|Year : 2019 | Volume
| Issue : 2 | Page : 141-149
Embryonic aneuploidy after preimplantation genetic screening: Age- and indication-matched comparative study between Indian and Spanish population
Aditi P Kotdawala1, Pere Mir2, Javier Herrero3, Rajni Khajuria4, P G. L. Lalitkumar5, Manish R Banker1
1 NIF, Ahmedabad, Gujarat, India
2 Igenomix, Valencia, Spain
3 IVI RMA Global, Spain
4 Igenomix, New Delhi, India
5 Head, Embryology Programme, NIF, Ahmedabad, Gujarat, India
|Date of Web Publication||17-Jun-2019|
Ms. Aditi P Kotdawala
Nova IVI Fertility, 108, Swastik Society, Navarangpura, Ahmedabad - 380 006, Gujarat
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Recent studies show that there are differences in female fertility in different ethnic groups with ovarian aging and IVF treatment outcomes. Advanced maternal age is a known risk factor for miscarriage, accounting largely due to genetically abnormal fetus. Aims and Objectives: This study investigates if there are any differences in rates of embryo aneuploidy based on age and indications for preimplantation genetic screening (PGS) between Indian and Spanish women. Materials and Methods: This multicenter study was carried out at fertility centers in India and Spain. Data from autologous IVF cycles of women <45 years age (Spanish: 39.4 ± 3.8 years; Indian: 35.3 ± 4.6 years) were included. A total of 37,962 embryos from 7009 IVF cycles from Spain and 1894 embryos from 308 IVF cycles from India, having similar clinical indications, underwent similar IVF treatment protocol. The embryos were analyzed by PGS using either a day-3 or day-5/6 embryo biopsy. Results: Both Indian and Spanish ethnic population showed a reduction in aneuploidy rate in day-5/6 biopsy compared with day-3 biopsy (Spanish: 53.3% vs. 81.1%, P < 0.01; Indian: 50% vs. 75%, P < 0.02). There was a significant decrease in highly abnormal or chaotic embryos in trophectoderm biopsies compared with day-3 biopsies (Spanish: 2% vs. 16.1%, P < 0.01; Indian: 2.5% vs. 17.7%, P < 0.01). Both the populations showed similar trend in aneuploidy rate with maternal age. The results showed no significance between aneuploidy rate compared within different age groups and indications. However, there was a significant reduction in the miscarriage rate in Spanish population in day-3 biopsy compared with Indian population (10.7% vs. 19.8%; P < 0.05; 95% confidence interval [0.0044–0.0712]). There were no differences in the clinical outcomes compared between the two populations. Conclusion: This study shows that the aneuploidy rates between Indian and Spanish women of the same age group undergoing IVF treatment do not differ. An in-depth analysis to compare the types of anomalies reported with PGS in both the population will be of much interest.
Keywords: Aneuploidy, array comparative genomic hybridization, chromosome aberrations, ethnic comparison, preimplantation genetic screening
|How to cite this article:|
Kotdawala AP, Mir P, Herrero J, Khajuria R, Lalitkumar P G, Banker MR. Embryonic aneuploidy after preimplantation genetic screening: Age- and indication-matched comparative study between Indian and Spanish population. J Hum Reprod Sci 2019;12:141-9
|How to cite this URL:|
Kotdawala AP, Mir P, Herrero J, Khajuria R, Lalitkumar P G, Banker MR. Embryonic aneuploidy after preimplantation genetic screening: Age- and indication-matched comparative study between Indian and Spanish population. J Hum Reprod Sci [serial online] 2019 [cited 2020 May 29];12:141-9. Available from: http://www.jhrsonline.org/text.asp?2019/12/2/141/260492
| Introduction|| |
Evolution of larger and detailed databases has led to the realization of the effect of ethnicity-specific variations in both diagnostic and therapeutic assisted reproductive technology (ART). This has led to further researches and re-evaluation of normal or average baseline values based on patients' ethnic origin. In an observational study including nationwide data, Purcell et al. have concluded that infertile Asian women have significantly fewer pregnancies than Caucasian women undergoing in vitro fertilization belonging to similar age, ovarian reserve, and infertility diagnosis along with similar treatment protocols and apparently similar responses to the treatment at the time of embryo transfer. Another study by Langen et al. has also reported odds of Asian patients having a live birth after undergoing IVF with a blastocyst transfer were almost half those of similar Caucasian patients. In our earlier publication including 5549 women of Indian and Spanish origins, we have found that ongoing pregnancy rate was significantly lower among Asian ethnic group following IVF-ICSI suggesting that ethnicity, like age, is also a major and independent predictor of IVF outcome. We have also reported in our previous study including a total of 465 women of Indian and Spanish origin that similar ovarian reserve markers and ovarian response were observed in women with a 6-year age difference in favor of the Spanish women, suggesting ethnic differences in ovarian aging.
