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Table of Contents
ORIGINAL ARTICLE
Year : 2019  |  Volume : 2  |  Issue : 2  |  Page : 66-73

The use of preimplantation genetic testing for aneuploidy and mitochondrial DNA scoring helps in improving assisted reproductive techniques outcome


Department of Reproductive Medicine, Mother and Child Hospital, New Delhi, India

Date of Submission28-Oct-2019
Date of Acceptance01-Nov-2019
Date of Web Publication31-Jan-2020

Correspondence Address:
Dr. Jasneet Kaur
Mother and Child Hospital, D.59 Defence Colony, New Delhi - 110 024
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/tofj.tofj_13_19

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  Abstract 


Background: Aneuploidy is a leading cause of implantation failure and miscarriage. Preimplantation genetic testing for aneuploidy (PGT-A) with mitoscore enables screening of viable euploid embryos, thereby improving in vitro fertilization (IVF) outcome.
Aim of the study: The aim of this study is to determine if the use of PGT-A in patients with valid indications can help improve assisted reproductive techniques outcome.
Materials and Methodology: All patients undergoing IVF-PGT-A cycles between April 2016 and March 2018 were included (n = 42) in the study. Patients were compared with a control group consisting of fresh or frozen blastocyst transfers selected by morphology, during the same period (n = 226). Trophectoderm samples were subjected to chromosome analysis and mitoscore assessment using next-generation sequencing. Single-embryo transfer was done according to transfer priority determined by mitoscore.
Statistical Analysis: The Chi-square test was used for comparisons between the study groups with respect to percentages. A value of P <0.05 was considered as statistically significant.
Results: The indications for PGT-A in our patients were advanced maternal age 33%, followed by recurrent pregnancy loss 25%, recurrent implantation failure 19%, previous history of aneuploidy 16%, and severe male factor 6%. An ongoing pregnancy rate (OPR) of 61% versus 48% (P = 0.0049) was achieved with PGT-A versus controls, respectively. Thirty-two percent of patients did not have any euploid embryos for transfer.
Conclusion: Offering PGT-A with mitoscore for valid indications seems to be an impressive tool to increase implantation and OPRs and helps in counseling patients for further course of treatment.

Keywords: Advanced maternal age, aneuploidy, mitoscore, preimplantation genetic testing, recurrent implantation failure


How to cite this article:
Mahajan N, Kaur J. The use of preimplantation genetic testing for aneuploidy and mitochondrial DNA scoring helps in improving assisted reproductive techniques outcome. Onco Fertil J 2019;2:66-73

How to cite this URL:
Mahajan N, Kaur J. The use of preimplantation genetic testing for aneuploidy and mitochondrial DNA scoring helps in improving assisted reproductive techniques outcome. Onco Fertil J [serial online] 2019 [cited 2020 Oct 26];2:66-73. Available from: https://www.tofjonline.org/text.asp?2019/2/2/66/277440




  Introduction Top


After four decades of in vitro fertilization (IVF), implantation failure still remains a major challenge to assisted reproductive techniques (ART) success. Human implantation is a multifaceted, finely orchestrated event requiring the presence of a healthy embryo, a receptive endometrium, successful embryo-endometrial cross-talk, and maternal immune protection of the allograft. Embryonic factors, particularly karyotype abnormalities are considered to play a prime role in implantation failure and early pregnancy loss. Aneuploid embryos are known to exhibit developmental arrest in vivo, failure to implant and an inability to sustain growth in utero, with majority ending in miscarriage. Transferring euploid embryos after IVF has been shown to improve implantation and pregnancy rates.[1] Unfortunately, embryo morphology does not reflect embryo ploidy, and the detection of karyotype abnormality requires chromosomal analysis of cells obtained through an embryo biopsy either on day 3 or day 5 of development in vitro.

Initially, preimplantation genetic testing for aneuploidy (PGT-A) was performed principally on the cleavage stage (day 3) embryos, one or two blastomeres were biopsied and analyzed with fluorescence in situ hybridization (FISH).[2] The use of FISH for embryo selection diminished when studies showed that these embryos had a higher incidence of mosaicism and a reduced implantation potential post biopsy.[3],[4] Advent of advanced molecular technology and refinements in blastocysts culture led to a renewed interest in PGT-A for embryo selection. PGT-A now involves biopsy of trophectoderm (TE) cells from day 5/6 embryos, followed by the analysis with comprehensive cytogenetic methods such as next-generation sequencing (NGS).[5],[6],[7] The use of NGS can significantly improve the ability to identify aneuploidy embryos and those with mosaic abnormalities or segmental errors.[8],[9],[10]

