGT-M, and can be classified in two groups, depending on whether sequencing data for the monogenic locus are derived with a targeted or a genome-wide method.

The three approaches of targeted sequencing presented here are merely an illustration of the wide range of possibilities. A more affordable solution is to increase the read depth across the site of the pathogenic variant (minimum 100×) only. In one report, the pathogenic variant loci were captured in a preamplification reaction with pathogenic variant specific primers while the concurrent inclusion of a specially designed primer pool allowed for parallel aneuploidy screening via real-time quantitative PCR [4]. The MARSALA method (mutated allele revealed by sequencing with aneuploidy and linkage analyses) works in a similar way: an aliquot of MALBAC-based WGA products undergoes targeted amplification and the mixture of WGA and targeted enriched templates is subsequently sequenced at low depth (0.1–2×), yielding targeted SNP haplotyping results for PGT-M together with genome-wide PGT-A data [55]. Chamayou and colleagues developed a universal strategy for concurrent PGT-M for cystic fibrosis and PGT-A [56]. Part of the MDA-based WGA products undergo multiplex PCR for coverage of the *CFTR* gene (pathogenic variants and SNPs), generating a targeted DNA library. Another aliquot of the WGA products is processed for comprehensive chromosome analysis, generating a genome-wide DNA library. Both DNA libraries are subsequently sequenced. In these three examples, the targeted amplification is coupled with the need for a locus-specific preclinical workup.

Genome-wide NGS is regarded as the most powerful platform for future PGT, as it will offer simultaneously genotype and chromosome copy number data with increased accuracy, reliability, and resolution, allowing a generic protocol for monogenic disorders (including the detection of de novo pathogenic variants and repeat expansions) and numerical and structural chromosomal aberrations (including balanced rearrangements). As the current cost is too high, the main approach has been to decrease the number of reads by reducing the complexity of the libraries. Some of these strategies are listed below (non-exhaustive).

The OnePGT solution, commercialized by Agilent, is a NGS-based generic application for PGT-M, PGT-SR, and PGT-A [8]. The method takes advantage of reduced-representation genome sequencing in offering a single workflow, starting from MDA-based WGA and followed by library preparation with double restriction enzyme digestion and enrichment for fragments in a specific range of length; sequencing data interpretation relies on the haplarithmisis algorithm with concurrent chromosome

copy number detection and SNP linkage-based haplotyping. The PGT-M module requires a minimum of 1.6× coverage and cosequencing of samples from the parents and a valid reference family member.

Haploseek is another universal workflow offering an economical analysis of PGT-M and PGT-A within a 24-hour protocol, starting from PicoPLEX-based WGA and genome-wide low coverage sequencing (0.3–1.4×) [7]. The information of high quality whole genome haplotypes of the couple and a reference obtained through SNP array is then integrated with the sample sequencing data and a hidden Markov model is used to predict whether haplotypes of the samples are shared with the reference or not.

In another NGS approach, a target enrichment gene panel with nearly 5000 Mendelian disease-associated genes (TruSight One sequencing panel) was applied on MDA-based WGA products, thereby offering direct testing of family pathogenic variant(s) plus indirect pathogenic variant detection through haplotyping of SNPs together with chromosome copy number detection through the log ratio of signal intensities, i.e., PGT-M and PGT-A together in a single workflow [57].

It is clear that the implementation of concurrent PGT-M and PGT-A offers further possibilities but may also generate incidental findings and more complex genetic information which we currently do not fully understand. This entails many ethical discussions and challenges for genetic counseling, because we lack knowledge to support data interpretation.

#### **6. Clinical Outcome**

It is difficult to compare clinical outcomes between individual centers because of varying factors such as maternal age, indication type, or differences in genetic testing and IVF procedures. Proper benchmarking against consortia data is neither straightforward, since reports of large data collections have been lagging. The most recent publication is about summary PGT data for 2015 from the ESHRE European IVF-Monitoring Consortium. This data set, which had been collected from 23 countries, showed a pregnancy rate of 39.7% per fresh embryo transfer cycle and a rate of 41.0% per frozen embryo transfer, over all PGT indications [58].

A high diagnostic efficiency is a prerequisite for any laboratory wishing to perform PGT as this potentially increases the clinical outcome. This is particularly true for indications with limited success rate a priori, such as for PGT-M with HLA typing where the chance of finding a genetically compatible embryo was shown to be only 16% [59]. This yielded a live birth delivery rate of 30.3% per transfer, showing that PGT with HLA typing is a valuable procedure where the high complexity and limited delivery rate are balanced by the successful transplantation outcome and the positive impact on families.

Apart from technical errors due to ADO and contamination, possible causes of misdiagnosis involve human errors such as mislabeling or incorrect embryo transfer. The misdiagnosis rate as published by the ESHRE PGT consortium is generally very low (<0.1%) [27]. The true misdiagnosis rate is difficult to assess as many embryo transfers yield no pregnancy or birth and only a minority of children have pre- or postnatal testing. PGT centers are encouraged to re-analyse part of the non-transferred embryos to provide an in-house estimate of the misdiagnosis rate.

#### **7. Children Follow-Up**

It was hypothesized that assisted reproductive technologies, especially the more invasive techniques like ICSI, would increase the risk for birth defects. Studies showed that IVF and ICSI are associated with a small but statistically significant increase in congenital anomalies at birth, compared with the general population [60,61]. No difference was observed between ICSI and IVF [62].

Follow-up data on children conceived after PGT with ICSI and embryo biopsy procedures are still limited. Neonatal follow-up studies did not report a higher rate of major congenital malformations after PGT when compared to IVF/ICSI [63–65]. The study from our center compared neonatal data of 995 live born children after PGT with the outcome data of 1507 live born children after ICSI and showed that embryo biopsy at cleavage stage did not add a significant risk to the overall medical condition of newborn children nor did it change the risk for major malformations [63]. These results were in line with studies carried out in other centers using embryo biopsy at cleavage stage [64,65]. The recent retrospective study in which a cohort of 1,721 children born after PGT with blastocyst biopsy and cryopreservation was compared with an IVF/ICSI control group showed no significant difference in neonatal outcome, indicating that neither embryo biopsy at blastocyst stage nor cryopreservation added further risk to the health of PGT children [66].

The follow-up of PGT children at a young age has been studied in smaller cohorts. Developmental neurological and cognitive assessment and follow-up on psychomotor and social functioning showed that PGT pre-schoolers were comparable with controls born after ICSI or after spontaneous conception [67–70]. Another study on body composition and blood pressure showed no adverse outcomes for 6-year-old children born after PGT (with day 3 embryo biopsy followed by blastocyst transfer) compared to children born after ICSI without embryo biopsy [71]. In summary, the follow-up results have so far been reassuring but further monitoring of the safety of PGT and the long-term health of the children remains necessary.

#### **8. Conclusions and Future Perspectives**

Major advancements have been introduced in the area of PGT and assisted reproduction over the years, making PGT a well-established, accurate, and safe clinical procedure. The implementation of genome-wide methods has allowed more standardization and uniformity for the genetic laboratories. However, additional genetic findings other than the requested genetic condition—such as chromosomal mosaicism in embryos tested for a monogenic disorder— have posed new dilemmas for genetic counseling and embryo transfer policy making. As the cost of sequencing continues to decline, PGT moves technically towards a sequencing-based, all-in-one solution for PGT-M, PGT-SR, and PGT-A. As there is more awareness among patients about the risks of transmitting genetic disorders and since the number of diseases with identifiable genetic cause(s) continues to rise, the total number of treatments as well as the list of indications for PGT is likely to expand. Whether the scope of PGT indications will broaden in the future from diagnosis of monogenic disorders to also predicting the risk of polygenic disorders (PGT-P) represents an emerging ethical challenge for PGT practice. It is clear that the rapid technological advances should be balanced with ethical reflection and thorough discussions.

**Author Contributions:** M.D.R. wrote the review. Both authors participated in critical reading and editing of the review. Both authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

#### *Review*

## **Use of Customizable Nucleases for Gene Editing and Other Novel Applications**

#### **Pradeep Reddy 1,\*, Felipe Vilella 2,3, Juan Carlos Izpisua Belmonte <sup>1</sup> and Carlos Simón 2,3,4,5**

Received: 25 July 2020; Accepted: 20 August 2020; Published: 22 August 2020

**Abstract:** The development of novel genome editing tools has unlocked new opportunities that were not previously possible in basic and biomedical research. During the last two decades, several new genome editing methods have been developed that can be customized to modify specific regions of the genome. However, in the past couple of years, many newer and more exciting genome editing techniques have been developed that are more efficient, precise, and easier to use. These genome editing tools have helped to improve our understanding of genetic disorders by modeling them in cells and animal models, in addition to correcting the disease-causing mutations. Among the genome editing tools, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system has proven to be the most popular one due to its versatility and has been successfully used in a wide variety of laboratory animal models and plants. In this review, we summarize the customizable nucleases currently used for genome editing and their uses beyond the modification of genome. We also discuss the potential future applications of gene editing tools for both basic research and clinical purposes.

