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Introduction
Assisted reproductive technologies (ART) allow for the treatment of most infertile couples with the aim of securing a healthy birth. The success of in vitro fertilization (IVF) cycles depends on various factors and is generally evaluated by implantation efficiency, clinical pregnancy, and live birth. These results are influenced by the ovarian response to stimulation, oocyte quality, embryo culture, transfer selections, and the age of the patient [1].
It is well established that embryonic aneuploidy is prevalent in IVF cycles, especially in women of advanced maternal age (AMA) [2]. Most embryos with an abnormal number of chromosomes are not compatible with life [3], and these fatal genetic defects are responsible for implantation failure and early miscarriage after the transfer of a morphologically good-quality embryo. This prevalence increases with age; the estimated aneuploidy rate increases from 25% for oocytes from women under 35 years old to more than 75% for oocytes from women aged >40 years [4]. On these and many other bases, one of the most important challenges for the embryologist is to discern which embryo is the most appropriate to transfer.
Preimplantation genetic testing (PGT) is a procedure used to identify genetic abnormalities in embryos created by IVF and can be used as a tool to select embryos of good quality for embryo transfer (ET), which, in theory, should improve implantation rates, decrease miscarriage rates (MR), and reduce the time to achieve a successful pregnancy. However, studies that directly compare the outcomes of cycles with and without PGT are scarce. Moreover, there are doubts about the benefits of using PGT for certain pathologies. There are many data points on the benefits of using PGT in a distinctive cohort of patients characterized by AMA, recurrent implantation failure (RIF) [5], recurrent pregnancy loss (RPL), severe male infertility, or elective single ET [6,7]. However, there are many controversies regarding this topic; thus, it needs to be clarified.
Therefore, to fully validate the advantages of PGT for aneuploidy in terms of an increased chance of successful pregnancy and live birth, and to provide an update on the efficacy of PGT for aneuploidy (PGT-A) in clinical outcomes, we aimed to conduct a systematic review in which we summarized all published studies.
Materials and methods
This systematic review was registered with the International Prospective Systematic Review Registry of the National Institute of Health Research (PROSPERO). Protocol and registration number: PROSPERO 2022 CRD42022354697. This systematic review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 guidelines for reporting systematic reviews [8]. Institutional Review Board approval was not required because the present study was a review.
1. Study selection
An electronic database search was conducted using PubMed, the Cochrane Library, ClinicalTrials.gov, Scopus, Embase, and Google Scholar. The authors used a combination of the following terms: “preimplantation genetic testing”, “PGT-A”, “pregnancy”, and “live birth”. The last screening date was October 1, 2022.
No restrictions or search filters (publication status, type of article, or language of publication) were applied to verify all potentially relevant studies.
The search strategy for the PubMed electronic database was as follows: using the advanced search builder in PubMed, the following combinations of keywords were used: (preimplantation genetic testing) AND (PGT-A) AND (pregnancy) AND (live birth), and no filters or limits were used.
In addition to PubMed, the Cochrane Library electronic database was searched. The search combinations were as follows: (preimplantation genetic testing) AND (PGT-A) AND (pregnancy); no filters or limits were used.
The electronic databases Scopus and Google Scholar were searched using the following words: ((preimplantation AND genetic AND testing) AND (PGT-A) AND (live AND birth) AND (pregnancy) AND (embryo AND biopsy)).
The search was also conducted in the ClinicalTrials.gov electronic database using an advanced search combination of “preimplantation genetic testing” and “pregnancy”.
Additionally, the search was conducted using MeSH terms in PubMed ((((«Preimplantation Diagnosis”[MeSH]) AND “Pregnancy”[MeSH]) AND “Pregnancy Rate”[MeSH]) AND “Live Birth»[MeSH]) and in the Cochrane Library (MeSH descriptor: [Preimplantation Diagnosis] explode all trees).
The search was conducted independently by three investigators (L.O., E.K., and S.I.) and the search results were saved to a reference manager (Zotero version 6.0.8; Corporation for Digital Scholarship, Fairfax, VA, USA). After searching, all articles were reviewed based on their titles and abstracts. All studies were selected, and each potentially relevant study was obtained in full text and independently assessed for inclusion by the authors. Additionally, a manual search of the references of the articles was performed to identify additional studies of interest. Any disagreements regarding the inclusion or exclusion of the preselected studies and other disagreements during the review process were resolved with the help of a fourth author (L.P.).
