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 Table of Contents  
Year : 2020  |  Volume : 4  |  Issue : 1  |  Page : 32-41

In vitro fertilization with single-Nucleotide polymorphism microarray-based preimplantation genetic testing for aneuploidy significantly improves clinical outcomes in infertile women with recurrent pregnancy loss: A randomized controlled trial

1 Shanghai Ji Ai Genetics and IVF Institute, Obstetrics and Gynecology Hospital, Fudan University, Shanghai 200011, China
2 Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Obstetrics and Gynecology Hospital, Fudan University, Shanghai 200011, China
3 Shanghai Ji Ai Genetics and IVF Institute; Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Obstetrics and Gynecology Hospital, Fudan University, Shanghai 200011, China

Date of Submission13-Aug-2019
Date of Decision11-Sep-2019
Date of Acceptance29-Nov-2019
Date of Web Publication2-Apr-2020

Correspondence Address:
Xiao-Xi Sun
Shanghai Ji Ai Genetics and IVF Institute, Obstetrics and Gynecology Hospital, Fudan University, No. 588 Fangxie Road, Huangpu District, Shanghai 200011
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/2096-2924.281852

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Objective: To evaluate the effect of preimplantation genetic testing for aneuploidy (PGT-A) in infertile patients with recurrent pregnancy loss (RPL).
Methods: A prospective randomized clinical trial was performed in a university-affiliated fertility center in Shanghai, China. Patients in the PGT-A group underwent blastocyst biopsy followed by single-nucleotide polymorphism microarray-based PGT-A and single euploid blastocyst transfer, whereas patients in the control group underwent routinein vitro fertilization/ICSI procedures and frozen embryo transfer of 1–2 embryos selected according to morphological standards.
Results: Two hundred and seven infertile patients with RPL were included in this study and randomly assigned to either the control or the PGT-A group. Baseline variables and cycle characteristics were comparable between the two groups. The results showed that PGT-A significantly improved the ongoing pregnancy rate (55.34% vs. 29.81%) as well as the live birth rate (48.54% vs. 27.88%) and significantly reduced the miscarriage rate (0.00% vs. 14.42%) on a per-patient analysis. A significant increase in cumulative ongoing pregnancy rates over time was observed in the PGT-A group. Subgroup analysis showed that the significant benefit diminished for patients who attempted ≥2 PGT-A cycles.
Conclusions: PGT-A significantly improved the ongoing pregnancy and live birth rate, while reduced miscarriage rate in infertile RPL patients. However, the significance diminished in patients attempting ≥2 cycles; thus, further studies are warranted to explore the most cost-effective number of attempts in these patients to avoid overuse.

Keywords: Assisted Reproductive Treatment, Clinical Outcomes, Preimplantation Genetic Testing for Aneuploidy, Recurrent Pregnancy Loss

How to cite this article:
Sui YL, Lei CX, Ye JF, Fu J, Zhang S, Li L, Peng XD, Zhang YP, Chen GW, Sun XX. In vitro fertilization with single-Nucleotide polymorphism microarray-based preimplantation genetic testing for aneuploidy significantly improves clinical outcomes in infertile women with recurrent pregnancy loss: A randomized controlled trial. Reprod Dev Med 2020;4:32-41

How to cite this URL:
Sui YL, Lei CX, Ye JF, Fu J, Zhang S, Li L, Peng XD, Zhang YP, Chen GW, Sun XX. In vitro fertilization with single-Nucleotide polymorphism microarray-based preimplantation genetic testing for aneuploidy significantly improves clinical outcomes in infertile women with recurrent pregnancy loss: A randomized controlled trial. Reprod Dev Med [serial online] 2020 [cited 2021 Sep 17];4:32-41. Available from: https://www.repdevmed.org/text.asp?2020/4/1/32/281852

  Introduction Top

According to the guidelines of the Practice Committee of the American Society for Reproductive Medicine (ASRM)[1] and the European Society of Human Reproduction and Embryology,[2] a diagnosis of recurrent pregnancy loss (RPL) can be considered after loss of greater than or equal to two pregnancies, which challenges patients and clinicians both technically and emotionally. RPL affects 1%–5% of women, while higher prevalence is seen in in vitro fertilization (IVF) centers, for these patients often seek the help of assisted reproductive technology (ART).[3]

