Preimplantation Genetic Diagnosis Research Articles

Four Decades of Carrier Detection and Prenatal Diagnosis in Hemophilia A: Historical Overview, State of the Art and Future Directions.

Hemophilia A (HA), a rare recessive X-linked bleeding disorder, is caused by either deficiency or dysfunction of coagulation factor VIII (FVIII) resulting from deleterious mutations in the F8 gene encoding FVIII. Over the last 4 decades, the methods aimed at determining the HA carrier status in female relatives of HA patients have evolved from phenotypic studies based on coagulation tests providing merely probabilistic results, via genetic linkage studies based on polymorphic markers providing more accurate results, to next generation sequencing studies enabling highly precise identification of the causative F8 mutation. In parallel, the options for prenatal diagnosis of HA have progressed from examination of FVIII levels in fetal blood samples at weeks 20-22 of pregnancy to genetic analysis of fetal DNA extracted from chorionic villus tissue at weeks 11-14 of pregnancy. In some countries, in vitro fertilization (IVF) combined with preimplantation genetic diagnosis (PGD) has gradually become the procedure of choice for HA carriers who wish to prevent further transmission of HA without the need to undergo termination of pregnancies diagnosed with affected fetuses. In rare cases, genetic analysis of a HA carrier might be complicated by skewed X chromosome inactivation (XCI) of her non-hemophilic X chromosome, thus leading to the phenotypic manifestation of moderate to severe HA. Such skewed XCI may be associated with deleterious mutations in X-linked genes located on the non-hemophilic X chromosome, which should be considered in the process of genetic counseling and PGD planning for the symptomatic HA carrier. Therefore, whole exome sequencing, combined with X-chromosome targeted bioinformatic analysis, is highly recommended for symptomatic HA carriers diagnosed with skewed XCI in order to identify additional deleterious mutations potentially involved in XCI skewing. Identification of such mutations, which may profoundly impact the reproductive choices of HA carriers with skewed XCI, is extremely important.

SCM is potential resource for non-invasive preimplantation genetic testing based on human embryos single-cell sequencing

The ongoing development of assisted reproductive technologies has provided hope to individuals struggling with infertility, promising the potential for a healthy pregnancy. One significant innovation in field of pre-implantation genetic screening (PGS) requires the biopsy of embryos or oocytes, which has potential implications for the health and development of the resultant offspring. Therefore, a non-invasive approach to preimplantation genetic screening is highly sought after. The clinical application of non-invasive preimplantation genetic testing (ni-PGT) is currently limited, with its sensitivity and specificity requiring further investigation. In this study, we used 218 human embryos for single-cell whole genome amplification (WGA), along with ni-PGT of blastocoele fluid (BF) and spent culture medium (SCM). Whole blastocyst (WB), trophectoderm biopsy (TB), and inner cell mass (ICM) from embryo biopsies were used as controls to track genomic signal alterations. Our results showed that the overall genome similarity between SCM and ICM was higher than that of BF. Apart from the Y chromosome, both SCM and ICM demonstrated numerous variant sites across other chromosomes.Further categorization of gene variants in these two sample types revealed that missense variants were the most prevalent, single nucleotide polymorphisms were more common than insertions or deletions, and C > T was the dominant single nucleotide variants in both ICM and SCM. Lastly, we found that the mutant genes in SCM and ICM had different biological functions and pathways. This study indicates that SCM provides a more effective source of embryonic DNA for preimplantation genetic screening, offering a novel reference point for genetic screening research.

Novel economical, accurate, sensitive, single-cell analytical method for mitochondrial DNA quantification in mtDNA mutation carriers.

Although a variety of analytical methods have been developed to detect mitochondrial DNA (mtDNA) heteroplasmy, there are special requirements of mtDNA heteroplasmy quantification for women carrying mtDNA mutations receiving the preimplantation genetic diagnosis (PGD) and prenatal diagnosis (PD) in clinic. These special requirements include various sample types, large samplenumber, long-term follow-up, and the need for detection of single-cell from biopsied embryos. Therefore, developing an economical, accurate, high-sensitive, and single-cell analytical method for mtDNA heteroplasmy is necessary. In this study, we developed the Sanger sequencing combined droplet digital polymerase chain reaction (ddPCR) method for mtDNA quantification and compared the results to next-generation sequencing (NGS). A total of seventeen families with twelve mtDNA mutations were recruited in this study. The results showed that both Sanger sequencing and ddPCR could be used to analyze the mtDNA heteroplasmy in single-cell samples. There was no statistically significant difference in heteroplasmy levels in common samples with high heteroplasmy (≥ 5%), low heteroplasmy (< 5%), and single-cell samples, either between Sanger sequencing and NGS methods, or between ddPCR and NGS methods (P > 0.05). However, Sanger sequencing was unable to detect extremely low heteroplasmy accurately. But even in samples with extremely low heteroplasmy (0.40% and 0.92%), ddPCR was always able to quantify them. Compared to NGS, Sanger sequencing combined ddPCR analytical methods greatly reduced the cost of sequencing. In conclusion, this study successfully established an economical, accurate, sensitive, single-cell analytical method based on the Sanger sequencing combined ddPCR methods for mtDNA heteroplasmy quantification in aclinical setting.

Multi-level Fgf4- and apoptosis-dependent regulatory mechanism ensures the plasticity of ESC-chimaeric mouse embryo.

The preimplantation mammalian (including mouse and human) embryo holds remarkable regulatory abilities, which have found their application for example in the preimplantation genetic diagnosis of human embryos. Another manifestation of this developmental plasticity is the possibility of obtaining chimaeras by combining either two embryos or embryos and pluripotent stem cells, which enables the verification of the cell pluripotency and generation of genetically modified animals used to elucidate gene function. Using mouse chimaeric embryos (constructed by injection of embryonic stem cells into the 8-cell embryos) as a tool, we aimed to explore the mechanisms underlying the regulatory nature of the preimplantation mouse embryo. We comprehensively demonstrated the functioning of a multi-level regulatory mechanism involving the FGF4/MAPK signalling as a leading player in the communication between both components of the chimaera. This pathway, coupled with the apoptosis, cleavage division pattern, and cell cycle duration controlling the size of ESC component and giving it a competitive advantage over host embryo blastomeres, provides a cellular and molecular basis for regulative development, ensuring the generation of the embryo characterised by proper cellular composition.

Reproductive Counseling and Care in Cystic Fibrosis: A Multidisciplinary Approach for a New Therapeutic Era.

