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Year : 2019  |  Volume : 2  |  Issue : 2  |  Page : 74-78

Preimplantation genetic testing for aneuploidy and subsequent blastocyst transfer improves pregnancy outcome in advanced maternal age patients

Reproductive Medicine Unit, Ferticity Fertility Clinics, New Delhi, India

Date of Submission05-Nov-2019
Date of Acceptance14-Nov-2019
Date of Web Publication31-Jan-2020

Correspondence Address:
Dr. Surleen Kaur
Ferticity Fertility Clinics, 12, Navjeevan Vihar, Malviya Nagar, Delhi - 110 017
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/tofj.tofj_14_19

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Context: Advanced maternal age (AMA) is an important parameter that negatively influences the pregnancy rate in assisted reproductive technology because the rate of aneuploidy in the embryos increases with the patient age.
Aims: The aim of this study is to evaluate whether the transfer of an euploid embryo selected by preimplantation genetic testing for aneuploidy (PGT-A) would improve the pregnancy outcome in AMA patients.
Settings and Design: This was a retrospective controlled study.
Materials and Methods: A total of 250 patients who had undergone frozen transfers of day 5/6 blastocysts were recruited. Patients who had morphologically selected euploid blastocyst transfer were grouped under PGT-A (n = 47) and patients who had only morphologically selected blastocyst transfer were grouped under non-PGT-A (n = 203). The patients were further grouped as test group (AMA patients, ≥35 years of age) and control group (young patients, <35 years of age) in PGT-A and non-PGT-A arm. The comparison was performed between all of the groups. The primary outcome measure was serum beta-human chorionic gonadotropin level for pregnancy confirmation after 14 days of embryo transfer.
Statistical Analysis Used: The Chi-square test was used to analyze pregnancy outcomes.
Results: Overall pregnancy rate was found to be significantly increased in the PGT-A group in comparison to the non-PGT-A group irrespective of the age (78.5% vs. 59.7%, P = 0.00001). No significant difference were observed in Young versus AMA group of PGT-A arm (79.2% vs. 77.8%, P = 0.62). Interestingly, the study showed a significant increase in the pregnancy outcome of the AMA patients of PGT-A group (77.8% vs. 57.8%, P = 0.002) as compared to the non-PGT-A group.
Conclusions:> The present study demonstrates that the transfer of PGT-A-selected euploid embryo significantly improves the pregnancy outcome in AMA patients.

Keywords: Advanced maternal age, assisted reproduction technology, frozen embryo transfer, preimplantation genetic testing of aneuploidy (PGT-A), trophectoderm biopsy

How to cite this article:
Rawat B, Kaur S. Preimplantation genetic testing for aneuploidy and subsequent blastocyst transfer improves pregnancy outcome in advanced maternal age patients. Onco Fertil J 2019;2:74-8

How to cite this URL:
Rawat B, Kaur S. Preimplantation genetic testing for aneuploidy and subsequent blastocyst transfer improves pregnancy outcome in advanced maternal age patients. Onco Fertil J [serial online] 2019 [cited 2021 Jun 16];2:74-8. Available from: https://www.tofjonline.org/text.asp?2019/2/2/74/277441

  Introduction Top

Advanced maternal age (AMA) is increasingly becoming a critical social as well as a clinical issue as a larger proportion of women are delaying childbirth. Such delays may be associated with increased women empowerment, highly effective contraceptive strategies, change in lifestyle as well as the growing popularity of assisted reproductive technologies (ART). This increasing trend may pose as a challenge for fertility specialists as an increasing number of women seeking pregnancy are older than 35 years which is the cutoff to consider a patient of AMA.[1] The 35-year age limit is mainly based on the high embryo aneuploidy rate which increases dramatically from a baseline of 30%–90% in women in their late 30s to late 40s.[2] High aneuploidy rate of the embryo may be due to the gradual depletion of the ovarian reserve (OR) and/or alteration of processes such as dysfunctional cohesins, reduced stringency of spindle assembly checkpoint, shortening of telomeres, and impaired mitochondrial metabolic activity, thus modulating the competence level of the oocytes/embryos.[3],[4],[5],[6],[7],[8] Hence, the management of AMA must be based on the correct evaluation of woman's OR as well as the screening of aneuploid embryos which may be the reasons for not being able to conceive.