The failure of assisted reproduction cycle is usually attributed to different factors such as poor embryos or the lack of receptive intrauterine environment. There have been multiple reports in past few years that have showed with a variety of new generation arrays, that majority of IVF and early pregnancy failures, particularly associated with advanced age and severe male factor, are explainable by embryo aneuploidy., In a retrospective study including 256 patients, Kort et al. have interestingly reported that there may be minor difference in blastocyst formation rates; there is no difference in aneuploidy or euploid blastocyst transfer rates between Asian and Caucasian patients that would explain the discrepancy in IVF outcomes observed between these patient populations.
To further evaluate whether the lower reproductive outcome in Indian women was simply due to a quantitative reduction in ovarian reserve or due to poor quality or both, our current study aims to investigate whether there are differences in rates of embryo aneuploidy as per different age groups and indications in Indian and Spanish women.
| Materials and Methods|| |
This is a retrospective, multicenter study carried out between September 2013 and December 2015 at five different centers in India and clinics in Spain. The study included all autologous IVF cycles of patients of <45 years age which were screened using preimplantation genetic screening (PGS) for 24-chromosome analysis. Mean maternal age in patients from Spanish group was 39.4 (±3.8), and 35.3 (±4.6) for the group of patients from India. In the group of patients from Spain, a total of 37,962 embryos from 7009 IVF cycles were analyzed; and in the group of patients from India, 1894 embryos from 308 IVF cycles were analyzed. The patients were grouped as per their age into four groups: <35 years, 35–37 years, 38–40 years, and 40–44 years. Clinical indications for PGS included recurrent miscarriages (RM), defined as two or more miscarriages of unknown etiology; repeated implantation failure (RIF) defined as three or more previous IVF failures; male factor (MF) defined as males with poor semen parameters below the WHO normal values; previous trisomic pregnancy (PTP) defined as couples having a history of a PTP; advanced maternal age (AMA) defined as women of 35 years of age or older; and patients with more than one clinical indication for PGS, named mixed indications group.
Ovarian stimulation protocol
All the patients underwent controlled ovarian stimulation from the second day of their periods on the flexible antagonist protocol. When at least two follicles reached a size of 17 mm in diameter, a trigger (6500 IU 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 cycles were performed in different IVF centers using two different culture protocols, wherein embryos were either grown sequentially in Vitro life G-Plus series IVF medium (Vitrolife, Goteborg, Sweden) or COOK culture system (COOK, Sydney) was used in MINC tri-gas incubators (COOK, Sydney).
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 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 polymerase chain reaction (PCR) tubes containing 2 μL ×1 phosphate buffer saline (PBS). For blastomere washing and handling, 1% PBS-polyvinylpyrrolidone (PVP) was used.
Embryo biopsy performed on day 5
On day 3 following fertilization, all embryos were subjected to laser-assisted hatching, and all nonarrested embryos that were with herniating trophectoderm (TE) on day 5 or 6 underwent laser-assisted TE biopsy for PGS. After PGS, biopsied blastocysts were individually frozen. At least 5–8 cells were biopsied from the TE. Similar to day-3 biopsies, individual biopsies were placed in 0.2 mL PCR tubes containing 2 μL ×1 PBS. For TE biopsy washing and handling, 1% PBS-PVP was used.
For day 3 embryo biopsies, the results of array comparative genomic hybridization (aCGH) were made available on day 5. When a fresh embryo transfer was planned, 1 or 2 properly developed euploid embryos were transferred on day 5 depending on the availability and number of chromosomally normal embryos. Luteal phase was supported by micronized progesterone 400 mg twice a day, and β-hCG was tested 2 weeks later.
