Preimplantation genetic testing for aneuploidy optimizes reproductive outcomes in recurrent reproductive failure: a systematic review

Introduction Recurrent reproductive failure (RRF) is a common pregnancy complication, imposing great physical, emotional and financial burden for the suffered couples. The leading cause of RRF is believed to be aneuploid embryo, which could be solved by preimplantation genetic testing for aneuploidy (PGT-A) in theory. With molecular genetic development, PGT-A based on comprehensive chromosomal screening (CCS) procedures and blastocyst biopsy is widely applied in clinical practice. However, its effects in RRF were not defined yet. Methods A systematic bibliographical search was conducted without temporal limits up to June, 2023. Studies about the effects of PGT-A based on CCS procedures and blastocyst biopsy in RRF were included. Results Twenty studies about the effects of PGT-A based on CCS procedures and blastocyst biopsy in RRF were included. It revealed that PGT-A could optimise the reproductive outcomes of RRF sufferers, especially in those with advanced age. However, in patients with multiple occurrences of pregnancy losses, the benefits of PGT-A were limited. Discussion More randomized controlled trials with large sample size are required to evaluate the benefits of PGT-A in RRF sufferers and identify which population would benefit the most.


Introduction
Recurrent reproductive failure (RRF), a common pregnancy complication, mainly comprises recurrent pregnancy loss (RPL) and recurrent implantation failure (RIF) (1).RPL refers to ≥2 pregnancy losses or miscarriages (recurrent miscarriage, RM) before 20-24 weeks of gestation (2), affecting 1-2% of all couples (3), while RIF is defined as ≥3 failed embryo transfers with good-quality in in vitro fertilization (IVF) (4), affecting about 10% of couples undergoing IVF treatment (5).RRF had brought great physical and mental pressure to the suffered couples, which linked to increased risk of infertility and pregnancy loss (6).
Aneuploidy is a critical cause of RRF (7).In RPL, aneuploidy is identified in at least 55% of products of RPL sufferers' conception (8), while the embryo is thought to be responsible for 30-50% of RIF (9).Therefore, euploid embryo transfer (ET) is speculated to optimize the reproductive outcomes of RRF.Fortunately, euploid embryos could be selected by preimplantation genetic testing for aneuploidy (PGT-A).Originally, PGT-A was achieved by fluorescence in situ hybridization procedure (10), which was highly limited since it assessed only nine out of 24 chromosomes simultaneously with low resolution (11).Multiple major professional societies recommended against its general use ( 12), as PGT-A based on the FISH procedure failed to improve reproductive outcomes in clinical practice (13,14).With molecular genetic advances, comprehensive chromosomal screening (CCS) procedures and blastocyst biopsy were developed in PGT-A.The commonly used CCS procedure encompasses array comparative genomic hybridization (aCGH), quantitative real-time PCR (qRT-PCR) and next-generation sequencing (NGS), etc. (12).CCS procedures not only analyzed the number of all chromosomes, but also segmental abnormalities.As blastocysts could better tolerate the insults of biopsy with an increased accuracy rate, blastocyst biopsy was found to yield overall improved reproductive outcomes than cleaved embryo biopsy or polar biopsy (12,15,16).Therefore, PGT-A based on CCS procedure and blastocyst biopsy is widely used in clinical practice.However, no consensus has been reached about its effects in RRF sufferers.The review aims to investigate if PGT-A is beneficial for RRF sufferers, and identify suitable population by sub-group analysis.

Search strategy and study selection
Databases of PubMed, Embase, and Cochrane Central Register of Controlled Trials were searched with the following terms: (recurrent OR repeated OR habitual) AND (pregnancy loss OR spontaneous abortion OR miscarriage OR fetal wastage) AND (preimplantation genetic diagnosis OR preimplantation genetic screening OR euploid OR preimplantation genetic test) from inception to June, 2023.The inclusion criteria were as follows.Published in English in peerreviewed journals; irrespective of study-design; studies focusing on the impact of PGT-A based on CCS procedures and blastocyst biopsy on the reproductive outcomes of RRF sufferers.Commentaries, letters, reviews, conference abstracts, and irrelevant studies were excluded.Studies in which PGT-A assay not based on CCS procedure and blastocyst biopsy were also excluded.