Another challenge in AMA women, who are at high risk of maternal morbidity and mortality, is to achieve a singleton pregnancy under the ART management. This can be ensured by transferring single blastocyst as they drastically lower miscarriage rate and limit multiple pregnancy rates.[9] However, since the rate of aneuploidy in embryos increases with an increase in maternal age, achieving embryo implantation and pregnancy rate may get challenging, as transferring such embryos may result in spontaneous abortions. In view of this, Preimplantation genetic testing for aneuploidy (PGT-A) is a tool for embryo testing and has led to an overall improvement in clinical outcomes by screening and identifying aneuploid/euploid embryos in a cohort produced during an in vitro fertilization (IVF) cycle.[10] One of the cited indications for PGT-A includes patients with AMA (≥35 years).[10] Overall, in AMA women, a higher clinical pregnancy rate as well as live birth rate was observed in cases with a day-5 biopsy with subsequent transfer (50%–60%) as compared to day-3 biopsy and a subsequent transfer (24%–35%).[11] However, the main limitation for PGT-A is chromosomal mosaicism, which may occur due to the error in chromosomal segregation postfertilization. As a result, PGT-A-detected euploid embryos may result in implantation failure or spontaneous abortions. Therefore, it is essential to evaluate the clinical outcomes after the transfer of PGT-Aselected euploid blastocyst compared to the non-PGT-A blastocyst transfer in AMA women.

Aim of the study

The primary objective of this study is to investigate whether the transfer of PGT-A-selected euploid blastocyst improves the pregnancy outcome in IVF cycles for patients with AMA.

  Materials and Methods Top

Type of study

This was a retrospective controlled study.

Inclusion criteria

Patients with only frozen embryo transfer (FET) were included in this study. Further, all of the PGT-A cases in which one or more euploid embryos available for transfer were included in the study. The informed consent was obtained from the patient to conduct and analyze the data for this study.

Exclusion criteria

Patients with all abnormal PGT-A embryos and male patients diagnosed with azoospermia or undergone any surgical sperm retrieval were excluded from the study.


The stimulation protocol was followed by downregulation with GnRH antagonist (Cetrolix, Intas Pharmaceuticals Ltd, India) protocol depending on patient's age, body mass index, OR, and previous response. In patients with AMA, a combination of recombinant follicle-stimulating hormone and Menopur was given for 10–12 days to stimulate the ovaries to produce enough numbers of the oocyte. The follicular development was monitored by transvaginal sonography along with estradiol levels. Once the follicles reach to the size of 18–22 mm, the human chorionic gonadotropin (hCG) 10,000 IU was administered. Oocyte retrieval was performed 34–36 h of hCG trigger under general anesthesia.

The oocyte retrieval was done with the help of transvaginal ultrasonography under general anesthesia by suction. Follicular fluid was screened under stereo zoom microscope. The cumulus–oocyte complex was transferred to G-IVF, fertilization media (Vitrolife, Kungsbacka, Sweden) for culture at 37°C and 6% CO2 for 3–4 h. Oocytes were then denuded with the use of enzymatic (Hyase-10x; Vitrolife, Kungsbacka, Sweden) and mechanical processes. All the mature oocytes (metaphase II) were used for intracytoplasmic sperm injection (ICSI). The semen sample was obtained on the day of oocyte retrieval. Semen samples were collected from all the patients through masturbation and processing was done by double density gradient. The morphologically normal spermatozoa were selected for the ICSI. Microinjected mature oocytes were incubated in droplets of the medium under culture oil in a  Petri dish More Details at 37°C and 6% CO2 and 5% O2. Fertilization was observed 16–18 h postinjection. Normal fertilization was confirmed by the presence of two distinct pronuclei and two polar bodies. Embryos were cultured using the standard incubation protocol. The embryo assessment was performed on day 5/6, and only Grade I fully expanded or hatching blastocysts were selected either for vitrification in the case of non-PGT-A group or for biopsy followed by vitrification in the case of PGT-A group. The biopsy was performed once enough number of trophectoderm (TE) cells herniated from the blastocyst. Five to eight cells of herniated TE were aspirated using biopsy micropipette (Origio, Denmark). The biopsy specimen was removed by either “pulling” method or either “flicking” method gently by giving laser pulsation on the joints of TE cells. The biopsied specimen was rinsed in several drops of wash buffer to prevent DNA contamination and then loaded into polymerase chain reaction (PCR) tubes. The PCR tubes were then stored and transported to the genetic laboratory at −20°C for testing. Collapsed blastocysts after biopsy were incubated for expansion and were then vitrified using kitazato vitrification kit (Kitazato BioPharma, Tokyo, Japan) and were stored in cryotops in liquid nitrogen.