**Keywords:** genome editing; genetic diseases; embryos

#### **1. Introduction**

The ability to precisely manipulate the genome revolutionized not only molecular biology, but also opened several areas of biotechnology that are useful for research, agriculture, and medicine. Initial gene targeting primarily depended on homologous recombination to insert exogenous DNA sequences in the human cells [1]. The establishment of mouse embryonic stem (ES) cells led to the application of homologous recombination in pluripotent cells to modify the genome and generation of genetically engineered mice [2–4]. Initially, the efficiency of gene targeting using homologous recombination was extremely low, which was solved to an extent by enriching the edited cells using the antibiotic selection cassette. Interestingly, Russell and Hirata observed that recombinant adeno-associated virus (rAAV) vectors with homology arms proved efficient in modifying the chromosomal target sequences [5]. The mechanism behind this observation is not entirely clear. However, the single-stranded DNA of the AAV genome and higher transduction rates might be the reason for higher homology recombination.

Double-strand breaks (DSBs) on DNA are repaired using three different mechanisms, namely non-homology end joining (NHEJ), microhomology-mediated end joining (MMEJ), and homologous recombination (HR). Due to the nature of the repairs, NHEJ and HR are referred to as error-prone

and error-free, respectively. NHEJ is active during all the cell cycle phases, whereas HR occurs only during the S-G2 phase of the cell cycle. For this reason, NHEJ is active in all the cell types, both dividing and non-dividing. During the repair of DSBs, the donor template with homology arms can get inserted at the site of repair using homology-directed repair (HDR) and dramatically increases the gene targeting efficiency [6]. Based on this method, rare cutting endonucleases such as the 18-bp cutter I-SceI have been used to introduce DSBs to increase the gene targeting efficiency [7]. Despite the presence of several natural meganucleases with unique recognition sites, the chances of finding a cutting site at the desired location are low. Nevertheless, modifications to meganucleases allowed some of the challenges to be overcome [8].

The discovery of customizable nucleases that can be programmed to induce DSBs at desired loci on the genome dramatically increased the efficiency of homologous recombination, leading to another revolution in gene editing with much broader implications in several different fields. This review aims to provide an overview of the recent developments in and applications of engineered nucleases that have helped lay the groundwork for their use not only for genome editing in various animal models, but also to correct the genetic mutations in human cells for clinical use. We also discuss the additional applications of genome editing tools in addition to modifying the genome, such as modulating the expression of genes and live-cell imaging. Finally, we review the use of genome editing tools in human embryos.

#### **2. Customizable Nuclease**

#### *2.1. Zinc-Finger Nucleases (ZFNs)*

ZFNs are a class of DNA-targeting components with two monomer subunits containing DNA-binding and cleavage domains. Each monomer is composed of three zinc fingers, which recognize nine base pairs and a *Fokl* endonuclease domain. The DNA-binding zinc fingers can be engineered to bind to specific regions on the genome. *Fokl* is enzymatically active as a dimer; therefore, two ZFN subunits are designed for each target sequence to facilitate the dimerization and cleavage [9]. The dimerization requirement of *Fokl* and binding of fingers to the target sequence have increased ZFNs attractiveness as a tool for introducing DSB at desired locations on the genome, which is mostly repaired by NHEJ pathway, resulting in the disruption of the amino acid sequence and function of the gene [10,11]. ZFNs have been widely used to create mutations in a wide variety of organisms, including, drosophila, zebrafish, mouse, rats, sea urchins and frogs [11]. ZFNs have also been employed for therapeutic purposes, such as disrupting the *CCR5* gene to interrupt the expression of co-receptor and prevent HIV infection [12]. Moreover, homologous recombination with donor template DNA at the target site has also been achieved in mice and rat embryos [13,14]. However, gene targeting did not succeed in all the animals, such as zebrafish, despite a high rate of DNA cleavage and mutagenesis, potentially due to differences in DNA repair mechanisms [11]. ZFNs are smaller in size and are presumed to possess low immunogenic properties due to their similarity to mammalian transcription factors. Nevertheless, the complexity and cost of designing the domains are disadvantages of ZFNs.

#### *2.2. Transcription Activator-Like E*ff*ector Nucleases (TALENs)*

Similar to ZFNs, TALENs have a customizable DNA-binding domain and *FokI* nuclease domain. The TALE-DNA-binding domain is composed of conserved repeats domains derived from transcription activator-like effectors (TALEs) secreted by *Xanthomonas* bacteria to alter the transcription in host cells [15]. Each TALE repeat binds to a specific single base of DNA, and the number of repeats corresponds to the length of the target site. Two TALE repeats with target site binding domains fused with the catalytic domain of *FokI* endonuclease are used to create a DSB. TALENs exhibit higher specificity and efficiency than ZFNs. For this reason, TALENs have been widely used for genetic manipulation in different organisms [16]. Additionally, two pairs of TALENs have been used on the same chromosome to generate large deletions [17]. Moreover, TALENs have also been employed to

introduce the donor sequences into the genome [18]. The single base pair recognition of TALE allows greater flexibility in designing the TALENs; however, the repeat arrays of TALE presents a technical challenge in cloning the identical sequences and also delivering in viral vectors. Additionally, the large size and immunogenicity of TALENs limit their clinical applications.

#### *2.3. CRISPR-Cas System*

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) nuclease is a recently identified system in prokaryotes with adaptive immunity against viruses and plasmids. Cas9 complexes with CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) to form an endonuclease that can recognize and cleave foreign genetic sequences. The DNA binding occurs using a 20-base pair DNA sequence in the crRNA that complements the targeting region next to protospacer adjacent motif (PAM) that triggers Cas9 to create DSB. During the repair of DSB, primarily by NHEJ, small indels are created at the target site, which results in deletions, insertions or frameshift mutations leading to loss of function of the gene. The sequence of crRNA can be replaced with any synthetic target sequence without modifying the other components. The crRNA and tracrRNA were fused to form a single chimeric RNA (sgRNA) and complexed with Cas9 to induce site-specific DNA cleavage [19]. The applicability of CRISPR-Cas9 for genomic modifications was demonstrated in human cells [20–23]. The replacement of only the targeting sequence in the RNA component and higher targeting efficiency made the CRISPR-Cas9 an attractive genome editing tool. Moreover, the simplicity in design and cloning facilitated the adoption of the system to various labs around the globe. Notably, the size of the Cas9 nuclease also advantageously allowed the delivery of the system in vivo using adeno-associated viruses (AAVs) [24–29]. Recently, additional Cas9 orthologues that recognize different PAM sequences have been discovered [30]. The CRISPR-Cas9 has been widely used for genetic modification in several different organisms, including plants [31,32]. Importantly, the clinical potential of CRISPR-Cas9 to correct the genetic mutations that result in the manifestation of the diseases is demonstrated in several different animal models [31]. However, the presence of antibodies against certain types of Cas enzymes and a distinct cellular and molecular changes in the host raise concerns [33–36]. Thus, large-scale screening for antibodies in different populations and further studies in larger animals are required to reach a conclusion.

In an adult mammal, the majority of the cells in the body are post-mitotic, with a few exceptions like cells in the liver. The classical HDR mechanism is generally inefficient in non-dividing cells, which limits the possibility of performing a knock-in in these cells. We developed an NHEJ-based homology independent strategy using CRISPR-Cas9, named homology-independent target integration (HITI), for the integration of transgene in non-dividing cells [29,37]. Unlike the HDR-based CRISPR-Cas9 approach, the transgene that is integrated using the HITI method lacks homology regions but harbors Cas9 cleavage sites similar to the targeting sequence on the genome. The cleavage by Cas9 creates blunt ends on the transgene and at the targeting site, while the NHEJ pathway allows the integration of transgenes during the repair of genomic DSB. As the NHEJ pathway is active during all cell cycle phases, HITI opened the doors for targeted gene knock-in in non-dividing cells, including neurons and muscle [29]. However, HITI cannot repair existing mutations; therefore, it is limited in its ability to correct point or frameshift mutations. Recently, we developed another versatile knock-in method called intracellular linearized single homology arm donor mediated intron targeting integration (SATI) for gene knock-in in intronic regions using donor vector containing a single homology arm with a Cas9 cleavage site [38]. SATI has a bipotential capacity to use HDR and NHEJ DSB repair machinery, facilitating in targeting a broad range of mutations in different cell types. SATI was successfully used for gene knock-in in vivo to correct a dominant point mutation that causes premature aging in mice [38]. Similarly, several other gene-editing methods utilizing CRISPR-Cas9 and MMEJ were developed during the last couple of years for both in vitro and in vivo application [37].