Present guidelines for the evaluation and management of RPL mainly target known specific causes, while over 40% of RPL remains unexplained and is often treated empirically. Research has shown that embryo aneuploidy is associated with 50%–60% of pregnancy loss cases,[4] particularly in cases of early-stage pregnancy loss that occur within the first 12 weeks of gestation.[5] This has led to the theory that preimplantation genetic testing for aneuploidy (PGT-A) of embryos acquired by ART should improve the clinical outcomes in such patients.

PGT-A, previously preimplantation genetic screening or preimplantation genetic diagnosis of aneuploidy (PGD-A),[2] has been controversial since its inception. This is particularly true after a large-scale randomized clinical trial (RCT)[6] and a meta-analysis [7] showed that PGT-A, which is primarily based on cleavage stage biopsy and fluorescence in situ hybridization (FISH), significantly impaired pregnancy and live birth rates. This finding led to professional societies discouraging its use.[8],[9],[10] Inappropriate time of biopsy, inadequate FISH techniques, as well as incorrect patient selection might have contributed to the failure of PGT-A in the past.[11] However, recent studies showed that the new generation of PGT-A, which relies on blastocyst biopsy followed by efficient and accurate comprehensive chromosomal screening methods, such as array comparative genomic hybridization, polymerase chain reaction (PCR)-based detection, single-nucleotide polymorphism (SNP) microarray analysis, or next-generation sequencing (NGS), may significantly improve clinical outcomes in patients with RPL. These advances in chromosomal screening methods once again aroused interest in PGT-A among clinicians.[12],[13],[14],[15],[16] Since the prevalence of aneuploidy and the number of euploid embryos available for transfer should be considered when choosing the appropriate population, patients with RPL could benefit from the presently used method of PGT-A.

To the best of our knowledge, no well-powered prospective RCT has been performed to compare the clinical outcomes of IVF with or without PGT-A in patients with RPL.[2] Since the standard care for unexplained RPL is expectant management,[17] we consider it is inappropriate to recruit patients with RPL to merely undergo IVF/ICSI treatment without PGT-A. To avoid ethical problems, we included infertile patients with RPL, who were suitable for both IVF/ICSI and IVF/ICSI + PGT-A. The purpose of this study was to evaluate the impact of blastocyst biopsy and SNP microarray-based PGT-A on the clinical outcomes in infertile patients with RPL undergoing IVF.

  Methods Top

Study population and research design

This was a single-center, prospective RCT focusing on patients with RPL who were scheduled for IVF or ICSI between August 1, 2014, and July 31, 2016, in our hospital. All patients were initially diagnosed with RPL according to the guidelines of the Practice Committee of ASRM.[1] Factors contributing to infertility were revealed in subsequent medical evaluations, or patients were diagnosed as infertile after an expectant period of 1 year without pregnancy. After routine evaluation before IVF/ICSI, the first patient was enrolled in the study on August 29, 2014. The inclusion criteria were as follows: (1) normal uterine and adnexal ultrasonography; (2) antiphospholipid antibody (-) and antinuclear antibody (-); and (3) normal parental karyotype. The exclusion criteria were as follows: (1) anatomical factors that may result in infertility or spontaneous abortion, such as uterine malformation, hydrosalpinx without surgery, endometriosis, adenomyosis, submucous myoma or nonsubmucous myoma exceeding 4 cm, and intrauterine adhesions; (2) thyroid dysfunction without treatment or increased levels of CA 125; (3) acute genitourinary inflammation or the presence of any sexually transmitted diseases; and (4) previous pregnancy loss due to chromosomal abnormalities.

Patients who met the inclusion and exclusion criteria and provided voluntary informed consent were randomly allocated to the treatment or control groups prior to ovarian stimulation. A computer-based randomization table was prepared by the principal investigator, and the randomization arms were printed out and placed into sealed, sequentially numbered opaque envelopes. The envelopes were opened on the day that a qualified patient entered an oocyte retrieval (OR) cycle and were used to allocate each participant to the PGT-A or control group. Patients in the PGT-A group underwent blastocyst biopsy followed by SNP microarray-based PGT-A, whereas those in the control group had embryos selected according to morphological standards.