With the advent of highly effective modulator therapies, many people with cystic fibrosis (CF) are living longer, healthier lives. Pregnancy rates for women with CF more than doubled between 2019 and 2021, reflecting increases in both planned and unplanned pregnancies. For men with CF, CF-associated infertility can be mitigated with assistive reproductive technology, yet patient knowledge of these challenges and options is variable. Preconception and prenatal counseling for individuals with CF and for parents of children with CF who wish to expand their families requires nuanced discussions to promote informed reproductive decisions, drawing from a combination of standard practice recommendations and CF-specific assessments. This review article synthesizes the current literature and practice recommendations regarding reproductive counseling and care in CF, outlining the role of genetic counseling, carrier screening, teratogen counseling, in vitro fertilization and pre-implantation genetic diagnosis, and careful assessment and management of cystic fibrosis-related diabetes when present. Via a multidisciplinary, patient-centered approach, clinicians can support adults with CF and parents of children with CF as they make informed reproductive decisions and embark on family planning.

A Comprehensive Review of Pregnancy in Sickle Cell Disease.

Sickle cell hemoglobinopathies encompass a range of qualitative and quantitative hemoglobin disorders that are inherited genetically. This group of disorders includes sickle cell beta thalassemia, sickle cell trait, and sickle cell disease (SCD). Globally, SCD is the most common disorder. Even epidemiological data suggests the majority of diseases, as well as traits, are concentrated in Sub-Saharan Africa, North-East Africa, the Middle East, and India. The physiological changes in pregnancy predispose to an increased risk of catastrophic events like a vaso-occlusive crisis, thromboembolic events, and their related sequelae, leading eventually to villous infarction, necrosis, and fibrosis leading to compromising uteroplacental circulation. Conversely, the mother may exhibit exacerbated symptoms of gestational hypertension, placental abruption, preterm labor, and venous thromboembolism. Although this disease is manageable, it has the potential to adversely impact maternal and child health on a national level. The chances of severe complications in the pregnant state affecting both mother and fetus attract due attention of health services towards redefining and researching this disease and its management frequently. The literature review on the following situation advocates the general treatment to be observed under the headings of preconceptual care, strengthened antenatal care, strict intranatal care, and compliant post-natal care. Preconceptually, genetic screening of couples, with education on the adverse effects of the disease, comes as the first line of management. Newer facilities like preimplantation genetic diagnosis and celocentesis may even allow for early diagnosis as well as help patients who do not wish to terminate the pregnancy by selective transfer of unaffected embryos. This may be combined with an extensive evaluation of the psychosocial aspect and socioeconomic status of couples who administer vaccines as prophylaxis for preventable diseases. Strengthening antenatal care is associated with routine blood investigations for every registered antenatal patient with adequate awareness about the conditions that precipitate the crisis. All patients should be prophylactically treated with appropriate doses of aspirin, iron, folic acid, and multivitamins. Radiological examinations by ultrasonography may be used to monitor placenta previa, abruption, or preterm labor. Later in pregnancy, it should be recommended to performbiophysical profiling and assessment of umbilical artery flow. Intranatal care deals with strict-term institutional delivery of all sickle cell-diseased mothers with a preference for vaginal delivery. Post-natal care requires a precise assessment of blood loss during labor to initiate transfusion therapy as soon as needed. Exclusive breastfeeding, with the importance of early initiation of it, must be emphasized. Screening of neonates as quickly as possible must be done for hemoglobinopathies. Through this review, authors are trying to make aware of the complications that can be faced during pregnancy in SCD patients, its prevention, and its treatment according to various new guidelines and research available.

Outcomes of assisted reproductive technology procedures performed on weekdays versus weekends: a retrospective cohort study.

To evaluate if assisted reproductive technology (ART) outcomes are different based on whether procedures - oocyte retrieval, insemination, embryo biopsy, or embryo transfer - are performed on a weekday versus weekend/holiday. Retrospective cohort study of all patients ≥ 18years old who underwent oocyte retrieval for in vitro fertilization or oocyte banking (n = 3,197 cycles), fresh or natural-cycle frozen embryo transfers (n = 1,739 transfers), or had embryos biopsied for pre-implantation genetic testing (n = 4,568 embryos) in a large academic practice from 2015-2020. The primary outcomes were as follows: oocyte maturity for oocyte retrievals; fertilization rate for insemination; rate of no result on pre-implantation genetic testing for embryo biopsy; and live birth rate for embryo transfers. The average number of procedures performed per embryologist per day was higher on weekends/holidays than weekdays. For oocyte retrievals performed on weekdays vs. weekends/holidays, there was no difference in oocyte maturity rate (88% vs 88%). There was no difference in the fertilization rate (82% vs 80%) in cycles that had intracytoplasmic sperm injection performed on weekdays vs. weekends/holidays. No difference was found in the no result rate for embryos biopsied on weekdays vs. weekends/holidays (2.5% vs 1.8%). Finally, there was no difference by weekday vs. weekend/holiday in the live birth rate per transfer among all transfers (39.6% vs 36.1%), or when stratified by fresh (35.1% vs 34.9%) or frozen embryo transfer (49.7% vs. 39.6%). We found no differences in ART outcomes among women who had their oocyte retrievals, inseminations, embryo biopsies, or embryo transfers performed on weekdays versus weekends/holidays.

Impact of trophoectoderm biopsy for preimplantation genetic testing on serum β-hCG levels, time of delivery and birthweight following frozen embryo transfer cycles

Aim: This study investigated whether trophoectoderm (TE) biopsy adversely impacts serum β-human chorionic gonadotropin (hCG) level on the 15th day of embryo transfer (ET), delivery week and birthweight, between biopsied and unbiopsied embryo groups, in a cohort of women who delivered a singleton baby, following frozen–thawed ET. Methods: All women having had a live birth after blastocyst ETs following frozen ET cycles with preimplantation genetic testing (PGT) were included. A control group was selected among women who had a live birth following single frozen blastocyst transfer without PGT-A at the same period in our clinic Results: One hundred fifteen and 173 cycles with- and without-PGT, respectively, were included. Serum β-hCG level on the 15th day after ET was comparable between the groups (p = .336). Average birthweight of the babies born following biopsied embryos were significantly lower (3200 vs. 3380; p = .027). Women who received trophectoderm biopsied embryos had a significantly higher probability of having a baby weighing ≤1500 g and 1500–2500 g (p = .022) or ≤2500 g (p = .008). Proportion of preterm delivery was significantly higher in the biopsy group (p = .023). However, after adjusting for potential covariates, trophectoderm biopsy did not seem to increase the risk of preterm birth (OR 1.525; 95% CI, 0,644–3.611; p = .338) Conclusions: TE biopsy does not seem to impact serum β-hCG level on the 15th day after ET. Average birthweight is lower when a biopsied embryo was transferred. After adjusting for potential covariates, trophectoderm biopsy does not seem to increase the risk of preterm birth

Serum progesterone concentration on pregnancy test day might predict ongoing pregnancy after controlled ovarian stimulation and fresh embryo transfer.