Transfer of a chromosomally normal embryo of high morphological grade, however, cannot ascertain a positive outcome suggesting the role of additional factors. One factor under evaluation as a biomarker of embryo viability is the mitochondrial (mt) DNA content of the embryo. It is believed that mitochondria in early embryos support embryonic development up until the blastocyst stage.[11] Mitochondrial dysfunction is associated with an increase in mitochondrial proliferation.[12] An increase in mtDNA copy number in early embryos, therefore, is reflective of metabolic stress and a lowered ability to implant. Recent studies have found a correlation between the implantation potential of a euploid embryo and its mitochondrial DNA content.[13],[14] The mitochondrial score (Ms) “MitoScore” is a value that represents the normalized mitochondrial DNA content in euploid embryos. It is the ratio of mtDNA/nuclear (n) DNA and is used as an indicator of the mitochondrial copy number per cell.[15] Studies suggest that the use of NGS for aneuploidy detection along with the use of mitoscore for selecting the best viable embryo improves the implantation rate (IR), decreases miscarriage rate (MR), multiple pregnancy rate (MPR), and decreases time to pregnancy (TTP).[16],[17]

We tried to look at the impact of PGT-A and mitochondrial scoring using “Mitoscore” on IVF outcome in patients of advanced maternal age (AMA), recurrent implantation failure (RIF), recurrent pregnancy loss (RPL), severe male factor (MF), and previous aneuploid pregnancies in our clinic.

Study design

The study involves a retrospective cohort study.

Setting

The present study was conducted in tertiary ART center.


  Materials and Methodology Top


All IVF cycles from April 2016 to March 2018 were reviewed for inclusion. All cycles, in which one or more embryos were available for TE biopsy on day 5/6 and underwent chromosome screening by NGS, were included in the study. The control group consisted of all patients with a fresh or frozen blastocyst transfer during the same period, selected by morphology alone. Mitochondrial scoring was done for all euploid embryos. Indications for PGT-A included RIFs, AMA, RPL, severe MF, and history of aneuploidy in previous pregnancies. The IR, clinical pregnancy rate (CPR), ongoing pregnancy rate (OPR), and MRs were compared between the study and control groups. The groups were also stratified by age for comparison; <35, 35–40, and >40 years.

Stimulation protocol

Gonadotropin-releasing hormone (GnRH) antagonist protocol was followed for IVF in all the patients enrolled in our study. Dose of gonadotropin was decided based on the patient's age, body mass index (BMI), ovarian reserve, and previous response. Ovarian stimulation was done with recombinant follicle-stimulating hormone (follitropin-alfa Gonal-f®, EMD Serono, Inc.,) for the first 5 days followed by Menopur (highly purified HMG-Ferring Pharmaceutical Ltd.). In patients with AMA, a combination of rFSH and Menopur was used in the ratio of 2:1. GnRH antagonist (Cetrolix, Intas Pharmaceuticals Ltd.) was started according to the flexible protocol. Injection human chorionic gonadotropin (hCG) 10,000 IU or triptorelin 0.2 mg s/c was given as ovulation trigger. Oocyte retrieval was performed 34–36 h later under general anesthesia, using transvaginal ultrasound guidance.

Laboratory protocol

ICSI was performed in all cases. Fertilization was assessed 17–20 h after microinjection, and embryo growth was recorded every 24 h. Embryos were cultured using standard incubation and evaluated on day 5, TE biopsy was performed on either day 5 or day 6 depending on embryonic development. A biopsy pipette was used to aspirate three to five cells after perforating the zona pellucida with a few laser pulses. The biopsy specimen was removed with gentle traction and laser pulsation, rinsed in several drops of wash buffer, and then loaded into polymerase chain reaction tubes containing lysis buffer. The specimen was labeled and transferred to the genetic laboratory (Igenomix) for the analysis.

The TE samples obtained were subjected to whole-genome amplification followed by comprehensive chromosome analysis through NGS using the Ion Repro Seq PGS Kit (NGS) (Thermo Fisher Scientific, USA). Data analysis of the sample was performed using the Ion Reporter software that generates a graph representing the copy number variation of the sample analyzed compared to the reference bioinformatics baseline. If no DNA was detected in the cells a re-biopsy was performed if the TE was of good quality.

