Preimplantation genetic testing

INTRODUCTION/TERMINOLOGY — Preimplantation genetic testing (PGT, formerly known as PGD or preimplantation genetic diagnosis) involves in vitro fertilization (IVF); biopsy of the blastocyst; or less commonly polar body/bodies (I and II) for genetic testing; and then transfer of selected fresh or frozen-thawed embryos into the uterus based on the results of genetic testing. The four types of PGT are:

●Preimplantation genetic testing for aneuploidy (PGT-A) (formerly called preimplantation genetic screening) – The goal of PGT-A is to identify embryos with de novo aneuploidy and subchromosomal deletions and duplications in the embryo(s) of parents presumed to be chromosomally normal. Theoretically, avoiding transfer of these embryos will reduce the risk of miscarriage and complications related to pregnancy failure and improve the probability of conceiving a pregnancy that results in a live birth.

PGT-A can be used to select the sex of the transferred embryo, which is controversial when performed for nonmedical reasons and prohibited in some countries. (See 'Patients who wish to avoid a sex-linked disorder in offspring' below and "Sex selection".)

●Preimplantation genetic testing for monogenic (single-gene) disorders (PGT-M) – The goal of PGT-M is to establish a pregnancy that is unaffected by specific genetic characteristics, such as a known heritable pathogenic variant carried by one or both parents. It is also used to select embryos for transfer that have specific characteristics, such as a particular sex or compatible human leukocyte antigen complex type.

PGT-A is also commonly performed since leftover DNA is available for analysis [1].

●Preimplantation genetic testing for polygenic disorders (PGT-P) – The goal of PGT-P is to establish a pregnancy that is at low risk of being affected by polygenic disorders that occur later in life, such as hypertension and diabetes. A polygenic risk score is calculated for each embryo using deep whole genome sequencing and haplotyping. Those with the lowest risk scores are prioritized for embryo transfer. Polygenic disorders are more common than monogenic disorders and their inheritance is more complicated.

●Preimplantation genetic testing for structural rearrangements (PGT-SR) – The goal of PGT-SR is to establish a pregnancy that is unaffected by a structural chromosomal abnormality (translocation) in a couple with a balanced translocation or deletion/duplication. Contemporary technology may distinguish noncarrier embryos from balanced carriers [2].

IVF is required as part of this process even though many of these couples have no known difficulties conceiving a pregnancy. IVF is expensive, intrusive, and associated with risks, such as ovarian hyperstimulation syndrome and a higher frequency of some adverse pregnancy outcomes compared with natural conceptions. The inconvenience, risks, and expense of PGT limit the utilization of this technology to couples at risk of transmitting a genetic disease or those attempting to select for specific characteristics in their offspring.

This topic will discuss issues related to PGT (PGT-M, PGT-SR, and PGT-A). IVF is reviewed separately:

●(See "In vitro fertilization: Overview of clinical issues and questions".)

●(See "Assisted reproductive technology: Pregnancy and maternal outcomes".)

PROCEDURE

Obtaining DNA for analysis

Blastocyst or cleavage stage biopsy — The blastocyst, which is the developmental stage achieved by five to six days following fertilization, usually contains more than 100 cells (figure 1). To obtain genetic material for analysis, a laser, acid Tyrode solution, or a sharpened glass needle is used to create an opening in the zona pellucida when the embryo reaches the blastocyst stage [3-6]. Trophectoderm cells (ie, cells from the outer layer of the blastocyst destined to form the placenta) are extracted using a small pipette plus gentle suction or by gently compressing the blastocyst to extrude the cells through the opening. Usually, five to eight cells are removed to limit disruption to the developing fetoplacental unit, but two to three dozen cells can be removed for genetic analysis without compromising blastocyst viability. Compared with other techniques for obtaining preimplantation DNA, blastocyst biopsy is the least disruptive to subsequent development while providing the most DNA for testing, which reduces the possibility of diagnostic errors [7,8].

Biopsy can be performed on day 3 of development (cleavage stage). Embryo transfer of day 3 embryos used to be the preferred practice in in vitro fertilization (IVF). Approximately 50 percent of embryos survive in culture to day 3 of development (cleavage stage: four to eight cells [blastomeres]). If in vitro culture time is extended to day 5 or 6, only approximately 25 percent of embryos survive to form good-quality blastocysts in vitro. Therefore, extended culture to the blastocyst stage for PGT may increase the number of patients who have no embryos suitable for biopsy or transfer and may result in fewer embryos available for testing, transfer, or cryopreservation. Additional issues of extended culture include a small increase in the risk of monozygotic twinning, an increased probability of having a male child, and the potential for a small increase in the risk of epigenetic modifications leading to increased risk for adverse neonatal outcomes [9]. However, the use of day 5 embryos is increasing in IVF programs, even when PGT is not performed. The rationale for this change is that per cycle live birth rates as high or higher than with cleavage stage transfer can be achieved with transfer of fewer (one or two) blastocysts. Additionally, many programs are now cryopreserving all embryos created from each cycle of stimulation with the goal of transferring embryos to the uterus in a cycle without the high estrogen levels created during ovarian stimulation.

Issues related to IVF are reviewed in detail separately. (See "In vitro fertilization: Overview of clinical issues and questions" and "In vitro fertilization: Procedure" and "Assisted reproductive technology: Pregnancy and maternal outcomes".)

Polar body biopsy — In countries where biopsy of an embryo is not permitted, genetic analysis of polar bodies (figure 2) is an option since their genetic composition is predictive of the genetic composition of the oocyte [10]. As long as it is performed by an experienced individual, removal of polar bodies is not harmful to the oocyte since they are a byproduct of cell division.