2. Eligibility criteria and main outcomes
The inclusion criteria for the present systematic review were: women of reproductive age who underwent assisted reproduction, array comparative genomic hybridization (aCGH) or next-generation sequencing (NGS)-based PGT-A. Fresh embryo transfer was considered in groups without PGT-A intervention.
Studies containing information on other molecular methods used to assess chromosomal content were excluded. The use of aCGH or NGS is recommended by the European Society of Human Reproduction and Embryology (ESHRE) [9].
Randomized and non-randomized clinical trials published in English were included. Papers in languages other than English, case reports, preclinical studies, reviews, opinion articles, and studies published as abstracts were excluded.
The primary analysis aimed to assess the risk ratios of the clinical pregnancy rate (CPR) and live birth rate (LBR). CPR was defined as the number of clinical pregnancies expressed per 100 initiated, aspirated, or embryo transfer cycles. LBR was defined as the number of deliveries resulting in at least one live birth and is expressed per 100 cycle attempts [3].
Secondary analyses evaluated MR, implantation (IR), ongoing pregnancy/live birth, and spontaneous abortion rates. MR is defined as the spontaneous loss of intrauterine pregnancy before 22 weeks of gestational age [3].
The implantation rate was calculated as the number of gestational sacs visualized by transvaginal ultrasonography (number of implanted embryos) divided by the total number of embryos transferred. The ongoing pregnancy/live birth rate was defined as the number of ongoing pregnancies after the presence of a fetal pole with fetal heart tones and/or live births, divided by the total number of embryos transferred.
We grouped these results as “per embryo transfer” and “per patient”. “LBR per embryo transfer” refers to the LBR calculated based on the number of live births per embryo transfer procedure. “LBR per patient” refers to the LBR calculated based on the number of live births per individual patient undergoing fertility treatment. “CPR per embryo transfer” refers to the percentage of ET procedures that result in a clinical pregnancy. “CPR per patient” refers to the overall percentage of patients who achieve a clinical pregnancy following ET. “MR per embryo transfer” refers to the percentage of embryos that resulted in miscarriage following a transfer procedure. “MR per patient” refers to the likelihood of miscarriage in individual patients undergoing fertility treatment.
3. Quality assessment
A risk of bias assessment was performed for each of the included studies using the Cochrane Handbook for Systematic Reviews of Interventions [10]. Three review authors independently evaluated the quality of the selected studies. Any discrepancies between the reviewers were resolved through discussion or consultation with the fourth review author (L.P.).
Following the Cochrane Handbook for Systematic Reviews of Interventions, the risk of bias (RoB) 2 tool [11] was used to assess the risk of bias in randomized controlled studies, and risk of bias in non-randomised studies-of interventions (ROBINS-I) [12] was used for non-randomized studies (prospective controlled, prospective cohort, retrospective studies, and other types of studies). Additionally, these tools were used to assess the risk of bias arising from reporting biases resulting from missing synthesis results.
4. Statistical analysis
For quantitative synthesis, a meta-analysis (forest plot) was performed using the RevMan 5.4. (Cochrane Collaboration, London, UK) (recommended by the Cochrane Society). According to the Cochrane Handbook for Systematic Reviews of Interventions, an I2 value of 0 indicates no observed heterogeneity, whereas I2 values from 30% to 60% represent moderate heterogeneity, I2 values from 50% to 90% represent substantial heterogeneity, and I2 values from 75% to 100% represent considerable heterogeneity. Meta-analyses with heterogeneity greater than 75% were excluded.
Results
The entire search strategy and results are presented in the flow diagram (Fig. 1). The initial search yielded a total of 321 articles. After a MeSH search, 120 reports were identified: 76 from PubMed and 44 from the Cochrane Library. After removing duplicates and searching the titles and abstracts of the articles, 296 publications were selected. Therefore, 75 reports remained for full-text screening and analysis based on our inclusion criteria.