As per laboratory analysis, an embryo is considered normal when it has no deviations from the reference bioinformatics baseline for any of the 24 chromosomes. An embryo is considered abnormal by the presence of aneuploidy when there are points that are diverted into the upper (gain +) or lower part (loss −) of the graph. The presence of aneuploidies for more than five chromosomes on the same specimen is interpreted as a complex abnormal embryo.

Mitoscore assessment

Mitoscore was assessed in the euploid embryos as per the Igenomix protocol,[15] to determine transfer priority. The ratio of mtDNA/nDNA was classified as the Ms and used as an indicator of the mitochondrial copy number per cell. Blastocysts were divided according to their MitoScore into Mitoscore A (MsA): high implantation potential, Mitoscore B (MsB): average implantation potential, and Mitoscore C (MsC): low implantation potential. All embryos were vitrified 1–2 h postbiopsy and were subsequently thawed before embryo transfer.

Single-embryo transfer (SET) of a euploid embryo was done in all except one case under ultrasound guidance, in accordance with the transfer priority guided by the mitoscore value. In the control group, majority of the patients (97.5%) had two embryos transferred.

Outcome variables assessed

Implantation rate

Implantation rate was defined as the number of gestational sacs observed divided by the number of embryos transferred (expressed as a percentage).

Clinical pregnancy rate

Clinical pregnancy rate is the number of pregnancies (diagnosed by ultrasound visualization of one or more gestational sacs) per 100 initiated embryo transfer cycles.

Biochemical pregnancy rate

Biochemical pregnancy rate is defined as the proportion of cycles resulting in a transient elevation in hCG level in blood or urine, without ultrasound confirmation of a gestational sac per transfer.

Miscarriage rate

Miscarriage rate is defined as a pregnancy failure after a previously documented gestational sac on transvaginal ultrasound divided by the total number of clinical pregnancies.

Live-birth rate/ongoing pregnancy rate

Live-birth rate/ongoing pregnancy rate is defined as the number of cycles resulting in at least one live-born child delivered at >24 weeks' gestation of all transfers performed.

Statistical analysis

All data analyses were performed using the SPSS program for Windows version 21.0. A (SPSS Inc., Chicago, IL, USA). Chi-square test was used for comparisons between the study groups with respect to percentages. P < 0.05 was considered to be statistically significant.


  Results Top


PGT-A was offered to 42 patients. In 26% of patients (11/42), biopsy could not be performed due to poor quality of the embryos (inadequate TE cells) or failure to reach the blastocyst stage. A total of 94 blastocysts generated by 31 patients were biopsied. The mean number of blastocysts per patient available for embryo biopsy in our study group was 3.03.

The most common indication for PGT-A in our patients was AMA (10/31) 33%, followed by RPL (8/31) (26%), RIF (6/31)(19%), previous history of aneuploidy (5/31) (16%), and severe MF (2/31) (6%) [Figure 1]. The mean age of the patients who underwent PGT-A was 35.6 years. Majority 68% (21/31) of the patients undergoing PGT-A were >35 years old, whereas 32% were <35 years. Of 94 embryos biopsied, 45% (42/94) were euploid and underwent mitoscore assessment. Rebiopsy was performed in (1/31) 3.2% of the patients due to no DNA detected and on rebiopsy, the embryo was aneuploid. All patients who conceived had a mitochondrial score (Mt) of 35 or less. In 32% (10/31) of our patients all embryos biopsied were aneuploid, 80% of these patients were above the age of 35 years [Figure 2].
Figure 1: Diagrammatic representation of the indications for performing preimplantation genetic testing for aneuploidy

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Figure 2: Diagrammatic representation of the age distribution of patients with nontransferable embryos

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SET was done in all except 1 patient who elected to have a double embryo transfer. We achieve an IR of 15/18 (83%), a CPR of (15/18) 83% and an OPR of 11/18 (61%). We, however, had a high MR in women >35 years despite performing PGT-A.