Polar body biopsy (PBB) is only useful for evaluation of maternally inherited pathogenic variants or meiotic errors during oocyte development. The genetic composition of the oocyte can be inferred from analysis of the first and the second polar body [11,12]. If the mother is heterozygous for a known variant allele, division of the diploid primary oocyte at meiosis I may result in a normal ("wild-type") secondary oocyte and a first polar body with the same variant allele as the mother (figure 2). PBBs that demonstrate normal alleles are presumed to be associated with oocytes that have the variant alleles: These oocytes would not be used to achieve pregnancy. Conversely, PBBs demonstrating the variant alleles are presumed to be associated with oocytes with normal alleles, so these oocytes would then be used for IVF.

PBB has several limitations:

●PBB is not useful if the mother is affected (homozygous) by an autosomal recessive disorder and the father is a carrier since all of the mother's oocytes and polar bodies will carry the pathogenic variant. Blastocyst biopsy to assess both maternal and paternal genes in the developing embryos is necessary.

●The genetic phenomenon of recombination ("crossing over") occurs during meiosis between homologous chromosomes. If a single crossing-over event involves the region containing the candidate gene, the two chromatids of the chromosome would become heterozygous, and thus, biopsy of only polar body I would not permit an accurate prediction of the genetic composition of the oocyte. This problem can be addressed by removal and analysis of both polar body I and II (figure 2) [13]. The likelihood of recombination and diagnostic error is increased for genes close to the telomeric (distal) region of the chromosome since genes closer to the centromeric (central) region are far less likely to cross over. Improved PGT for monogenic (single-gene) disorders (PGT-M) protocols that include closely linked markers, short tandem repeats, or simple sequence repeats may alleviate those problems.

●The oocyte must either be fertilized or cryopreserved before the results of PBB are available, which leads to unnecessary and costly testing of the 30 percent of oocytes that will either not fertilize successfully (ie, develop two pronuclei) or will not undergo postfertilization development.

●PBB has limited ability to predict the genetic status of the embryo since analysis of polar bodies does not detect abnormalities that develop after syngamy (ie, fusion of the oocyte and sperm pronuclei), such as mitotic abnormalities.

●PBB may diagnose a meiotic aneuploidy after meiosis I that goes on to self-correct after meiosis II (figure 2).

Noninvasive and/or minimally invasive PGT — Noninvasive and/or minimally invasive PGT is an emerging technique that has the potential to reduce embryo damage caused by biopsy and lower the cost of PGT since less labor is required [14,15]. This approach involves collecting cell-free DNA released from preimplantation embryos, including fluids in the blastocysts, spent culture medium [16], or both [17]. Challenges that are being addressed include lower detection rates and DNA contamination from maternal cells [18]. However, it may become an alternative for helping to select the single best embryo for transfer.

Cryopreservation of the embryo or oocyte — After biopsy, the embryo or oocyte usually must be vitrified (frozen) until results of the genetic analysis are available since the comprehensive genetic techniques used for genomic analysis often cannot be completed in time to allow fresh transfer. Pregnancy outcomes with vitrified and warmed embryos are as good as or better than with fresh embryos, and maternal morbidity, such as ovarian hyperstimulation, may be less since the transfer will not occur during a stimulated cycle. Additionally, the patient may be able to undergo multiple transfers of single embryos from all the embryos obtained during a single ovarian stimulation cycle. (See "In vitro fertilization: Procedure", section on 'Cryopreservation'.)

It is also possible to thaw frozen unbiopsied embryos, perform PGT, and then repeat cryopreservation [19]. If results obtained from the initial biopsy are not conclusive, previously biopsied embryos can be thawed, rebiopsied, and refrozen until the results of repeat testing are available. Viability of embryos after the second vitrification and warming cycle appears to be slightly lower than that of embryos biopsied and frozen only once.

Genetic analysis

Techniques — PGT is based on analysis of either a single cell (polar body or day 3 blastomeres) or a few cells (day 5 or 6 biopsy). Contemporary genetic analysis involves DNA amplification using a variety of strategies (eg, whole-genome amplification, polymerase chain reaction [PCR] amplification), followed by use of one of several available platforms for analysis of the amplified DNA [20]. Array comparative genomic hybridization (CGH) was a widely used platform for a comprehensive chromosome screening because of its commercial availability but is being replaced by next-generation sequencing (NGS) for whole chromosome aneuploidy, segmental alterations, and mosaicism evaluation (figure 3). Efforts have been focused on increasing NGS coverages and on modifying bioinformatic algorithms to improve sensitivity and specificity.

Other less frequently used platforms include single-nucleotide polymorphism (SNP) array and quantitative real-time PCR. Karyomapping, an application of SNP array technology, is now commonly used for detection of single-gene defects. It is technically feasible to perform exome sequencing or even whole genome sequencing on biopsied embryos; however, these techniques are still considered experimental, are labor intensive, and are relatively more costly. These platforms have not been directly compared in randomized trials. (See "Tools for genetics and genomics: Cytogenetics and molecular genetics" and "Prenatal diagnosis of chromosomal imbalance: Chromosomal microarray".)

False negatives and positives — False-negative errors for many diseases have occurred following PGT. Inadvertent amplification of contaminating extraneous DNA can lead to false-negative results and the transfer of abnormal embryos thought to be normal. Errors can also result when one allele for the gene of interest fails to amplify ("allele drop-out") or amplifies poorly ("partial amplification"). Both types of technical failure have been described and have resulted in misdiagnoses leading to the transfer of an affected embryo believed to be normal or the discarding of a normal embryo thought to be affected. For autosomal dominant diseases, testing the family members of the patient carrying the pathogenic variant and using linked markers will reduce the false-negative rate.

