Different Strategies of Preimplantation Genetic Testing for Aneuploidies in Women of Advanced Maternal Age: A Systematic Review and Meta-Analysis

Background: Preimplantation genetic testing for aneuploidies (PGT-A) is widely used in women of advanced maternal age (AMA). However, the effectiveness remains controversial. Method: We conducted a comprehensive literature review comparing outcomes of IVF with or without PGT-A in women of AMA in PubMed, Embase, and the Cochrane Central Register of Controlled Trials in January 2021. All included trials met the criteria that constituted a randomized controlled trial for PGT-A involving women of AMA (≥35 years). Reviews, conference abstracts, and observational studies were excluded. The primary outcome was the live birth rate in included random control trials (RCTs). Results: Nine randomized controlled trials met our inclusion criteria. For techniques of genetic analysis, three trials (270 events) performed with comprehensive chromosomal screening showed that the live birth rate was significantly higher in the women randomized to IVF/ICSI with PGT-A (RR = 1.30, 95% CI 1.03–1.65), which was not observed in six trials used with FISH as well as all nine trials. For different stages of embryo biopsy, only the subgroup of blastocyst biopsy showed a higher live birth rate in women with PGT-A (RR = 1.36, 95% CI 1.04–1.79). Conclusion: The application of comprehensive chromosome screening showed a beneficial effect of PGT-A in women of AMA compared with FISH. Moreover, blastocyst biopsy seemed to be associated with a better outcome than polar body biopsy and cleavage-stage biopsy.

P–581 The day of blastocyst biopsy and the chromosomal constitution. Evaluation of 5125 embryos by Next Generation Sequency (NGS)

Study question Is there a relationship between the day of blastocyst biopsy and the results NGS analysis? Summary answer Embryos biopsied on day 6 or 7 are associated with the increased probability of being an aneuploidy embryo and less likely to be mosaic embryo. What is known already There is controversy about whether an embryo that reaches the blastocyst stage on day 5 has a higher chance of being euploid than embryos which are biopsied later. In our study, chromosome constitution was evaluated by next-generation sequencing (NGS)-based preimplantation genetic testing for aneuploidy (PGT-A) and confounding factors were eliminated. Study design, size, duration Data was collected retrospectively from June 2016 to January 2020 Participants/materials, setting, methods In total, 5125 blastocyst (day 5=2914, day 6 N = 2154 and day7 N = 57), generated from 1318 cycles were analysed with PGT-A. The chromosome constitution for each embryo was classified as euploid, aneuploid and mosaic. A multilevel model was made and associations betwwen variables by logistic regression were adjusted according to maternal age, SART blastocyst grade, fertilization method, biopsy operator and blastocyst stage. Main results and the role of chance The mean maternal age was 36.2 ± 4.2. Euploid rate was 62.1% and 37.9% (day 5 and day 6–7 respectively), aneuploidy rate was 47.0% and 53.0% (day 5 and day 6–7, respectively), mosaicism rate was 59.6% and 40.4% (day 5 and day 6–7, respectively) (p < 0.001). Embryos biopsied on day 6–7 have a significantly lower probability to be euploid and mosaicism than embryos biopsied on day 5 ((OR = 0.76 [0.68–0.86]); (OR = 0.84 (0.73 – 0.96) respectively) (p < 0.001). On the contrary, embryos biopsy on day 5 were significantly more likely to be euploid than day 6–7 (OR = 1.63[1.42–1.86]) (p < 0.001). Limitations, reasons for caution The results observed in this study should be confirmed using a larger number of samples. For the NGS analysis, a chromosome with a variation between 20 to 80% was considered mosaic. Wider implications of the findings: The present study revealed that embryos that reach blastocyst classified as full to hatched on day 5 are more like to be euploid compared to slow growing embryos. Trial registration number non-clinical trials

P–360 Blastocyst biopsy day does have an impact on clinical pregnancies in different frozen embryo transfer (FET) regimens: natural cycle (NC) versus hormone replacement therapy (HRT)