An age-wise comparison of implantation and pregnancy rates was made in the study group and control group [Table 1] and [Table 2]. Comparison of IVF outcome in the study and control group revealed a statistically significant improvement in IR and CPR for all ages in the PGT-A mt group [Table 3] and [Figure 3]. In women <35 years, we had an IR of 83% versus 35% (P = 0.0001) and a CPR of 83% versus 61%s (P = 0.005) in the study and control group, respectively. In women aged 35–40 years, we found an IR of 82% versus 33% (P = 0.001) and CPR of 82% versus 53% (P = 0.001) in the study versus control group; similarly in women >40 years, we found the IR of 50% versus 21% (P = 0.001) and CPR of 50% versus 33% (P = 0.0147) in the study versus control groups. We did not find a statistically significant increase in the OPR in women over 40 years in the PGT-A group - 25% versus 17% (P = 0.1649), probably because of low numbers in the study group.
Table 1: Clinical outcomes of cycles in which PGT-A was performed according to their age distribution

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Table 2: Clinical outcomes of cycles (control group) according to their age distribution

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Table 3: Age wise comparison of the IR, PR, OPR and MR in women with and without PGT-A

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Figure 3: Comparison of the assisted reproductive techniques outcome (in %) among the control and preimplantation genetic testing for aneuploidy group

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  Discussion Top


Chromosomal abnormalities arising during fertilization and embryo development are believed to be a major cause of implantation failure and early pregnancy loss. Reports suggest that the selection and transfer of euploid embryos in IVF improves implantation and pregnancy rates and leads to a reduction in miscarriage.[1]

The identification of euploid embryos is an invasive procedure necessitating the removal of a blastomere or TE cells from the embryo for chromosomal analysis. The procedure was initially performed by blastomere biopsy on cleavage stage embryos using the FISH technique, but this technique did not prove to be very efficient due to its inability to screen all the chromosomes.[2],[18],[19] Randomized control trials (RCTs) evaluating FISH gave controversial results, with few studies reporting an increase in live birth while the majority showed no benefit.[2],[3],[4] Improvement in genomics led to the introduction of array-CGH and NGS which allows screening of all 24 chromosomes from TE cells. TE biopsy allows more cells (~5–10 cells) – therefore more DNA – to be taken from the embryo for the analysis, without compromising the inner cell mass (ICM). It avoids the pitfalls associated with FISH and reduces the misdiagnosis rate.[20],[21]

The first studies using comprehensive chromosome screening technologies showed that aneuploidies may occur in any of the 24 chromosomes in preimplantation embryos and not the few chromosomes analyzed using FISH, emphasizing the need to screen all the chromosomes.[18],[21],[22],[23] NGS offers several advantages over array-CGH including enhanced detection of mosaicism in multicellular samples and enhanced detection of partial or segmental aneuploidies,[24],[25],[26] which has led to an improvement in the OPR/live birth rate (LBR) and reduction in biochemical pregnancies.[16],[17]

PGT-A thus would help in enhancing embryo selection and improving IVF outcome in a subset of patients having an increased incidence of numerical chromosome abnormalities, for example, patients with AMA, recurrent miscarriage, RIF, severe MF, and the previous history of aneuploidy. The addition of mitoscore to PGT-A aids in selecting the embryo with the maximum implantation potential,[15] encouraging the use of SET in IVF. SET reduces the morbidity and mortality associated with multiple gestations.[27]

In our study group, embryo selection for transfer was done using PGT-A screening for euploidy and mitochondrial scoring for transfer priority. The most common indication for PGT-A in our patients, was AMA (10/31) 33%, followed by RPL (8/31); (25%), RIF (6/31); (19%), previous history of aneuploidy (5/31); (16%), and severe MF (2/31); (6%). The preimplantation genetic screening (PGS) worldwide survey done across 386 IVF units from 70 countries found that the most common indication for PGS was RIF (32%) followed by RPL (31%) and AMA (27%). A few units (6%) offered PGS to all their patients.

We achieved an overall IR of 15/18 (83%), a CPR of 15/18 (83%) and an OPR of 11/18 (61%), whereas in the control group, we had an IR of 149/439 (34%) (P = 0.0001), CPR 132/226 (58%) (P = 0.0001), and an OPR of 108/226 (48%) (P = 0.049). This improvement is in accordance with studies of Yang et al.[27] and Scott et al.[28] Their studies were done on patients with a favorable prognosis, while ours included patients with RIF, RPL, and AMA. In the RCT conducted by Yang et al. 2012 in “favorable-prognosis” patients, pregnancy rates were significantly higher in the group that underwent PGT-A in comparison to the group, in which embryo selection for transfer was based on morphology alone (69.1% vs. 41.7%) (P = 0.009). Similarly, the RCT by Scott et al. 2013 in patients with “favorable prognosis” reported significantly higher IRs (79.8% vs. 63.2%) (P = 0.002) and delivery rates (84.7% vs. 67.5%) (P = 0.01) in the women who underwent PGT-A versus control group. On the other hand, Dahdouh et al., in 2015, reported that the use of PGS improved embryo selection allowing increase in elective SET practice and sharply declining the MPRs, but the OPRs between PGS and control groups were similar.[29]