Fifty-six articles did not meet the inclusion criteria for various reasons, as detailed in Fig. 1, and were excluded from the study. The most common reasons for exclusion were that the article concerned a conference abstract [13-19], absence of relevant inclusion criteria [20-49], absence of published study results [50,51], absence of a control group [52-63], and some studies [64-68] were review articles.
Additionally, 51 articles were found in the references of the 19 articles included in the qualitative analyses, all of which met the eligibility criteria. However, none of these studies were included in the systematic review because they were duplicates found earlier. Therefore, 19 studies [69-87] were retained for qualitative synthesis.
Five publications were randomized studies and 14 were nonrandomized studies. The process of inclusion and exclusion is detailed in the PRISMA flow diagram shown in Fig. 1. The characteristics of the included studies are provided in Table 1. The outcomes of the studies included in the meta-analysis are presented in Table 2.
According to the Cochrane Handbook, three reviewers (L.O., E.K., and S.I.) assessed the risk of bias in each of the included studies using RoB 2 for randomized control trials and ROBINS-I for non-randomized trials. Disagreements were resolved through discussion with a fourth author (L.P.)
Visualization tools were created using ROBVIS App (National Insitute of Health Research, Newcastle upon Tyne, UK) [88]. This application created “traffic light” graphs of domain-level judgments for each result and weighted bar graphs of the distribution of risk-of-bias judgments within each bias domain.
Based on these tools, randomized controlled trials had a low risk of bias and nonrandomized trials had a moderate risk of bias (Fig. 2).
1. Clinical pregnancy rates per embryo transfer
Eight studies reported results on CPR per ET cycle in women aged 35 years or older (risk ratio [RR], 1.44; 95% confidence interval [CI], 1.19-1.75; P=0.0002). Heterogeneity in this comparison was 55% (Fig. 3A). In this group, PGT-A improved CPR.
2. Live birth rates per embryo transfer
Five studies reported results on LBR per ET cycle in women 35 years old or younger (RR, 1.32; 95% CI, 1.11-1.57; P=0.002). The live birth rate per ET improved after PGT-A compared to controls. Heterogeneity in this comparison was 72% (Fig. 3B).
3. Live birth rates per patient
Two studies reported results on LBR per patient in women 38 years of age or younger (RR, 0.97; 95% CI, 0.87-1.09; P=0.59). No significant differences were found between the two groups. Heterogeneity in this comparison was 30% (Fig. 4A).
Two studies reported results on LBR per patient in the over-35-year-old age group; in this comparison, PGT-A improved live birth rates (RR, 1.65; 95% CI, 1.18-2.30; P=0.004). The heterogeneity of this comparison was 0% (Fig. 4B).
Among patients aged <35 years, PGT-A resulted in a higher LBR per ET than in those who did not undergo PGT-A. However, no significant differences were observed in the number of live births per patient in this age group. In patients aged >35 years, PGT-A improved live birth rates compared to those without PGT-A. Overall, PGT-A appears to have a more positive effect on live birth outcomes in older patients.
4. Effect of PGT-A on the live birth rate in patients with a poor prognosis
This meta-analysis of eight studies compared the LBR in patients with a history of previous miscarriage, RPL, or RIF (RR, 1.47; 95% CI, 1.14-1.90; P=0.003). The heterogeneity of this comparison was 68%. Thus, PGT-A improved LBR in this cohort of patients (Fig. 5A).
5. Miscarriage rates per embryo transfer cycle
Four studies reported results on MR per cycle of ET in women 35 years old or younger (RR, 0.80; 95% CI, 0.49-1.31; P=0.37). Consequently, no significant difference was observed between the PGT-A and control groups. Heterogeneity in this comparison was 26% (Fig. 5B).
Seven studies reported results on MR in the over-35-year-old age group. No significant differences between the two groups (RR, 0.72; 95% CI, 0.41-1.27; P=0.26). Heterogeneity in this comparison was 62% (Fig. 5C).