Progesterone (P4) is essential for pregnancy. A controlled ovarian stimulation (COS) leads to a iatrogenic luteal defect that indicates a luteal phase support (LPS) at least until pregnancy test day. Some clinicians continue the LPS until week 8 or later, when P4 is mainly secreted by syncytiotrophoblast cells.Measuring serum P4 on pregnancy test day after a fresh embryo transfer could help to identify women who might benefit from prolonged LPS. In women with LPS based on P4 administered by the rectal route, P4 concentration on pregnancy test day was significantly higher in patients with ongoing pregnancy than in patients with abnormal pregnancy.This monocentric retrospective study used data on 99 consecutive cycles of COS, triggered with human chorionic gonadotropin, followed by fresh embryo transfer resulting in a positive pregnancy test (>100 IU/L) (from November 2020 to November 2022). Patients undergoing preimplantation genetic screening or with ectopic pregnancy were excluded. All patients received standard luteal phase support (i.e. micronized vaginal progesterone 600 mg per day for 15 days). The primary endpoint was P4 concentration at day 15 after oocyte retrieval (pregnancy test day) in women with ongoing pregnancy for >12 weeks and in patients with miscarriage before week 12 of pregnancy.The median P4 concentration [range] at pregnancy test day was higher in women with ongoing pregnancy than in women with miscarriage (55.9 ng/mL [11.6; 290.6] versus 18.1 ng/mL [8.3; 140.9], p = 0.002). A P4 concentration ≥16.5 ng/mL at pregnancy test day was associated with higher ongoing pregnancy rate (OR = 12.5, 95% CI 3.61 - 43.33, p <0.001). A P4 concentration ≥16.5 ng/mL at pregnancy test day was significantly associated with higher live birth rate (OR = 11.88, 95% CI 3.30-42.71, p <0.001).After COS and fresh embryo transfer, the risk of miscarriage is higher in women who discontinue luteal support after 15 days, as recommended, but with P4 concentration <16.5 ng/mL. The benefit of individualized prolonged luteal phase support should be evaluated.

P-773 No impact of cleavage-stage or blastocyst-stage embryo biopsy on growth and health in children up to 2 years of age

Abstract Study question Does embryo biopsy for preimplantation genetic testing (PGT) followed by fresh or vitrified-warmed embryo transfer affect children’s health up to 2 years of age? Summary answer Cleavage- or blastocyst-stage embryo biopsy followed by fresh or vitrified-warmed-embryo transfer had neither impact on anthropometry at birth, infancy or childhood nor on health outcomes. What is known already Literature data regarding neonatal outcomes after embryo biopsy for PGT show mixed results due to small sample sizes and/or the heterogeneity in terms of embryo biopsy (cleavage- or blastocyst-stage) and type of embryo transfer (fresh or frozen-thawed). Even fewer data exist on the impact of embryo biopsy on children’s health beyond infancy, including growth. Study design, size, duration This single-center cohort-study compared outcomes in singletons conceived after cleavage- or blastocyst-stage embryo biopsy either in a fresh or vitrified-warmed transfer cycle with results after non-biopsied embryo transfer between 2014 and 2018. Pregnancies after IVM, oocyte vitrification or oocyte/embryo donation were excluded. Eligible singletons living in Belgium were invited for examination at 3-6 months and 2 years. Anthropometric measurements at birth, infancy and childhood and health outcomes including surgeries, medication use and hospitalisations are reported. Participants/materials, setting, methods Birth characteristics were available for 630 and 222 children after cleavage-stage and blastocyst-stage embryo biopsy and for 1532 children after transfer of a non-biopsied embryo. Follow-up data were available for 426, 131 and 662 children, respectively. The impact on children’s health following embryo biopsy in vitrification and fresh cycles was the primary outcome. Other outcomes were the impact of timing of biopsy and of vitrification. Subgroup analysis according to infertility background was additionally performed. Main results and the role of chance Regarding the impact of embryo biopsy, either at the cleavage or blastocyst stage, no differences in anthropometrics were found at birth, infancy or childhood in vitrified-warmed transfer cycles compared to outcomes in non-biopsied vitrified-warmed transfer cycles, even after adjustment for neonatal, treatment and maternal characteristics. Likewise, no impact of embryo biopsy (cleavage stage) was found in fresh transfer cycles. The timing (day 3 or day 5/6) of embryo biopsy did not affect anthropometrics at birth, infancy or childhood. However, children born after cleavage-stage embryo biopsy followed by a vitrified-warmed transfer cycle had larger birth sizes than children born after cleavage-stage biopsy followed by a fresh transfer. Weight and height gain from birth to infancy and from infancy to early childhood were comparable in all biopsied and non-biopsied groups. Reassuringly, comparable rates of major congenital malformations, (severe) developmental problems, hospital admissions, surgical interventions and of chronic medication use up to the age of 2 years were observed in biopsied and non-biopsied groups. Subgroup analysis in children born to parents with an infertility diagnosis showed that birth parameters were not different in children born after embryo biopsy. Limitations, reasons for caution As the majority of the PGT cycles in our center are performed for a genetic indication only, the number of children born after embryo biopsy in infertile parents is rather limited and the results should therefore be interpreted cautiously. Wider implications of the findings Our findings add reassurance that embryo biopsy does not adversely affect the health of offspring up to 2 years of age. This is of particular importance given the widespread shift to blastocyst-stage embryo biopsy in PGT. Trial registration number Not applicable

P-241 Cumulative live birth rates after embryo transfers at Day-2/3 versus Day-5/6: a comparative nationwide cohort study