For subsequent FET cycles, endometrium was prepared either by giving estrogen supplements to the patient. After obtaining adequate endometrial thickness, progesterone was started and FET was planned. On the day of transfer, thawing was done during early morning using kitazato thawing kit (Kitazato BioPharma, Tokyo, Japan). Blastocysts were incubated for 2 h before transfer to check the survival. One to two frozen-thawed blastocysts were transferred to the patient using a Cook catheter (Sydney IVF Embryo Transfer Set, Cook, Bloomington, IN, USA). Fourteen days later, the patient underwent urine pregnancy test and pregnancy was determined based on serum β-hCG level. The pregnancy rate was calculated based on serum β-hCG level (>25 mIU/mL) and were compared between the groups.


Statistical analysis was performed using Statistical Package for the Social Sciences version 21.0.A (SPSS Inc., Chicago, IL, USA). Variables are presented as absolute numbers and percentage. The Chi-square test was used to analyze the pregnancy outcome. Differences were considered significant at P< 0.05.

  Results Top

In this study, a total of 250 numbers of patients were recruited. All these patients met the inclusion criteria. Of 250, the number of patients who had undergone biopsy followed by euploid blastocyst transfer were grouped under PGT-A (n = 47), and the rest of the patients where the blastocyst transfer was performed on the basis of morphology alone were grouped under non-PGT-A (n = 203). The test group of a study includes AMA patients with ≥35 years of age (n = 104), and the control group includes young patients with <35 years of age (n = 146). The AMA test group and young control group patients were further divided under non-PGT-A and PGT-A arm, and the comparison was performed between the groups [Figure 1].
Figure 1: Flow chart representing the number of patients (n) enrolled in each group

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The overall pregnancy rate was found to be significantly increased in the PGT-A arm as compared to the non-PGT-A arm, irrespective of the age (78.5% vs. 59.7%, P = 0.00001) [Figure 2]. This indicates that the transfer of PGT-A-selected euploid embryo increases the overall pregnancy outcome. There is a significant decrease in the overall pregnancy rate of AMA group in comparison to the control group where outcomes of both PGT-A-selected euploid and non-PGT-A embryo transfers were analyzed (67.8% vs. 70.4%, P = 0.0215) [Figure 3]. Further, when the comparison was made between the age groups, the results revealed significant difference in Control versus AMA group of non-PGT-A arm (61.5% vs. 57.8%, P = 0.003), and no significant difference was observed in Control versus AMA group of PGT-A arm (79.2% vs. 77.8%, P = 0.62) which indicates that PGT-A improves the rate of pregnancy in AMA group to the extent that is comparable to the young age group [Figure 4]. As expected, improved rate of pregnancy was observed in the control group of PGT-A and non-PGT-A arm (79.2% vs. 61.5%, P = 0.00001) [Figure 4]. Interestingly, study showed a significant increase in pregnancy outcome of the main group of the study that is AMA group under PGT-A arm in comparison to the non-PGT-A arm (77.8% vs. 57.8%, P = 0.002) [Figure 4].
Figure 2: Comparison of overall pregnancy outcome in non-PGT-A and PGT-A groups irrespective of the patient age

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Figure 3: Comparison of overall pregnancy outcome in control (young) and advanced maternal age patients

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Figure 4: Comparison of pregnancy outcome between control versus advanced maternal age group of patients in non-PGT-A and PGT-A arm

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  Discussion Top

The present study shows that there is a significant increase in the pregnancy rate in AMA patients following the transfer of PGT-A-selected euploid blastocyst as compared to the AMA patients where non-PGT-A blastocysts that were selected only on the basis of morphology alone were transferred.