During the study interval, patients were allowed a maximum of 5 ORs and multiple embryo transfers (ETs) until ongoing pregnancy, unless they decided to withdraw on their own accord. Patients with available embryos who had not returned for transfer within 12 months after the last OR were also regarded as having voluntarily withdrawn from the study.

Controlled ovarian hyperstimulation, oocyte retrieval, and embryo culture

An antagonist protocol for controlled ovarian hyperstimulation was employed for all participants. Treatment with recombinant human follicle-stimulating hormone (Gonal-f, Merck Serono, Switzerland) or human menopausal gonadotropin (Livzon Pharmaceutical Group Inc., China) was initiated on the 2nd or 3rd day of the menstrual cycle at a usual starting dose of 150–300 IU/day. Dosage was adjusted for age, body mass index, antral follicle count, follicle-stimulating hormone, anti-Müllerian hormone (AMH), and ovarian reaction in any previous cycles. Serum estradiol (E2), progesterone, and luteinizing hormone levels were measured using an electrochemiluminescent immunoassay, and the follicles were monitored using transvaginal ultrasound starting on the 6th day of gonadotropin administration. An average of 0.25 mg/day gonadotropin-releasing hormone antagonist (Cetrotide, Merck Serono, Switzerland) was administered to the patient when a dominant follicle reached 14 mm in size or serum E2 level reached 350 pg/mL. This treatment continued until the leading follicle reached 18 mm or two follicles reached 16 mm in size. Subsequently, 5,000–10,000 IU of human chorionic gonadotropin (Livzon Pharmaceutical Group Inc., China) was administered as a trigger, and oocytes were retrieved after 36 h. ICSI was performed for all patients in the PGT-A group to avoid contamination, whereas IVF or ICSI was used in the control group based on the male partner's semen quality. Embryos were cultured to blastocyst in the PGT-A group. For patients in the control group, those with ≥4 day-3 embryos (Grade 1 or 2) were offered blastocyst culture with informed consent, whereas this was not mandatory, because the blastocyst culture history of the control patients were also taken into consideration if they had. Zona perforation was performed on all day-3 embryos intended blastocyst culture.

Trophectoderm biopsy and genetic screening

Embryos that developed to the expanded blastocyst stage with differentiation of the inner cell mass and trophectoderm (TE) were selected for biopsy. TE cells herniating through the zona perforation were gently aspired into a biopsy pipette (COOK Ireland Ltd., Limerick, Ireland), and the cells were pulled gently away from the remainder of the blastocyst with laser assist if necessary. The biopsied cells were immediately rinsed in phosphate-buffered saline (PBS), loaded into a PCR tube with 2 μL PBS, and shipped on ice to the genetics laboratory where SNP microarray analysis was performed according to the manufacturer's instructions (Infinium HD Assay Ultra Protocol Guide, Illumina Inc., USA).[18] Blastocysts were vitrified immediately after biopsy.

Embryo transfer protocol, endometrium preparation, and luteal support

Frozen ET (FET) was adopted. Patients in the PGT-A group had single euploid blastocyst transfer (SET), whereas those in the control group had two untested embryos transferred in an ET cycle (DET). For intention-to-treat (ITT) analyses and the calculation of cumulative clinical outcomes (per OR and per patient), the following two special situations of ET were allowed: (1) when patients in the PGT-A group strongly requested the transfer of two euploid blastocysts in an ET cycle after being informed of the risks or (2) when patients in the control group had only one embryo available but no further OR cycle was intended such that a single embryo was transferred.

Endometrium preparation entailed either a natural protocol for participants with regular menstrual cycles or a programed cycle using oral contraceptives and estrogen supplementation for the other participants.

Luteal support with progesterone injection (60 mg/d) was given after the endometrial lining reached 8 mm and serum E2 levels reached 150 pg/mL. Day-3 or day-5 embryos were transferred on the 4th or 6th day of progesterone supplementation, and the procedures were performed with a catheter (Wallace, Smits-Medical, Dublin, Ireland) under ultrasound guidance.