In patients at the risk of OHSS where agonist trigger was used, well-developed euploid embryos were vitrified and transferred in a subsequent HRT cycle.
For TE biopsy patients, all blastocysts were frozen individually after the biopsy, as the results of aCGH cannot be available in time to carry out the fresh embryo transfer. In the following menstrual cycles, frozen-thawed embryo transfer as per earlier mentioned protocol was carried out if euploid embryos were available.
DNA amplification and array comparative genomic hybridization protocol
To analyze day-3 embryo biopsies, a single cell from each embryo was amplified using the SurePlex DNA amplification system (Illumina, Inc., San Diego, CA, USA), according to the manufacturer's protocol. To analyze TE biopsies, all the cells of the biopsy were handled altogether as a single unit. For both types of biopsies, the same aCGH protocol was followed. 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 microarrays (v2.0 and v3.0, Illumina, Inc., San Diego, CA, 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 comparative 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 (Illumina, Inc., San Diego, CA, USA) was used for data processing. The 24 sure microarray product description (February 8, 2012, document version 2.3, and model number 408501-00) describes 10 Mb effective resolution for 24 sure 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 could be scheduled on day 5 when the embryo biopsy was carried out on day 3.
The outcomes of the study, i.e., pregnancy rate per transfer (defined as the percentage of clinical pregnancies with a fetal heartbeat out of total number of embryo transfers carried out in each group); implantation rate (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 out of total number of pregnancies achieved) were calculated.
A proportion test was performed to compare results between groups. For this test, the null hypothesis tested was that the proportions in each group were the same and the alternative hypothesis was that the probability of success in the first group is not equal to the probability of success in the second group. A confidence interval (CI) for the difference of proportions of 95% was used.
| Results|| |
The overall embryo aneuploidy rate was similar in India and Spanish population (73.4% and 80%, respectively). However, to properly compare aneuploidy rates, results were divided by type of embryo biopsy. In the two populations, aneuploidy rates in TE biopsy were significantly lower compared to day-3 biopsies. For Spanish patients, aneuploidy rate in day-3 biopsies was 81.1%, and it was 53.3% for TE biopsies (P < 0.0001). A similar decrease in aneuploidy rate was observed in the Indian population, from 75% in day-3 biopsies to 50% in TE biopsies (P = 0.0158) [Table 1]. However, comparing the type of abnormalities found, there was a huge decrease in highly abnormal or chaotic embryos in TE biopsies compared to day-3 biopsies. in the Spanish group, it decreased from 16.1% to 2% (P < 0,001) and in Indian group from 17.7% to 2.5% (P = 0.0024).
|Table 1: Types of aneuploidies found in both populations for the two types of embryo biopsies|
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In the two types of embryo biopsy groups, aneuploidy rates were also analyzed per different age groups. Results in the two populations showed the same trend with increased number of aneuploidies as maternal age increases. For day-3 embryo biopsies, aneuploidy rates in the group of patients from Spain ranged from 64.2% in patients below 35 years of age up to 91.1% in patients with the age group of 41–44 years; and in India patients, it ranged from 70.5% in patients below 35 years of age up to 90.8% in patients with age group of 41–44 years [Supplementary Table 1]. For TE biopsies, aneuploidy rates in the group of patients from Spain ranged from 40.7% in patients below 35 years of age up to 75.6% in patients with age group of 41–44 years; and in Indian patients, it ranged from 39.3% in patients below 35 years of age up to 87.5% in patients with age group of 41–44 years [Supplementary Table 2]. No statistical differences were found in any of these subgroups.
Preimplantation genetic screening indication
In the two types of embryo biopsy groups, aneuploidy rates were also analyzed according to the different clinical indications. Regarding day-3 embryo biopsies, aneuploidy rates were very similar for Spanish group in all the indications (ranging from 67% in MF group to 68.6% in patients with mixed indications), except for AMA group which had the highest aneuploidy rate of 86.6%. In day-3 cases of the Indian group, differences were more pronounced but not statistically significant (ranging from 66.2% in RM group to 79.1% in PTP group), and similar to Spanish group, AMA patients had the highest aneuploidy rate of 86.1% [Supplementary Table 3].