Conclusion
1. Our results
In this meta-analysis, we evaluated the effectiveness of PGT-A in IVF/intracytoplasmic sperm injection (ICSI) cycles in patients of different ages and found that PGT improved the efficiency of ART, increasing clinical pregnancy and LBR, especially in women of AMA and with a poor prognosis. However, no benefits were demonstrated when applied to younger women. The advantages of these groups of patients can be explained by the higher rates of aneuploidy, which is rational because it is well established that embryonic aneuploidy is the main genetic factor influencing human reproductive success, not patient age alone. Poor oocyte quality in these patients can be explained by cytoplasmic errors, particularly in mitochondrial function [89,90]. According to our results in the specific population under 35 years of age, PGT-A did not reduce the MR as expected, which can be attributed to different factors. MR increase with age, and although this population is still relatively young, some age-related factors that are not related to chromosomal abnormalities may contribute to miscarriages. For example, pregnancy complications such as gestational diabetes or preeclampsia are more common in older mothers [91-93].
It is also important to highlight that the PGT-A is not a perfect test, and there can be technical limitations that lead to false results. For example, mosaic embryos (with both normal and abnormal cells) may be incorrectly classified as abnormal and not transferred, or vice versa. Additionally, some chromosomal abnormalities, particularly those that affect only a small portion of the chromosome, may not be detected by PGT-A [77,94,95]. Additionally, studies that have looked at the effectiveness of PGT-A in reducing MR in this age group may have had small sample sizes, which may limit the generalizability of the results.
In a study conducted by Anderson et al. [96] in 2020, the authors suggested that age did not appear to be a factor when considering embryo implantation and live birth rates between treatment groups.
However, according to published data, aneuploid embryos account for at least 10% of human pregnancies and the incidence can exceed 50% in women over 35 years of age [97].
However, these findings remain controversial. One of the included studies, the Single Embryo Transfer of Euploid Embryo (STAR) study trial [79], was highly debated. For example, the published reanalysis of this study revealed significant shortcomings in its statistical analyses [98]. Thus, the STAR study revealed that PGT-A did not beneficially affect IVF outcomes. Moreover, based on the Preimplantation Genetic Diagnosis International Society (PGDIS) 2019 analysis of this trial, not even reaching statistical significance (P=0.053), the authors did not hesitate in reporting that “a significant increase in ongoing pregnancy rate” was observed [99].
2. Stage of embryo biopsy and its influence on embryo development
One of the factors that can affect the results is that the stages of embryo biopsy in the enrolled studies were different; some of them involved biopsy in the cleavage stage, while others involved biopsy in the blastocyst stage. However, the role of biopsy duration remains controversial. According to the ESHRE recommendations on the most appropriate day, blastocyst biopsy is performed on days 5-7 post-insemination, according to the rate of development, once the inner cell mass is clearly visible [100].
Some authors have suggested that blastomere biopsy can lead to potential embryonic damage, higher levels of abnormality, and mosaicism. In a randomized controlled trial conducted by Scott et al. [101] in 2013, the effect of pre-implantation genetic testing for monogenic diseases biopsy on developing embryos was assessed, and the results demonstrated a relative 39% reduction in IR in the cleavage-stage biopsy group compared to controls without a reduction in the trophectoderm (TE) biopsy group. Only the D5 biopsy group showed a statistically significant increase in the LBR per ET. In a recent study, Sarkar et al. [102] assessed whether embryo biopsy for PGT-A affected the birth weight or preterm birth rate. The authors reported that trophectoderm biopsy for PGT-A did not increase the risk of small for gestation age, low birth weight, or preterm birth in IVF pregnancies [102].
3. Comprehensive chromosome screening (CCS)
The types of PGT methods used for complete chromosome screening (aCGH and NGS) in the included studies differed, which may have affected the results. NGS is the newest technique used for incorporation into second-generation PGT. Various studies that validated the precision of the NGS approach for embryonic CCS have demonstrated 100% consistency in the diagnosis of aCGH [103,104]. NGS and aCGH results were compared. In a retrospective cohort study, Friedenthal et al. [105] compared the IR, ongoing pregnancy/LBR, biochemical pregnancy rate, and spontaneous abortion between NGS and aCGH groups. Preimplantation genetic screening using NGS significantly improves IR and LBR compared with PGT using aCGH in single-thawed euploid embryo transfer cycles, which might be attributed to the advantages of NGS in detecting small chromosomal deletions, duplications, and mosaicism [105].