In addition to the use of CRISPR-Cas9 for gene deletion and insertion, Cas9 nickase and catalytically deficient Cas9 (dCas9) are fused with deaminases for the conversion of single nucleotides. The cytosine

base editor (CBE) is the first base editor developed that can convert C to T [39]. The commonly used third-generation CBE consists of a cytidine deaminase (APOBEC1), Cas nickase, and a uracil glycosylase inhibitor (UGI). In mammalian cells, an average of 37% permanent conversion at the target site is reported [39]. Nishida et al. also reported another cytosine base editing system using cytidine deaminase 1 (CDA1) [40]. Later, adenine base editor (ABE) was developed by replacing the cytidine deaminase with adenine deaminase (TadA) that can convert A to G [41]. The ABE comprises heterodimeric proteins (wild-type non-catalytic TadA monomer and evolved TadA\* monomer) and Cas9 nickase in a single polypeptide chain. Base editors are successfully used in plants, zebrafish, mice and human embryos [30]. Additionally, RNA base editors (RBE) are created by fusing nucleobase deaminase with Cas13 protein to convert A to inosine (I) or C to uracil (U) in the targeted RNA [42].

Interestingly, three independent groups have recently reported the development of dual adenine and cytosine base editors by combining both the deaminases with Cas9 nickase [43–45]. Using the dual deaminase base editor, both A to G and C to T substitutions were achieved at the target site. The dual deaminase base editors will offer new possibilities that were not possible with single-deaminase base editors such as targeting multi-nucleotide variants and CA/TG-rich transcription factor binding sites [43]. Additionally, Zhao et al. developed glycosylase base editors that can cause C to A transversions in bacteria and C to G transversions in mammalian cells [46]. The glycosylase base editors contain Cas9 nickase, a cytidine deaminase and uracil-DNA glycosylase. In mammalian cells, editing efficiency of 5–53% was observed.

Base editors do not generate DSBs and the chances of undesired changes such as indels are low. However, the base editors sometimes show off-target specificity [47]. Moreover, the presence of multiple C and A nucleotides close to the target base called "bystander base" can result in multiple base conversions, which can affect the targeting efficiency of base editors. Notably, Arbab et al. reported the creation of a machine learning model called BE-Hive using experimental data from more than 38,000 target sites in human and mouse cells [48]. This tool facilitates in predicting the base editing efficiency at the target site, especially in the presence of bystander nucleotides.

Anzalone et al. reported a prime editing approach similar to base editors, where Cas9 nickase was fused to an engineered reverse transcriptase, which is guided by a two-part guide RNAs termed "search" and "replace". The "search" guide localizes the Cas9 to target loci, where it cuts the DNA and the "replace" guide sequence is used by reverse transcriptase to make complementary DNA to integrate at the cut site [49]. Since most of the genetic diseases occur due to point mutations, the therapeutic potential of base and prime editors for the treatment of human disorders is currently being explored [50–52].

Interestingly, the CRISPR-Cas complex was also found to be encoded in a class of bacterial Tn7-like transposons [53]. Due to the site-specific integration of Tn7 transposon downstream of the conserved genomic sequence in *Escherichia coli*, researchers have hypothesized that transposon encoded CRISPR-Cas promoted this process [53]. In a recent study, Klompe et al. demonstrated how transposons have used the RNA-guided DNA targeting mechanism for site-specific integration without the need of DSBs and homologous recombination [54]. The unique features of this system might help to overcome the issues that arise from potential DSBs at off-target sites and the requirement of a long-homology arm, which limits the size of the target sequence. However, the system has not been tested in human cells and the efficiency of integration of template DNA at the target site remains unknown [55].

In addition to the targeting of DNA, Cas9 is also directed to target the single-stranded RNA (ssRNA) matching the guide RNA sequence when PAM is presented in trans as a separate DNA oligonucleotide (PAMmers). The PAMmers were able to stimulate the Cas9 endonuclease activity on ssRNA, similar to DNA cleavage [56]. This approach was used to eliminate the toxic microsatellite repeat expansion RNA in myotonic dystrophy type 1 patient cells [57]. Later, a naturally existing CRISPR-Cas13 system with RNA-targeting endonuclease activity was discovered in bacteria. Cas13 has four family members (Cas13a-d) and each demonstrates specific base preferences; for example, Cas13a requires a

protospacer flanking sequence consisting of a single A, U or C base pair [58]. Moreover, Cas13 subtypes differ in CRISPR RNA structure, direct repeat sequence and size. Although all exhibit promiscuous ribonuclease activity upon target recognition, Cas13d RNA cleavage is observed only in bacteria and not in the mammalian cells [59]. This unique characteristic of Cas13d allowed targeting RNAs in mammalian cells [59,60].

#### **3. Specificity and O**ff**-Target E**ff**ects of Customizable Nucleases**

The specificity of nucleases used for genome editing is one of the essential criteria for success in both basic and translational research. The customizable nucleases are engineered to target the region of interest on the genome using sequence information; however, the shorter-targeting sequence length used for the identification and few mismatches might allow the binding of nucleases at off-target sites. The concern of off-target applies to all the customizable nucleases in general. For therapeutic use of nucleases, they need to demonstrate higher specificity, activity, and gene modification ability. They should also be easier to deliver. Along this line, re-engineering strategies have been implemented for Zinc finger proteins and TAL effectors to improve the targeting precision [61–63]. Since Zinc finger nucleases and TALENs have been actively used for a long time, they demonstrate a long track record in safety, including their use in patients for therapeutic purposes. Conversely, CRISPR is relatively new; due to the simplicity in its design and ease of use, it is widely used, but we are still understanding the specificity and repair outcomes [63]. In the initial studies using CRISPR-Cas9, depending on the number of mismatches in the guide RNA, the rate of off-target effects were observed [20,64,65]. Later, a Cas9 nickase mutant paired with two guide RNAs was used to create DSBs, which led to reduction of off-target effects by 50 to 1000-fold [66]. Similarly, base and prime editing approaches can be a good alternative way to correct the disease-causing mutations without introducing DSBs and thereby avoid unintended changes. Nevertheless, the development of advanced bioinformatics tools and high-throughput sequencing methods will help in screening and identification of guide RNAs that are specific and do not have any off-targets [67]. In addition, the use of cell- or tissue-specific promoters to restrict the expression of CRISPR in combination with the local delivery, short-term expression, and use of selective AAV serotype can also help to minimize the negative effects.

#### **4. Applications of Customizable Nucleases**

#### *4.1. Modeling Genetic Diseases in Cells*

The traditional approach of genetic modification in the cells is less efficient, especially in performing targeted gene knockout or knock-in of fluorescent reporters. The customizable nucleases allowed the generation of a variety of genetic modifications in mammalian cells to model diseases, including cancer, metabolic and neurodegenerative diseases [68]. Stem cells are routinely used in the laboratories as they can be differentiated to any cell type using a defined medium, but genome editing by homologous recombination is less efficient and possesses limited use in the modeling diseases. The generation of pluripotent stem cells from patient somatic cells by reprogramming opened the possibilities of generating personalized induced pluripotent stem cells (iPSCs) [69]. iPSCs are routinely used to generate disease causing mutations to mimic the disease phenotypes in cell culture, which aid in understanding the cellular and molecular changes that occur during the disease progression [70]. Also, the engineered iPSCs can be differentiated to other cell types and used them for large-scale drug screenings and CRISPR knockdown screenings to identify new gene networks that play a role in disease progression or prevention. Moreover, pluripotent stem cells are widely used to generate different types of organoids [71,72]. Interestingly, gene-editing tools are also used in the in vitro organoid system to model human diseases to understand the etiology of the disease as well as for drug screening [73]. Notably, the genetically engineered cells have been used to generate several different types of organoids, including the brain, intestine, liver, kidney and lung [70].

#### *4.2. Correction of Disease-Causing Mutations*

The correction of genetic mutations to treat human diseases has proven an exciting potential application for the genome editing tools. In this line, ZFNs, TALENs, and the CRISPR-Cas system have been used to correct mutations in cells in vitro and transplanting them back to the patients. ZFNs have been successfully employed in mammalian cells to deliver the vectors expressing the ZFN-coding sequences by transfection of DNA or infection with viruses [11]. The higher specificity and mutagenesis rate of ZFN provides an advantage for therapeutic applications. ZFNs have been used to knock out the *CCR5* gene in T-cell precursor cells isolated from HIV patients to avoid the infection of HIV-1 [12]. Currently, clinical trials are ongoing that use this approach with modifications in the delivery of ZFNs to T-cells and improvements in the engraftment of infused cells [74]., Moreover, the genetic mutation that causes β-thalassemia and sickle cell disease in hematopoietic stem cells has been corrected using ZFNs [75,76]. Similarly, TALENs are also used in chimeric antigen receptor (CAR) T-cell therapy, where the T-cells are genetically modified to produce artificial receptors that recognize a specific protein on the tumor cells. Many products based on TALEN-edited cells have begun clinical trials, especially those focused on immuno-oncology [74].