Statistical analysis

Data from our IVF center showed that the ongoing pregnancy rate in patients with RPL after IVF/ICSI was around 30%. According to our power analysis, we had to enroll 95 women in both treatment groups to detect an absolute increase of 20% in the ongoing pregnancy rate with the use of PGT-A (power = 80%). The threshold for statistical significance was set at 0.05.

The primary outcome was ongoing pregnancy, which was defined as a viable intrauterine pregnancy 12 weeks after ET. Implantation, clinical pregnancy, live birth, miscarriage, and time to pregnancy were included as secondary outcomes. Time to pregnancy was calculated from the first OR cycle until the ET day of a later assured ongoing pregnancy. Per-patient, per-OR, and per-ET cycles were analyzed separately, and all outcomes were assessed according to the ITT principle. Cycles were recorded as “failed” when no embryo was available for transfer after OR.

Continuous variables were assessed for normality of distribution using the Shapiro–Wilk test and were presented as the mean ± standard deviation or median (interquartile range). Results were compared using Student's t-test or the Mann–Whitney U-test. Categorical variables were shown as counts (percentages), and the Chi-squared and Fisher's exact tests were applied as appropriate. The cumulative ongoing pregnancy rates in the PGT-A and control groups were plotted using Kaplan–Meier curves and compared using the Breslow test. Logistic regression analyses were used to assess the associations between PGT-A treatment and clinical outcomes. Effects were presented as relative risk (RR) with 95% confidence interval (95% CI). Potential confounding variables among basal and cycle characteristics, including age, AMH level, duration of infertility, previous ovarian stimulation cycles, number of embryos vitrified, and the number and stage of embryos transferred, were evaluated by multivariate logistic regressions for pregnancy outcomes per patient.[19],[20],[21] Covariates were retained in the final adjusted models if they were significantly associated with outcome parameters (P < 0.05). Statistical analysis was performed with SPSS version 19.0 software (IBM Corporation, NY, USA) and a two-sided P < 0.05 was considered statistically significant.

Ethics approval and study registration

This study was approved by the Ethics Committee of the Shanghai Jiai Genetics and IVF Institute and registered at www.clinicaltrials.gov with the registration ID number NCT02223221. Written informed consent was obtained from each couple after counseling regarding PGT-A.

  Results Top

Participant flow

A total of 286 patients with RPL at our clinic were screened for participation during the study period, among whom 207 agreed to participate and were randomly assigned to the PGT-A and control groups (103 and 104 patients, respectively). The CONSORT diagram of participant flow is shown in [Figure 1].
Figure 1: CONSORT 2010 flow diagram

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Baseline and cycle characteristics

No patient had PGT-A cycles before allocation. There were no significant differences in baseline variables between the two groups [Table 1] and [Table 2].
Table 1: Comparisons of baseline characteristics between PGT-A group and control group

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Table 2: Comparisons of cycle characteristics between PGT-A group and control group

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In the control group, 42 patients underwent day-3 ET, and 62 patients underwent blastocyst transfer. Because of the reasons listed in the “ET Protocol,” 12 patients in the PGT-A group underwent 19 DET cycles, whereas 20 patients in the control group underwent 27 SET cycles [Table 1] and [Table 2]. Patients and cycles that were not following the transfer protocol were included in the outcome analysis for the ITT principle, but the results were adjusted by statistical methods [Table 3].
Table 3: Comparison of the clinical outcomes of PGT-A and control groups

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Outcome analysis

Clinical outcomes of the PGT-A and control groups are shown in [Table 3]. Patients in the PGT-A group showed significantly higher implantation, clinical pregnancy (per ET), ongoing pregnancy (per ET, per OR, and per patient), and live birth rates (per ET, per OR, and per patient) compared with patients in the control group. For patients who achieved ongoing pregnancy, the median time to pregnancy was not significantly different between the two groups [Table 3]. However, Kaplan–Meier analysis showed that patients in the PGT-A group had achieved higher cumulative pregnancy rates in a significantly shorter time interval [Figure 2], P = 0.001].
Figure 2: The cumulative ongoing pregnancy rate over time

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Subgroup analysis according to the stage of embryos transferred showed that patients in the PGT-A group had significantly improved implantation rate, ongoing pregnancy rate (per ET), live birth rate (per ET), and decreased miscarriage rate (per ET, per OR, and per patient) when compared with patients in the control day-5 subgroup. However, the clinical outcomes showed no significant difference between the day-3 and day-5 subgroups among the control patients [Table 3].