Study screening and data extraction
Following an initial search and duplicates removed, a total of 1,063 literatures were screened by two authors independently (XK and XL).An overview of and screening process is presented in Figure 1.The selected studies were comprehensively examined, and the relevant data were extracted according to our developed data extraction spreadsheet by authors (YM and YL).Information selected included author's name, publication year and country of the study, study year, study aim, sample size, methodology, sample characteristics, and outcome measures.Any discrepancies would be resolved by discussion until consensus was reached.The primary outcomes of interest were live birth rate (LBR), defined as the percentage of couples achieving a live birth after 24 weeks' gestation.Secondary outcomes of interest included implantation rate (IR), clinical pregnancy rate (CPR)/ ongoing pregnancy rate (OPR), biochemical pregnancy loss (BPL) rate, and miscarriage rate (MR).This study was exempted from Institutional Review Board approval, as it was a systematic review.We adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines (17).

Study characteristics
Unfortunately, only two prospective studies were retrieved.The other 18 included studies were all retrospective.Meta-analysis was precluded owing to their great heterogeneity.Table 1 showed the basic clinical characteristics of the included studies.These studies were all published between 2014 and 2023, and most (n = 13) published between 2019 and 2023.The geographical spread of these studies was as follows: seven from China, five from the United States, three from India, two from Italy, and one from Japan, Greece, and Latvia, respectively.In the present review, the study population consisted of RRF sufferers using PGT-A, whereas the control population comprised of RRF sufferers not using PGT-A or those without RRF history using PGT-A.RPL/RIF PGT-A refers to RPL/RIF sufferers who underwent PGT-A.RPL/RIF NO PGT-A refers to RPL/RIF sufferers who did not undergo PGT-A.NO RPL/RIF PGT-A refers to patients without a history of RPL/RIF who underwent PGT-A.The reproductive outcomes were presented in Table 2.
It should be noted that mosaic embryos were commonly observed according to the results of PGT-A based on CCS.Mosaicism is defined as the presence of ≥2 cell populations with different chromosomal constitutions within the same embryo (37), which may be contributed by many factors such as mitotic errors, amplification bias, contamination and the PGT-A provider, etc. (38).It was reported that the low-range mosaic embryos (<50%) showed a higher ongoing pregnancy rate and lower miscarriage rate, while a high-range mosaicism detection (>50%) was associated with whole chromosome aneuploidy in a significant proportion of cases.Therefore, cutoff 50% of mosaicism was recommended as a reference in clinical management (39).According to previous literature, the rate of mosaic embryo was  (27).This suggested that mosaicism rate did not always consistent with maternal age.
3.3 PGT-A could optimize the reproductive outcomes in the general population with RRF 3.3.1 Patients using PGT-A had better reproductive outcomes than those not using PGT-A in RRF In 2019, Kim et al. (21) retrospectively reviewed the reproductive outcomes of RPL sufferers undergoing their first single embryo transfer (ET).Results found that RPL sufferers using PGT-A (n = 660) had significantly higher CPR (73 vs. 61%, p = 0.01), LBR (62 vs. 41%, p < 0.01), and reduced clinical pregnancy loss rate (15 vs. 32%, p < 0.01), compared with RPL sufferers not using PGT-A (n = 101).In the same year, Lei et al. (30) reported that RPL sufferers using PGT-A (n = 212) had acquired higher LBRs per cycle start (26.6 vs. 15.4%,p = 0.0004) and transfer (44.9 vs. 25.1%,p < 0.0001), and lower MR (15.7 vs. 34.6%,p = 0.0007) than those not using PGT-A (n = 294).In 2020, retrospective study of Mantravadi et al. (25) reported that the MR (9.68 vs. 23.33%,p = 0.0610) was lower in patients with idiopathic RPL using PGT-A (n = 82) than those not using PGT-A (n = 30), although the LBR and take home baby rates were not significantly different.Also in 2020, Sato et al. ( 20) made a multi-center prospective study which revealed that no significant differences were observed in the LBR and the MR in RPL sufferers given or not given PGT-A.However, PGT-A improved the LBR per embryo transfer in the RPL (52.34) retrospective study revealed that in the RM group, a significant decrease of early pregnancy loss rate (18.1 vs. 75%, p = 0.001) and significant increase in LBRs per transfer (50 vs. 12.5%, p = 0.002)/per patient (36 vs. 12.5%, p = 0.026) were observed in the PGT-A group compared with the non-PGT-A group   ).However, the use of an euploid embryo does not significantly decrease MR (12.4 vs. 12.2%, p = 1.000).