Similar to ZFN and TALEN, T-cells have been genetically edited using CRISPR to knockdown immune checkpoint inhibitor programmed cell death-1 (PD-1), which is upregulated during activation of T-cells to reduce the autoimmune reaction and aid cancer cells in evading the immune system [77]. Moreover, CRISPR is also used to induce exon skipping of defective exons in Duchenne muscular dystrophy (DMD), to inactivate faulty genes that lead to the manifestation of disease including amyotrophic lateral sclerosis and Huntington disease, correct a genetic mutation that causes premature aging and eliminate the entire chromosome in aneuploid stem cells [32,78,79]. Importantly, gene-editing tools have already been used to correct the mutation in β*-globin* gene in patient hematopoietic stem cells [31].

ZFNs and CRISPR-Cas9 have been used to edit the HIV-1 genome and block its expression in T-cells, microglia and promonocytes [80–82]. CRISPR-Cas9 has been used to directly target and disrupt the reverse-transcribed products of lentivirus RNA generated during their life cycle [83]. Although the use of nucleases to edit the HIV-1 genome from human cells may not be considered a correction of disease-causing mutations, the HIV-1 genome in the cells produces new viral particles that lead to the development of acquired immune deficiency syndrome (AIDS).

The recent development of the NHEJ based genome-editing approach in non-dividing cells now allows for correcting mutations that cover a wide range of diseases, including Parkinson's disease and amyotrophic lateral sclerosis. HITI was employed to correct a mutation that causes inherited degenerative eye disease, retinitis pigmentosa, in rats [29]. Likewise, SATI was used to knock-in a normal copy of *Lmna* and prevent the expression of mutated copy, which led to the extension of the lifespan of progeria mice [38]. Furthermore, other NHEJ and CRISPR-Cas9 based approaches were used to knock-in a normal copy of the gene to rescue diseases such as tyrosinemia type I in mice [37]. Notably, several clinical trials are ongoing using CRISPR for the treatment of genetic diseases, including a blood disorder, eye disease and muscular dystrophy [31]. Likewise, CRISPR single-base editors are now used for the correction of single-base mutations or to disable the expression of the mutant gene [52]. Although CRISPR-based gene editing was identified recently, it has been tested in almost all cell types.

#### *4.3. Generation of Animal Models*

The traditional approach for creating gene-edited animals requires a time-consuming method that involves the use of modified ES cells. The generation of modified ES cells is a time and labor-intensive process. Moreover, the low success rate of obtaining the founder mice with a higher contribution of injected ES cells to the germline also hindered the process. Furthermore, the lack of ES cells for certain animals, including rats, also limited the generation of genetically modified animals of different species. However, the development of customizable nucleases has helped to overcome the need for ES cells by

performing the gene modifications directly in the zygotes. ZFNs were the first nucleases widely used for targeted mutagenesis and gene replacement, beginning with fruit fly and nematodes and progressing to other organisms such as silkworm and zebrafish, by injecting the ZFN pair at the early stages of zygote or embryo development. Notably, ZFNs opened the door to the possibility of creating genetically modified rats, which was not possible with other approaches [84]. In mice and rats, ZFNs have been able to induce both mono and biallelic gene disruption and homologous recombination with donor DNA at the target site. Furthermore, the germ cells carried the modifications. Similar to ZFNs, TALENs have been used to disrupt the expression of target genes in different animal models, including mice, rats, frog, zebrafish, pig and fruit fly [16]. In most of these studies, a single TALEN pair was used to create DSB to interrupt the function of the targeted gene. Moreover, to generate large deletions or chromosomal inversions, two pairs of TALENs were used to target the same chromosome [85,86]. The higher genome editing efficiency of TALEN is also used for generating animal models that mimic human diseases such as hypercholesterolemia.

Currently, the CRISPR-Cas system is the most favored customized nuclease and is widely used for performing in vivo genetic modification. Importantly, the CRISPR-Cas system allows the possibility of the simultaneous manipulation of several different genes, thereby dramatically reducing the amount of time required for the generation of double- or triple- knockout animals [87]. Moreover, in addition to mice, CRISPR has been successfully used in rats and other large animals, including dogs, pigs and monkeys [88]. In addition to performing gene knockout in vivo, the CRISPR-Cas system is also used to perform knock-in of reporters not only in dividing cells but also in non-dividing cells such as neurons [29,37]. Likewise, CRISPR-Cas9 along with NHEJ and MHMEJ-based methods have been used for transgene knock-in in mouse and monkey embryos [89]. Wang et al. reported the generation of non-human model of Hutchinson-Gilford progeria syndrome (HGPS) in monkey embryos using base editor to introduce C to T conversion in *LMNA* gene [90]. However, the microinjection of nucleases into zygotes requires special equipment and skill, which are not always available in all labs. To solve this problem, alternative methods are being developed, such as the injection of nucleases into the oviduct of pregnant females, followed by in vivo electroporation to deliver the components into zygotes [91].

#### *4.4. Targeting Mitochondrial Genome*

Among the different nucleases, so far, ZFNs and TALENs are the only ones so far that have been successfully used to target the mitochondrial genome. Unlike the nuclear genome, mitochondrial DNA (mtDNA) is mostly maternally inherited. The number of mitochondria and mtDNA can vary between different cell types and tissues. Moreover, multiple copies of mtDNA exist, ranging from a few hundred to thousands in each cell based on their energy demand. The mutations in the mtDNA lead to the degeneration of tissues and organs with high energy demand, including muscle and neurons, resulting in the manifestation of mitochondrial disease phenotypes [92]. Due to a lack of an efficient DNA repair mechanism in the mitochondria and the presence of multiple copies of both mutated and non-mutated mtDNA, the strategy of selective elimination of mutated mtDNA and re-population of normal mtDNA has been undertaken [93]. ZFNs and TALENs are localized into the mitochondria using a mitochondria localization signal and they selectively degrade the mtDNA with the disease-causing mutation in the cells obtained from mitochondrial disease patients [94–96]. Moreover, in a recent study, both ZFN and TALENs were delivered in vivo to reduce the mtDNA with a disease-causing mutation in muscle and heart and rescue the disease phenotypes in a mitochondrial disease mouse model [97,98]. Additionally, TALENs were also used in the mice embryos to target specific mitochondrial haplotypes and prevent their transmission to the next generation [99].

However, the CRISPR-Cas system has not been reported to be successfully used for targeting mtDNA. The major hurdle for the application of CRISPR-Cas system to mtDNA editing has been the successful import of gRNAs into the mitochondrial matrix, partially due to the inefficient RNA import mechanism present in the mitochondria of mammalian cells [100]. Although a few publications report the use of CRISPR-Cas9 to target mtDNA, the results presented in the studies lack proper

experimental evidence and have not yet been independently validated by other groups [101,102]. Interestingly, a new study reports a CRISPR-free mitochondrial base editing approach, which is accomplished using an interbacterial cytidine deaminase toxin fused to TALE domains [103]. The cytidine deaminase was split into two halves to avoid the toxicity and is inactive until brought together at the target site. Using this new approach, the CG to TA base conversions in mtDNA are possible without the need of DSBs and five different genes on mtDNA were edited without any off-target effects [103]. Though this approach is promising to introduce base changes in the mtDNA, a strong preference for 5 -TC-3 is reported, which dramatically limits the number of target regions. In fact, only one mitochondrial mutation 8356T>C found in humans can fulfill this requirement. Nevertheless, the modification of current cytidine deaminase may allow us to overcome this requirement. Currently, we lack animal models for mitochondrial disease due to the inability to edit the mtDNA. However, a CRISPR-free mitochondrial base editing approach can be used to generate animal models by introducing pathogenic mutations in the mtDNA.

#### **5. Applications Beyond Genome Editing**

#### *5.1. Epigenetic Modifications*

Although initial applications of nucleases mostly focused on gene editing, they were quickly redirected for other purposes, such as gene regulation. Zinc-fingers and TALEs were fused to transcriptional activators, among the different activators, VP64 and p65 proved the strongest when targeted upstream of the transcription start site and in promoter regions [104]. Similarly, the catalytic inactive dCas9 was fused with different transcriptional activators, repressors, modifiers or fluorophores to modulate the expression of the target gene by modifying the epigenetic status at the promoter regions [30]. To repress the gene expression, dCas9 was fused with the transcription repressor domain Krüppel-associated box (KRAB), which induces heterochromatin formation and changes in chromatin structure. dCas9-KRAB has been shown to silence genes, non-coding RNA and proximal and distal enhancer elements [30]. For gene activation, multiple transcription activation domains were fused to dCas9 [105] or recruited using the dimerized MS2 bacteriophage coat proteins that bind to the minimal hairpin aptamer on the gRNA tetraloop and stem-loop 2 [106]. Using this approach, multiple genes were simultaneously activated at the same time for cellular reprogramming of somatic cells to pluripotent state or direct differentiation of fibroblasts to neurons [30]. Furthermore, other epigenetic modifications, such as the acetylation and methylation of DNA have also been performed by fusing histone acetyltransferase p300 or catalytic domain of methylcytosine dioxygenase TET1 to dCas9 [104]. Recently, a study achieved dose-dependent activation of genes was achieved using CRISPR and chemical epigenetic modifiers that recruit endogenous chromatin machinery [107].