The true risk for obtaining a clinically relevant false-negative result can be determined by prospective auditing and reporting of outcomes of all conceptions after PGT, but no large prospective studies have been conducted yet [21].

Transfer of embryos with mosaicism — Mosaicism arises from postfertilization mitotic errors and leads to distinct cell populations with both euploid and aneuploid chromosomal constitutions. Embryo mosaicism is potentially the largest source of false-positive or false-negative errors and may reduce the overall probability of pregnancy from a single-assisted reproductive technology cycle. Random biopsies of a small sample of cells may not represent the actual chromosomal constitution of the remainder of the dynamic embryo.

The stage of development of the embryo where the mitotic error occurred will determine the proportion of abnormal cells in the embryo. Earlier errors will lead to higher percentages of abnormal cells. Since aneuploid cell lines divide more slowly than euploid cells, "self-correction" may occur so that the percentage of abnormal cells in an embryo may decrease over time [22]. At the blastocyst stage, approximately 17 percent of biopsied embryos were mosaic to some degree in one study [23]. At 10 and 13 weeks of gestation, 1 to 2 percent of chorionic villus sampling samples show persistence of mosaic cell lines. Mosaicism has been identified throughout pregnancy.

Current NGS-based testing methodologies are able to detect low percentages of these abnormal cell populations (as low as 20 percent of the total biopsied specimen). In contrast to older, less sensitive techniques, the percentage of mosaicism likely accurately reflects the mosaic status of the embryo. With previous, less sensitive technologies, such as quantitative PCR, array CGH, and SNP array, mosaic embryos were previously either diagnosed as "normal" and transferred or as "abnormal" and either discarded or not used for transfer. When these abnormal embryos were transferred due to the absence of any normal embryos, the implantation and birth rates of the mosaic embryos were affected by the extent of mosaicism, and healthy children were born without evidence of the persistent abnormal cell line [24]. It appears that the implantation rate of these embryos is decreased compared with normal embryos only when the abnormal cell line comprises >50 percent of the cell population sampled but those that implant still result in the birth of healthy children [25]. Thus, placing arbitrary thresholds on the percent of abnormal cells that can result in a healthy child is without scientific merit and will reduce the pool of available embryos for transfer, as well as the overall probability of pregnancy.

Criteria for aneuploidy in embryos for transfer — No universal criteria exist for selecting identified mosaic or abnormal embryos for transfer. We consider a combination of factors:

●Whether the patient has any remaining euploid embryos

●Whether the patient cannot or will not make more embryos

●The percent of abnormal cells in the biopsy

●The potential effect of the abnormality if it persists

●The quality of the embryo

Many clinics have developed their own criteria for selecting embryos for transfer. In a survey of assisted reproductive technology clinics in the United States, the most common threshold for considering an embryo euploid was >80 percent normal cells, and the most common threshold for considering an embryo aneuploid was >80 percent abnormal cells; however, substantial variation existed among clinics [26]. Over 60 percent of respondents had or would transfer a mosaic embryo if only mosaic embryos were identified, one-fourth reported they had no mosaicism threshold for transfer, and nearly 40 percent were unsure of their threshold. One group used outcome data from 1000 mosaic embryo transfers to develop a prioritization scheme for mosaic embryo selection [25] and found that embryos classified as mosaic experienced higher miscarriage rates than euploid embryos, with a particularly high frequency shortly after implantation [27]. However, infants born of mosaic embryo transfers had similar birth weight, length of gestation, and frequency of congenital anomalies as infants of euploid embryo transfers.

The Preimplantation Genetic Diagnosis International Society updated their position statement on the transfer of mosaic embryos in 2021 based on recent scientific evidence [28]. As published earlier [29], this recent evidence also revealed that mosaic embryos (whole or segmental) could develop into healthy pregnancies [30]. The Society advised prioritizing transfer of embryos with a normal euploid result over those with mosaic results, but stated that transfer of mosaic embryos appeared to have low or minimal risk of negative outcomes beyond the background risk for any pregnancy.

The practice committee of the American Society for Reproductive Medicine has developed guidelines regarding counseling patients who are considering transfer of a mosaic embryo based on trophectoderm biopsy [31]. They concluded that, "there is no evidence-based method available to determine which embryos with mosaic results have the best chance of successful pregnancy, or which may have the lowest risk of an undesired outcome," however, it seems reasonable to counsel patients that transfer should be prioritized to those embryos that would have the best outcome if the abnormal cell line identified persisted in the fetus. While genetic testing of a pregnancy conceived after the transfer of an embryo identified as mosaic is strongly encouraged, there is no evidence to date that the abnormal cell line has persisted in these children.

The ASRM also recommends genetic counseling before testing of embryos for aneuploidy so patients can be informed of the incidence of mosaic results as well as the reporting criteria used by the testing laboratory. They should also receive counseling by a "clinical genetics specialist who has specific knowledge of perinatal and pediatric outcomes associated with chromosomal mosaicism."

SAFETY — Evaluation of clinical outcomes of pregnancies that underwent PGT is limited by the relatively small number of living offspring produced as a result of this technology, the short length of time for follow-up since birth, and a skewed follow-up of PGT for monogenic (single-gene) disorders (PGT-M) and PGT for structural rearrangements (PGT-SR) compared with PGT for aneuploidy (PGT-A), which comprise different populations. The following is a synopsis of available data:

●Implantation and live birth rates – Blastocyst-stage biopsy has no measurable impact on implantation and live birth rates compared with nonbiopsied controls [7]. Cleavage-stage biopsy (at the 6- to 10-cell stage) may reduce implantation rates, but this is based on a single, biased study where poor-quality embryos were biopsied on day 3, and good-quality embryos were grown to day 5 and biopsied at that time.