For TE biopsy cases, aneuploidy rates were similar for Spanish group in all the indications (ranging from 42.3% in patients with mixed indications to 50% in patients with PTP), except for AMA group which had a higher aneuploidy rate of 66.1%. In the Indian group, there were no cases of PTP patients with TE biopsy. For the rest of clinical indications, lowest aneuploidy rate was identified for three different indications: 27.6% for RIF, 36.4% for MF, and 37.5% for RM; while the two remaining clinical indications showed the highest aneuploidy rate: 78.1% for AMA and 81.8% for patients with mixed indications [Supplementary Table 4].
Regarding the clinical outcome of the two study groups, results were divided by the two types of embryo biopsies and then compared per clinical indication. All of them were summarized in [Table 2], and they were comparable in global, but some exceptions could be found.
|Table 2: Reproductive outcome in both populations for preimplantation genetic screening cases with trophectoderm biopsy|
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Day-3 embryo biopsy
For the PGS cases performed on day-3 embryo biopsies, overall pregnancy rate was similar in Spanish and Indian group (54.5% vs. 49.8%, respectively). These results when divided by the type of PGS indication also showed similar results for all of the clinical indications. The main difference was seen in the RM group (56.4% for Spanish group and 32.3% for Indian group) but also not statistically significant. Similarly, implantation rate was also higher in the Spanish group but not statistically significant (48.2% vs. 39.4%). The main difference in implantation rate among all the clinical indications was found in RM patients, where the implantation rate for Spanish group was 46.4% and for Indian group was 26.9%. The only statistically significant difference was found in the overall miscarriage rate comparing Indian and Spanish data for day-3 embryo biopsies (19.8% vs. 10.7% respectively; P = 0.0386; 95%-CI [0.0044–0.0712]). However, regarding the same data divided by PGS clinical indication, the main difference was found in RIF patients but without reaching a statistically significant difference (23.9% vs. 9.9%; P = 0.0642) [Table 3].
|ble 3: Reproductive outcome in both populations for preimplantation genetic screening cases with day-3 embryo biopsy|
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For all the PGS cases performed on TE biopsies, overall pregnancy rate was similar in both study groups (62.4% vs. 66.7%), and it was also similar for all the PGS indications. The main difference in pregnancy rate was found in the AMA subgroup (64.2% vs. 25.0%) but was not statistically significant. Implantation rate, albeit higher for the Indian group in overall (62.5% vs. 52.7%), was quite similar in both groups for all the PGS clinical indications. Only three miscarriages were present in the Indian group for TE biopsies: one miscarriage in AMA group and two more miscarriages in RIF group. No conclusive statistical analysis can be performed with this low sample size [Table 2].
Finally, the clinical outcome of PGS cases performed in the two types of embryo biopsy was compared for the two populations. In the same population, pregnancy rate was higher for TE biopsies (62.4% for Spanish group and 66.7% for Indian group) compared to day-3 biopsies (54.5% for Spanish group and 49.8% for Indian group) but was not statistically significant. Similarly, implantation rate was higher for TE biopsies in both scenarios. In Spanish population, implantation rate was 52.7% for TE biopsies and 48.2% for day-3 embryo biopsies; and for Indian population, implantation rate was 62.5% for TE biopsies and 39.4% for day-3 embryo biopsies.
| Discussion|| |
Aneuploidy is frequently attributed for the failure of IVF treatment. It is observed that different ethnic populations respond differently to IVF treatment and thus even though there is no clear etiology, they have varying IVF outcomes. In a preliminary study on ethnicity-specific aneuploidy rates by Kort et al., it was observed that after controlling for maternal age, embryo aneuploidy does not differ between Asian and Caucasian patients.
