Aneuploidy screening by array comparative genomic hybridization improves success rates of in vitro fertilization: A multicenter Indian study.

OBJECTIVE: To evaluate the usefulness of preimplantation genetic screening (PGS) using array comparative genomic hybridization (aCGH) in the Indian population.
MATERIALS AND METHODS: This is a retrospective, multicenter study including 235 PGS cycles following intracytoplasmic sperm injection performed at six different infertility centers from September 2013 to June 2015. Patients were divided as per maternal age in several groups (<35, 35-36, 37-38, 39-40, and >40 years) and as per indication for undergoing PGS. Indications for performing PGS were recurrent miscarriage, repetitive implantation failure, severe male factor, previous trisomic pregnancy, and advanced maternal age (≥35). Day 3 embryo biopsy was performed and analyzed by aCGH followed by day 5 embryo transfer in the same cycle or the following cycle. Outcomes such as pregnancy rates (PRs)/transfer, implantation rates, miscarriage rates, percentage of abnormal embryos, and number of embryos with more than one aneuploidy and chaotic patterns were recorded for all the treated subjects based on different age and indication groups.
RESULTS: aCGH helped in identifying aneuploid embryos, thus leading to consistent implantation (range: 33.3%-42.9%) and PRs per transfer (range: 31.8%-54.9%) that were obtained for all the indications in all the age groups, after performing PGS.
CONCLUSION: Aneuploidy is one of the major factors which affect embryo implantation. aCGH can be successfully employed for screening of aneuploid embryos. When euploid embryos are transferred, an increase in PRs can be achieved irrespective of the age or the indication.

INTRODUCTION

Human embryo development occurs through a process that encompasses reprogramming, sequential cleavage divisions, mitotic chromosome segregation, and embryonic genome activation. Chromosomal abnormalities in the germ cells and/or preimplantation embryos may arise during such developmental processes and are a major cause of spontaneous miscarriage or birth defects. Aneuploidy, an alteration in number of chromosomes, is one such chromosomal aberration which is commonly observed in early stage human embryos.[1] Trisomic and monosomic embryos account for at least 10% of human pregnancies and for the women nearing the end of their reproductive lifespan, the incidence of aneuploidy may exceed to 50%.[2]

It is usually perceived that aneuploidy rates are higher in oocytes and embryos in women with advanced maternal age (AMA) which results in increase in spontaneous abortions, thereby reducing ongoing implantation rates (IRs).[3] It might be associated with both maternal meiosis I and meiosis II nondisjunction events.[4] Recent studies in humans and model organisms have shed light on the complexity of meiotic defects, providing evidence that the errors associated with advancing age in human females is not attributable to a single factor but, to an interplay between oogenetic defects and other endogenous and exogenous factors.[2]

Aneuploidy might also be a contributing factor in other infertile populations; for example, despite other potential causes, an abnormal embryonic karyotype was found to be the most frequent cause of recurrent miscarriage (RM).[5] In the same study, the percentage of cases with RM of truly unexplained origin was limited to 24.5%. While the diagnosis of repetitive implantation failure (RIF) remains a clinical challenge, embryonic aneuploidy has been proposed as one of the leading causes behind it.[67]

Even, male factor (MF) infertility, resulting from sperm chromosomal abnormalities, is considered to be due to an impairment of the meiotic process.[89] This could also be supported by a higher incidence of abnormal karyotypes described in the miscarriages of couples undergoing intracytoplasmic sperm injection (ICSI).[10]

In the last few years, preimplantation genetic screening (PGS) has been more broadly performed before transfer of embryos in in vitro fertilization (IVF) programs. It helps to identify the aneuploid embryos, so as to reduce the implantation and miscarriage complications during the pregnancy. There have been multiple studies explaining the use of fluorescence in situ hybridization (FISH) for PGS. A meta-analysis compiling nine studies showed no usefulness of FISH attributed to the limited number of chromosomes that can be screened using FISH.[11] However, there has been a recent study with different conclusions, showing the usefulness of FISH for patients with AMA and RIF.[12]