Abstract Study question Are there significant differences between cumulative live birth rates (CLBR) after a short and an extended embryo culture when comparisons are performed per cycle? Summary answer The CLBR per cycle was significantly lower in the Day-5/6 group and higher per embryo transfer as compared with the Day-2/3 group. What is known already Previous evidence has shown the probability of live birth following a fresh embryo transfer in In Vitro Fertilization (IVF) is higher after extended embryo culture (E-EC: Day-5/6) than after a shorter embryo culture (S-EC: Day-2/3). A further controversy literature has arisen on cumulative pregnancy rates, related to the higher risks of embryo transfer cancellation after E-EC compared with S-EC. Moreover, all of these studies used slow freezing/thawing embryo protocols with a higher survival for Day-2/3 embryos than Day-5/6 embryos. Finally, a recent study combining vitrification highlighted CLBR improvements after E-EC but CLBR were expressed per embryo transfer, not per cycle. Study design, size, duration A French national register study including all IVF cycles with at least one cleaved embryo at day-2 performed in France from 01/2016 to 12/2018 was carried out (n = 165,808 cycles), after exclusion of gamete donation cycles, attempts with preimplantation genetic diagnosis, surgical spermatozoa, viral context, and for which the embryo freezing was performed by slow-freezing. We chose to include only cycles from IVF centers with standardized CLBR similar or above to the national mean (99%CI). Participants/materials, setting, methods From the biomedicine Agency’s registry, two groups were identified: cycles with embryo(s) at Day-2/3 with/without embryo transfer (S-Group) and cycles with all embryos cultured to Day-5/6 with/without embryo transfer (E-Group). The CLBR defined as delivery of at least one live-born infant in fresh or in subsequent frozen-thawed cycles were compared. Only the first delivery was considered. Regression model adjusted for women age, parity, Day-2 embryos, IVF-method and attempt rank was used. Main results and the role of chance In total, 48,293 attempts fulfilled the inclusion criteria. Among them, 31,769 cycles were included in the S-Group and 16,524 in the E-Group. Overall, the CLBR per cycle was significantly lower in the E-Group compared with the S-G group (32.9% versus 33.5%; aOR: 0.89, 95%CI [0.86-0.93], p&amp;lt;.0001). A significant higher rate of fresh embryo transfer cancellation was observed in in the E-Group compared with the S-Group (19.8% versus 3.1%, p &amp;lt; .0001). However, when we performed further comparisons restricted to data from IVF centers with LBR strictly significantly above to the national mean LBR, the CLBR were similar between both groups (36.4% versus 38.5% in S-Group and E-Group, respectively; aOR: 0.97 [0.90-1.04], p = 0.402). Finally, the CLBR in E-group was significantly higher than in S-group when expressed per embryo transfer (aOR: 1.15 [1.10-1.21], p&amp;lt;.0001). Limitations, reasons for caution Main limitations are: the retrospective design and the 1 year follow-up that could not include all frozen embryo cycles performed. Wider implications of the findings Overall, the nationwide results per cycle suggest that extended embyo culture until blastocyst stage, even used in combination with vitrification, could not improve live birth rates. Trial registration number not applicable

P-713 Preimplantation DNA methylation screening (PIMS) illustrates the outcome of assisted reproductive technology

Abstract Study question It is unknown whether using DNA methylation can improve the outcome of ART and can exclude the imprinted gene diseases. Summary answer Our study provides a criterion on selecting blastocyst according to DNA methylome, which can improve live birth rate and decrease birth defect rate. What is known already How to select the embryos is one key factor that can determine the successful rate of ART. As aneuploidy exists in 20-30% of blastocysts, preimplantation genetic screening (PGS) has been widely used, which increases the live birth rate. Unfortunately, the live birth rate remains around 50% even with the help of PGS. DNA methylation is known to play important function during embryogenesis. Previous study has shown that a large proportion of human embryos have abnormal DNA methylome. However, whether DNA methylation pattern can affect the clinical outcome of ART has not been investigated in clinics. Study design, size, duration To determine whether PIMS method can increase live birth rate during ART, the whole genome DNA methylation of 3-5 biopsied cells from trophectoderm were measured. The methylation level and chromosome copy number variation (CNV) were analyzed by using the methylome data. elective single embryo transfer (eSET) will be performed for embryos with euploid chromosome. The live birth rate, pregnant rate and abortion rate will be examined. 182 families including 800 blastocysts were enrolled in PIMT. Participants/materials, setting, methods Eligible couples include advanced maternal age, recurrent pregnancy loss with unknown reason, recurrent implantation failure with unknown reason, severe oligoteratozoospermia. Eligible couples must have two or more good-quality blastocysts. We call a blastocyst as good quality if the blastocyst is 4BC or better according to the Gardner morphologic criteria on day 5-6 of embryo culture. Main results and the role of chance No one has ever performed clinical trial to investigate the impact of DNA methylation of blastocyst on the outcome of artificial assisted reproductive (ART). Here, we performed PIMS using the biopsied trophectoderm of 800 blastocysts. Chromosome copy number variation and DNA methylation pattern were analyzed by using methylome data. 162 of euploid embryos were transferred. Our data show that significant variation of DNA methylation level of blastocysts can be observed in both younger women (£37 years old) and older women (&amp;gt;37 years). Blastocysts with global methylation level around 0.26 have the highest birth and pregnancy rate, but the lowest abortion rate. The higher the difference methylation value from 0.26 is, the lower the birth rate becomes. Furthermore, by using artificial intelligence method according to different methylated regions between the embryos succeeding in live-birth and those failing in live-birth, we can robustly predict the clinical outcome of blastocysts. PIMS method can find the abnormality of DNA methylation states in the imprinted control regions which can cause the imprinted diseases. Our study provides a criterion on selecting blastocyst according to DNA methylome during ART, which can potentially improve the live birth rate and decrease the birth defect rate. Limitations, reasons for caution Because of the low coverage of single cell DNA methylome, PIMS method cannot cover all imprinted control regions. Wider implications of the findings PIMS method can potentially find more human diseases which is caused by DNA methylation mutation. Trial registration number NCT03642574

P-160 Trophectoderm biopsy techniques with and without laser use and their contribution to PGT-A results in sibling embryos