4. Embryo mosaicism
Embryo mosaicism occurring during mitotic division of the embryo, giving rise to chromosomally different cell lines, is one of the main sources of error when performing PGT-A [106-111]. Several studies have shown that mosaic embryos theoretically have a reduced IR and an increased risk of miscarriage, pregnancy complications, and clinically affected live births [112].
According to the PGDIS 2021 statement [112], embryos with a mosaicism rate lower than 20% can be considered euploid (and transferable), whereas embryos with more than 80% abnormal cells are classified as aneuploid. The remaining (20-80%) can be classified as mosaics. However, establishing the thresholds between which embryos can be considered transferable remains controversial. In a criticism of the earlier PGDIS 2019 position statement [99], the authors declared that accurate percentages of aneuploid DNA could not be calculated because of the inability to determine how many cells were damaged during biopsy, contributing to the fractional loss of DNA content and sample contamination [113].
A single trophectoderm biopsy of on average 5-6 cells, as is currently the practice in PGT-A at the blastocyst stage, cannot mathematically represent the whole embryo. Gleicher et al. [114] established two mathematical models to assess the probabilities of false-negative and false-positive results of an average 6-cell biopsy from approximately 300-cell TE. Both models revealed that even under the best-case scenario, assuming an even distribution of mosaicism in TE (because mosaicism is usually clonal and a highly unlikely scenario), a biopsy of at least 27 TE cells would be required to achieve minimal diagnostic predictability from a single TEB [114]. The data did not support an equal distribution of mosaicism throughout the trophectoderm and suggesting that mosaicism levels may be highly dependent on the biopsy [115]. Therefore, the trophectoderm alone cannot reliably represent the inner cell mass for biological reasons. Given the nature of the biology of mosaicism genesis and propagation, any biopsy piece analyzed as mosaic may not accurately reflect the surrounding trophectoderm or the rest of the embryo [116].
It is important to assess the efficacy of PGT using the “per patient” indicator to avoid excluding patients with poorer prognosis whose embryos may never reach ET [99]. Preliminary data suggested a 50% aneuploidy rate at the blastocyst stage (and even higher rates at cleavage stages) and with further gradual self-correction downstream [117]. According to PGDIS 2019, given the current knowledge base, discarding embryos based on a single TE biopsy appears shortsighted, and represents another misunderstanding of embryo biology [99].
However, the transfer of blastocysts in which mosaic aneuploidies have been found should only be considered following expert advice and appropriate genetic counseling for patients. It is recommended that clinicians inform patients that there is currently no evidence-based method available to determine which embryos with mosaic results have the best chance of resulting in a successful pregnancy or which may have the lowest risk of an undesired outcome [118,119]. The question of the correlation between transfer of (under PGDIS definition) “mosaic” embryos and reduced implantation and/or increased rates of miscarriage needs further investigation, and the current available data clearly dispute these propositions [99].
Patient counseling should include a discussion of various possible explanations for the mosaic results of the PGT-A and potential outcomes. In clinical medicine, the responsibility of establishing validated evidence in support of a proposed treatment and/or test rests with the proponents of the treatments or tests, mandating that such evidence exists before such treatments or tests are integrated into routine clinical practice [100].
Embryo mosaicism is another limitation of this study. The transfer threshold differed among the enrolled studies, which could also affect the results of these studies.
5. Fresh and frozen embryos were transferred
Both fresh and frozen embryos were used in the studies included in our meta-analysis. There are still many concerns regarding the effect of cryopreservation on the health of children born and the outcome data after frozen ET. In their systematic review, Maheshwari et al. [120] analyzed obstetric and perinatal outcomes after fresh or thawed frozen ET and found that frozen-thawed ET was associated with better perinatal outcomes than fresh IVF embryos. Based on these findings, we assume that the differences in the included studies may be confounding factors affecting the results.