We have shown, for the first time in vivo, that Cas9 and transcription activation complexes can be recruited to the target loci using modified guide RNAs to activate the expression of endogenous genes [108]. Using this approach, we treated acute kidney disease, diabetes and muscular dystrophy in mouse models [108]. Later, a transgenic mouse model expressing a modified dCas9 system was used in vivo to activate multiple neurogenic endogenous genes to directly convert astrocytes into functional neurons [109]. Recently, Matharu et al. delivered dCas9-VP64 using AAVs to rescue the obesity phenotypes in a haploinsufficient mouse model [110]. Moreover, CRISPR-Cas12a was fused to a transcriptional activation domain to enable multiplexed knockout and transcriptional activation in vivo [111]. Similarly, the CRISPR system was also used to repress the expression of genes in vivo. In the last few years, several studies have successfully delivered dCas9 fused with different transcriptional repressors to reduce the expression of genes that play a role in reducing brain function [112], lower circulating low-density lipoprotein (LDL) concentration [113], and to correct retinitis pigmentosa phenotypes [114]. All these studies demonstrate the therapeutic potential of CRISPR based epigenetic modifications in vivo in ameliorating disease phenotypes [115].

#### *5.2. Live Cell Genome Imaging and Large-Scale Genetic Screenings*

Among the different repurposes, nucleases were also used for labeling specific regions of chromatin using fluorescent proteins. Before the identification of CRISPR-Cas system, ZFN and TALE proteins were used to recruit the fluorescent protein to the regions such as telomeres and centromeres for live imaging in cells [116,117]. Higher targeting efficiencies of Cas9 and dCas9 have replaced the other nucleases. Moreover, in another study, the improvement to the gRNA scaffold containing multiple MS2 binding modules facilitated the increase of the fluorescent signal with as few as four gRNAs. This improvement facilitated better labeling of low-repetitive and non-repetitive regions and tracking of the transcriptionally active and inactive regions [118]. Among the different nucleases, CRISPR-Cas system is the popular choice for performing high-throughput screens. Additionally, the CRISPR-Cas system is also employed in large-scale functional screening using hundreds of gRNAs in the cells and is efficient compared to the traditional short hairpin RNA (shRNA) system [119]. This approach allows large-scale genetic screening in an unbiased way to understand the role of a gene on a specific phenotype [106,120,121]. CRISPR-Cas system is also used to perform large-scale knockout screens to identify causative drivers of cancer in a native tissue environment [122,123]. Notably, this type of screen can be performed in a regular laboratory with access to cell culture, and no automated robotics are required. However, it involves a significant amount of sequencing, data analysis, and validation of data [124].

#### *5.3. Use of CRISPR to Detect Nucleic Acids for Diagnostic Purposes*

The use of CRISPR for molecular diagnosis has provided another exciting development during the last couple of years. Initially, the promiscuous ribonuclease activity of Cas13a upon target recognition of the surrounding RNA molecules was used to develop a molecular detection platform termed specific high-sensitivity enzymatic reporter unlocking (SHERLOCK). This platform was used for the detection of the Zika and Dengue viruses [125]. Moreover, a new version of SHERLOCK (SHERLOCKv2) was developed that can simultaneously detect multiple targets in the same reaction using different Cas enzymes and fluorescent reporters [126]. The SHERLOCK system was also shown to identify single-nucleotide polymorphisms (SNPs) on the viral genome [127]. Similarly, Cas12a possesses a non-specific cleavage of ssDNA molecules upon recognition of targeted sequence; using this feature, a DNA detection platform termed DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR) was developed [128]. Interestingly, both SHERLOCK and DETECTR approaches have been used for the detection of SARS-CoV-2 from nasal swabs during the recent COVID19 pandemic [129]. Notably, in the case of both the approaches, the results can be obtained in less than one hour and can be observed on a lateral flow strip. Furthermore, SHERLOCK and DETECTR methods are specific and sensitive in detecting the nucleic acids at low concentrations. These features make them ideal as care diagnostic tools that can be deployed and used with minimal setup during pandemics such as COVID19.

Interestingly, the RNA targeting property of CRISPR was also tested to target the SARS-CoV-2 genome. A CRISPR-Cas13-based strategy, PAC-MAN (prophylactic antiviral CRISPR in human cells), was shown to target and degrade RNA from SARS-CoV-2 sequences and H1N1 IAV load in respiratory epithelial cells [130]. In this study, the PAC-MAN was tested in an in vitro setting; however, the successful translation of this approach in vivo requires a non-viral delivery vehicle. Nanoparticles can be used to deliver mRNAs encoding CasRx/gRNAs directly into the lungs in an aerosol format using a nebulizer, are an ideal choice. Nevertheless, directly targeting the virus RNA genome is a promising strategy to prevent viral replication without relying on the body's immune system, which is an important factor for patients with low immunity or older individuals that cannot fight the infection and develop antibodies efficiently [131,132].

#### **6. Gene Editing in Human Embryos**

Genetic mutations in the germline of parents are passed down to the next generation. Some of the mutations can be lethal and affect embryo development that may lead to the early termination of pregnancy. In some less severe cases, the mutation can lead to the development of disease later during life. With the advancements in diagnosis and availability of cutting-edge medical interventions, it is now possible to treat or prevent the progression of many diseases. However, we still lack effective methods for treating the majority of the diseases caused by the genetic mutations inherited from the parents, so the correction of these mutations in the early stage of embryo development has been considered. Nevertheless, genome editing in somatic cells and germline differs greatly, not only in terms of DNA repair mechanism, but also in long-term consequences. Genome editing in somatic cells involves the modification of patient cells to cure a disease, which can be performed by isolating the cells and transplanting them back after correcting the mutation. However, for germline editing, the correction needs to be performed during the early stages of embryo development, and all the cells from the embryo may carry the modification, including the germ cells, which will also impact the future generations. Alternatively, it is possible that only some cells in the embryo undergo correction, which can lead to the generation of a mosaic embryo.

In the last few years, several research groups have performed gene editing in human embryos. Until now, more than seven different studies have been carried out using human embryos to test the cleavage efficiency and off-targets using CRISPR-Cas system [133–136]. Interestingly, during the correction of a pathogenic mutation in human embryos, DSBs have been repaired using endogenous HDR mechanism and wild-type allele as a template, which differs from the HDR efficiency observed in pluripotent stem cells [135]. In addition to the correction of pathogenic mutations, gene editing has also been used performed in human embryos to understand the role of pluripotent transcription factor OCT4 during early embryogenesis [137]. Moreover, base editing technology has also been used in the human embryos to correct pathogenic mutations [138,139]. Interestingly, better correction efficiency and higher homozygotic nucleotide conversion with no overlapped mutations have been observed at two-cell and four-cell human embryos compared to zygote [139].

Interestingly, the development of a long-term in vitro culture system for human embryos (until 14 days) [140,141], opened up possibilities of culturing the gene-edited embryo to better understand the early developmental problems. Moreover, pluripotent stem cells are now cultured in vitro on different cellular matrices to allow them to self-organize and generate structures called synthetic embryos that are similar to normal embryos and mimic the early developmental program of natural embryos [142]. Recently, using a single stem cell type, extended pluripotent stem cells of mouse origin, and a 3D-differentiation system has been used to generate blastocyst-like structures [143]. A similar approach is currently being refined to create synthetic embryos using human pluripotent stem cells. We can foresee that in the near future, the synthetic embryos might help to replace the use of natural human embryos for basic research purposes, especially for gene editing, to generate various disease models. Notably, the successful generation of a synthetic human embryo can, to some extent, avoid the ethical issues surrounding the use of human embryos for basic research purposes [144,145].

Until recently, the goal of genome editing in human embryos was intended to better understand the efficacy of gene correction and early developmental problems without implanting the edited embryos. However, to prevent HIV infection, one researcher in China to prevent HIV infection attempted to modify the *CCR5* gene in the human embryos that were later transferred to a human resulting in the birth of twin babies. This controversial experiment reignited an international debate on the necessity and ethical issues on genome editing in human embryos. Currently, in several different countries, a moratorium exists on genome editing in human embryos for clinical purposes. According to the guidelines developed by the National Academy of Science, clinical trials for heritable genome editing can be permitted when performed adhering to the regulatory framework and fulfilling a list of criteria that includes, among others things, the absence of reasonable alternatives, the prevention of the transmission of serious disease, the restriction of the conversion of genes to the versions already

prevalent in the population, plans for long-term and multigenerational follow-up, and oversight to prevent the use for other purposes [146,147]. Reports from the World Health Organization and International Commission on the Clinical Use of Human Germline Genome editing organized by US National Academy are due later this year. In general, pre-implantation genetic diagnosis (PGD) can be used to select un-mutated embryos free of mutation for implantation and avoid genome editing. However, the selection-based approaches can be a challenge for families who produce a lower number of embryos or when one of the partners carries a homozygous autosomal dominant mutation [148].