Besides FISH, other techniques such as oligo arrays, single nucleotide polymorphism arrays, quantitative polymerase chain reaction (qPCR), and bacterial artificial chromosome arrays are also used for PGS.[13141516] However, the effectiveness of array comparative genomic hybridization (aCGH) technology in PGS has been reported by some studies.[1718] In recently published reviews, aCGH was described as a reliable and accessible diagnostic approach to assess 24-chromosome aneuploidy.[1920]

The present study is the first of its kind from India evaluating the reproductive outcomes of the IVF patients after performing PGS using aCGH for 24-chromosomes on the embryos to be transferred. It also highlights the impact of performing PGS using aCGH for raising the pregnancy rates (PRs) in couples undergoing IVF.

MATERIALS AND METHODS

Study design

This was a retrospective, multicenter study carried out at six different infertility centers, from September 2013 to June 2015. The patients were grouped as per their age as <35, 35–36, 37–38, 39–40, and >40 years. Clinical indications for PGS included RM (two or more miscarriages of unknown etiology), RIF (three or more previous IVF failures), MF (poor semen parameters), previous trisomic pregnancy (PTP) (couples with a PTP), and AMA (35 years of age or older).

All the patients underwent controlled ovarian stimulation from the second day of their periods on the flexible antagonist protocols. When at least two follicles reached a size of 17 mm in diameter, a trigger (250 mg human chorionic gonadotropin [hCG] or 0.2 mg triptorelin, if there was a risk of hyperstimulation) was administered, and oocyte retrieval was scheduled 35 h later. ICSI was performed in all the cases. Fertilization was assessed 17–20 h after microinjection, and embryo growth was recorded every 24 h. The comprehensive chromosomal screening cycles were performed in different IVF centers using two different culture protocols; wherein embryos were either grown sequentially in Vitrolife G-Plus Series IVF Medium (Vitrolife, Goteborg, Sweden) or COOK Culture System (COOK, Sydney, Australia) was used with tri-gas incubators.

The study was carried out as per the provisions of the Declaration of Helsinki.

Embryo biopsy performed on day 3

Embryos were placed on a droplet containing Ca2+/Mg2+ free medium (G-PGD, Vitrolife, Goteborg, Sweden/LifeGlobal, Guilford, CT, USA), the zona pellucida was perforated using LASER technology (OCTAX, Herborn, Germany), and one/two blastomere was withdrawn from each embryo. Only embryos with five or more nucleated blastomeres and <25% fragmentation were biopsied. Individual blastomeres were placed in 0.2 ml PCR tubes containing 2 μl phosphate-buffered saline. For blastomere washing and handling, 1% polyvinylpyrrolidone was used.

Embryo transfer

The results of aCGH were available on day 5. If a fresh embryo transfer was planned, one or two properly developed euploid embryos were transferred on day 5. Luteal phase was supported by micronized progesterone 400 mg twice a day; β-hCG was tested 2 weeks later. In patients at the risk of ovarian hyperstimulation syndrome where agonist trigger was used, well-developed euploid embryos were vitrified, and transferred in a subsequent hormone replacement therapy cycles.

DNA amplification and array comparative genomic hybridization protocol

A single cell from each embryo was amplified using the Sureplex DNA Amplification System (BlueGnome, Cambridge, UK). Amplicon quality was assured by gel electrophoresis (Lonza, Rockland, USA). Sample and control DNA were labeled with Cy3 and Cy5 fluorophores following the manufacturer's instructions. Labeling mixes were combined and hybridized on 24 sure arrays (V2 and V3, Illumina, USA) for 6–12 h. Each probe used was specific to a different chromosomal region and occupied a discrete spot on the slide.

Chromosomal loss or gain was revealed by the color adopted by each spot after hybridization. The technique involved the competitive hybridization of differentially labeled test and reference DNA samples. Fluorescence intensity was detected using a laser scanner (Power Scanner, TECAN, Mannedorf, Switzerland), and BlueFuse Multi Software was used for data processing (BlueGnome, Cambridge, UK). The 24sure microarray product description (February 8, 2012, document version 2.3 and model number 408501-00) describes 10 Mb effective resolution for 24sure using BlueFuse software, the minimum size specified for segmental aneuploidies. The entire protocol was completed in <24 h and therefore, embryo transfer and vitrification of surplus euploid embryos were scheduled for day 5.