Abstract Study question Do the methods used in trophectoderm biopsy and the use of laser affect the genetic results of the harvested cells and the embryos thus examined? Summary answer The different methods of trophectoderm biopsy showed no differences in terms of euploidy rate and the specific type of chromosomal abnormalities. What is known already Next generation sequencing (NGS) is a comprehensive method for the genetic screening of preimplantation embryos and is now favored by many clinics. The method can detect structural abnormalities on all chromosomes and offers better reproductive success compared to earlier tests. However, as the test is very sensitive, biopsy of the embryo and treatment of the biopsied embryos and harvested cells are important to improve diagnostic success. Contamination of the biopsied sample can affect the results of the genetic test and lead to misdiagnosis. Study design, size, duration A prospective, randomized controlled trial of 44 couples undergoing infertility treatment with pre-implantation genetic screening (PGT-A, NGS) at the British Cyprus IVF Hospital between October 2021 and December 2022. Participants/materials, setting, methods PGT-A cases with more than two blastocysts were included in the study. Randomization was performed on days 5 and 6 of embryo development and embryos were divided into two groups before the biopsy. In group 1, embryos were biopsied using the flicking method without laser assistance. In group 2 embryos, cells were removed by pulling the desired cells with 3 to 5 laser shots at the edge of the selected cell group. Main results and the role of chance PGT-A results of 288 blastocyst-stage embryos from 44 cases were analyzed. 136 embryos were in group 1 (flicking) and 152 to group 2 (laser). The embryos in the groups were siblings, so the basic patient characteristics, number of eggs retrieved, sperm characteristics, and early embryonic details were identical between the groups. The mean number of retrieved cells was 6.19 ± 0.915 and 6.14 ± 0.849 (p = 0.764), the number of euploid embryos was 49/122 (40.2%) and 57/137 (41.6%) (p = 0.814), the number of embryos with whole chromosome aneuploidy was 46/73 (63%) and 58/80 (72.5%) (p = 0.209) and embryos with complex aneuploidy (more than two abnormal chromosomes) were 30/73 (41.1%) and 40/80 (50%) (p = 0.268) in group 1 and group 2, respectively. Limitations, reasons for caution As this is a preliminary research result, the small sample size is the major limitation. Mosaicism, which is common in preimplantation embryos and has a significant impact on the results, was not investigated. Wider implications of the findings Although the present data showed no differences between the two biopsy procedures in terms of PGT-A test results, invasive embryo biopsy procedures can damage embryos or misdirect genetic test results. Therefore, validation and universalization of the method for embryo biopsy are needed, which is emerging in recent publications. Trial registration number not applicable

P-632 Micronized progesterone is better than GnRH antagonist to prevent LH surge in oocyte donation cycles

Abstract Study question Micronized progesterone or GnRH antagonist: which is better to control the LH surge in the egg donation process? Summary answer Micronized progesterone is better. Is just as effective as GnRH antagonist and offers three additional advantages: cheaper, more convenient, and saves intermediate controls. What is known already GnRH antagonists have been widely considered to prevent LH surge. When fresh embryo transfer is not advised, micronized progesterone has been shown to be an effective alternative with some other very interesting advantages. If efficacy would prove to be similar, there will be three obvious advantages for the preferential use of micronized progesterone over the antagonist protocol: oral or vaginal administration is preferred over subcutaneous injection, cost would be much lower, and progesterone can be used from day one and avoid inconvenient intermediate controls. This would be particularly interesting in egg preservation, preimplantation genetic screening, and oocyte donation programs. Study design, size, duration We retrospectively analyzed our oocyte donation program from 2018 to 2021. All 358 cycles under short cycle protocol were studied. 67 donors were under the Antagonist cycle; age 24,7 years (18 – 32) with normal Body Max Index (22,2 Kg/m2 range 17-29) 291 donors were under the micronized progesterone cycle; age 24,5 years (18-31) with normal Body Max Index (22,3 Kg/m2 range 18-29) Participants/materials, setting, methods 67 donors were under the Antagonist cycle: LH suppression was accomplished by subcutaneous injections of Ganirelix 0.25 mg starting when follicles &amp;gt;14mm or E2 levels &amp;gt;400 pg/ml and continued until GnRH triggering. No patients were cancelled. 291 donors were under the micronized progesterone cycle: endogenous LH suppression was accomplished by vaginal administration of micronized progesterone (200 mg) once a day at bedtime, from stimulation day 1 and continuing until GnRH triggering. Four patients were cancelled. Main results and the role of chance 67 donors were under the GnRH antagonist cycle. 1115 oocytes were retrieved (16,6 range 2-39). All but two donors had egg vitrification: 65 patients, 916 oocytes (14,1 range 1-30) so 82% could be preserved. (916 / 1115). From 67 cycles, 916 vitrified oocytes were obtained (average 13,7). 291 patients were under the micronized progesterone cycle. Four patients were cancelled (no response in two, follicular asynchrony in one and personal reasons in one. No LH surges were observed). From 287 egg collections, 4683 oocytes were retrieved (16,3 range 2-49). All but two patients had vitrification: 285 patients, 3784 oocytes (13,3 range 2-37) so 81% could be preserved. (3784 / 4683). From 291 cycles, 3784 vitrified oocytes were obtained (average 13,2) Both groups were similar: total oocytes recovered per patient (16,3 vs 16,6) egg maturation (81 vs 82%) and global results (13,2 vs 13,7) Micronized progesterone offers three additional advantages: is obviously cheaper than GnRH antagonist. Also, vaginal route is commonly preferred to subcutaneous injections. And allows to offer a more flexible donor program making unnecessary to set some intermediate follicular controls. CONCLUSION: Micronized progesterone offers similar results than GnRH Antagonist with three additional advantages: is cheaper, is more convenient and intermediate controls can be avoided. Limitations, reasons for caution Although they are most likely similar, fertilization and pregnancy rates after both protocols should be deeply analyzed. Wider implications of the findings Micronized progesterone would also be particularly useful in egg preservation cycles and preimplantation genetic screening programs. Trial registration number not applicable

P-719 Evaluation of genetic risk of apparently balanced chromosomal rearrangement carriers by breakpoint characterization