#### **7. Future Perspectives**

Customizable nucleases have opened new possibilities in the treatment of genetic mutations. Promising results from in vitro experiments and animal models demonstrate the potential application of CRISPR-Cas system in both basic research and clinical settings. However, before these nucleases can be used in clinics, several improvements must be achieved, including more precise targeting efficiency, lower off-targets, fewer unintended changes and a good delivery vehicle that can target a wide range of tissues when delivered in vivo. Certainly, significant progress on these requirements has been made. Notably, the repair efficiency has been improved by delivering Cas protein, and the availability of different types of Cas nucleases isolated from other prokaryotes and nickases are used to avoid DSBs and reduce the off-target activity [31]. Moreover, to prevent the unintended changes at the targeting site during the correction, the intronic region upstream of the mutation site is targeted to perform gene knock-in; this approach will offer greater flexibility in designing the gRNAs, and any additional changes will potentially not affect the gene function [38]. Similarly, the use of delivery vehicles other than viruses, including nanoparticles, are being tested to deliver the CRISPR-Cas system efficiently and safely. Another factor that might affect the clinical use of CRISPR-Cas system is the pre-existing antibodies against Cas proteins due to their bacterial origin, which could lead to inflammation and lower stability of Cas proteins [31]. However, more evidence is needed to determine the minimal levels of Cas protein that can activate the immune system. Together, these improvements will aid in using the CRISPR-Cas system to correct the mutations in vivo and cure the genetic disease. Notably, people with some genetic variants are more susceptible to the development of neurological disorders and some cancers. Moreover, with age, the number of mutations in somatic cells increases dramatically, and some of these mutations can lead to the development of cancer [149]. The availability of an efficient CRISPR-Cas system might facilitate the modification of specific regions of the genome in the future to prevent the development of diseases, for example, a variant of the gene that encodes for less functional protein or familial mutation that can lead to the development of the neurodegenerative disease. Moreover, the development of efficient genome editing tools, including the base editors with high specificity and no off-target effects, will open the possibilities of using them in human embryos to avoid the transmission of the disease-causing mutation. Similarly, the newly developed CRISPR-free mitochondrial base editing approach promises tremendous potential in the future to correct the pathogenic mutations in the mtDNA present in the unfertilized oocytes or embryos and prevent the transmission of mitochondrial diseases to the next generation.

In addition to the application of customizable nucleases for gene editing, their use in modulating the expression of the genes by changing the epigenetic marks on the promoters offers great potential. Interestingly, genetic diseases are not only caused due to the mutations in a gene, but also due to the lower expression of said genes that can affect the function of tissues and organs. Notably, during aging, dysregulation of epigenetic marks leads to the decreased expression of several different genes that are important for the normal function of cells and tissue, which eventually leads to the manifestation of disease phenotypes [150–152]. Since customizable nucleases were shown to modulate the expression of target gene without modifying the genomic sequences, they can be an attractive method in clinical settings to treat various diseases. Notably, we have shown that the use of CRISPR-Cas system may allow the activation of endogenous genes in vivo and reverse disease phenotypes [108]. In vivo gene activation using a CRISPR-Cas system can also overcome several limitations posed by traditional gene

therapy, including the size and number of transgenes that can be delivered. Age-associated disorders are not caused due to dysregulation of a single gene or pathway, and in such cases, multiple genes may need to be activated at the same time. For this reason, a multiplex system needs to be developed in which several gRNAs can be simultaneously delivered to every cell and activate multiple genes that will help to ameliorate the cellular hallmarks of aging and restore the function of the tissues. Furthermore, the use of tissue or cell-specific promoters will help to restrict the expression of Cas enzyme or gRNAs and prevent unintended gene activation in other tissues.

In conclusion, during the last couple of years, we have not only witnessed the discovery and development of new genome editing approaches, but also their implementation to treat various diseases. Currently, many clinical trials are underway that use the newly developed gene-editing tools, and in the next few years, some of them will be eventually used in clinics not just for the treatments of genetic diseases, but also to prevent or treat viral infections such as SARS-CoV-2.

**Author Contributions:** Writing-review and editing, P.R., F.V., J.C.I.B., and C.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Moxie Foundation and Department of Defense (grant number W81XWH-17-1-0552) to J.C.I.B. Miguel Servet Program Type II of ISCIII [CPII18/00020], and a FIS project [PI18/00957] to F.V.

**Conflicts of Interest:** All the authors declare no conflict of interest.

#### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Characteristics of the IVF Cycle that Contribute to the Incidence of Mosaicism**

#### **Lorena Rodrigo 1,\*, Mónica Clemente-Císcar 2, Inmaculada Campos-Galindo 1, Vanessa Peinado 1, Carlos Simón 3,4,5,6 and Carmen Rubio <sup>7</sup>**

Received: 18 August 2020; Accepted: 26 September 2020; Published: 30 September 2020

**Abstract:** Highly sensitive next-generation sequencing (NGS) platforms applied to preimplantation genetic testing for aneuploidy (PGT-A) allow the classification of mosaicism in trophectoderm biopsies. However, the incidence of mosaicism reported by these tests can be affected by a wide number of analytical, biological, and clinical factors. With the use of a proprietary algorithm for automated diagnosis of aneuploidy and mosaicism, we retrospectively analyzed a large series of 115,368 trophectoderm biopsies from 27,436 PGT-A cycles to determine whether certain biological factors and *in vitro* fertilization (IVF) practices influence the incidence of overall aneuploidy, whole uniform aneuploidy, mosaicism, and TE biopsies with only segmental aneuploidy. Older female and male patients showed higher rates of high-mosaic degree and whole uniform aneuploidies and severe oligozoospermic patients had higher rates of mosaicism and only segmental aneuploidies. Logistic regression analysis identified a positive effect of female age but a negative effect of embryo vitrification on the incidence of overall aneuploid embryos. Female age increased whole uniform aneuploidy rates but decreased only segmental aneuploidy and mosaicism, mainly low-mosaics. Conversely, higher ovarian response decreased whole uniform aneuploidy rates but increased only segmental aneuploidies. Finally, embryo vitrification decreased whole uniform aneuploidy rates but increased mosaicism, mainly low-mosaics, compared to PGT-A cycles with fresh oocytes. These results could be useful for clinician's management of the IVF cycles.

**Keywords:** PGT-A; NGS; aneuploidy; mosaicism; segmental; vitrification; ovarian response; female age

#### **1. Introduction**

Current genetic analysis platforms used in preimplantation genetic testing for aneuploidy (PGT-A) are highly sensitive and, when applied to trophectoderm embryo biopsies (TE), identify mosaicism when not only euploid or aneuploid cells, but a combination of both are present in the TE biopsy. Embryonic mosaicism is mitotic in origin and is caused by improper separation and segregation of chromosomes during cell division [1]. Next-generation sequencing (NGS)-based platforms are

most commonly used in PGT-A programs; however, differences in platform sensitivity and specificity, the threshold established for data interpretation and the cut-offs applied for low-level mosaicism classification affect the percentage of mosaicism reported among genetic laboratories and the number of euploid embryos deemed suitable for transfer [2]. Other factors related to biopsy technique, the number of cells biopsied, and the conditions surrounding the cell loading can also affect the results [3]. Studies by Popovic et al. [4,5] on the inner cell mass (ICM) and TE analysis from aneuploid and mosaic embryos suggest limitations in the accuracy of diagnosing mosaicism in PGT-A due to difficulty distinguishing technical bias from biological mosaicism. As a result, the use of validated NGS platforms and the development of algorithms to improve mosaicism diagnosis have become priorities for many genetic laboratories [6–8].

Ovarian stimulation protocols during the IVF cycle can influence the incidence of euploid embryos [9]. Additionally, the fertilization method [10] and conditions in the clinical IVF laboratory, such as the embryo culture media, pH, oxygen, osmolality, temperature or plastics, are linked to increased aneuploidy and mosaicism [3]. Accurate mosaicism determination describes incidences of euploid/aneuploid mosaicism from 31% at the cleavage stage to 4–5% at the blastocyst stage [11,12]. Maternal age is a major influence on aneuploidy, but primarily related to incidence of uniform aneuploidies. Isolated mosaicism may be independent of maternal age, but Rubio et al. [13] described a slight decrease in mosaicism in women over 37 compared to younger patients. The same study, however, did not observe an effect of the ovarian response and vitrification of oocytes on mosaicism rate. Finally, the use of PGT-A in couples with compromised semen quality shows higher rates of mosaic embryos with low sperm concentrations [14,15].