Pregnancy outcomes

The outcomes of the study, i.e., PR per transfer (defined as the percentage of clinical pregnancies with a fetal heartbeat); IR (defined as the percentage of embryos transferred resulting in an implanted gestational sac); and miscarriage rate (defined as the percentage of clinical pregnancies that were spontaneously miscarried before week 12 of pregnancy) were calculated.

Statistics analysis

Chi-square test was used for comparisons between study groups with respect to percentages. P < 0.05 was considered to be statistically significant.

RESULTS

Overall, 235 PGS cycles were included in the study. When data were to be analyzed as per the female age groups, a total of five cases were excluded from the study as embryo transfer had yet to be planned for them. However, while analyzing the data as per indication groups, three more cases (out of 230) were excluded as PGS was carried out for them due to patients’ choice (unspecific indication). The parameters included in the PGS analysis are given in Table 1.

Table 1

Parameters of the cycles included in preimplantation genetic screening

The mean age of the women included in the analysis was 35.3 years. A total of 1401 embryos were analyzed and in 1342 (95.8%) of them, amplification and further analysis were successful. The details of the parameters according to the age groups are given in Table 2.

Table 2

Clinical outcomes of 230 comprehensive chromosome screening cycles according to the different age groups

In 160 cycles, at least one euploid embryo was available for transfer, with a PR of 51.4% per transfer, and 36.9% per cycle. The overall miscarriage rate was found to be 15% in the different age groups. The mean number of embryos analyzed was similar for all the indication groups. The percentage of aneuploid embryos was comparatively lower for all the indications below 35 years of age (67.6% in RM, 69.7% in RIF, 81.5% in PTP, and 75.3% in MF) as compared to the AMA group (79.8%).

These results had an impact on the percentage of cycles with at least one euploid embryo for transfer, making it lower for the AMA group (61.2%) compared to the other indications. However, once embryo transfer was achieved, the chances of successful pregnancy and implantation were similar for all the mentioned indications, with a range between 30.8% and 46.2% for PRs per transfer and between 33.3% and 42.9% for IRs.

However, the PR per PGS cycle was lower for the RM group (31.3%) compared to rest of the groups.

Interestingly, Table 3 shows a different distribution of chromosomal abnormality types according to the indication. The chromosome distribution showed a chaotic pattern which was relatively homogenous among indications. The most remarkable difference was observed for the percentage of embryos with aneuploidy for more than one chromosome, which was comparatively higher in the AMA and MF groups (34.5% for both) as compared to the other indications (range: 19.7%–26.2%).

Table 3

Clinical outcomes of 227 comprehensive chromosome screening cycles according to the different indications

This percentage increased with maternal age, ranging from 32.1% in 40 years to 55.8% in 42 years of age. Therefore, the overall incidence of aneuploidy ranged from 63.4% to 95.6%.

Figure 1 represents the relationship between aneuploidy rate and the maternal age. It represents the rate of aneuploidy among different age groups with three different types of aneuploidy, namely, aneuploid embryos, embryos with chaotic pattern, and embryos with >1 aneuploidy.

Figure 1

Relationship between aneuploidy rates and maternal age

The present data, given in Table 4, showed much better results for all the age groups in terms of PRs and IRs compared with the patients who did not get PGS done for the euploid embryo selection during the same period at our clinics.