Abstract Study question What are breakpoint characteristics of ABCR carriers? What are the genetic risks of ABCR carriers caused by chromosome rearrangements? Summary answer We analyzed the characteristics of ABCR's breakpoints and found that heterogeneous deletion of some AD disease-related genes may not result in clinical phenotype. What is known already ABCRs are common chromosomal abnormalities. People with ABCRs generally lack any visible abnormal features; however, during childbirth, the abnormal pairing and missegregation of homologous chromosomes can lead to the production of unbalanced gametes, resulting in spontaneous abortion or the birth of children with chromosomal disorders. Prenatal diagnosis or preimplantation diagnosis can effectively prevent the birth of children with unbalanced chromosome rearrangements, but no substantial data has been collected to report on the characteristics and potential genetic risks of the chromosome breakage caused by the rearrangement. Study design, size, duration This is a retrospective study of 2441 breakpoints in 1219 ABCR carriers who underwent Preimplantation Genetic Testing (PGT) at the Reproductive and Genetic Hospital of Citic-Xiangya from August 1, 2016 to December 31, 2021. Participants/materials, setting, methods All patients in this study sought PGT treatment due to primary or secondary infertility caused by ABCRs. To characterize the chromosome breakpoints, the MicroSeq technique, which involves chromosome microdissection and next generation sequencing, was employed. Additionally, gap-junction PCR and Sanger sequencing were utilized to further validate some of the breakpoints. Main results and the role of chance In this study, we retrospectively analyzed 2441 chromosome rearrangement breakpoint MicroSeq localization data from 1219 ABCR carriers, including 2364 breakpoint regions in 1182 reciprocal translocations, 68 breakpoint regions in 34 inversions, and 9 breakpoint regions in 3 insertional translocations. We not only identified 8q24.13, 11q11.23, and 22q11.21 as previously reported hotspots of chromosomal balanced rearrangement breaks, but also identified the 10 Mb region of 12q24.13-q24.3 as a possible rare region of balanced rearrangement breaks. the average range of breakpoints identified by MicroSeq technology was only within 3.8 Kb, which clarified 95.82% (2339/2441) of balanced chromosomal rearrangements as to whether they broke genes or not. We identified 960 genes that were interrupted, of which 53 genes with autosomal dominant mode of inheritance (AD) were interrupted. Clinical phenotypic evaluation of these patients carrying interrupted AD genes identified that some of the AD genetic interruptions may not have a clinical phenotype. Limitations, reasons for caution MicroSeq cannot precisely pinpoint breakpoints to the gene or single base level. Moreover, carriers of ABCRs were not comprehensively assessed for other clinical characteristics, and the alteration in function of the known Haploinsufficiency gene could not be confirmed. Additionally, the position effect of the breakpoints has not been evaluated. Wider implications of the findings This investigation offers a reference for evaluating the pathogenicity and genetic risks of protein-truncation mutations in some AD genes. Trial registration number Not applicable

O-050 The impact of embryo biopsy on obstetric and neonatal outcomes

Abstract Background Preimplantation genetic testing of embryos (PGT) was performed first in 1990 to prevent the transmission of X-linked disease. Since then, the technique has improved to include couples who are both carriers of the same autosomal recessive disorder, to those with balanced translocations having recurrent pregnancy loss, and to women with advanced maternal age who are at higher risk for having aneuploid embryos. Originally, PGT involved biopsy of the polar body or of one blastomere of a cleavage stage embryo for diagnosis. The contemporary practice of PGT involves the culture of embryos to the blastocyst stage (5-7 days after fertilization), with biopsy of several trophectoderm cells that would become the placenta, transport of biopsy tissue off-site for genetic analysis, and cryopreservation of blastocysts until biopsy results are available. A single euploid embryo is then transferred into the uterus as a frozen embryo transfer. Objective We sought to determine if removal of these cells for preimplantation genetic testing was associated with adverse obstetrical or neonatal outcomes after frozen-thawed single embryo transfer compared to frozen-thawed single embryo transfer without biopsy. Further, we review other studies of contemporary practice of PGT to draw conclusions about the safety of the practice. Study design We linked assisted reproductive technology surveillance data from the Society for Assisted Reproductive Technology Clinic Outcome Reporting System to birth certificates and maternal and neonatal hospitalization discharge diagnoses in Massachusetts, USA, from 2014-2017, considering singleton births after frozen-thawed single embryo transfer. We compared outcomes of cycles having embryo biopsy (n = 585) to those having no biopsy (n = 2,191), adjusting for mother’s age, race, education, parity, body mass index, birth year, insurance, and all infertility diagnoses (Sites CK et al, Am J Obstet Gynecol 2021;225:285.e1-7). Results With no biopsy used as the reference, we found no differences between no biopsy and biopsy groups with respect to preeclampsia, pregnancy-induced hypertension, placental disorders (placental abruption, placenta previa, placenta accreta, placenta increta, and placenta percreta), preterm birth, low birthweight, cesarean delivery, gestational diabetes mellitus, or maternal or infant length of stay after delivery. Comparing our outcomes to 4 other studies of contemporary PGT practice of frozen-thawed embryo transfers (Zhang WY Fertil Steril 2019, Li M Am J Obstet Gynecol 2020, Makhijani R Hum Reprod 2021, He H Fertil Steril 2019), our data are consistent with no effect of biopsy on low birth weight, pregnancy-induced hypertension, gestational diabetes, placenta previa, and placenta accreta. A slight decrease in preterm delivery (RR 1.10, CI 1.02-1.18), slight reduction in cesarean section (RR 0.90, CI 0.82-0.99), and increase in intrauterine growth restriction (RR 1.21, CI 1.06-1.38) are reported with biopsy in a systematic review (Hou W, Fertil Steril 2021). The increase in risk for intrauterine growth restriction with biopsy was influenced by one study (Li M Am J Obstet Gynecol 2020). Conclusion Embryo biopsy of trophectoderm cells for PGT in contemporary practice appears to be safe generally with respect to maternal and neonatal outcomes. There is no clear increase in diagnoses related to placentation (preeclampsia, pregnancy-induced hypertension, placental disorders, preterm birth, low birthweight), cesarean delivery, gestational diabetes mellitus, or maternal or infant length of stay after delivery. Further study of a possible increase in intrauterine growth restriction with biopsy is indicated.

P-703 The role of maternal polymorphic karyotype variants among embryonic ploidy and mosaicism status in patients undergoing IVF treatments