Study question Do euploid blastocysts biopsied on day (D) 5 or D6 differ in clinical pregnancy rates when single FET are performed in NC or HRT cycles? Summary answer In single FET cycles, euploid D5 blastocysts have higher clinical pregnancy rates than D6 in NC, while outcomes are comparable in HRT cycles. What is known already: The synchronization between the endometrium and the embryo development is fundamental for a successful implantation. When performing FET with euploid blastocysts biopsied on D5 or D6, higher clinical pregnancy rates have been reported with D5 blastocysts, however contradictory findings were described due to the study design heterogeneity and endometrial preparation (EP) protocol variabilities. In FET cycles, no consensus has been defined of the superiority of NC over HRT cycles when euploid blastocysts are transferred. Consequently, the question remains unanswered if the clinical pregnancy rates of single euploid FET with D5 or D6 blastocysts differ when the EP protocol remains constant. Study design, size, duration A single center observational study was performed between June 2017 and November 2020, including 1027 single euploid FET with blastocysts biopsied on D5 or D6. All patients with primary or secondary infertility who underwent a FET in a NC or HRT EP protocol, with blastocysts graded ≥ BL3CC (Gardner scoring system) prior to biopsy were included. Vitrified-warmed blastocysts that did not re-expand within 1-hour post-warming were excluded from the analysis. Participants/materials, setting, methods In NCs, vaginal progesterone (P4) (Endometrin®) was administrated (3x100mg) after endocrinological confirmation of ovulation until pregnancy test. For HRT cycles, oral estradiol administration was started on day 2 (4 mg) and increased to 6mg on D5 of the cycle. When endometrial thickness was ≥6 mm, P4 was given (3x100mg) until pregnancy test. All FET were performed on D5 after start of P4 administration. Clinical pregnancy was recorded as the presence of an intrauterine gestational sac. Main results and the role of chance Women’s mean age was 33.8 ± 5.5 years. A total of 651 FETs were performed with D5 euploid blastocysts (37.6% in NC and 62.4% in HRT) and 376 with D6 (43.1% in NC and 56.9% in HRT). Clinical pregnancy rate in NC was higher with D5 blastocysts compared to D6 (66.9% vs 50.0%; OR = 0.494, 95% CI = 0.322–0.758; p < 0.001), while no significant differences were found when vitrified-warmed blastocysts were transferred in HRT cycles (64.3% vs 58.4%; OR = 0.781, 95% CI = 0.548–1.112; p = 0.164). Additionally, clinical miscarriage was significantly higher with D5 euploid blastocysts transferred in NC (D5=10.9% vs D6=3.7%, OR = 0.239, 95% CI = 0.044–0.837; p = 0.019). In HRT, miscarriage outcomes were similar between D5 and D6 euploid blastocysts (D5=18.7% vs D6=20.8%, OR = 0.781, 95% CI = 0.548–1.112; p = 0.164), but significantly higher (p < 0.001) than in NC. From a multinomial logistic regression model including age, blastocyst quality and day of biopsy as confounding factors, the clinical pregnancy rate was significantly affected by D6 blastocyst biopsy (OR = 0.571, 95% CI = 0.360–0.906, p = 0.017) and inner cell mass (ICM) grade A (OR = 3.941, 95% CI = 1.149–10.402; p = 0.006) or B (OR = 2.601, 95% CI = 1.146–5.907, p = 0.022) in NC. In HRT cycles, exclusively ICM was statistically significant (OR = 2.555, 95% CI = 1.214–5.381, p = 0.015 and OR = 2.397, 95% CI = 1.286–4.470, p < 0.001 for grade A and B, respectively). Limitations, reasons for caution The current results are based on an observational retrospective study. Live birth and perinatal outcomes should be considered in a further analysis to evaluate the performance of the NC vs HRT protocols when D5 or D6 euploid blastocysts are transferred in FET cycles. Wider implications of the findings: While the clinical pregnancies of D5 and D6 euploid blastocysts are comparable in HRT protocols only, the miscarriage rates seem to be significantly increased as compared to NC. Further studies are required to personalize EP protocols based on the day of blastocyst biopsy in order to improve clinical outcomes. Trial registration number No

O-204 Is mosaicism affected by an embryologist’s experience in biopsy?