●Fetal anomalies – The rate of fetal anomalies is not higher than that with in vitro fertilization (IVF) alone [32-39].

●Short-term neonatal outcomes – While the outcomes of pregnancies resulting from biopsied embryos (either for PGT-A or PGT-M) generally have been reassuring [40-42], some studies have reported higher risks for maternal hypertensive disorders of pregnancy (6.9 percent with PGT versus 4.7 percent with IVF alone [43], 10.5 percent with PGT versus 4.1 percent with IVF alone [44]), higher risks for small for gestational age newborns (12.4 percent with PGT versus 4.5 percent after IVF alone [43]), and preterm birth (14.1 versus 12.5 percent; adjusted odds ratio [OR] 1.20; 95% CI 1.09–1.33 [45]). However, the authors of these studies did not adjust for other variables, such as the method of endometrial preparation for the frozen embryo transfer (ie, natural or programmed), which may influence these outcomes.

●Childhood development – Children up to five to six years old exposed to PGT have generally been reported to have similar cognitive and psychomotor developmental outcomes as children not exposed to PGT or conceived naturally [46-49].

Continuing surveillance of this technology is obviously needed. The International Preimplantation Genetic Diagnosis Consortium, the Society for Assisted Reproductive Technology, and the PGT Consortium of the European Society of Human Reproduction and Embryology are providing the needed resources for monitoring clinical outcomes of PGT pregnancies.

COUNSELING — Couples who are considering PGT for monogenic (single-gene) disorders (PGT-M), PGT for structural rearrangements (PGT-SR), or PGT for aneuploidy (PGT-A) must be aware of its limitations, as well as the small risk of misdiagnosis, which can be due to biological or technical reasons or human error. Therefore, counseling by an experienced individual prior to proceeding is extremely important. The following points are key elements of the counseling process and informed consent [50]:

●The PGT result is not definitive – Confirmatory conventional prenatal testing with chorionic villus sampling or amniocentesis is recommended for all pregnancies conceived after PGT-M, PGT-SR, or PGT-A to rule out misdiagnosis due to mosaicism and other factors (eg, technological or human error, spontaneous pregnancy) [51]. Misdiagnosis can be a falsely normal or falsely abnormal result. Amniocentesis is the preferred method for genetic testing during pregnancy since it evaluates the chromosomal status of the fetus rather than the placenta, and it more accurately evaluates methylation of imprinted chromosomes for disorders in which this information is needed.

●PGT-M – Causes of misdiagnosis (defined as the birth of a child who carries the pathogenic variant being screened for) include unprotected sexual intercourse concurrent with the in vitro fertilization (IVF) cycle resulting in fertilization and implantation of an ovulated egg that was not retrieved rather than a successful implantation of the transferred embryo, transfer of the wrong embryo and other human errors, sampling error (ie, sampled cells are not representative of the eventual embryonic genotype), failure of amplification of one of the parental alleles, contamination by parental or extraneous DNA, and postzygotic mitotic changes [21]. Best estimates of true error rates are difficult to elucidate and will only be identified through transfer of an embryo thought to be free of disease that implants and is tested during pregnancy or on products of conception from miscarriage. The actual error rates will depend on the disease being tested for and associated linked markers.

•For patients undergoing PGT-M or PGT-SR, alternative approaches to avoiding the birth of an affected child should be discussed, such as use of donor gametes free of the pathogenic variant, conventional prenatal testing and termination of affected pregnancies, and adoption.

•Genetic counseling should be provided so that patients fully understand their risk of having an affected child, the impact of the disease on that child and family, and the benefits and limitations of all available options for preimplantation and prenatal diagnosis.

●PGT-A – The best estimate of the rate of misdiagnosis for the biopsy and genetic analysis techniques utilized should be discussed. These estimates are elusive since aneuploid embryos misdiagnosed as euploid often do not implant or fail very early in development, thus the misdiagnosis will not be identified by confirmatory prenatal testing. If a euploid embryo is misdiagnosed as aneuploid, no pregnancy will be attempted, and the embryo will likely be discarded so the misdiagnosis will not be identified.

•Sampling "errors" may occur because biopsy of one or more cells at the blastocyst stage may not be representative of the entire trophectoderm; trophectoderm ploidy does not reliably represent the inner cell mass, which develops into the embryo because of mosaicism; and ploidy may self-correct after the blastocyst stage [52,53]. Based on data from the STAR trial, it is estimated that up to 50 percent of embryos that will be discarded because of aneuploidy of the sampled cells have the potential for implantation and pregnancy [54].

•The possibility of false-positive results, which may lead to discarding potentially normal embryos, thus lowering the chances for pregnancy, should be discussed. False-positive results may arise as a result of random biopsy of a small cluster of cells in an embryo with over 100 cells or in the way a laboratory interprets mosaic results. False-positive results are different from sampling error.

•Although not documented with blastocyst biopsy, there is still a theoretic possibility that the embryo biopsy procedure itself may negatively affect the viability of the embryo and lower the probability of pregnancy. Embryo survival to the blastocyst stage in vitro may also be lower than that in an in vivo environment, lowering the probability of pregnancy in patients with limited quantities of available day 3 embryos.

•The fate of embryos not transferred either due to lack of viability or an abnormal result should be decided upon prior to undergoing IVF. (See "In vitro fertilization: Procedure", section on 'Cryopreservation'.)