Multivariate logistic regression models were applied to adjust for the different transfer methods and the potential confounding variables among basal and cycle characteristics, and the results showed significantly improved clinical pregnancy rate (adjusted RR [aRR]: 3.64 [1.49, 8.88], P = 0.00), ongoing pregnancy rate (aRR: 6.20 [2.50, 15.40], P = 0.000), and live birth rate (aRR: 4.90 [2.00, 11.92], P = 0.00) in the PGT-A group compared with that in the control group [Table 3].

Besides the embryo stage and the number of embryos transferred per cycle, other potential confounding factors, including age, AMH, duration of infertility, previous ovarian stimulation cycles, and the number of embryos vitrified,[19],[20],[21] were also evaluated using multivariate logistic regressions for pregnancy outcomes calculated by per patient. Following adjustments, the results showed that PGT-A was an independent factor that significantly improved clinical pregnancy rates (aRR: 2.28 [1.17, 4.44], P = 0.02), ongoing pregnancy rate (aRR: 4.62 [2.34, 9.12], P = 0.00), and live birth rate (RR: 3.60 [1.81, 7.13], P = 0.00) [Table 3].

For patients undergoing their first attempt after allocation, significantly improved ongoing pregnancy and live birth rates and significantly decreased miscarriage rate were displayed in the PGT-A group [Figure 3]a. However, for patients who failed to achieve ongoing pregnancy after the first OR cycle and attempted two or more cycles, no significantly improved clinical outcomes were observed [Figure 3]b.
Figure 3: (a) Subgroup analysis: comparisons of pregnancy outcomes (per patient) between the two groups of patients undergoing (a) the first attempt (*P < 0.001, - P = 0.005) and (b) two or more attempts after allocation (*P > 0.05)

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  Discussion Top

To the best of our knowledge, this is the first RCT to provide class I evidence of the beneficial effect of PGT-A on ongoing pregnancy and live birth while avoiding recurrent miscarriage among infertile patients with RPL. In addition, PGT-A improved the efficacy of IVF/ICSI in infertile patients with RPL, with a shorter time interval to achieve a higher cumulative pregnancy rate. However, the significant benefit diminished when patients underwent two or more OR cycles.

Improved clinical outcomes after preimplantation genetic testing for aneuploidy

According to a worldwide survey,[22] most respondents believe that PGT-A may increase live births among patients with RPL despite the ongoing debate and lack of robust evidence. In contrast, there are researchers who believe that caution should be exercised against the premature introduction of PGT-A.[11],[23] Some experts are of the opinion that PGT-A, as a method of diagnosis, would not change the ongoing pregnancy and live birth rate per OR cycle or per patient. This is because a freeze-all-transfer-all protocol could achieve the highest pregnancy rate once OR and fertilization are completed.[24] A virtual trial shows that PGT-A, whether by avoiding potentially aneuploid embryos or merely ranking potentially diploid embryos to be transferred first, is not expected to be superior for live birth when every available embryo is considered. Furthermore, the premature termination of a clinical trial is likely to be biased in favor of genetic testing.[25] Among patients with RPL who are carriers of chromosomal abnormalities, the use of PGD is theoretically beneficial. However, a recent systematic review demonstrates similar LBR, time to subsequent conception, and miscarriage rates through natural conception and IVF-PGD.[26] We believe that the result of a full cycle clinical trial should be more convincing than any theoretical inference.