Abstract Study question Is there a relationship between the chromosomal status of biopsied embryos and the presence of polymorphic variants in the female karyotype in IVF cycles? Summary answer The chromosomal status of biopsied embryos is prejudiced because of the presence of specific maternal polymorphic variants (qh-, qh+ and ps+) or combinations of them. What is known already Due to the increasing average age of patients who undergo IVF cycles with their own oocytes, more cycles are performed with embryo biopsy for preimplantation genetic test of aneuploidies (PGT-A). Thus, advanced maternal age has been defined as the main factor causing embryonic chromosomal abnormalities. However, few studies have established direct relationships with other factors. The prevalence of chromosomal polymorphic variants has a higher incidence in the infertile population, which is why there is a need to know the role they play in the genetic inheritance of embryos. Study design, size, duration Retrospective evaluation of a cohort of women who underwent autologous IVF cycles and karyotyping. The sample included 162 IVF cycles with embryo biopsy on D5 and/or D6 for PGT-A, performed between July 2017-December 2021: control group (CG) with normal karyotype (86) and study group (SG) with presence of polymorphisms (76). We studied the connection between maternal karyotype polymorphisms and molecular genetic outcomes in terms of euploid, mosaic, and aneuploid blastocyst rates. Participants/materials, setting, methods The analysis of the karyotype of women, prior to the IVF cycle, was performed using the guidelines of the International System for Human Cytogenetic Nomenclature (ISCN). The trophoectoderm biopsies on D5 and/or D6 blastocysts were analysed by NGS using the Illumina platform (VeriSeq Illumina®, San Diego, CA, USA). The association between the different polymorphic variants in the female karyotype and chromosomal status of embryos was analysed using R (v. 4.2.0) statistical software. Main results and the role of chance We analyzed the results of 483 embryos from 162 IVF-PGT-A cycles. Mean female age was 39.05±2.72 (CG) and 38.97±2.90 (SG). Mean number of mature oocytes (MII) was 7.16±3.94 (CG) and 8.01±5.33 (SG). Mean number of biopsied embryos was 2.97±2.15 (CG) and 2.93±1.88 (SG). No statistically significant differences were found between the CG and SG groups in terms of the rate of euploid embryos: 26.99% vs 21.50% (p = 0.3), transferable mosaic embryos: 8.90% vs 6.76% (p = 0.5), aneuploid embryos: 61.98% vs 69.96% (p = 0.12) and non-informative embryos: 2.13% vs 1.78% (p = 0.2), respectively. However, an increase in the rate of chromosomally abnormal embryos was observed when analysing each polymorphism individually. In females carrying the qh+ variant, we found a statistically significant decrease in the euploid embryo rate: 10.97% vs 26.00% found in CG (p = 0.036). Moreover, the variants qh+ and ps+ increased the rate of aneuploid embryos: 84.17% vs 63.48% in CG (p = 0.013) and 73.52% vs 61.30% in CG (p = 0.021), respectively. Furthermore, it is found that the qh- variant increased the rate of transferable mosaic embryos: 20.00% vs 8.90% in CG (p = 0.025). The results were corrected for confounding variables such as maternal age, oocyte origin and male factor variables, including polymorphic variants in the male karyotype. Limitations, reasons for caution Presence of unmeasured variables that may influence the results. Larger prospective studies including homogeneous cohorts are needed in order to corroborate our initial results. Wider implications of the findings The results show that polymorphic variants in the female karyotype may have a direct effect on the embryonic ploidy and mosaicism status. Therefore, it would be advisable for patients who undergo IVF-PGT-A cycles to have a karyotype performed and be informed of the implications that it may entail. Trial registration number Not applicable

P-186 Combination of proteomics and automatic scoring using artificial neural networks to detect aneuploid embryos

Abstract Study question How powerful is the combination of artificial neural networks that combine embryo's protein profile with its automatic score provided by time-lapse videos to predict ploidy? Summary answer An artificial neural network (ANN) that considers proteomics and automatic score assigned by deep learning achieves 71% accuracy in distinguishing between euploid and aneuploid embryos. What is known already Currently the most widely used technique for detecting chromosomal abnormalities involves biopsy of the developing embryo. However, it has several disadvantages related to invasiveness, technical difficulty, high economic costs, etc. Therefore, different non-invasive techniques are being studied for the detection of aneuploid embryos. The discovery of cell-free DNA (cfDNA) released by the embryo to the culture media during its development marked the beginning of a new era of noninvasive PGT (niPGT) but some factors require adaptation for the analysis. Also, artificial Intelligence (AI) represents a valuable alternative to developing new models for predicting PGT outcomes without disturbing the embryo. Study design, size, duration This study included 294 samples of culture medium from 81 treatments of the PGT-A program. Out of the total, 23 were control samples (medium in which no embryos had been cultured) and 271 were samples where there was a developing embryo. Embryos were cultured until the blastocyst stage in EmbryoScope systems (Vitrolife, Sweden) with single-step medium (Gems, Genea) and automatically scored by iDAScore v2 algorithm from 1 to 9.9. Participants/materials, setting, methods The spent culture medium was collected on day 5/6 of embryo development and chromosome analysis was performed using next-generation sequence technology (Juno Genetics, Valencia). The relative concentrations of 92 proteins were analysed using Proseek Multiplex Assays (Olink Bioscience) using 1 μl of each sample. The final assay readout was presented as Normalized Protein eXpression (NPX) values. Finally, we developed our own ANN algorithms considering protein profile and automatic embryo score. Main results and the role of chance For euploid embryos (n = 101), 35 protein samples analysed had different NPX values between conditioned and control media*. The relative concentration was reduced for 11 proteins (consumption by the embryo) and 24 proteins increased their levels (secretion). For aneuploid embryos (n = 170), 33 protein samples analysed had different NPX values between conditioned and control media*. The relative concentration was reduced for 4 proteins and 29 proteins increased their levels. Out of the total, only 6 proteins had on average different concentrations between normal and abnormal embryos*: MCP_1, IL_17A, CXCL1, IL18, IL_22RA1 and CSF_1. Additionally, the automatic embryo score provided by iDAScore v2 algorithm was higher for euploid embryos than for aneuploid embryos (5.9 ± 2.7 vs. 5.1 ± 2.6)*. For ploidy prediction, three architectures of ANN were developed with different input data ANN1 (six discriminatory proteins), ANN2 (automatic embryo score) and ANN3 (six discriminatory proteins and automatic embryo score). Our dataset was divided into 68% training, 16% validation and 16% test. The accuracy, sensibility and specificity for the test phase were as follows: 63.6%, 76.9% and 44% for ANN1; 61%, 11.8% and 95.8% for ANN2; and 71.1%, 62.5% and 77.3% for ANN3. Limitations, reasons for caution Only one laboratory by using single step culture medium from one brand was involved in this study. Wider implications of the findings Our study showed a new approach to avoid transferring aneuploid embryos in cases where embryo biopsy is not performed. In addition, further studies on this field may result in a new non-invasive methodology for detecting aneuploidy. Trial registration number PI21/00283

P-228 Reliability of blastomere versus trophectoderm biopsy in preimplantation genetic testing for mitochondrial DNA disorders

