Preimplantation Genetic Diagnosis Research Articles

CREATING SAVIOR SIBLINGS: MORAL OR IMMORAL?

A savior sibling is a child conceived through in-vitro fertilization for the purposes of saving a sibling, who needs an organ or cell transplant because of a fatal genetic disorder. The procedure involves pre-implantation genetic diagnosis (PGD), which analyzes the embryos to detect potential genetic disorders prior to implantation. This is to reduce the possibility of passing such disorders to offspring. However, the creation of savior siblings also raises issues of morality. The idea that a child is born for the sole purpose of saving the lives of others, even if siblings, is not acceptable to some sections of society. This is because such a child is being born not for the sake of having it, but merely to be used as an instrument by the parents to save their sick child. Different countries, such as Malaysia, the UK, U.S. and Australia take various positions on whether or not to permit PGD. This article examines the rationale for the conduct of PGD, and, specifically, whether or not the act of creating a savior child can be considered as immoral. Importantly, the examination is undertaken with a focus on Immanuel Kant’s moral theory. The notion of the end in itself by Immanuel Kant demands that we always treat humanity not only as a means to an end, but also as an end in itself. Impliedly, parents should not conceive a child solely for the purposes of serving as a donor to a sick sibling, but should also genuinely desire that child. In other words, they should also respect the savior sibling and recognize its inherent value. In this way, the savior sibling is treated not merely as a means, but also as an end. By implication, the creation of savior siblings is not unethical as such, and should be permitted. A more significant factor that should be considered is how they are treated by parents after birth. This article uses a qualitative research methodology and relies on scholarly writings, such as books, journal articles, as well as laws and decided cases.

ESHRE 2023 Abstract Highlights

The following selected highlights draw attention to numerous interesting timely abstracts presented at the European Society of Human Reproduction and Embryology (ESHRE) 39th Annual Meeting, covering topics such as obstetric and neonatal outcomes following embryo biopsy, the role of genital tract infection and pelvic surgery in endometriosis, and the impact of fertility and preservation in patients with cancer.

Single closed-tube quantitative real-time PCR assay with dual-labelled probes for improved sex determination of equine embryos

In addition to fulfilling many breeders’ curiosity, equine embryonic sex determination can have a profound commercial impact. However, the application of currently described assays for equine embryonic sexing has rendered variable diagnosis and validation rates, with sensitivity being the main problem. In addition, while pregnancy results of in vivo-flushed equine embryos following a needle aspiration biopsy equal those of non-biopsied embryos, the effect on in vitro-produced embryos is unknown. Here, we aimed to develop a highly sensitive and specific assay for equine sex determination that can be directly performed on few embryonic cells, and to test the effect of a needle aspiration biopsy on the viability of the in vitro-produced embryo. To this end, a multiplex quantitative real-time PCR (qPCR) assay with dual-labelled probes was designed to allow the simultaneous generation of both male-specific and control fragments in a single closed-tube reaction, avoiding potential sample loss or contamination. To improve sensitivity, multicopy and polymeric genes were chosen to be specifically amplified, i.e., eight copies of Y-chromosomal ETSTY5 as male-specific and four autosomal UBC monomers as control fragment. Specificity was enhanced by the equine-specific character of ETSTY5 and by using dual-labelled probes. The assay was optimised with equine male and female genomic DNA and demonstrated a 100% accuracy and a >95% qPCR efficiency down to 10 pg of DNA. The assay was subsequently applied to determine the sex of 44 in vitro-produced embryos, collecting trophectoderm biopsies by means of a needle aspiration biopsy and herniating cells. Of all trophectoderm biopsies and herniating cell samples (n = 54), 87% could be diagnosed. Assay results were validated on a second sample obtained from the biopsied embryo (n = 18) or, by ultrasound-based sex determination of the foetus (n = 7) following the transfer of the biopsied embryo to a recipient mare, with about half of the embryos being fillies and colts. The needle aspiration biopsy procedure did not impair initial pregnancy rate or early pregnancy losses as compared to non-biopsied embryos. In conclusion, we report a safe, reliable, fast, and cost-effective assay for equine sex determination which was validated for the sex determination of in vitro-produced embryos based on few embryonic cells, and needle aspiration biopsy did not impair the embryo's viability. The assay and safe biopsy strategy hold potential for other applications.