Blastocyst biopsy for PGT-A provides enough information whether the embryo is euploid or aneuploid by assessing the genetic makeover of an embryo. PGT-A plays a crucial role in selecting those embryos for transfer which have a normal karyotype and hence a higher capability of implantation. Recently, Capalbo et al. demonstrated that blastocyst biopsy with array comparative genomic hybridization is a reliable method for detecting euploid embryos for transfer and proved that PGT-A is a reliable tool for selecting euploid embryos, especially for AMA patients.[12] Another study conducted by Schoolcraft and Katz-Jaffe assessed the outcome of comprehensive chromosome screening (CCS)-based PGT-A versus conventional morphology-based selection in AMA women undergoing single blastocyst transfer.[13] This study revealed that ongoing pregnancy rate of the CCS group was significantly higher than for the morphology-based group (60.0% vs. 43.8%, P< 0.05), highlighting the importance of PGT-A in AMA patients.[13] Rubio et al. demonstrated a significant increase in live birth rates of the AMA patients in the preimplantation genetic screening (PGS) group when day-3 biopsy followed by blastocyst transfer in comparison with the non-PGS group.[14] Further, there are studies that have emphasized the significance of TE biopsy, followed by single-embryo transfer in AMA patients.[15],[16] However, there are few studies comparing the live birth rates in AMA patients with TE biopsy followed by euploid embryo transfer. In this regard, Lee et al. have supported that the application of TE biopsy and PGT-A could improve the live birth rate in women aged 40–43 years.[17] In the present study, TE biopsy with PGT-A appeared to be associated with an increased pregnancy outcome in AMA patients in terms of serum β-hCG levels; however, differences in implantation rates and live birth rates were not analyzed.

The ultimate goal of an ART treatment is to accomplish healthy live birth, followed by transferring one or two euploid embryos. To accomplish this goal, we require a tool to select a euploid embryo from the cohort of the same to minimize the chance of transferring an aneuploid embryo. PGT-A may prove to be such a tool which would aid in accomplishing high pregnancy rates and reduced miscarriage rate even in cases of single-embryo transfers, thereby avoiding health risk associated with multiple gestational pregnancies. Conducting PGT-A in AMA women may also reduce the number of IVF cycles required to accomplish a positive clinical outcome and potentially reduce the time to pregnancy as well as the cost of extra cycles. However, PGT-A has some limitations, such as testing for aneuploidy of blastocysts is time-consuming, requiring blastocyst development, cryopreservation of biopsied blastocyst, and embryo transfer in the next cycle. Moreover, not all the embryos of the cohort may be examined as the procedure is invasive and expensive. Furthermore, most of the AMA women experience a decline in oocyte numbers; hence, it becomes difficult to cultivate embryos up to the blastocyst stage to perform PGT-A.[18] Due to these factors, the application of PGT-A is still debatable, and therefore, assessment of the necessity and appropriate counseling of the patients is of utmost importance.

  Conclusions Top

In conclusion, we found that PGT-A improved the likelihood of a successful pregnancy rate compared with conventional morphology-based embryo selection. The benefits of PGT-A in AMA patients seem to outnumber the putative limitations, and currently, it is the only efficient strategy that might limit the age-related reproductive risks in these women. To further understand the implications of applying PGT-A in AMA women, more studies with the evaluation of implantation rate and live birth in larger sample sizes are required.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