Our research allowed multiple attempts (with a maximum of five OR cycles) and transfers of all available embryos until ongoing pregnancy unless the patients withdrew on their own accord, which is similar to what is observed with the use of PGT-A in a clinical setting. Furthermore, we did not limit the age of patients with RPL, and randomization was achieved before ovarian stimulation to avoid the inclusion of only those patients with a good prognosis who reached blastocyst biopsy and/or ET. This ensured that all possible cycle outcomes were captured, and the true efficacy of PGT-A in infertile patients with RPL could be determined.

Since not all patients had a good prognosis, we did not restrict control patients to blastocyst culture or SET to acquire the best result of IVF. This may be a potential weakness of this study. We have also noticed that the embryo stage (day 3 or 5) and the number of embryos transferred (1 or 2) could potentially confound our study, so we have tried to account for these using subgroup analyses and logistic regressions. The clinical outcomes favored the use of PGT-A when compared with day-5 subgroup of the controls. Meanwhile, analysis with multivariate logistic models also showed significantly improved clinical outcomes in the PGT-A group with all listed potential confounders adjusted, which reflected the true clinical effect of PGT-A on RPL.

We cautiously speculated the reasons behind the improved outcomes calculated per OR and per patient in the PGT-A group:[15],[16] (1) adverse clinical outcomes in the control group, such as miscarriage or implantation failure, had adverse psychological impacts on control patients; (2) recurrent intrauterine operations for abortion impaired endometrial receptivity, which reduced the probability of pregnancy even if euploid embryos were transplanted in subsequent ET cycles; and (3) an aneuploid embryo interfered with the implantation potential of a euploid embryo when both embryos were transferred together. However, these conjectures need to be confirmed by future studies.

For patients who failed to reach ongoing pregnancy after the first OR and attempted greater than or equal to two cycles, the PGT-A group showed improved pregnancy outcomes, but without statistical significance [Figure 3]b. Among patients who underwent 4 or 5 OR cycles, PGT-A did not help with achieving ongoing pregnancy [Figure 1]. This is a reasonable finding since RPL is a multifactorial disorder influenced by anatomical, endocrine, immunological, and thrombophilic factors besides embryo euploidy, suggesting that these patients might have factors other than aneuploidy that influence pregnancy outcomes and cannot benefit from repeated PGT-A cycles. Future studies are warranted to explore the potential etiology of RPL in these patients, as well as the best treatment strategies. Furthermore, the effect of PGT-A diminished with increased cycles, so further studies are expected to explore the most cost-effective number of attempts.

Relatively high failed-cycle rate after preimplantation genetic testing for aneuploidy

Although improved clinical outcomes were seen after PGT-A, the high proportion of cycles (50.27%, 92/183) and patients (24.27%, 25/103) who underwent PGT-A but failed to reach transfer cannot be ignored. While the purpose of PGT-A is to select euploid embryos, the culture-to-blastocyst transition may cause the loss of euploid embryos that cannot develop to day-5 in vitro. Meanwhile, biopsy and vitrification may potentially damage the embryos, resulting in decreased efficacy of OR cycles and aggravated financial burden for patients. Counseling patients with RPL regarding the potential treatment options should include sharing the success rates with PGT-A per euploid embryo transferred as well as information about the number of embryos transferred per cycle.[27] Therefore, a model to predict the possibility of failed cycles is needed to improve the effectiveness and avoid overuse of PGT-A.

Preimplantation genetic testing for aneuploidy and elective single embryo transfer

Multiple pregnancies increase the risk of miscarriage, premature birth, stillbirth, and complications of pregnancy and is one of the adverse outcomes of ART.[28] According to the guidelines by the ASRM, transfer of a euploid embryo has the most favorable prognosis and should be limited to one in patients of any age.[29] However, the practice of eSET is often neglected in IVF clinics since infertile patients often volunteer to take the risks of multiple pregnancies and clinicians are willing to transfer multiple embryos with the hope that there will be a higher chance of pregnancy.[30],[31],[32]