Table 4

Clinical outcome comparison of patients with or without preimplantation genetic screening

DISCUSSION

Errors in the meiotic divisions in human cells cause various anomalies in chromosomal content in the embryos, namely, aneuploidy. It leads to a reduction in PRs by reducing IRs, increasing miscarriage rates, and other complications during pregnancy.[162223]

Interestingly, the euploid and the aneuploid embryos cannot be distinguished from one another by regular morphological evaluation methods.[24] Hence, there is a need of a reliable method to assess aneuploidy in the embryos. Independent of the type of platform used, the technique selected for screening all the 24 chromosomes should offer reliable and timely results, and should only be applied in clinical programs after validation with an already well-established technique.[25]

PGS through various techniques such as FISH, qPCR, or aCGH can be performed to monitor the chromosomal anomalies in the embryos. However, aCGH technique offers a 100% exhibition of aneuploidy in embryos by evaluation of all the 24 chromosomes and eases out on cumbersome steps involved in FISH, such as fixation on microscopic slides.[25] The current study confirmed the benefits of PGS using aCGH to screen the aneuploidy in the embryos to be used in the IVF processes. Baart et al., 2006 evaluated the efficiency of PGS in detecting the aneuploidy and mosaicism in the embryos by reanalyzing them on day 3 and day 5 stage in the younger females (<38 years). They concluded that mosaicism and aneuploidy impacted the results of PGS, but the latter is still an effective and reliable tool in analyzing the aneuploidy in the embryos before transferring them into females during IVF. The conclusions by Baart et al. have shown that rates of mosaicism and aneuploidy in the embryos from young IVF patients were similar to those published for older women by Staessen et al.[2627] However, in the present study, the incidence of aneuploidy rate increased as the age increased which supported the previous reports where similar trend was observed for patients having more than one aneuploid embryo.[92829]

The high aneuploidy rate of 75% observed in the present study is comparable to the results in the study by Rabinowitz et al., where they found 72.3% aneuploidy rate with the cleavage stage embryos.[930]

Franasiak et al. in 2014 concluded that the higher rates of aneuploidy specifically trisomies and monosomies were obtained in elder women with an age more than 40 years.[31] Similarly, Gutiérrez-Mateo et al. observed 63.2% aneuploidy rate for cleavage stage embryos which was found to be increasing with the maternal age.[32] They found aCGH to be a robust and specific (with low error rates of 1.2%) approach to assess the aneuploidy in the embryos to be used in IVF, thus leading to improved rates of implantation and pregnancy success. aCGH was found to detect about 42% more abnormalities and 13% more abnormal embryos than other PGS techniques such as FISH.[32]

Aneuploidy is also considered to be one of the main causes leading to implantation failures in the patients undergoing IVF treatment.[162425] The same was established through this study since the embryos screened to be having normal chromosomal numbers were successfully implanted, resulting in pregnancy. The similar conclusion was drawn by Rodrigo et al., who conducted similar study with the objective to evaluate the usefulness of PGS by aCGH to identify normal embryos from the aneuploid ones.[9]

It has been observed in the current study that the IRs were higher (40.7%) and miscarriage rates were lower (6.1%) in the AMA groups. Understandably, the patients in the higher age group had more chances of no embryos available for the transfer, but the patients who had embryo transfer possible exhibited the similar pregnancy and IRs compared to the younger age group patients. Younger age group patients showed an increase in the miscarriage rate, which could be due to other uterine factors.[9] Donoso et al. concluded the same in their study in 2007.[28]

In addition, the percentage of aneuploid embryos was the highest in AMA than all the other indications, in both the present study (79.8%) and the study conducted by Rodrigo et al. (85.3%). Rodrigo et al. observed a range of 31.3%–60% in the rates of pregnancy/cycle with the highest percentage in MF and the lowest in RM. However, this range was 19.3%–52% in the current study with the highest rate in MF and the lowest in RM.[9]

In PTP couples, published data described an increased risk of recurrent aneuploid conceptions, particularly in women under 37 years of age.[33] However, the current study indicates comparatively low conception rate in this group when day 3 biopsy is performed. This can be due to low sample number in the group.

The best clinical results after PGS are observed in MF couples (PR 60.0% and IR 42.9%). This type of 24-chromosome PGS seems to be a very promising indication for this patient group.

CONCLUSION

A 24-chromosome PGS using aCGH is a robust method to assess the aneuploidy in the embryos. PGS can further lead to transfer of good quality euploid embryos, resulting in an improved implantation and pregnancy/transfer rate and reduced miscarriage rates.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.