Patients with RRF using PGT-A had no inferior reproductive outcomes than patients without RRF history
In 2019, Kim's (21) above mentioned study revealed that the Cin patients with RPL CPR using PGT-A (n = 660) was comparable (73 vs. 72%, p = 0.01) to that of infertile patients using PGT-A without RPL history (n = 3,975), although the clinical pregnancy loss rate was higher (15 vs. 12%, p < 0.01).In the same year, Wang et al. (23) conducted a retrospective cohort study that enrolled patients who had their first IVF cycle with PGT-A.It revealed that the positive β-HCG (65.9 vs. 68.9%),ongoing pregnancy (43.9 vs. 53.6%),and total pregnancy loss rates (33.3 vs. 21.4%)did not significantly differ in patients with RPL (n = 41) compared with patients without a history of miscarriage (n = 183).Bhatt et al. 's (29) above mentioned study also revealed no difference was observed in the reproductive outcomes between patients with RPL using PGT-A and those with tubal factors.In 2020, Lee et al. ( 22) retrospectively compared the reproductive outcomes in patients who experienced RIF (n = 82), RM (n = 82), and oocyte donors (OD) (n = 45) using PGT-A.Results showed that the LBR were similar among patients RIF, and OD groups (51.6 vs. 55.9 vs. 57.1%).These studies suggested that patients with RRF using PGT-A had comparable reproductive outcomes than patients without RRF history.However, study of Murugappan et al. (19) concluded that PGT-A could not improve the reproductive outcomes of patients with RRF.This retrospective study, which included 112 RPL patients desired who preimplantation genetic screening and 188 patients who chose expectant management (without further examination and treatment), revealed that the rates of CPR, LBR, and MR were similar between the PGT-A and expectant management groups.Moreover, the median time to pregnancy was even longer in the PGT-A group than in the expectant management group (6.5 vs. 3.0 months).However, it should be noted that in this paper patients with expectant management were used as a control, while the control group usually refers to RRF sufferers who do not use PGT-A or patients without RRF history who underwent PGT-A in other papers (40).

PGT-A could improve the reproductive outcomes in all age groups of RRF, especially in the advanced age group
In 2014, Greco et al. (18) conducted a retrospective study to investigate the effects of PGT-A in patients with RIF aged <36 years.The results revealed that the IR (68.3 vs. 22%, p = 0.001) and CPR (68.3 vs. 21.2%,p = 0.001) were significantly increased in the RIF PGT-A group (n = 43) than RIF NO PGT-A group (n = 33).On the other hand, the RIF PGT-A group had similar IR (68.3 vs. 70.5%,p = 1) and CPR (68.3 vs. 70.5%,p = 1), compared with NO RIF PGT-A group.Du et al. (36) also reported that the CPR was significantly increased in RIF PGT-A group (n = 59) compared with RIF NO PGT-A group (n = 119) (71.19 vs. 56.30%,p = 0.039) in RIF patients aged <38 years old.The OPR was also higher in the PGT-A group than the RIF without PGT-A group (55.93 vs. 45.38%p = 0.214), although the difference was not significant.These results indicated that PGT-A could optimize the reproductive outcomes in patents <38 years old.
Keiichi Kato (41) conducted a retrospective study which enrolled 32 patients who underwent PGT-A (18 in the RIF protocol and 14 in the RPL protocol) and 2,556 patients with IVF treatment at the same period for women aged 35-42 years in 2023.Results revealed that RPL patients with PGT-A had acquired increased LBR per ET (80.0 vs. 0, p = 0.005) and reduced MR (20.0 vs. 100.0%,p = 0.0098), compared with RPL sufferers without PGT-A.In the RIF sufferers, the PGT-A group also had better reproductive outcomes with higher LBR per ET [90.0 vs. 69.2%(p = 0.2313)], and lower MR (0 vs. 10.0%,p = 0.3297), although the difference was not significant.In 2021, Tong et al. (17) retrospectively compared the reproductive outcomes in RIF patients who underwent PGT-A between the younger (<38 years) and advanced (>38 years) age groups.Results revealed that there were no significant differences in the IR (39.1 vs. 51.0%),CPR (39.1 vs. 48.0%),and MR (4.3 vs. 7.8%) per ET between the two groups.In the same year, the above mentioned study conducted by Bhatt (30) revealed that in RPL sufferers, the adjusted odds ratio comparing IVF-FET with PGT-A vs. without PGT-A for live birth outcome was 1.31 (95% CI: 1.12, 1.52) for age < 35 24) above mentioned study revealed that the benefits of PGT-A were limited in patients with >2 failed PGT-A cycles (who failed to achieve ongoing pregnancy).In the same year, another retrospective multi-center cohort study by Cozzolino et al. (32) also reported that PGT-A could significantly improve the IR and OPR in the moderate RIF group (>3 implantation failures).However, the IR and OPR were not different in the severe RIF group (>5 implantation failures).Similarly, Ni et al. 's (26) retrospective study concluded that compared with the control group (patients using PGT-A after one spontaneous abortion with abnormal genetic testing results in aborted villus tissues and women with ≤2 IFs and ≤ 1 BPL, n = 103), patients with ≥4 previous early miscarriages (n = 56) had a significantly increased early miscarriage rate (6.58 vs. 31.11%,p < 0.001) and a decreased live birth rate (53.49 vs. 34.18%,p = 0.007) after euploid transfer.These studies demonstrated that PGT-A's effectiveness was limited in patients with multiple pregnancy losses.