POTENTIAL CANDIDATES FOR PGT-M

Patients known to be at increased risk of offspring with a specific medically actionable condition — PGT for monogenic (single-gene) disorders (PGT-M) enables patients to establish pregnancies that are unlikely to be affected by specific medically actionable genetic conditions that their offspring are known to be at risk for (eg, cystic fibrosis, sickle cell disease, Duchenne muscular dystrophy, hemophilia, spinal muscular atrophy). This is important for patients who would, for a variety of reasons, be unable or unwilling to terminate an affected pregnancy diagnosed by prenatal diagnostic techniques (eg, diagnostic amniocentesis, chorionic villus sampling) but could discard affected preimplantation embryos or oocytes. Surveys of patients and their partners who are at high risk of transmitting a genetic disorder report that 30 to 74 percent would prefer PGT-M to prenatal diagnosis procedures followed by termination of affected fetuses [55].

Patients undergoing PGT-M may ask about the feasibility of implanting embryos that do not carry pathogenic variants, such as BRCA, that increase the offspring's risk of developing specific adult-onset disorders. The ethics committee of the American Society for Reproductive Medicine has opined that PGT-M for adult-onset conditions is: "ethically justifiable when the conditions are serious and when there are no known interventions for the conditions or the available interventions are either inadequately effective or significantly burdensome. For conditions that are less serious or of lower penetrance, PGT-M for adult-onset conditions is ethically acceptable as a matter of reproductive liberty" [56].

The UK Human Fertilisation and Embryology Authority has granted licenses for PGT-M for over 600 inherited disorders, including familial adenomatous polyposis, hereditary breast and ovarian cancer associated with BRCA1 and BRCA2 pathogenic variants, and Lynch syndrome caused by pathogenic mismatch repair gene variants.

Patients who wish to give birth to a child with a compatible HLA type for stem cell therapy of a sibling — Stem cell transplantation has significantly improved the prognosis for many life-threatening diseases. PGT-M has been used successfully to identify human leukocyte antigen (HLA) complex-compatible, unaffected embryos to permit treatment of an affected sibling by cord blood transfusion or bone marrow transplantation [57]. One such example is children affected with Fanconi anemia. Identification of a disease-free embryo that is also HLA compatible with the affected child and can provide cord blood stem cells for transplantation will significantly improve the probability of survival of the affected sibling. (See "Collection and storage of umbilical cord blood for hematopoietic cell transplantation".)

One limitation of this approach is that few of the embryos tested (approximately 16 percent) will be both unaffected and an HLA match for the affected sibling.

Patients who wish to avoid a sex-linked disorder in offspring — Ideally, PGT-M is used to identify unaffected males so that these embryos, as well as noncarrier females, could be preferentially transferred into the uterus. In a large European database, Duchenne muscular dystrophy and hemophilia were the most common medical conditions where preimplantation sex selection was utilized [58].

However, sometimes the pathogenic variant causing the disease is known to be on a sex chromosome but has not yet been identified or the disease preferentially affects children of one sex (ie, autism has a 4:1 male-to-female ratio). PGT-M for the Y chromosome enables the clinician to limit transfer of embryos to only those of the sex unaffected or less severely affected by the disorder [59].

Sex selection for other reasons (eg, personal preference, balancing the family unit) is more controversial, prohibited in some countries, and reviewed separately. (See "Sex selection".)

Patients who wish to avoid passing a suspected but unconfirmed autosomal dominant disease to offspring — One of the most difficult situations encountered in centers performing PGT-M is the request for testing embryos for late-onset autosomal dominant diseases (eg, Huntington chorea). Patients often present when one of their parents or another first-degree relative is diagnosed with the disorder, and they are made aware that they have a 50 percent chance of carrying the pathogenic variant. Many patients do not want to know if they are carriers of the pathogenic variant for personal and insurance reasons; however, if they inherited it from their parent, they want to make sure that they do not pass it on to their offspring.

In these situations, PGT-M with "nondisclosure" can be performed. The patient undergoes an egg retrieval, the eggs are fertilized, but the results (number of eggs, embryos, the number of embryos transferred, the results of the genetic testing) are not divulged to the patient or their partner because disclosing this information could reveal their genetic status for the disorder. Unaffected embryos are selected for transfer. If there are no normal embryos available, sometimes a "mock" embryo transfer with no actual embryos transferred is performed.

A challenging ethical situation occurs when the patient is found not to be a carrier of the pathogenic variant, based on the results of the initial cycle of in vitro fertilization (IVF) and PGT. Providing this information would constitute disclosure, thus some clinicians would not inform the patient. This obliges the patient to plan and undertake future pregnancies using IVF and PGT, including assumption of the costs and risks involved.

POTENTIAL CANDIDATES FOR PGT-SR

Patients with a balanced translocation at risk of recurrent pregnancy loss from an unbalanced translocation — Approximately 2 to 4 percent of patients experiencing recurrent pregnancy loss will have one partner with an identifiable balanced translocation as a potential cause of the losses. Due to the manner in which homologous chromosomes align and recombine (cross over) during meiosis I, these individuals can produce gametes that are unbalanced (with either missing or extra genetic material), and embryos derived from these unbalanced gametes will usually miscarry.

PGT for structural rearrangements (PGT-SR) appears to significantly reduce the frequency of miscarriage in these couples [60-62]. In one literature review including six studies, the rate of miscarriage without PGT-SR ranged from 26 to 64 percent versus 5 to 15 percent with PGT-SR [62]. This review also observed that use of PGT-SR appeared to shorten the time it took to conceive a pregnancy that resulted in a live birth [62].