Similar to the results of other studies, the implantation rate of embryos was significantly higher in the PGT-A group (48.51%)[12],[16],[33] than in the control group. Meanwhile, patients in the PGT-A group showed significantly higher ongoing pregnancy and live birth rates despite fewer numbers of transferred embryos. Twin pregnancy rates shown in [Table 4] were not significantly different between two groups, that were result from 19 DET cycles in the PGT-A group and 129 DET in the control group, again proving the high implantation rate with PGT-A. Therefore, we can confidently adopt PGT-A followed by eSET to improve the chance of delivering a healthy term singleton without compromising the overall pregnancy and delivery rates in this population.
Table 4: Comparisons of the neonatal outcomes of PGT-A and control groups

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Controversy and limitations of preimplantation genetic testing for aneuploidy practice

Blastocyst culture, embryo biopsy, and vitrification may lead to epigenetic alterations, imprint interferences,[34],[35] or preterm delivery.[36],[37] The epigenetic influence of PGT-A was beyond the scope of this research, and the increased risk of PGT-A on preterm delivery was not confirmed in our study [Table 4]. Studies have shown that FET has clinical results similar to those with fresh ET.[38],[39] Meanwhile, significantly improved live birth rate and time to pregnancy in the PGT-A group confirmed its safety and efficiency.

As a clinical diagnostic test, misdiagnosis with PGT-A might result in the transfer of chromosomally abnormal or aneuploid embryos. Because of the technical difficulty in handling delicate cells and the fact that the cells can be tested only once, PGT-A practice places a high demand on clinicians working in embryological and genetic laboratories. However, one study observed highly consistent and reproducible laboratory and clinical outcomes when all of the embryologists received identical training with similar equipment.[40] A limitation reported with the use of microarrays is the inability to correctly identify copy number in cases of consanguinity.[41] In our IVF center, amniocentesis were performed on all patients who got pregnant after PGT-A, and the results of amniocentesis were in full accord with PGT-A analyses, showing high accuracy of the technique.

In addition to the technical challenge, mosaicism adds further complexity to the diagnosis using PGT-A and the transfer strategy due to the following reasons: (1) embryos diagnosed as aneuploid might be discarded, even though the inner cell mass were completely or mainly euploid, thus yielding a false-positive diagnosis; (2) some embryos diagnosed as euploid could be mosaic and possibly affect their implantation potential; (3) the majority of mosaic blastocysts fail to implant or miscarriage, but a minority produce viable pregnancies and live births, which is of particular importance for women with poor prognoses having difficulty in obtaining euploid blastocysts.[42],[43],[44] In our study, the adopted SNP microarray platform could detect mosaicism when >30% of TE biopsy samples were aneuploid, and these mosaic blastocysts were not considered for transfer. However, healthy babies born after intrauterine transfer of mosaic blastocysts challenged the theoretical basis of PGT-A.[45] The Preimplantation Genetic Diagnosis International Society, with limited data available, released guidelines regarding the effect of mosaicism on PGT-A. These guidelines suggested a biopsy of five cells at the blastocyst stage, followed by validated NGS-based PGT-A to improve the diagnostic accuracy. These guidelines also reported that at least 20%–80% of aneuploid cells had mosaicism. The guidelines also gave advice regarding the transfer of mosaic embryos,[46] while appealing for research on the prevalence of mosaicism in preimplantation embryos, the proportion of aneuploid cells, and the impact of abnormal chromosomes on future development.

Finally, PGT-A improves clinical outcomes and efficiency by providing supplemental genetic information improves ovarian function to produce euploid embryos. Emerging technologies such as autologous mitochondrial transfer or cytoplasm transfer may be useful but are still under investigation and remain the subject of ethical disputes.[47]

In conclusion, SNP microarray-based PGT-A significantly improved clinical outcomes in infertile patients with RPL. However, the benefit was not significant for patients who had undergone two or more PGT-A cycles. Further studies are warranted to explore the effect of multiple attempts using PGT-A and to develop a model to predict the probability of acquiring euploid blastocysts such that the doctors and patients can have appropriate anticipation when intending a PGT-A cycle and to prevent overuse of this technique.

Financial support and sponsorship

Shanghai Shenkang Hospital Development Center Program (SHDC12017105).

Conflicts of interest

There are no conflicts of interest.

  References Top

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  [Table 1], [Table 2], [Table 3], [Table 4]

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