Discussion
Overall, our results indicated that PGT-A based on blastocyst biopsy and CCS procedures could optimize the reproductive outcomes of patients with RRF, which could be expected as the incidence of chromosomal abnormalities was higher in these patients.However, it should be noted that PGT-A cannot identify all possible genetic abnormalities or developmental defects.It could not guarantee successful pregnancy, which requires embryo with good quality and endometrial receptivity (42).As known, RPL and RIF are both complex and multifactorial condition.It is critical that RPL or RIF sufferers be properly evaluated to identify all possible causes and treated individually.Furthermore, they should be recommended to have prenatal diagnosis during the pregnancy period (27,35).Their offspring should also have postnatal follow-up (25).In the future, the reproductive outcomes of RRF may be furtherly improved by artificial intelligence which could play a role in the following aspects: ultrasound monitoring of folliculogenesis, endometrial receptivity, embryo selection based on quality and viability, and prediction of post implantation embryo development, etc. (42).
In sub-analysis, we found that PGT-A was effective in RRF patients of any age, especially in patients with advanced aged.This made sense as the aneuploidy rate increased in patients with advanced maternal age in PGT-A cycles (16,17,20).However, it should be noticed that chances of women with advanced age getting blastocysts are less as their ovarian reserve is decreased.Deng et al. (43) reported that in patients with poor ovarian response, PGT-A cycles had less chance to reach embryo transfer compared with those not using PGT-A (13.7 vs. 70.6%,p < 0.001), and no difference were observed in the LBR per oocyte retrieval in cycles using or not using PGT-A (6.6 vs. 5.4%, p = 0.814).31 PGT-A cycles were needed to avoid one clinical miscarriage.Therefore, PGT-A should be cautiously used for the population with advanced maternal age with poor ovarian response.Also, our results revealed that >3 instances of previous RRF or > 2 cycles of PGT-A cycles limited the benefits of PGT-A.This could be explained by the fact that no difference was observed in the prevalence of chromosomal abnormalities in couples with 2 and ≥ 3 pregnancy losses (44, 45), and PGT-A only selected euploidy embryos instead of changing the embryo pool (46).On the other hand, some other factors also affected the successful pregnancy rate other than an aneuploid embryo, such as thrombophilia, immunology, metabolic/ endocrinological abnormalities, and anatomical abnormalities (47).Therefore, the benefits of PGT-A were not obvious in RRF with multiple pregnancy losses.
The primary limitation of this review was the paucity of highquality studies which only included two prospective studies.Second, most studies did not show concomitant factors, such as AMH, previous times of pregnancy loss and other endocrine and immune disorders.As already known, AMH is an independent variable of increased aneuploidy embryo rate (27,48).And previous occurrences of pregnancy loss and other endocrine and immune disorders were also closely associated with reproductive outcomes during the PGT-A cycles.Therefore, a direct comparison was challenging, because of the heterogeneity in patient cohorts of these studies.

Conclusion
Overall, PGT-A was beneficial for patients with RRF, especially in advanced aged patients.However, in patients with decreased ovarian reserve, the benefits of PGT-A may not be obvious as the probability of getting an euploid embryo was lower.In addition, PGT-A may have limited benefits for patients with multiple occurrences of pregnancy loss.

FIGURE 1 The
FIGURE 1The search strategy diagram.

TABLE 1
Basic clinical characteristics of the included studies.