Abstract Study question Can a single blastomere or trophectoderm (TE) biopsy accurately reflect the heteroplasmy levels of the whole embryo for mitochondrial DNA (mtDNA) disorders? Summary answer Heteroplasmy levels of single blastomere and TE are comparable to those of the rest of the embryo, including the inner cell mass (ICM). What is known already Oocytes of women carrying mtDNA mutations can display varying mutation loads – percentage of mutated mtDNA copies. Preimplantation genetic testing (PGT) aims at reducing the risk of having affected children by screening for embryos with minimal mtDNA heteroplasmy. The technique involves either a blastomere biopsy on cleavage stage embryos or TE biopsy from blastocysts, followed by genetic analysis to select embryos suitable for transfer. However, it remains unclear whether there is a uniform segregation of heteroplasmic mtDNA during the early stages of embryogenesis, and therefore whether the mutation load from blastomere or TE biopsies are representative of the whole embryo. Study design, size, duration We investigated the suitability of PGT for mtDNA disorders by comparing the mutation load between a single blastomere recovered at the eight-cell stage, TE cells and rest of the same blastocyst (n = 15) in a mouse model heteroplasmic for the m.5024C&amp;gt;T mutation. We also compared the levels of heteroplasmy between TE and ICM of heteroplasmic human blastocysts (n = 5) and between blastomeres of arrested cleavage stage embryos (n = 4) donated by two mtDNA disease patients. Participants/materials, setting, methods To obtain heteroplasmic mouse embryos, we mated female mice carrying a mitochondrial tRNAAla mutation (m.5024C&amp;gt;T) with wild-type males. Whereas in human experiments, we analysed preimplantation embryos from two different carriers of the m.3243A&amp;gt;G mutation, associated with MELAS syndrome, who showed 16% and 32% mutation load in their peripheral blood. After embryo biopsy, NGS was performed in three independent technical replicates and profiles were compared between samples from the same embryo to assess heteroplasmy levels. Main results and the role of chance Analysis of heteroplasmic mouse embryos showed a strong correlation in mtDNA heteroplasmy between blastomere and the corresponding embryo (r2 = 0.76), and between TE and the rest of the blastocyst (r2 = 0.94) (n = 15). Moreover, a strong correlation was observed between the biopsied blastomere and TE from the same embryo (r2 = 0.90). Heteroplasmy levels in the embryos analysed ranged from 55.3% to 88.3%, and the mean overall mutation load was 73.7 ± 7.9%. Development of biopsied embryos was comparable to those of non-manipulated mutant (n = 10) and wild-type embryos (n = 10) (blastocyst rate = 85.7%). For human blastocysts (n = 5), statistical analysis showed a strong correlation in heteroplasmy levels between ICM and TE portions (r2 = 0.98). Moreover, a modest correlation was observed among blastomeres of cleavage stage human embryos (r2 = 0.60) (n = 24 from 4 embryos). The mutation load in the embryos analysed ranged from 10.3% to 57.4%, and the mean overall heteroplasmy was 42.8 ± 12.2% in cleavage stage embryos and 38.6 ± 17.3% in blastocysts. Overall, concordance in heteroplasmy levels between single blastomeres, TE portions and corresponding blastocyst was established in human and mouse embryos. Limitations, reasons for caution These results should be further validated by increasing the sample size and by the inclusion of different mtDNA mutations. Furthermore, current data are suggestive, but not definitive, to guarantee that the mtDNA mutation load in the embryo will remain constant throughout life. Wider implications of the findings Taken together, results suggest that TE biopsy may be better than blastomere biopsy for mtDNA disorders. Moreover, biopsy at the blastocyst stage is likely less harmful for embryo development. Following, we will also investigate how heteroplasmic mtDNA segregates at the peri-implantation stages after the resumption of mtDNA replication. Trial registration number Not applicable

P-533 Comparison of Endometrial Receptivity Array (ERA) with Preimplantation genetic testing (PGT) versus ERA alone in improving outcomes in Recurrent implantation failure.

Abstract Study question Whether addition of Preimplantation Genetic Testing for aneuploidy (PGT-A) to Endometrial Receptivity Array (ERA) helps in improving success rates in Recurrent Implantation Failure (RIF) patients. Summary answer Addition of PGT-A intervention to ERA in RIF patients did not improve outcomes. What is known already RIF causes great emotional stress and financial burden for the infertile couple. Successful implantation requires a synchronous interaction between a competent blastocyst and a receptive endometrium. Though ERA, as a measure to assess window of implantation (WOI), is around a decade old, its role in improving success rates in patients with RIF is still controversial. PGT-A, as a tool for selecting euploid embryos, though has shown to be beneficial in women with advanced maternal age, its role in improving success rates in RIF needs further evaluation. Study design, size, duration This is a multicenter retrospective cohort study carried out from September 2015 till September 2022. 285 patients with RIF with previous failed transfers with ≥ 3 good quality embryos were included. Participants/materials, setting, methods 285 patients with RIF were divided into 2 groups. Group 1 underwent PGT-A and Personalised Embryo Transfer (pET) guided by ERA and Group 2 underwent only ERA followed by pET. In Group 1 patients, embryo biopsy was done on day 5 and day 6 blastocyst before cryopreservation and NGS platform was used to detect euploid embryos. Both groups then underwent ERA followed by pET in the next cycle. Main results and the role of chance In total of 285 RIF patients studied, 59.2% patients showed displaced WOI. 91.7% ERA reports were pre-receptive. Group 1 included 137 patients and Group 2 had 148 patients &amp; baseline characteristics were similar in both the groups. Group 1 had more self transfer cycles, i.e. 76.7% self and 17.2% donor oocytes, as compared to Group 2 with 40.2% self and 30.7% donor oocyte cycles. The implantation rate (IR), live birth rate (LBR), miscarriage rate and cumulative LBR were similar in both the groups. Higher Pregnancy rate (PR) in Group 2 (70.9% vs 57.83%) could be attributed to higher donor cycles in this group. Multiple gestation rate was also higher in Group 2 (31.49% vs 11.21%) as 2 blastocysts were transferred in most cycles in group 2 compared to Single embryo transfer(SET) in most cycles in Group 1. As PR was higher in ERA group, subgroup analysis of self gamete transfers and donor oocyte transfers was also done, which showed similar PR, IR and LBR in both the groups. Hence, PGT failed to show any value in improving pregnancy outcomes in RIF. Limitations, reasons for caution Retrospective study design. Wider implications of the findings The role of PGT in improving clinical outcomes in RIF patients needs to be studied in larger prospective studies. In our study, ERA as an intervention improved outcomes in RIF. Trial registration number not applicable