Mills M, Rindfuss RR, McDonald P, te Velde E, ESHRE Reproduction and Society Task Force. Why do people postpone parenthood? Reasons and social policy incentives. Hum Reprod Update 2011;17:848-60.  Back to cited text no. 1
Franasiak JM, Forman EJ, Hong KH, Werner MD, Upham KM, Treff NR, et al. The nature of aneuploidy with increasing age of the female partner: A review of 15,169 consecutive trophectoderm biopsies evaluated with comprehensive chromosomal screening. Fertil Steril 2014;101:656-30.  Back to cited text no. 2
Cheng JM, Liu YX. Age-Related Loss of Cohesion: Causes and Effects. Int J Mol Sci 2017;18. pii: E1578.  Back to cited text no. 3
Capalbo A, Hoffmann ER, Cimadomo D, Ubaldi FM, Rienzi L. Human female meiosis revised: New insights into the mechanisms of chromosome segregation and aneuploidies from advanced genomics and time-lapse imaging. Hum Reprod Update 2017;23:706-22.  Back to cited text no. 4
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de Lange T. How telomeres solve the end-protection problem. Science 2009;326:948-52.  Back to cited text no. 6
Van Blerkom J. Mitochondrial function in the human oocyte and embryo and their role in developmental competence. Mitochondrion 2011;11:797-813.  Back to cited text no. 7
Cimadomo D, Fabozzi G, Vaiarelli A, Ubaldi N, Ubaldi FM, Rienzi L. Impact of Maternal Age on Oocyte and Embryo Competence. Front Endocrinol (Lausanne) 2018;9:327.  Back to cited text no. 8
Ubaldi FM, Cimadomo D, Vaiarelli A, Fabozzi G, Venturella R, Maggiulli R, et al. Advanced Maternal Age in IVF: Still a Challenge? The Present and the Future of Its Treatment. Front Endocrinol (Lausanne) 2019;10:94.  Back to cited text no. 9
Lee CI, Wu CH, Pai YP, Chang YJ, Chen CI, Lee TH, et al. Performance of preimplantation genetic testing for aneuploidy in IVF cycles for patients with advanced maternal age, repeat implantation failure, and idiopathic recurrent miscarriage. Taiwan J Obstet Gynecol 2019;58:239-43.  Back to cited text no. 10
Scott RT Jr, Upham KM, Forman EJ, Zhao T, Treff NR. Cleavage-stage biopsy significantly impairs human embryonic implantation potential while blastocyst biopsy does not: A randomized and paired clinical trial. Fertil Steril 2013;100:624-30.  Back to cited text no. 11
Capalbo A, Ubaldi FM, Cimadomo D, Maggiulli R, Patassini C, Dusi L, et al. Consistent and reproducible outcomes of blastocyst biopsy and aneuploidy screening across different biopsy practitioners: A multicentre study involving 2586 embryo biopsies. Hum Reprod 2016;31:199-208.  Back to cited text no. 12
Schoolcraft WB, Katz-Jaffe MG. Comprehensive chromosome screening of trophectoderm with vitrification facilitates elective single-embryo transfer for infertile women with advanced maternal age. Fertil Steril 2013;100:615-9.  Back to cited text no. 13
Rubio C, Bellver J, Rodrigo L, Bosch E, Mercader A, Vidal C, et al. Preimplantation genetic screening using fluorescence in situ hybridization in patients with repetitive implantation failure and advanced maternal age: Two randomized trials. Fertil Steril 2013;99:1400-7.  Back to cited text no. 14
Ubaldi FM, Capalbo A, Colamaria S, Ferrero S, Maggiulli R, Vajta G, et al. Reduction of multiple pregnancies in the advanced maternal age population after implementation of an elective single embryo transfer policy coupled with enhanced embryo selection: Pre- and post-intervention study. Hum Reprod 2015;30:2097-106.  Back to cited text no. 15
Wu MY, Chao KH, Chen CD, Chang LJ, Chen SU, Yang YS. Current status of comprehensive chromosome screening for elective single-embryo transfer. Obstet Gynecol Int 2014;2014:581783.  Back to cited text no. 16
Lee HL, McCulloh DH, Hodes-Wertz B, Adler A, McCaffrey C, Grifo JA.In vitro fertilization with preimplantation genetic screening improves implantation and live birth in women age 40 through 43. J Assist Reprod Genet 2015;32:435-44.  Back to cited text no. 17
Shapiro BS, Richter KS, Harris DC, Daneshmand ST. Influence of patient age on the growth and transfer of blastocyst-stage embryos. Fertil Steril 2002;77:700-5.  Back to cited text no. 18


  [Figure 1], [Figure 2], [Figure 3], [Figure 4]


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