POTENTIAL CANDIDATES FOR PGT-A

Older patients who desire eSET — Elective single embryo transfer (eSET) is defined as transfer of one good-quality embryo in cases in which at least two good-quality embryos are available. It is desirable because it dramatically reduces the frequency of multiple gestation compared with double embryo transfer. However, some patients, especially older patients, may choose to transfer more than a single embryo to increase their chances of pregnancy with a single in vitro fertilization (IVF) cycle. (See "Strategies to control the rate of high order multiple gestation", section on 'Single embryo transfer'.)

PGT for aneuploidy (PGT-A) is not required for eSET, and eSET without PGT-A in younger patients with an otherwise good prognosis for a successful pregnancy does not have a significant impact on live birth rates since the embryos of these patients are at relatively low risk for aneuploidy, and the pregnancy rate is quite high when a single untested embryo with good morphology is transferred [63,64].

Older patients, particularly those ≥37 years of age, are at higher risk of having an aneuploid embryo, so PGT-A in this population can help select a euploid embryo for eSET and increase the efficiency (live birth rate per transferred embryo) of IVF [23,64]. Since aneuploid embryos that have little chance of implanting or developing are discarded with PGT-A, the delivery rate per transfer is higher than with transfer of untested embryos [65,66].

PGT-A in older patients does not increase the overall probability of achieving pregnancy per oocyte retrieval procedure since testing only screens out potential aneuploid embryos for disposal. Since more embryos are available for transfer, the live birth rate per IVF cycle is similar to that with PGT-A [66]. Patients who do not undergo PGT-A generally have more surplus embryos for freezing and later use than patients who had their embryos tested. Furthermore, once all embryos (fresh and frozen) from a single oocyte retrieval procedure have been transferred over two or more cycles, patients who do not undergo PGT-A/eSET have similar cumulative live birth rates as patients who undergo PGT-A/eSET, but they also have higher rates of multiple gestation. False-positive errors from PGT may also lead to the discarding of potentially viable embryos that are erroneously excluded from transfer.

UNPROVEN INDICATIONS

PGT-A to improve pregnancy outcome for patients undergoing IVF — PGT-A is not recommended for routine use in all infertile patients [67,68]. It may reduce the probability of pregnancy overall since many embryos will be discarded in the PGT-A group, resulting in fewer embryos for subsequent transfer and a lower cumulative live birth rate. It does not reduce the risk of clinically relevant miscarriage since the majority of aneuploid embryos do not survive to implant. The reason selection for euploid embryos appears to not improve live birth rates may be that random biopsy may not accurately reflect the actual chromosomal constitution of the tested embryo.

Evidence:

●In a 2020 meta-analysis of randomized trials (13 trials, 2794 participants) comparing in vitro fertilization (IVF) with versus without PGT-A, IVF with PGT-A did not clearly improve the cumulative live birth rate (odds ratio [OR] 1.05, 95% CI 0.66-1.66), live birth rate after the first embryo transfer (OR 1.10, 95% CI 0.68-1.79), or miscarriage rate (OR 0.89, 95% CI 0.52-1.54) [69].  

●Additional details were provided by a 2019 multicenter, randomized trial that evaluated outcomes with PGT-A performed at the blastocyst stage [23]. Nearly 1000 patients were recruited, and 661 were randomized at the blastocyst stage when at least two blastocysts were available for biopsy. Patients randomized to the PGT-A group had all of their embryos biopsied and cryopreserved, whereas patients in the control group had their best embryo cryopreserved and the rest of their embryos biopsied and cryopreserved; transfer occurred for all patients in a subsequent programmed cycle.

The results did not demonstrate benefit of genetic testing of the embryo for any group. This is relevant since randomization should have occurred at the start of stimulation, as randomization once the patient already has at least two blastocysts will bias the results in favor of PGT. Interestingly, while 43 percent of embryos overall were deemed euploid, replacing either the most morphologically normal "euploid" embryo or an untested but morphologically "best" embryo resulted in the same outcome and no difference in miscarriage rate.

When analyzed by age (under 35 and 35 to 40 years), patients in both groups had similar outcomes and similar miscarriage rates when analyzed from randomization. Only when analyzed by embryo transfer procedure did patients over 35 have an improved implantation rate, but there was no difference in live birth rate per randomized patient. Additionally, PGT-A appeared to lower the number of infants born per IVF retrieval procedure and may have increased the number of cycles needed to achieve a live birth. This is likely due to a significant number of patients in the older group (17.2 percent) not having a transfer procedure due to lack of a single euploid embryo.

●A 2021 trial included 606 subfertile patients aged 20 to 37 years randomly assigned to PGT-A versus conventional IVF when they had at least three good quality blastocysts on day 5 after oocyte retrieval [42]. The cumulative live birth rate was lower in the PGT-A group (77.2 versus 81.8 percent; absolute reduction -4.6 percent, 95% CI -9.2 to 0). The cumulative pregnancy loss rate was also significantly lower in the PGT-A group (8.7 versus 12.6 percent). Obstetrical and neonatal complications were similar for the two groups.

Recurrent IVF implantation failure — Despite the transfer of multiple morphologically normal embryos, some patients do not achieve pregnancy. Some studies have observed a higher rate of aneuploidy in the preimplantation embryos of these patients compared with controls, which may account, at least in part, for their recurrent implantation failure [70,71]. However, there are potentially many reasons why an embryo does not implant or continue to develop.