On stratification by age, we found an increase in the IR and PR across all the age groups in the study group compared to the control group. In women aged 35–40 years old, we found an IR of 82% versus 33% (P = 0.001) and CPR of 82% versus 53% (P = 0.001), in women >40 years, we found an IR of 50% versus 21% (P = 0.001) and CPR of 50% versus 33% (P = 0.0147) in the study vs. control groups, respectively. Lee et al.[30] also suggested a statistically significant improved IR in women 40–43 years old (50.9% vs. 25.4%) and an improved live birth rate (45.5% vs. 19%) in women aged 38–40 years with the use of PGT-A (P = 0.002). A study by Rubio et al.[31] in women with AMA (38–41 years old) found the LBR to be significantly higher in the PGT-A group (52.9%) in comparison with women, in whom embryos were transferred based on morphology alone 24.2% (P = 0.0002). They also found a lower MR in the PGT-A group (2.7% vs. 39%, P = 0.0007). A study done by Ubaldi et al. 2015[32] in women with AMA having eSET compared ART outcomes in groups with and without PGT-A. A significant increase in the live birth rate per transferred embryo was found in the PGT-A group in comparison to the group, in which embryos were transferred on the basis of morphology alone; 17.0% versus 10.6% (P < 0.01). Kang et al. also reported an increase in CPR and LBR per ET in women over 37 years, but this advantage was lost when calculated per retrieval. The CPR, LBR, or decrease MR in women was not seen in women ≤37 years.[33] About 32% of patients in our study did not reach embryo transfer, 80% of them were >35 years. This is in concordance with Kang et al.'s study, in which they found that 32% of their patients >37 years did not have any euploid embryos. About 68% of their PGT-A patients had a transfer versus 95% in the control group (P = 0.001).

In our study, the MR in women >35 years was high despite having the transfer of euploid embryos; though, this difference was not statistically significant. This could be attributed to associated embryonic developmental errors, uterine factors, or immune factors.

The importance of selecting a euploid embryo with the maximum implantation potential by mitochondrial scoring is highlighted by the PGS web survey which revealed that 30% of chromosomally normal embryos fail to produce an ongoing pregnancy.[34] A study done by Fragouli et al., in 2017,[35] demonstrated that euploid blastocysts of good morphology, but with high mtDNA levels had a greatly reduced implantation potential. mtDNA quantification was done in 199 euploid blastocysts, transfer of single euploid embryo with a normal mitoscore had an OPR of 64%, while PR with elevated mtDNA levels was 0% (P < 0.0001). Diez-Juan et al.[15] also demonstrated that an increased mtDNA in euploid embryos is related to poor implantation potential and may be indicative of reduced metabolic fuel during oocyte maturation and increased “energetic stress” in the embryo. They proposed the use of “MitoScore” to prioritize embryo transfer. They found that Day-5 embryos with <18.19 (MsA) had an IR of 81%; those with 18.19–24.15 (MsB) had an IR of 50%; those with 24.15–50.58 (MsC) had an IR of 62%; and those with levels >50.58 (MsD) had an IR of 18%. Embryos with mt levels >60 never implanted.

Using PGT-A in combination with mitoscore helped us to improve the IVF outcome in patients who did not fall into the “favorable prognosis” group. It also gave us a strong base for counseling those patients with no euploid embryos.

The improved IVF results reported with the use of PGT-A have led to an increase in the use of this procedure, in the US over 20% cycles are PGT-A cycles. Werlin et al. in 2018 reported that MR is two to six times higher in pregnancies from embryos that are not subject to PGT-A.[36] This results in a significantly delayed time to conception, increased emotional stress of multiple embryo transfers, and a high dropout rate. However, one cannot deny that potential disadvantages of the procedure exist and universal use of PGT-A for embryo screening is discouraged. Issues related to cost, increased need for resources, accuracy of a mosaic diagnosis, and thereby the possibility of discarding embryos that may have resulted in healthy babies are some of the limitations of the procedure.[37] TE biopsy does not provide information of ICM and chromosomal mosaicism though low, remains an issue even at the blastocyst stage.[38]


  Conclusion Top


Offering PGT-A with mitoscore to patients for valid indications seems to be an impressive tool to increase implantation and OPRs and decrease TTP. PGT-A also helps in counseling patients, especially those with AMA having nontransferable embryos.

Limitations of our study

Limitations included small sample size.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

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