EXPLORING THE BUILDING BLOCKS OF LIFE: THE HUMAN GENOME PROJECT, ITS IMPACT IN TODAYS WORLD

The Human Genome Projects (HGP) conclusion altered a number of disciplines, including biotechnology, genetics, medicine, and forensics. Key outcomes included personalised medicine, gene therapy, and enhanced DNA profiling. The HGP increased our knowledge of hereditary illnesses and shed light on human evolution. The privacy of genetic information and the ethical application of knowledge were raised. The management and analysis of genomic data require the use of bioinformatics. The future of genetics and biology will be shaped by the HGPs substantial effects on healthcare, innovation, and the economy. In vitro fertilisation and preimplantation genetic diagnosis are two reproductive technology developments made possible by the HGP. Also, it sparked the creation of focused treatments for genetic diseases, completely altering the range of patient treatment options. These advancements have led to a new era of precision medicine and personalised healthcare, in addition to improving patient outcomes.

Current Applications and Controversies in Preimplantation Genetic Testing for Aneuploidies (PGT-A) in In Vitro Fertilization.

Preimplantation genetic testing for aneuploidy (PGT-A) has evolved over recent years, including improvements in embryo culture, biopsy, transfer, and genetic testing. The application of new comprehensive chromosome screening analysis has improved the accuracy in determining the chromosomal status of the analyzed sample, but it has brought new challenges such as the management of partial aneuploidies and mosaicisms. For the past two decades, PGT-A has been involved in a controversy regarding its efficiency in improving IVF outcomes, despite its widespread worldwide implementation. Understanding the impact of embryo aneuploidy in IVF (in vitro fertilization) should theoretically allow improving reproductive outcomes. This review of the literature aims to describe the impact of aneuploidy in human reproduction and how PGT-A was introduced to overcome this obstacle in IVF (in vitro fertilization). The article will try to analyze and summarize the evolution of the PGT-A in the recent years, and its current applications and limitations, as well as the controversy it generates. Conflicting published data could indicate the lacking value of a single biopsied sample to determine embryo chromosomal status and/or the lack of standardized methods for embryo culture and management and genetic analysis among other factors. It has to be considered that PGT-A may not be a universal test to improve the reproductive potential in IVF patients, rather each clinic should evaluate the efficacy of PGT-A in their IVF program based on their population, skills, and limitations.

A novel mutation on OCA2 gene detection and preimplantationgenetic diagnosis for oculocutaneous albinism type 2 (OCA2)

Oculocutaneous albinism (OCA) is a group of recessive genetic congenital disorders characterised by a disorder of melanin synthesis. OCA is currently classified into seven different types, of which OCA type 2 (OCA2 - OMIM 203200) ranks as the second most common type of albinism in the world and accounts for nearly 30% of albinism cases. Nowadays, there is currently no known way to treat OCA. Preimplantation genetic diagnosis (PGD) is the most effective method of disease prevention, which helps families carrying gene mutations to have healthy children. Objectives: To develop a procedure and conduct PGD for embryos of a family carrying the OCA2 mutation causing OCA2. Materials and methods: Research subjects are blood samples of four members of an OCA family, including father, mother, and two daughters; and 6 embryo biopsies on day 5 of this parents. Next-generation sequencing (NGS) was carried out on family members participating in the study. Based on the identified mutation results, primers for PCR amplification of the segments spanning the detected mutation were designed. Then, Sanger sequencing method is combined with linkage genetic analysis to detect pathogenic mutations on embryo biopsies after whole-genome amplification (WGA). Results: A novel mutation NM_000275.3:c.2323G>A (NP_000266.2: p.Gly775Ser) was detected on exon 22 of the OCA2 gene. The outcomes of PGD for 6 embryos of a family carrying the OCA2 mutant gene causing OCA2 were 3 affected embryos, 2 normal embryos, and 1 carriers.

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