Since the first pregnancies reported by Greco in 2015 [16] transferring mosaic embryos after PGT-A analysis, more clinicians are considering transfer in the absence of euploid embryos. Clinical outcomes are poorest for high-mosaic compared to low-mosaic embryo transfers [17]; therefore, mosaic degree classification is critical to the decision-making process for clinicians and patients. We evaluated the distribution of euploid and aneuploid cells in a large series of 115,368 TE biopsies to assess whether the distribution was affected by characteristics of the IVF cycle, including the indication to perform a PGT-A cycle, the use of fresh or vitrified oocyte/embryos, the ovarian response, sperm concentration, and maternal and paternal ages. Aneuploidies were analyzed considering uniform aneuploidies, low- and high-degree mosaicism, and segmental aneuploidy. Logistic regression analysis identified maternal age, ovarian response, and PGT-A cycles with vitrified embryos as the factors with the most influence on aneuploidy.

#### **2. Materials and Methods**

#### *2.1. Study Design*

This was a retrospective observational study carried out between October 2018 and December 2019 that included TE embryo biopsies from PGT-A cycles analyzed in our laboratory subsidiaries (Igenomix, Valencia, Spain). Every TE biopsy was classified as euploid or aneuploid. The aneuploidies observed in the TE biopsies were distributed among four categories: (i) only segmental aneuploidy, when only partial deletion/duplications were observed; (ii) low-mosaic, when one or two low-mosaic degree aneuploidies without additional uniform or segmental aneuploidies were observed; (iii) high-mosaic, when one or two high-mosaic degree aneuploidies or one low- and one high-mosaic degree aneuploidies without additional uniform or segmental aneuploidies were observed; or (iv) whole uniform aneuploidy, when at least one aneuploidy for a whole chromosome was observed in the TE biopsy, combined or not combined with additional segmental or mosaic aneuploidies.

The distribution of euploid and aneuploid results within the four categories of aneuploidies (whole uniform aneuploidy, low-mosaic, high-mosaic, and only segmental) were evaluated according to the following characteristics of the IVF cycle: the indication to perform the PGT-A cycle, the origin of the embryos, ovarian response, sperm count, and maternal and paternal age. Patients with altered karyotype, cycles with females aged ≥ 45 years at embryo biopsy, as well as ovum donation cycles where excluded from analysis.

#### *2.2. Study Population*

The study included 115,368 day 5, 6, or 7 TE biopsies from 27,436 PGT-A cycles. Clinical indications for PGT-A were: (1) advanced maternal age, ≥37 years old (AMA; *n* = 48,174 TE biopsies); (2) repetitive implantation failure with ≥2 failures (RIF; *n* = 6742 TE biopsies); (3) recurrent pregnancy loss with ≥2 miscarriages (RPL; *n* = 5244 TE biopsies); (4) male factor infertility with impaired sperm parameters and/or increase in the incidence of aneuploid sperm (MF; *n* = 6562 TE biopsies); (5) previous aneuploid conception (PAC; *n* = 512 TE); (6) mixed causes (MIX; *n* = 2199 TE biopsies); or (7) aneuploidy screening (AS; *n* = 45,935 TE biopsies). Female age in RIF, RPL, MF, PAC, MIX, and AS indications was <37 years old.

Information on embryo origin was available in 34,346 (29.8%) of the embryos analyzed, distributed as fresh oocyte origin (FO; *n* = 30,312 TE biopsies), vitrified oocyte origin (VO; *n* = 1649 TE biopsies), and vitrified embryo origin (VE; *n* = 2385 TE biopsies). A total of 14,092 (12.2%) TE biopsies had information regarding the ovarian response, considered as the total number of MII oocytes retrieved in the IVF cycle. These were distributed as: (i) ≤5 MII oocytes (*n* = 1677); (ii) 6–10 MII oocytes (*n* = 3302); (iii) 11–15 MII oocytes (*n* = 2646); (iv) 16–20 MII oocytes (*n* = 2373); and (v) >20 MII oocytes (*n* = 4094). The sperm count of the samples used in IVF cycles was available for 8360 (7.3%) of the TE biopsies analyzed, classified as: (i) normozoospermia with <sup>≥</sup>15 <sup>×</sup> 106 sperm/mL (N; *<sup>n</sup>* <sup>=</sup> 6428); (ii) moderate oligozoospermia between >5 <sup>×</sup> 106 and <15 <sup>×</sup> 10<sup>6</sup> sperm/mL (MO; *<sup>n</sup>* <sup>=</sup> 911); and (iii) severe oligozoospermia with <sup>≤</sup><sup>5</sup> <sup>×</sup> <sup>10</sup><sup>6</sup> sperm/mL (SO; *<sup>n</sup>* <sup>=</sup> 1021).

#### *2.3. Next-Generation Sequencing of TE Biopsies*

TE biopsies were performed on day 5, 6, or 7 of blastocyst growth. NGS analysis was conducted using an Ion ReproSeqTMPGS kit for 24 chromosome aneuploidy screening (Thermo Fisher Scientific, Waltham, MA, USA) and was performed on the Ion ChefTM and Ion S5 System instruments (Thermo Fisher Scientific). Data were analyzed with Ion Reporter Software using the human genome build (hg19) (Thermo Fisher Scientific). An internally validated algorithm was applied for automatic mosaicism calling. Low mosaic degree was determined when the TE biopsy had 30% to <50% of aneuploidy cells while a high mosaic degree was determined by 50% to <70% aneuploid cells. TE biopsies with <30% aneuploid cells were classified as euploid and those with ≥70% were classified as uniform aneuploid [13]. TE biopsies showing another uniform aneuploidy were not reported as mosaic but as uniform aneuploid. Partial deletions/duplications were determined by >10 Mb.

#### *2.4. Statistical Analyses*

Univariant analysis using pairwise comparisons between pairs of proportions with correction for multiple testing (based on Pearson's Chi-squared test) was applied to evaluate the distribution of embryo ploidy (euploid and aneuploid) and the category of aneuploidy (whole uniform aneuploidy, only segmental aneuploidy, low-mosaic degree, or high-mosaic degree) according to the indication to perform PGT-A (AMA, RIF, RPL, MF, PAC, MIX, or AS), the ovarian response (≤5 MII oocytes; 6–10 MII oocytes; 11–15 MII oocytes; 16–20 MII oocytes; >20 MII oocytes), the classification of sperm count (N, MO, or SO) and the embryo origin (FO, VO, or VE). To evaluate the distribution of the four categories of aneuploidies according to maternal and paternal age, a pairwise comparison between means (± standard deviation, SD) with corrections for multiple testing (based on *t*-test) was used.

Multivariant analysis was performed in 4885 TE biopsies for which all variables were present (4.4% of the informative embryos). A binary logistic regression model was applied to evaluate the variables affecting embryo ploidy and the four categories of aneuploidies. Maternal and paternal age, sperm concentration, and ovarian response defined as the number of mature oocytes retrieved (MII) were considered as quantitative variables and the embryo origin and the indication for the PGT-A were

considered qualitative variables in the model, considering FO for the embryo origin and AS for the indication for the PGT-A as references. The odds ratio for each coefficient and the confidence interval at 95% were computed in the case of a significant variable to analyze the effect of each parameter.

Statistical analysis was performed using R Free software (R Foundation for Statistical Computing, Vienna, Austria) and GraphPad InStat v3.10 for Windows (GraphPad Software, Inc., San Diego, CA, USA). Differences were considered statistically significant when *p* < 0.05, highly significant when *p* < 0.01, and extremely significant when *p* < 0.001.

#### **3. Results**

We retrospectively analyzed 115,368 TE biopsies and 111,860 were informative. From the informative TE biopsies, 46.1% were euploid and 53.9% were aneuploid, mainly due to whole uniform aneuploidies (42.6%) followed by mosaicism (6.2%) and TE biopsies with only segmental aneuploidies (5.1%) (Table 1). When analyzing the data per day of biopsy from day-5 to day-7, the percentage of euploid TE biopsies decreased significantly (49.4% in day 5; 42.1% in day 6; 35.7% in day 7; *p* < 0.0001) and the percentage of aneuploid embryos increased significantly (50.6% in day 5; 57.9% in day 6; 64.3% in day 7; *p* < 0.0001). The incidence of TE biopsies with whole uniform aneuploidy increased significantly from day-5 to day-7 (39.5%, 46.5% and 52.8 in day-5, day-6 and day-7, respectively; *p* < 0.0001). No significant differences in the percentage of TE biopsies with mosaicism or only segmental aneuploidies were observed in different biopsy day.

Note: a-b, a-c, d-e, d-f, g-h, g-i, j-k, j-l, k-l *p* < 0.0001; b-c, e-f, h-i *p* < 0.001; n-o *p* < 0.01; m-o *p* < 0.05; Fisher's Exact test and Welch's *t* test.