No data support a recommendation to perform PGT-A to enhance pregnancy rates in these patients. In two randomized trials that evaluated the impact of PGT-A on pregnancy rates in IVF patients with recurrent implantation failure, those undergoing PGT-A had similar implantation rates and pregnancy rates compared with the control groups that did not undergo PGT-A [72,73]. However, PGT-A may be reasonable in good-prognosis patients who have failed multiple prior embryo transfers and have a large number of high-quality embryos [52].

Advanced maternal age — The increasing prevalence of infertility and miscarriage with advancing maternal age is attributed, in large part, to the increasing prevalence of aneuploidy, including more complex aneuploidies (eg, involving two or more chromosomes), in embryos of older patients [74].

Available evidence does not support the use of PGT-A to increase the probability of a live birth in older patients undergoing IVF. In a 2011 meta-analysis including nine randomized trials, PGT-A using fluorescence in situ hybridization for genetic analysis reduced the live birth rate after IVF for patients of advanced maternal age (risk difference -0.08, 95% CI -0.13 to -0.03; live birth rate 26 percent after IVF without PGT-A, 13 to 23 percent with PGT-A) [75]. This was again seen in a 2019 large randomized trial using the most current technology [23]. Although PGT-A selected euploid embryos, potentially resulting in higher implantation rates and pregnancy rates per transfer, pregnancy rates per patient or per started cycle were lower since fewer embryos were available for transfer and fewer transfers were performed.

In a 2017 trial of patients aged 38 to 41 years with at least five metaphase II oocytes randomly assigned to PGT-A using comparative genomic hybridization or transfer of untested blastocysts, both groups had similar cumulative delivery rates per patient by six months after trial closure [65]. PGT-A resulted in a higher rate of live birth from the first embryo transfer (53 versus 24 percent) and overall fewer embryo transfers per live birth (mean 1.8 versus 3.7) because of lower miscarriage rates (3 versus 39 percent). However, fewer cycles in the PGT-A group resulted in embryo transfer (68 versus 91 percent). The authors concluded that PGT-A was advantageous in patients of advanced age because it reduced the emotional burden of miscarriage and the time from initiation of IVF to pregnancy resulting in a live birth (7.7 versus 14.9 weeks).

In contrast to this trial, which was restricted to patients with multiple metaphase II oocytes, PGT-A may prolong the time to conceive a pregnancy in patients who are undergoing multiple IVF cycles to have adequate numbers of embryos to perform PGT-A (ie, embryo banking). Although patients in whom all embryos were chromosomally abnormal in this trial avoided the psychological, physical, and economic burden of unsuccessful embryo transfers (miscarriage, implantation failure), there is a real possibility that some embryos considered abnormal by PGT-A testing, particularly mosaics, could have resulted in a healthy euploid child (false abnormal PGT-A) had they been transferred [76]. Patients with limited numbers of embryos must be made aware of this prior to testing. In addition, since most abnormal embryos will not continue to develop or implant if transferred into the uterus or would result in an early miscarriage without need for medical intervention, PGT-A to avoid transfer of abnormal embryos is not likely to reduce maternal medical or surgical morbidity associated with pregnancy loss. This was seen in the older population in the STAR trial; older patients did not have a different miscarriage rate or live birth rate with PGT-A compared with those in the control group per randomized patient [23].

Recurrent early pregnancy loss — The majority of conceptions that end in miscarriage and successfully karyotyped demonstrate a chromosomal abnormality, typically aneuploidy (loss or gain of an entire chromosome), but some are related to duplication or deletion of a portion of the chromosome (segmental aneuploidy) or to mosaicism [77,78]. Recurrent aneuploidy may account for some cases of recurrent pregnancy loss (RPL), regardless of maternal age [79,80].

In patients who have experienced RPL and have a history of proven aneuploid abortuses, PGT-A has been proposed to select euploid embryos for transfer, which might increase the implantation rate, lower the spontaneous loss rate, and thus improve pregnancy outcome. For patients with RPL who have had proven euploid miscarriages, PGT-A is unlikely to be of benefit.

Randomized trials using contemporary IVF and PGT techniques in patients with recurrent aneuploid pregnancy losses have not been performed. Although some observational studies have reported that RPL patients who underwent PGT-A had pregnancy rates comparable to those in control populations without RPL (eg, patients undergoing PGT-M for sex-linked disorders, IVF because of male factor infertility) [79-81], the probability of subsequent live birth in patients with RPL without intervention is already high, approaching 70 percent; thus, these studies do not prove the benefit of PGT-A in management of RPL [82,83]. It is theoretically possible that PGT-A may reduce the probability of miscarriage in patients with RPL due to aneuploidy and reduce the potential sequelae of recurrent miscarriage (uterine and emotional trauma); however, there is no evidence that PGT-A will increase the eventual take-home baby rate in these patients.

Improving implantation rates in patients under the age of 35 or using donor eggs from a younger donor — Available evidence suggests that PGT-A does not improve pregnancy outcome metrics in patients under the age of 35 or in cycles using donor eggs from a young donor [84-86].

Selecting for traits — IVF with embryo biopsy and genetic testing to select for characteristics or traits that do not affect the health or well-being of a child is generally discouraged. While some characteristics can be tested for, such as hair color or eye color, there are potential maternal risks associated with the IVF procedure, such as anesthesia, bleeding and infection from the retrieval, and ovarian hyperstimulation. Undergoing IVF when not required to achieve pregnancy exposes the mother to these risks with no significant health benefit to the child.