Study question Are there any correlations between blastocyst mosaicism rate and biopsy experience among embryologists? Summary answer Blastocysts biopsied by embryologists with ≥1 year of biopsy experience have significantly lower mosaicism rate compared to those with <1 year of biopsy experience. What is known already It has been reported that the incidence of blastocyst mosaicism is highly variable between centres (PGDIS, 2019). It is also suggested that the technical aptitude of the embryologist performing blastocyst biopsy may give rise to mosaicism. Thus, a retrospective study was conducted to investigate the relationship between blastocyst mosaicism rate and biopsy experience among embryologists in Alpha IVF. Study design, size, duration Thirteen competent embryologists who were trained in blastocyst biopsy were included in this study: 5 have ≥1 year of biopsy experience (Group A; Embryologist A-1, A-2, A-3, A-4, A-5); 8 have <1 year of biopsy experience (Group B; Embryologist B-1, B-2, B-3, B-4, B-5, B-6, B-7, B-8). Embryologists from Group A biopsied a total of 4795 blastocysts while those from Group B biopsied 4869 blastocysts from January 2018 to December 2019. Participants/materials, setting, methods TE biopsy was performed either on Day 5, 6 or 7 using the laser or flicking method. The biopsied cells had Preimplantation Genetic Testing for Aneuploidy (PGT-A) analysed using Next Generation Sequencing (Ion Torrent, USA) and chromosomal mosaicism analysis was done using ReproSeq Mosaic PGS w1.1 workflow. Mosaic blastocysts were reported when 20% - 80% of aneuploid cells are tested in the biopsied samples. Only successfully amplified biopsy samples were included in this study. Main results and the role of chance The mosaicism rate of blastocysts biopsied by embryologists from Group A and B were 17.8% and 19.8% respectively. Blastocysts from Group A showed significantly lower mosaicism rate compared to Group B (p = 0.01). The mosaicism rates of blastocyst biopsied by Embryologist A-1, A-2, A-3, A-4 and A-5 were 17.3%, 19.1%, 16.8%, 15.2%, and 18.9% respectively. The mosaicism rates of blastocyst biopsied by Embryologist B-1, B-2, B-3, B-4, B-5, B-6, B-7, and B-8 were 17.5%, 18.6%, 22.5%, 20.4%, 27.8%, 20.6%, 20.1% and 20.3% respectively. There were no significant differences in blastocyst mosaicism rate between embryologists within Group A (p > 0.05). Contrarily, in Group B, Embryologist B-5 had a significantly higher blastocyst mosaicism rate compared to the other embryologists within the same group (p < 0.05). Limitations, reasons for caution Since this study is retrospective in nature, the biopsy technique (either the laser or flicking method) was not controlled. Hence, further studies to analyse the differences between these 2 biopsy techniques should be carried out to confirm its effect on the occurrence of blastocyst mosaicism. Wider implications of the findings Our study demonstrates that blastocysts biopsied by embryologists with ≥1 year of biopsy experience have significantly lower mosaicism rate compared to those with <1 year of biopsy experience. This indicates that the skill and experience of an embryologist in biopsy may have an impact on the mosaicism rate. Trial registration number Not applicable

Prolonged interval time between blastocyst biopsy and vitrification compromised the outcomes in preimplantation genetic testing

Summary This study aimed to evaluate to what extent the different interval times between trophectoderm (TE) biopsy and vitrification influence the clinical outcomes in preimplantation genetic testing (PGT) cycles. Patients who underwent frozen embryo transfer (FET) after PGT between 2015 and 2019 were recruited. In total, 297 cycles with single day 5 euploid blastocyst transfer were included. These cycles were divided into three groups according to the interval times: <1 h group, 1–2 h group, and ≥2 h group. Blastocyst survival, clinical pregnancy, miscarriage, and ongoing pregnancy rates were compared. The results showed that, in PGT-SR cycles, survival rate in the ≥2 h group (96.72%) was significantly lower than in the <1 h group (100%, P = 0.047). The clinical pregnancy rate in the ≥2 h group was 55.93%, significantly lower than in the <1 h group (74.26%, P = 0.017). The ongoing pregnancy rates in the 1–2 h group and the ≥2 h group were 48.28% and 47.46%, respectively, significantly lower than that in the <1 h group (67.33%, P < 0.05). The miscarriage rate in the 1–2 h group was 18.42%, significantly higher than that in the <1 h group (5.33%, P = 0.027). In PGT-A cycles, the clinical pregnancy and ongoing pregnancy rates in the <1 h group were 67.44% and 53.49%, respectively, higher than that in the 1–2 h group (52.94%, 47.06%, P > 0.05) and the ≥2 h group (52.63%, 36.84%, P > 0.05). In conclusion, vitrification of blastocysts beyond 1 h after biopsy significantly influences embryo survival and clinical outcomes and is therefore not recommended.