#### *3.1. Univariant Analysis*

#### 3.1.1. Maternal and Paternal Age

Euploid TE biopsies showed significantly lower mean female and male age compared to aneuploid TE biopsies (34.8 ± 4.2 vs. 37.0 ± 4.4 for female, and 37.6 ± 6.0 vs. 39.4 ± 6.2 for male, respectively; *p* < 0.0001). When considering the categories of aneuploidies, mean female age was similar in the groups of euploid (34.8 ± 4.2) and low-mosaic degree (34.9 ± 4.2) TE biopsies and both were slightly but significantly higher compared to only segmental aneuploidy TE biopsies (34.6 ± 2.2; *p* < 0.001) and significantly lower compared to the high-mosaic degree (36.5 ± 4.3) and whole uniform aneuploid TE biopsies (37.5 ± 4.2) (*p* < 0.0001) (Figure 1a). Mean paternal age was similar in the euploid (37.6 ± 6.0), low-mosaic degree (37.8 ± 6.0), and only segmental aneuploidy (37.5 ± 6.0) TE biopsies and these were significantly lower compared to the groups of high-mosaic degree (39.0 ± 6.1) and whole uniform aneuploid (39.8 ± 6.1) TE biopsies (*p* < 0.0001) (Figure 1b). In summary, whole uniform aneuploidy had the highest mean maternal and paternal age, significantly increased compared to the other categories of aneuploid TE biopsies (*p* < 0.0001).

**Figure 1.** Box plot distribution of mean maternal age (**a**) and mean paternal age (**b**) expressed in years in the results categories of euploid, low-mosaic, high-mosaic, only segmental, and whole uniform aneuploid TE biopsies.

#### 3.1.2. Ovarian Response

Figure 2 shows the distribution of aneuploidies in TE biopsies according to the number of mature oocytes (MII) retrieved. The percentage of aneuploid TE biopsies decreased progressively as the number of MII oocytes increased. The percentage of whole uniform aneuploidies was significantly higher in patients with less than 11 MII oocytes compared to patients with more than 11 MII oocytes (*p* < 0.01). No significant differences in the percentage of TE biopsies with mosaicism or only segmental aneuploidies were observed within the ovarian response.

**Figure 2.** Distribution of chromosomal abnormalities in TE biopsies (euploid, low-mosaic, high-mosaic, only segmental, and whole uniform aneuploidy) according to categories of ovarian response considering the number of mature oocytes retrieved (≤5 MII oocytes; 6–10 MII oocytes; 11–15 MII oocytes; 16–20 MII oocytes; >20 MII oocytes). The mean number of embryos biopsied (SD) per case was 2.6 (1.9) for ≤5 MII oocytes category, 3.4 (2.1) for 6–10 MII, 4.5 (2.9) for 11–15 MII, 4.8 (2.7) for 16–20 MII and 6.5 (3.9) for >20 MII oocytes category.

Mean maternal age was significantly higher in the group with ≤5 MII (38.9 ± 2.7) compared to the other groups (38.1 ± 3.3 in 6–10 MII, 37.6 ± 3.5 in 11–15 MII, 38.0 ± 3.4 in 16–20 MII, 37.4 ± 3.8 in >20 MII; *p* < 0.0001). The group with 6–10 MII compared to the group with 16–20 MI, and the group with 11–15 MII compared to the group with >20 MII showed similar mean maternal ages.

#### 3.1.3. Classification of Sperm Count

Analysis of TE biopsies considering patient sperm count showed significantly higher incidence of aneuploid embryos in patients with N (63.8%) compared to patients with MO (57.6%, *p* < 0.001) and SO (54.2%, *p* < 0.0001) (Figure 3). Mean maternal age was also significantly higher in the N group (38.8 ± 3.1) compared to MO (37.4 ± 3.5) and SO (35.9 ± 3.8) (*p* < 0.001, among groups). The N group showed significantly higher incidence of embryos with whole uniform aneuploidy (53.8%) compared to MO (47.5%) and SO (41.5%) groups, *p* < 0.001. Although the percentage of embryos with mosaicism (6.1% in N; 6.4% in MO; 7.9% in SO) and only segmental aneuploidy (3.9% in N; 3.7% in MO; 4.9% in SO) was slightly increased in patients with SO, no statistical differences were observed among groups. However, the incidence of embryos with aneuploidies due to mosaicism or only segmental instead of uniform aneuploidy was significantly higher in the SO compared to the N group (12.7% vs. 10.0%, respectively; *p* < 0.05).

**Figure 3.** Distribution of the percentages of chromosomal abnormalities in TE biopsies (euploid, low-mosaic, high-mosaic, only segmental, and whole uniform aneuploidy) according to the classification of sperm count (normozoospermia, with <sup>≥</sup>15 <sup>×</sup> 10<sup>6</sup> sperm/mL; moderate oligozoospermia, between >5 <sup>×</sup> 10<sup>6</sup> and <15 <sup>×</sup> 106 sperm/mL; severe oligozoospermia, with <sup>≤</sup>5 <sup>×</sup> 10<sup>6</sup> sperm/mL). The mean number of embryos biopsied (SD) per case was 4.6 (3.2) for N category, 5.2 (4.1) for MO and 5.1 (3.7) for SO category.

#### 3.1.4. Embryo Origin

Figure 4 shows the distribution of aneuploidies in TE biopsies according to embryo origin. The percentage of euploid embryos was significantly higher in the VE group (46.5%) compared to FO (41.3%) and VO (39.2%) groups, *p* < 0.0001. Comparing the four categories of aneuploidies, the VE group showed significantly lower percentage of embryos with whole uniform aneuploidy (40.6%) compared to FO (47.5%) and VO (48.7%) groups, *p* < 0.0001. The percentage of embryos with only segmental aneuploidy was significantly higher in the VE groups (6.5%) compared to the FO group (5.2%), *p* < 0.05. No differences were observed in the incidence of mosaic embryos among the three

groups (6.0%, 6.9% and 6.3% in FO, VO, and VE groups respectively, NS). The VO group had the highest mean maternal age (38.7 ± 3.7), followed by the group of FO (36.6 ± 4.5), and the VE group showed the lowest maternal age (35.7 ± 4.6) (*p* < 0.0001 among groups).

**Figure 4.** Distribution of the percentages of chromosomal abnormalities in TE biopsies (euploid, low-mosaic, high-mosaic, only segmental, and whole uniform aneuploidy) according to the embryo origin (fresh oocyte, vitrified oocyte, and vitrified embryo). The mean number of embryos biopsied (SD) per case was 4.8 (3.3) for fresh oocyte category, 4.7 (3.4) for vitrified oocyte and 4.5 (3.5) for vitrified embryo category.

#### 3.1.5. Indication for PGT-A

PGT-A cycles with indication of AMA showed significantly lower percentage of euploid embryos (32.1%) compared to cycles with indication of RIF (54.2%), RPL (53.9%), MF (56.7%), PAC (54.9%), MIX (55.5%), and AS (56.8%), *p* < 0.0001 (Figure 5). Considering the four categories of aneuploidies evaluated, AMA showed significantly higher percentage of embryos with whole uniform aneuploidy (59.1%) compared to RIF (32.9%), RPL (32.5%), MF (29.6%), PAC (31.4%), MIX (31.5%), and AS (30.4%), *p* < 0.0001. Moreover, the percentage of whole uniform aneuploidy in MF and AS cycles was significantly lower than that observed in RIF and RPL cycles.

The percentage of embryos with only segmental aneuploidy was significantly lower in AMA cycles (3.5%) compared to RIF (6.4%), RPL (6.4%), MF (7.0%), PAC (8.1%), MIX (6.3%), and AS (6.1%), *p* < 0.0001, and significantly higher in MF compared to AS (*p* < 0.05). A similar trend was observed for the overall incidence of mosaicism, which was significantly lower in AMA (5.4%) compared to RIF (6.4%, *p* < 0.01), RPL (7.3%, *p* < 0.0001), MF (6.7%, *p* < 0.001), MIX (6.7%, <0.05%) and AS (6.7%, *p* < 0.0001). The percentage of embryos with low-mosaic degree was significantly lower in AMA (2.9%) compared to RIF (4.4%), RPL (5.1%), MF (4.7%), MIX (4.4%), and AS (5.7%) (*p* < 0.0001). However, TE biopsies with high-mosaic degree were significantly increased in AMA compared to AS (2.4% vs. 2.0%, respectively; *p* < 0.001). Mean maternal age was 40.1 ± 1.7 in AMA, 33.0 ± 3.1 in RIF, 32.8 ± 3.4 in RPL, 32.6 ± 3.6 in MF, 33.5 ± 2.9 in PAC, 33.1 ± 3.2 in MIX, and 33.1 ± 3.3 in AS.