Selecting for polygenic disorders (PGT-P) — Polygenic disorders occur when multiple genes interact to predispose an individual to a nonlethal disease that alters quality of life or requires intervention, such as diabetes and hypertension. PGT-P uses deep whole genome sequencing and haplotyping to assign each embryo a "polygenic risk score" for polygenic disorders that occur later in life. It enables preferential transfer of embryos less likely to be affected. A preclinical study reported 99.0 to 99.4 percent genotype accuracy at sites relevant to polygenic risk scoring [87]. The study included 110 day 5 trophectoderm biopsies. Limitations of the study include that genotype accuracy was determined from only 10 couples and assessed 12 medical conditions. Furthermore, the predictive probability of these analyses will be best only in populations where the sample tested closely matches the original population in this study and least predictive when the populations are different.

Although commercial laboratories have begun offering this new tool to patients, the use of PGT-P is controversial and should be approached cautiously as a number of potential problems exist [88-92]. Offspring health benefits are not proven. Parents need to understand the differences between relative and absolute predicted risk and the uncertainty involved in the estimate, which are complex concepts. They also need to understand the risk is based on the genotype, which cannot account for postnatal environmental and medical factors that could mitigate this risk. Based on the score, some patients may destroy potentially viable blastocysts or undergo extra cycles of ovarian stimulation to produce more blastocysts. Additionally, selecting a specific embryo based on this score may inadvertently select an embryo with a higher risk for another disorder that was not tested for.

SUMMARY AND RECOMMENDATIONS

●Types – There are multiple types of preimplantation genetic testing (PGT):

•Preimplantation genetic testing for monogenic (single-gene) disorders (PGT-M)

•Preimplantation genetic testing for structural rearrangements (PGT-SR)

•Preimplantation genetic testing for aneuploidy (PGT-A)

•Preimplantation genetic testing for polygenic diseases (PGT-P, a relatively new concept that is rarely performed)

All require in vitro fertilization (IVF), biopsy of the embryo, or less commonly, the polar body for genetic testing, and then uterine transfer of selected embryos based on the results of genetic testing. (See 'Introduction/terminology' above.)

●Candidates – The main reasons that patients choose PGT-M are to avoid having a pregnancy with a fetus affected by, or at risk for, a severe debilitating disease; to increase the parents' chances of having a human leukocyte antigen complex-compatible offspring; and for medically-indicated sex selection. (See 'Potential candidates for PGT-M' above.)

Couples with balanced translocations choose PGT-SR to reduce the risk of recurrent pregnancy loss (RPL) from unbalanced translocations.

●Counseling – Counseling should be provided to ensure that patients fully understand the benefits and limitations of PGT and other available options for prenatal diagnosis or other ways to avoid having a child with a genetic disorder. They should understand the limitations of the technique, the small but nonzero rate of false-positive and false-negative results, and the need for amniocentesis (preferred) or chorionic villus biopsy to confirm PGT findings. (See 'Counseling' above.)

Biopsy does not appear to decrease implantation and live birth rates or have any long-term harm on offspring. (See 'Safety' above.)

●Biopsy and vitrification – DNA for genetic analysis is usually obtained from biopsy of trophectoderm cells from a blastocyst on day 5 or day 6 after fertilization (see 'Blastocyst or cleavage stage biopsy' above). Biopsy of a polar body is also possible, but results are much more limited. (See 'Polar body biopsy' above.)

After biopsy, the embryo or oocyte usually must be vitrified (frozen) until results of the genetic analysis are available. It is possible to thaw frozen unbiopsied embryos, perform PGT, and then repeat cryopreservation, although this may compromise embryo viability. Rebiopsy of a previously frozen embryo to clarify ambiguous results from an initial biopsy is also possible. (See 'Cryopreservation of the embryo or oocyte' above.)

●Genetic analysis – Genetic analysis involves DNA amplification using a variety of strategies (eg, whole-genome amplification, polymerase chain reaction [PCR] amplification), followed by use of one of several available platforms for analysis of the amplified DNA. (See 'Genetic analysis' above.)

Inadvertent amplification of contaminating extraneous DNA can lead to false-negative results and transfer of abnormal embryos thought to be normal. Errors in PGT-M can also result when one allele for the gene of interest fails to amplify ("allele drop-out") or amplifies poorly ("partial amplification"). Both types of technical failure have been described and have resulted in misdiagnoses leading to the transfer of an affected embryo believed to be normal or the discarding of a normal embryo thought to be affected. Linkage analysis can help improve the accuracy of the results. (See 'False negatives and positives' above.)

●Utility

•PGT-M and PGT-SR can reduce the risk of a pregnancy with a fetus affected by a severe, debilitating genetic condition or a miscarriage due to unbalanced translocation. (See 'Potential candidates for PGT-M' above and 'Potential candidates for PGT-SR' above.)

•PGT-A to eliminate the transfer of aneuploid embryos has not been proven to improve pregnancy rate or outcome per started IVF cycle and may lower the overall probability of pregnancy due to errors and lower numbers of available embryos. Additionally, miscarriage rates are not reduced. (See 'PGT-A to improve pregnancy outcome for patients undergoing IVF' above.)

The major clinical utility of PGT-A is that multiple gestation rates can be significantly reduced without reducing delivery rates if a euploid result leads to single embryo transfer (eSET) rather than double embryo transfer. However, studies evaluating the transfer of one fresh blastocyst and, if no pregnancy occurs, one frozen/thawed blastocyst, report the same cumulate live birth rates as transferring a single blastocyst after PGT-A. (See 'Potential candidates for PGT-A' above.)

•Although commercial laboratories have begun offering PGT-P to patients, its use is controversial and should be approached cautiously as a number of potential problems exist. (See 'Selecting for polygenic disorders (PGT-P)' above.)