The current study was designed to further analyze whether comparison of differing aneuploidy rates among Indian and Spanish patients may explain the dissimilarity in IVF outcome for different age groups and the clinical indication responsible for the need of PGS. Even though the sample size in both populations analyzed for the study had a huge difference in terms of total number, there was no significant difference observed between embryo aneuploidy rate, evaluated either at cleavage stage or blastocyst stage in both the patient groups which matched with the previous findings. It can be deduced that irrespective of patient ethnicity, the aneuploidy rate follows the same trend of increasing with age. Similarly, types of aneuploidies were identified, whether there was involvement of single chromosome, multiple chromosomes, or there was a chaotic pattern of aneuploidy, in both ethnic groups whether cleavage stage or TE biopsy was carried out. The average number of blastocysts analyzed for both the ethnic populations was also similar which was in contradiction with the previous findings by Sharara et al., where they have concluded that South Asian women produced fewer blastocysts for biopsy, had a lower number of top-quality embryos, resulting in a lower clinical pregnancy rate compared to Caucasian women. However, in the current study, we have observed that for TE biopsy, patients had an equal chance of having at least one euploid embryo available for transfer in both the ethnic groups (75.3% vs. 75%, respectively, for Spanish vs. Indian population). While in cleavage stage biopsy group, Indian patients had very high chance of having at least one euploid embryo available for transfer compared to Spanish population (70.5 vs. 49.5, respectively), which may be due to a very small number of patients in Indian group compared to Spanish group (6677 vs. 288, respectively). The clinical outcomes, pregnancy rates, and implantation rates were also comparable for both the ethnic groups in both types of biopsies. However, the miscarriage rate was significantly higher in Indian population compared to the Spanish patients for both the cleavage stage as well the TE biopsy group even after euploid embryo transfer was carried out. This suggests that there is some nonploidy determinant of embryo viability and endometrial receptivity that may explain the poorer ongoing pregnancy rates observed for the Indian patients undergoing euploid embryo transfer compared to its Spanish counterpart. This has also been documented in the past stating Asian women have a decreased clinical pregnancy and live birth rate compared to their Caucasian counterpart. The indication-wise distribution of patients in both type of biopsies also showed similar kind of trend demonstrating no ethnic base for variation in clinical outcomes which are lower in Asian population compared to the Caucasian group [Table 2] and [Table 3]. For RM group of patients in cleavage stage biopsy, Indian population had comparatively higher chance to have euploid embryos transferred, but the implantation rates were not so high even after the euploid embryo transfer, and the miscarriage rates were high compared to the Spanish group. However, for all the other clinical indications, both the populations had more or less equal chance of having euploid embryo transfer and the implantation rates were also comparable. This indicates that once the patient has a chance to have a euploid embryo transfer, the chances of getting a clinically viable pregnancy are high, except for the RM subgroup. However, the RM sub-group from Indian population had good clinical outcomes when underwent TE biopsy compared to the Spanish counterparts with miscarriage rate going down to 0%. However, this can be due to a very small number of patients in this group. On the other hand, the AMA group which had good clinical outcomes with the cleavage stage biopsies showed very poor results with TE biopsy for Indian population compared to the Spanish group with a low pregnancy rate (25%) and very high miscarriage rate (100%). This can be due to the fact that the ovarian aging of Asian women is faster than the Caucasian women leading to poor blastocyst formation and decreased clinical outcomes.
The primary weakness of the current study is its retrospective nature and second, the disparity in sample size between two ethnic populations analyzed in the study, the Indian group being significantly lower in number compared to the Spanish group. Moreover, other baseline factors such as patient BMI, endometriosis, and previous reproductive treatment history were not taken into account which may have some impact on the implantation and miscarriage rates.
| Conclusion|| |
Thus, to conclude, this study shows that the aneuploidy rates between Indian and Spanish women of the same age group undergoing IVF treatment do not differ. The results also reinstate that TE biopsy performed on day 5/6 is superior to blastomere biopsy on day 3, as we see more number of euploid embryos in the former category. This is possibly due to the increased availability of time for embryos to undergo self-correction. The pregnancy rate is also higher with euploid embryos subjected to TE biopsy on day 5/6, compared with day-3 blastomere biopsy. The above results are similar both in Indian and Spanish population, ruling out any ethnic variations with the above results. Thus, TE biopsy for PGS could be strongly suggested, globally for better clinical outcome. The cause for increased prevalence of miscarriage seen in Indian population with euploid embryos biopsied on day 3 requires further studies. Furthermore, an in-depth analysis and comparison of different types of anomalies reported with PGS in Indian and Spanish population will be of much interest.
The authors would like to thank all the clinicians and embryologists at the IVF units included in this study. We would also like to thank Deven Patel (Regional Head-Embryology, Nova IVI Fertility, India), Carmen Rubio Lluesa (Igenomix, Valencia, Spain) and Juan Antonio García (Director IVI Madrid, IVI Director for India and GCC) for their support and guidance.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3]