6. Male factor
We did not consider the influence of male factor on infertility; however, some aneuploidies may be derived from sperm. Men with an abnormal karyotype and Y chromosome deletions tend to produce sperm with an unbalanced set of chromosomes. Several other factors such as varicocele, chemotherapy, age, and lifestyle can also negatively influence meiotic division during spermatogenesis [121]. Petousis et al. [122] demonstrated that the rate of abnormal spermatozoa after fluorescence in situ hybridization examination was significantly higher in male patients with infertility (55.8% vs. 15.0%) and that teratozoospermia was strongly correlated with the incidence of chromosome 17 aneuploidy. Recent studies examining the effect of advanced paternal age on sperm aneuploidy rates have found that men over 50 years of age have more DNA-damaged spermatozoa, a lower rate of blastocyst development, a higher overall rate of aneuploidy, and a higher rate of trisomy [123,124].
7. Cost-effectiveness
The PGT-A strategy becomes more cost-effective with age. Somigliana et al. [125] stated that it is not economically advantageous to use PGT in women aged <36 years of age. Sensitivity analyses that vary the cost of ET, the cost of genetic tests, the magnitude of the adverse effect of PGT-A on LBR, and overall LBR alter the efficacy thresholds to some extent but generally support the notion that PGT-A may be cost-effective in some specific subgroups [125-127].
Our systematic review and meta-analysis evaluated the effectiveness of PGT-A in IVF/ICSI cycles in patients of different ages and included 19 clinical trials that evaluated approximately 100,000 IVF cycles in quantitative synthesis. Furthermore, well-defined eligibility criteria that prioritized only studies using aCGH or NGS were used. Meta-analyses with moderate or low heterogeneity were included.
Nevertheless, there were limitations to our systematic review and meta-analysis. First, there was a lack of clinical studies with a low risk of bias. Thus, our meta-analysis included studies with both moderate and low risk. Additionally, we could not perform subgroup analysis in cases of high heterogeneity because of the small number of relevant clinical studies. The reasons for the high heterogeneity may include the inclusion of randomized and non-randomized studies, studies with low and moderate risk of bias, patients with poor prognosis with different pathologies, and different days of embryo biopsy in these studies (Table 1).
Second, our search strategy included studies published only in English; conference abstracts were excluded, limiting our electronic search. Additionally, not every study transferred mosaic embryos, and this was not mentioned by all of the authors. Owing to the known inaccuracy of PGT-A testing and the possible natural resolution of mosaicism, some authors have suggested that mosaic embryos should be considered normal and transferred [99]. Currently, this is not standard practice.
Moreover, the outcome “per embryo transfer” is controversial regarding PGT-A studies. For example, Rubio et al. [78] did not use a single ET. However, in the study published by Wilkinson [128], participants refused to perform ET in cases of poor prognosis.
The largest trial included in our systematic review and meta-analysis by Sanders et al. [76] was rebutted by Roberts et al. [129]. These authors argue that the comparator group must consist of treatments that could have had PGT-A if the option were available. Their analysis obtained estimates of the effect of PGT-A, which suggested an overall modest reduction in treatment success rates. The treatment effect of PGT-A was different, with an overall odds ratio for a live birth event of 0.82 (0.68-1.00) using >one transferrable embryo control and 0.80 (0.64-0.99) using >five embryo-created controls.
The next limitation is that PGT-A and NGS use frozen ET and should not be compared with fresh ET controls. Finally, it is more relevant to assess the effectiveness of PGT-A on cumulative LBR. However, there was an insufficient number of studies to perform meta-analysis.
Implications for future research may include modern techniques for non-invasive PGT. This method may play an enormous role in future fertility treatment, as damage to the embryo and the associated risks are negligible. Therefore, their use in routine practice should be investigated. In addition, although there are some doubts regarding time-lapse techniques, they should be further evaluated for evidence-based evaluation and decreased controversy. However, we need to consider not only embryos, but also gametes for better pregnancy rates. Thus, it is essential to develop gamete rejuvenation techniques to improve IVF outcomes in couples of advanced parental age.
Based on our systematic review and meta-analysis, we evaluated the effectiveness of PGT-A in IVF/ICSI cycles in patients of different ages and found that PGT improved the efficiency of ART, increasing clinical pregnancy and LBR, especially in women of AMA and those with a poor prognosis; however, no benefits were demonstrated when applied to younger women. Nevertheless, further research is needed to fully understand the effectiveness of PGT-A and to answer all questions regarding the importance of the validation, accuracy, and safety of PGT-A.
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